ENCYCLOPEDIA OF

FOOD AND HEALTH

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 ENCYCLOPEDIA OF
FOOD AND HEALTH
EDITORS-IN-CHIEF

BENJAMIN CABALLERO

PAUL M. FINGLAS

FIDEL TOLDRÁ

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EDITORS-IN-CHIEF

Benjamin Caballero is professor of International Health and of Maternal and Child Health
(Bloomberg School of Public Health), and professor of pediatrics (School of Medicine) at Johns
Hopkins University.
He obtained his MD from the University of Buenos Aires, his MSc in biochemistry from the
University of San Carlos, and his PhD in neuroendocrine regulation from MIT, in Cambridge, MA.
He started his academic career as assistant professor of pediatrics at Harvard Medical School and
director of the Nutrition Unit of Boston Children’s Hospital, and subsequently became the found-
ing director of the Center for Human Nutrition at Johns Hopkins University, in Baltimore.
Prof. Caballero has focused his research on child nutrition and health in developing countries. In
particular, he has explored the combination of undernutrition and overweight that has become
increasingly prevalent in low- and middle-income countries. He was a member of the Food and
Nutrition Board of the Institute of Medicine, National Academy of Sciences, USA, and of a number
of expert panels created by the Institute, including the Dietary Reference Intakes (DRI) Committee,
the Expert Panel on Macronutrient Requirements, and the Childhood Obesity Task Force. He was
also a member of the Dietary Guidelines for Americans Advisory Committee, of the Scientific
Advisory Board of the Food and Drug Administration (FDA), and of a number of advisory
committees of the National Institutes of Health (USA).
He is the editor-in-chief of the Encyclopedia of Food Sciences and Nutrition, a 10-volume work on
food production, consumption and biological effects. He is also editor-in-chief of the Encyclopedia of Human Nutrition, which received the
Book of the Year Award from the British Medical Association. His Guide to Dietary Supplements summarizes the current scientific basis for the
use of mineral and vitamin supplements. His book The Nutrition Transition: Diet and Disease in the Developing World explored the impact of
demographic and economic development on diet- and lifestyle-related diseases in developing countries. His book Obesity in China
summarizes research conducted in rural and urban China to track the impact of socioeconomic development on health outcomes. He is
also coeditor of a widely used textbook on human nutrition, Modern Nutrition in Health and Disease.
He is a member of the Spanish Academy of Nutritional Sciences, and a Fellow of the American Society for Nutrition and of the Royal
Society of Medicine (UK). Recent awards include the Donald Medearis Lectureship from the Massachusetts General Hospital/Harvard
Medical School, the Mataix Prize for lifetime achievements in nutrition science from the Spanish Academy of Nutritional Sciences, the Ancel
Keys Prize for achievements in international public health, and the Thompson–Beaudette Lectureship from Rutgers University.

Paul Finglas joined the Institute of Food Research in 1981 and is currently head of the Food
Databanks National Platform and Research Leader in Food and Health at the Institute (http://www.
ifr.ac.uk/science/platform/FD/default.html). He has, for most of his science career, been involved
in a wide range of research in food composition and analysis, and the nutritional effects of
micronutrients in food and health research. Paul has considerable experience of co-coordinating
both national and international projects (e.g., EuroFIR, TDS-EXPOSURE, Bacchus and QualiFY (all
EU FP7), and is currently of the spin-out EuroFIR AISBL, a non-profit international association
based in Belgium, from one of these projects. Paul has a broad range of experience in science
publishing and is currently editor of the journals Food Chemistry and Trends in Food Science and
Technology, and was one of the coeditors for the Encyclopedia of Food Science and Nutrition (2nd Ed.).
Paul has a degree in chemistry from Aston University in Birmingham and has published over 150
publications on a wide range of topics in food science and nutrition.

v
vi Editors-in-Chief

Fidel Toldrá holds a BSc in chemistry (1980), high degree on food technology
(1981) and PhD in chemistry from the University of Valencia (1984). Professor
Toldrá was a Fulbright postdoctoral scholar at Purdue University in West Lafayette
(US, 1985–86) and visiting scientist at the University of Wisconsin-Madison
(1991 and 1995), and the Institute of Food Research-Bristol (UK, 1987). Cur-
rently, he is research professor at the Instituto de Agroquı́mica y Tecnologı́a de
Alimentos (CSIC), in Paterna, Valencia (Spain). He is also associate professor of
food technology at the Polytechnical University of Valencia.
Prof. Toldrá has focused his research on food biochemistry and its relationship
with nutrition, quality and safety. He has filed 12 patents, directed 22 PhD thesis
and published over 245 manuscripts in recognized scientific journals and more
than 115 chapters of books. His h-index is 41. Prof. Toldrá has authored two
books and edited/co-edited more than 30 books for major publishers like CRC
Press, Wiley-Blackwell, Elsevier and Springer.
Prof. Toldrá is the European editor of Trends in Food Science and Technology
(2005–) and associate editor of Meat Science (2014–); he was the editor-in-chief of Current Nutrition & Food Science (2005–2012), section
editor of the Journal of Muscle Foods (2009–2010) and guest editor of 12 special journals issues. He is a member of the editorial boards of
Food Chemistry, Food Analytical Methods, Journal of Food Engineering, Journal of Food and Nutrition Research, The Open Nutrition Journal, The
Open Enzyme Inhibition Journal, Recent Patents in Agriculture, Food and Nutrition, Food Science & Nutrition and Current Opinion in Food Science.
He has been a member of the Scientific Panel on food additives, flavorings, processing aids and materials in contact with foods (periods
2003–2008) and the Scientific Panel on flavorings, enzymes, processing aids and materials in contact with foods (2008–2015) of the
European Food Safety Authority (EFSA) acting as Chairman of the Working groups on Irradiation (2009–2010), Processing Aids
(2011–2014) and Enzymes (2010–2015). He was a member of FAO/WHO group of experts to evaluate chlorine-based disinfectants in
the processing of foods (2008–2009). He was a member of the Executive Committee of the European Federation of Food Science and
Technology (EFFOST, 2002–2009). He is a Fellow of the International Academy of Food Science and Technology (IAFOST, 2008) and of the
Institute of Food Technologists (IFT, 2009–). He received the Iber Award on Food and Cardiovascular Diseases (1992), the Institute Danone
award in Food, Nutrition and Health (2001), the International Prize for Meat Science and Technology from the International Meat
Secretariat (2002), GEA award on RþD activity from the Valencian Community (2002), and the Distinguished Research Award (2010)
and Meat Processing Award (2014), both from the American Meat Science Association.
EDITORIAL ADVISORY BOARD

Siân Astley has worked extensively with individuals and organizations throughout Europe from a
variety of disciplines including research, food and biotech industries, and the media. She is the
author of more than 300 popular science articles for magazines and trade publications as well as 25
peer-reviewed papers, and she was awarded her Diploma in Science Communication in 2009
(Birkbeck University of London). After 14 years as a bench-scientist, Siân became
communications manager for NuGO, one of the first FP6 networks of excellence, and was the
European communications manager for the Institute of Food Research in Norwich (UK) until April
2012. Currently, she is the training and communications manager for the European Food Infor-
mation Resource (EuroFIR AISBL) supporting training within EU-funded research projects and
networks, and communication of research activities.

David J. Baer is a supervisory research physiologist with the US Department of Agriculture’s

Beltsville Human Nutrition Research Center located in Beltsville, Maryland. He serves as the
research leader for the Center’s Food Components and Health Laboratory and also serves as the
director of the Center’s Human Studies Facility.
Dr. Baer conducts controlled dietary intervention studies to investigate the relationship between
diet and the risk for chronic degenerative diseases, especially cardiovascular disease, cancer, and
diabetes in people. His research also includes studies on the health impacts of weight gain and
determining the calorie content of foods. Some of the dietary interventions he has investigated
include the effects of different types of protein, fats and fatty acids, fiber, margarine, butter, plant
sterols, salad dressings, nuts, whole grains, berries, alcohol, and tea on overall nutrition and health.
In addition to dietary intervention studies, Dr. Baer is involved in research studies to validate food
survey methodologies and in developing new methods for dietary assessment.
Dr. Baer earned his bachelor’s degree from the University of Illinois and his master’s and
doctorate in nutrition from Michigan State University.

vii
viii Editorial Advisory Board

Marina Carcea was awarded a master degree in agricultural sciences at the University of Pisa, Italy
“cum laude” in 1980, and a PhD in food science also “cum laude.”
She is currently a senior researcher in the Research Center on Food and Nutrition of the Council
for research in agriculture and analysis of agricultural economy (CRA-NUT formerly INRAN
National Research Institute on Food and Nutrition) and she was the director of the Cereals
Research Programme in INRAN. CRA-NUT is a primary research institute in Italy under the egis
of the Ministry of Agriculture. Dr. Carcea joined INRAN in 1989 after having worked in Italian and
English universities (Queen Elizabeth College, King’s College, and University of London) and after
a two-year contract with the Food and Agriculture Organization (FAO) of the United Nations (UN),
Rome.
She has a vast experience in the field of research on foods, cereals in particular. In recent years,
her main research interests have been: chemical characterization and study of the functional
properties of cereal components; study of the interactions between components and of the
interrelationships between the biochemical properties of components and the technological prop-
erties of the raw material and derived products; development of new, cereal-based products;
development of methods to assess technological parameters of the raw material; nutritional
value of cereals; and developments of protocols for quality assurance of cereals, food authenticity.
She has taken part and/or co-ordinated several research projects within national or international
programs (European Commission, FAO) involving several institutions. She is the author of more
than 160 scientific publications, mostly in international journals, eight book chapters, and two
scientific books. She delivered lectures on her research activity at about 150 national and international congresses and she seats in several
national and international committees (Italian Ministry of Agriculture, Codex Alimentarius, and European Commission) regarding food
and nutrition topics. She is also a member of the editorial board of scientific journals.
From 1994 to 2006, she has also been a lecturer of food science and technology at the University of Tor Vergata, Rome, Italy.
She is a founding member of AISTEC, the Italian Association of Cereal Science and Technology. Since 1996, she is an elected member in
the Executive Committee of the same association and since 2009, president of the association.
Since 2000, she is the Italian National Delegate of the International Association for Cereal Science and Technology (ICC) and she was also
the president of the same association for 2011–2012.
In 2004, she was the first woman to be awarded the International Harald Perten Prize for her excellent research achievements in the field
of cereal science and technology.
She is also a member of the Georgofili Academy in Florence, Italy.

Lawrence J. Cheskin graduated from Dartmouth Medical School and completed a fellowship in
gastroenterology at Yale–New Haven Hospital. He is an associate professor of health, behavior, and
society at the Johns Hopkins Bloomberg School of Public Health, with a joint appointment in
International Health–Human Nutrition, and in medicine (GI) at the Johns Hopkins University
School of Medicine. Dr. Cheskin is also a founder and director of the Johns Hopkins Weight
Management Center, a comprehensive treatment program for obesity.
In his research, Dr. Cheskin has studied the effects of medications on body weight, the gastro-
intestinal effects of olestra, how cigarette smoking relates to dieting and body weight, and the
effectiveness of lifestyle and dietary changes in weight loss and weight maintenance.
He is also the author of four books: Losing Weight for Good, New Hope for People with Weight
Problems, Better Homes and Gardens’ 3 Steps to Weight Loss, and Healing Heartburn. Dr. Cheskin has
appeared on television news programs and lectured to both professional and lay audiences on the
topics of obesity and weight control.
 Editorial Advisory Board ix

Nigel Cook is a graduate of the University of Dundee. After postdoctoral research in the Univer-
sities of Aberdeen and Leicester, he moved to the Central Science Laboratory (now the Food and
Environment Research Agency (FERA)) at the Food Science Laboratory, Torry, Aberdeen in Sep-
tember 1994, before relocating to new facilities in York. At FERA, he studies the transmission of
pathogens, particularly enteric viruses, through foods and the environment. He has a visiting
professorship at the Katholieke Universiteit Leuven in Belgium. He is a councilor of the Interna-
tional Association for Food and Environmental Virology. He is a project leader within the
standardization working group ISO TC34 SC9 WG6, currently developing a standard for detection
of Cryptosporidium and Giardia on berry fruits and leafy green vegetables. He was a coordinator of
the European Framework 7 project “Integrated monitoring and control of foodborne viruses in
European food supply chains (VITAL),” and a chair of COST Action 929 “A European Network for
Environmental and Food Virology” from 2006 to 2010. Between 2009 and 2014, he was a member
of various European Food Safety Authority’s Working Groups preparing opinions on the risk of
foodborne viruses, and represented the European Communities on the Codex Committee on Food
Hygiene Working Group developing Guidelines on the Application of General Principles of Food Hygiene
to the Control of Viruses in Food. He was a member of the UK Advisory Committee on the
Microbiological Safety of Food’s Viral Infections Subgroup. He was the founding editor of the
journal Food and Environmental Virology, published by Springer.
Luca Simone Cocolin graduated in 1994 in food science with a grade of 110/110 and remark
followed by food biotechnology PhD studies from 1995 to 1998. In February 1999, he defended
his thesis acquiring the title of PhD in food biotechnology. From 1998 to 2001, he received a
scholarship from the Friuli Venezia Giulia region (Italy). From November 1, 2001, he was an
assistant professor at the University of Udine, Faculty of Agriculture, Food Science Department,
Italy, and in October 1, 2006, he became an associate professor at the University of Torino, Italy. In
January 2014, he had the habilitation for full professor and from June 2015, he is the full professor
in food microbiology at the University of Torino.
From September 2008, he is an executive board member of the International Committee on
Food Microbiology and Hygiene (ICFM) part of the International Union of Microbiological
Societies (IUMS) (http://www.icfmh.org/). From January 2008, he is the editor-in-chief of the
International Journal of Food Microbiology and he is a member of the editorial board of Applied and
Environmental Microbiology, Food Analytical Methods, Frontiers in Food Microbiology, and Frontiers in
Nutrition and Food Science Technology. He regularly reviews paper for Food Microbiology, Meat Science,
Journal of Applied Microbiology, and Letters in Applied Microbiology. He is a co-author of more than 180
papers on national and international journals and he attended national and international con-
gresses with oral and poster presentations. On Scopus (www.scopus.com, consulted on March
2015) he has 172 documents reviewed, which were cited 3520 times, resulting in an h index of 33.
His scientific interests comprise:
development, optimization, and application of molecular methods for the detection, quantification, and characterization of foodborne
pathogens;
study of the microbial ecology of fermented foods (mainly sausage, cheese, and wine) by using culture independent and dependent
methods;
bioprotection: molecular characterization of bacteriocin production and its study in vitro and in situ;
selection of new putative probiotics from artisanal fermented foods; and
study of the human microbiome and its influence on human health.

Christopher Duggan, for the past 25 years, has been performing clinical trials in the fields of
pediatric nutrition, gastroenterology, and global health. His early work centered on the manage-
ment of diarrheal diseases in children, including trials that demonstrated the feasibility and efficacy
of oral rehydration solutions (ORS) for diarrhea management in the United States and globally. In
collaboration with colleagues at Harvard TS Chan School of Public Health and Muhimbili Uni-
versity of Health and Allied Sciences in Dar es Salaam, Tanzania, Dr. Duggan and colleagues are
evaluating the efficacy of micronutrient supplementation in infants and young children born to
women with or at risk of HIV infection. Recent studies include the development of new biomarkers
of environmental enteric dysfunction as well as the evaluation of nutritional status on neurodeve-
lopment. With colleagues at St John’s Research Institute in Bangalore, India, he is evaluating the
efficacy of maternal vitamin B12 supplementation on biochemical and clinical parameters during
pregnancy and infancy. He is a course co-director of the Bangalore, Boston Nutrition Collaborative
(http://bbnc.globalhealth.harvard.edu). Past and present research support has come from the
National Institutes of Health, the Gates Foundation, and the World Health Organization.
In addition to his global health research interests, he is a pediatric gastroenterologist and a
nutrition physician at Boston Children’s Hospital where he directs the Center for Nutrition (http://www.childrenshospital.org/nutrition).
He is a medical director of the Center for Advanced Intestinal Rehabilitation, one of the largest centers in the United States for the care of
children with intestinal failure/chronic diarrhea syndromes (http://www.childrenshospital.org/cair). He is also the course co-director of an
inaugural Harvard College course “Nutrition and Global Health” and mentors undergraduate, graduate, and postdoctoral students at HMS
and HSPH (http://www.hsph.harvard.edu/nutrition-and-global-health/).
He is a professor of pediatrics at Harvard Medical School and a professor in the Department of Nutrition at the Harvard TS Chan School of
Public Health (http://www.hsph.harvard.edu/faculty/christopher-duggan/).
x Editorial Advisory Board

Jed William Fahey’s current research concerns elucidating the mechanisms of how plants protect
themselves against unfavorable and stressful conditions, and how this understanding can be
translated to chemoprotection of eukaryotic mammalian systems. This work draws on elements
of natural product chemistry, enzymology, nutritional epidemiology, and clinical research in order
with isothiocyanates (e.g., sulforaphane) and glucosinolates. His work led to the discovery that
broccoli sprouts are an exceptionally rich and consistent source of phytochemicals that induce the
detoxification of carcinogens, and to the development of methods for their detection and for
assessing their metabolism in humans. He discovered that one of the inducers, sulforaphane, has
potent antibiotic activity against Helicobacter pylori, a causative agent of peptic ulcer and stomach
cancer, and followed up with trials in animals and in H. pylori-infected humans. Ongoing collab-
orations examine the effects of broccoli, Moringa, and the other plants and their phytochemicals
against a range of chronic diseases. Dr. Fahey has for years taught courses in chronic disease
prevention and nutrition at both medical and public health schools.

Manuel Franco is an associate professor at University of Alcalá in Madrid (Spain) where he leads
the social and cardiovascular epidemiology research group (http://www3.uah.es/cardiosocialepi/).
He is also an adjunct professor at Johns Hopkins University (Baltimore).
Prof. Franco’s work focuses on the social determinants of cardiovascular diseases and its major
risk factors as diet. His methodological interests include the measurement of the urban environ-
ment and large social and economic changes in relation to cardiovascular health. He is the lead
investigator of the Heart Healthy Hoods, study funded by the European Research Council, that will
study the urban environment in relation to cardiovascular health in Madrid (http://hhhproject.
eu/). This longitudinal study will be collecting neighborhood level data (via audits, Google Street
view, photovoice, and qualitative methods) and linking them to clinical outcomes collected from
patients enrolled at the City of Madrid primary healthcare clinics. Prof. Franco trained in Spain and
Germany to obtain his MD and obtained his PhD from Johns Hopkins Bloomberg School of Public
Health working with Dr. Ana Diez-Roux in the MESA study on food environment and dietary
patterns. He has published over 30 international high impact articles and collaborates with
universities in the United States, Europe, and Latin America.

Maria Glibetic is a research director of Centre of Research Excellence in nutrition research, Institute
for Medical Research in Belgrade, University of Belgrade, Serbia, and member of executive board of
directors for food data association EuroFIR AISBL. She is an experienced basic and nutritional
scientist with over 250 scientific publications and presentations. Maria has considerable experience
in leading national and international projects and since 2006, she participated in nine EU funded
projects including EuroFIR, EURRECA, BaseFOOD, CHANCE, BACCHUS, and ODIN. Maria and
her team are responsible for the creation of the first online national food database, for designing
food data management system, and for the development of different nutritional tools for intake
analysis. She was a principal leader of many nutrition intervention studies evaluating the plant
bioactive component effects on human cardiovascular health. She leads postgraduate department
for integrated nutritional sciences at University of Belgrade, where she teaches two courses.

Linda Harvey obtained her PhD from the University of East Anglia, UK. She is currently the head of
the Human Nutrition Unit at the Institute of Food Research, Norwich, UK. Her research interests
include micronutrient requirements, bioavailability, and metabolism.

Ronald Jackson received his bachelor’s and master’s degrees from Queen’s University and
doctorate from the University of Toronto. His time in Vineland, Ontario, and subsequent
sabbatical at Cornell University, redirected his interest in Botrytis toward viticulture and
enology. As part of his teaching duties at Brandon University, he developed the first wine
technology course in Canada. For many years he was a technical advisor to the Manitoba
Liquor Control Commission, developing sensory tests to assess candidates of its sensory
panel, and was a member of its external tasting panel. He is the author of Wine Science:
Principles and Applications, 4th edition (2014), Wine Tasting: A Professional Handbook, 2nd
edition (2009), Conserve Water, Drink Wine, and chapters and technical reviews in other
multiple books and encyclopedia. He is retired in Bronte, Ontario, but remains active
writing, cycling, doing yoga, and traveling, as well as being a fellow in the Cool Climate
Viticulture and Oenology Institute, Brock University, St. Catharines, Ontario, Canada.
 Editorial Advisory Board xi

Joe P. Kerry is a senior college lecturer and the head of the food packaging research
group in the School of Food and Nutritional Sciences at University College Cork
(UCC). He received his doctorate in microbiology at University College Galway in
1995. Prof. Kerry is also a qualified member of the Institute of Packaging. He is very
involved in national and international research projects both at fundamental and
applied levels. Primary research interests address various aspects of food packaging,
shelf-life stability, food composition, and numerous aspects of food quality, partic-
ularly in relation to muscle foods. He also has very strong links with industry and his
research team assists companies in relation to many aspects of new food product
development. He has over 220 publications in peer-reviewed international journals,
over 300 presentations at major international conferences, along with several other
significant publications. His expertise includes use and manipulation of modified
atmosphere packaging systems for use with foods, use of extrusion technology for the manufacture of food products/packaging materials,
and applications and sensor/new technology developments within the area of food packaging, especially in the area of smart packaging.

Frédéric Leroy, after studying bio-engineering at Ghent University, obtained a PhD in applied
biological sciences at the Vrije Universiteit Brussel in 2002, where he continued his academic career
at the research group of Industrial Microbiology and Food Biotechnology (faculty of sciences and
bio-engineering sciences). As associate professor, his lecturing activities include courses in food
science and technology (i.e., “Nutrition,” “Technology of animal products,” “Food microbiology
and ecology,” and “Quantitative and predictive microbiology”). Dr. Leroy’s research primarily
deals with the ecology and functional roles of bacterial communities in (fermented) foods, in
particular with respect to the generation of quality, safety, and/or nutritional and health advan-
tages. Focus is mostly on meat products, but other food systems are also being studied, including
fermented milks and sourdough breads. In addition, his research interests relate to elements of
tradition and innovation in foods, both from a technological and societal point of view.

Catherine M. Logue completed her undergraduate and postgraduate degrees in Ireland and earned
a PhD in meat microbiology from the University of Ulster, UK in 1996. Dr. Logue was a faculty
member at North Dakota State University from 1999 to 2011 rising through the ranks of assistant
to associate and full professor. In 2011, she re-located to Iowa State University’s College of
Veterinary Medicine and is a professor of veterinary microbiology and preventive medicine. Dr.
Logue is also the director of faculty and staff advancement and equity for the college. Her research
interests focus on foodborne pathogens of food animals and the contamination of meat and meat
products destined for human consumption. Her research studies the detection, isolation, and
characterization of a range of foodborne pathogens such as Salmonella, Campylobacter, Listeria,
Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA) in poultry, bovine, and
swine. She also focuses her research on antimicrobial resistance in commensals and pathogens of
production animals. She has been an author and a co-author on more than 90 peer-reviewed
papers and book chapters as well as more than 150 abstracts and presentations at national and
international meetings.

F. Xavier Malcata graduated in chemical engineering in 1986 from the University of Porto
(Portugal), received a PhD in chemical engineering/food science from University of Wisconsin,
Madison (USA) in 1991, and his habilitation in food science and engineering by Portuguese
Catholic University in 2002. He was the dean of College of Biotechnology of Portuguese Catholic
University, the chairman of Portuguese Society of Biotechnology, Portuguese representative at VI
and VII European Union Framework Programs of research and development, expert for European
Food Safety Agency, and a co-ordinator of Portuguese Engineering Accreditation Board in chemical
engineering for Northern Region. He is currently a full professor at University of Porto.
His major research interests have focused on technological improvement of traditional Portuguese
foods and upgrade of byproducts thereof, development of nutraceutical ingredients and functional
foods, design and optimization of enzymatic reactors for edible oil processing, characterization of plant
proteases toward cheese and whey cheese manufacture, production of starter and nonstarter cultures
from adventitious microflora, and optimized application of unit operations to food processing.
With an academic career of independent research and teaching for more than two decades,
Prof. Malcata published more than 450 papers in refereed journals worldwide, wrote 11 books, and
prepared more than 45 chapters for edited books. Among many international distinctions, he was
xii Editorial Advisory Board

recipient of Ralph H. Potts Memorial Award in 1991 by American Oil Chemists’ Society (AOCS, USA), Foundation Scholar Award – Dairy
Foods in 1998 by American Dairy Science Association (ADSA, USA), Young Scientist Research Award in 2001 by AOCS, Canadian/
International Constituency Investigator Award in physical sciences and engineering in 2002 and 2004 by Sigma Xi (USA), Danisco
International Dairy Science Award in 2007 by ADSA, Scientist of the Year Award in 2007 by European Federation of Food Science and
Technology (the Netherlands), Samuel C. Prescott Award in 2008 by Institute of Food Technologists (IFT, USA), International Leadership
Award in 2008 by International Association for Food Protection (IAFP, USA), Elmer Marth Educator Award in 2011 by IAFP, Distinguished
Service Award in 2012 by ADSA, and William V. Cruess Award in 2014 by IFT. He has been elected for the honor societies of food science
(Phi Tau Sigma, USA), scientific research (Sigma Xi, USA), and engineering (Tau Beta Pi, USA). He was also elected for fellow of IFT, ADSA,
AOCS, and International Academy of Food Science and Technology.
Gopinadhan Paliyath is a professor at the Department of Plant Agriculture, University of Guelph,
and the research program director for “Food for Health,” under the UG/OMAFRA partnership.
Dr. Paliyath is a biochemist and has an interest in various aspects of fruits and vegetables,
specifically the nutraceutical components and their mechanism of action. He obtained his BSc
Ed degree (botany and chemistry) from the University of Mysore, MSc degree (botany) from the
University of Calicut, and PhD degree (biochemistry) from the Indian Institute of Science, Banga-
lore. Subsequently, he did postdoctoral work at Washington State University, University of Water-
loo, and University of Guelph. Dr. Paliyath’s research is focused on the biochemistry of plant
senescence, specifically pertaining to postharvest biology and technology of fruits and vegetables.
Investigations on the role of phospholipase D (PLD) in membrane homeostasis and signal
transduction have led to advances in the understanding of the mechanism of membrane deterio-
ration that occur during stress and senescence. Another aspect of his research is focused on
understanding the mechanism of action of food components in disease prevention. The efficacy,
bio-accessibility, bioavailability, and molecular mechanisms of action of nutraceuticals in fruits
and processed products in relation to their cancer-preventive and anti-inflammatory actions are
being investigated using mammalian cell lines, and animal model systems.
Dr. Paliyath has developed technologies and products for enhancing the shelf life and quality of
fruits and vegetables based on PLD inhibition. R&D activities relevant to the industry sector
include: (1) optimization of an enhanced freshness formulation for application to various fruits,
vegetables, and flowers; (2) developing methods for nutraceutical carriers that would enhance the
functional food quality and delivery (e.g., stabilizing lycopene in tomato juice, sauce, etc., for health beneficial effects); and (3) developing
novel technologies to enhance the cancer-preventive ingredients in fruit products, etc. Patents awarded include: (1) # 6,514,914 (US) and
2,298,249 (Canada); (2) #7,198,811 (USA), 4141387-1 (Japan), 260738 (Mexico), 1469736 (Turkey), 028 284763 (China), and 223077
(India). The patents describe the use of nanoformulations based on hexanal and other generally regarded as safe (GRAS) ingredients for
enhancing the shelf life and quality of fruits, vegetables, and flowers by pre or postharvest treatments. These technologies are currently being
evaluated for extending shelf life and quality of mango in India and Sri Lanka with the assistance of the Canadian International Food
Security Research fund. The collaboration involves researchers from Canada, India (Tamil Nadu Agricultural University), and Sri Lanka
(Industrial Technology Institute).
Dr. Paliyath is also the research program director for the food and health theme-related activities under the OMAF/MRA/University of
Guelph research partnership. He serves on the editorial board of several journals. He is a member of American Chemical Society and
Canadian Society of Plant Biologists.
(Total-refereed publications in journals – 92; patents and intellectual properties – 2; disclosures – 4; chapters in books – 27; nonrefereed
contributions – 10; research reports – 28; conference proceedings – 88; edited books – 9; book reviews – 6 (Google Scholar: h index – 31,
i10 index – 68, citations – 4332; RG score – 35.63)).
Yolanda Picó is a full professor of nutrition and food science at the University of Valencia since
1998. She is currently the head of the research group on food and environmental safety of the
University of Valencia. Her research interests are the development of new analytical methods to
determine organic contaminants in food and the environment, identification of unknown com-
pounds by liquid chromatography–mass spectrometry, micro-extraction separations, and environ-
mental and food safety. To the date, she is the author of nearly 200 peer-reviewed papers, 170
scientific papers in journals of SCI, 25 book chapters, and editor of four books on food and
environmental safety.
 Editorial Advisory Board xiii

Vieno Piironen is a professor of food chemistry at the Department of Food and Environmental
Sciences, University of Helsinki, Finland. She received her PhD in food chemistry at the University
of Helsinki in 1987 and has approximately 35 years of experience in food research and education
on bachelor, master, and PhD levels. She has participated actively in international research and
education projects and networks. Her research has focused especially on chemical and nutritional
properties, reactions and analysis of lipids, vitamins, and other bioactive compounds. Research on
vitamins has been active from the beginning of 1980s. She has studied both lipid- and water-
soluble vitamins; their chemical and nutritional properties and importance in foods and diets as
well as factors influencing vitamin levels. In addition, development of analytical methods for
different vitamers as well as validation and harmonization of the methods through international
collaboration have been among the priorities. Recent collaboration projects have focused on
enhancing vitamin contents in cereal-based foods by plant breeding, utilization of vitamin-rich
grain fractions, and bioprocessing. Currently, the research focus lies on investigating microbial
in situ synthesis of folate, vitamin B12, and other B vitamins in cereal and legume matrices as a
means to improve nutritional quality of foods and to develop new food applications. In lipid
research, different lipid classes and their chemical and enzymatic reactions in food matrices are
studied. Diverse methods are used to study proceeding of oxidation from primary products to
monomeric oxides, volatiles, and polymerization products and to study possibilities to control
oxidation. Controlling enzymatic reactions leading to off-flavors in cereal and legume matrices is
also among the interests. Phytosterols and their conjugates have been studied as natural food
components belonging to the dietary fiber complex. On the other hand, questions related to sterol enrichment such as oxidation
susceptibility and mechanisms as well as factors affecting oxidation reactions have been of interest. She has also studied nutrients and
anti-nutrients in legumes and more recently started research on utilization of high value components in microalgae. She has approximately
160 papers in international journals and a number of other publications.
David Rodrı́guez-Lázaro is a doctor in veterinary medicine (DVM), specialized in food science
(BSc and MSc) and molecular microbiology (PhD). He is a senior scientist at ITACyL and an
assistant professor of microbiology at the University of Burgos. He has performed research stays in
the Danish Institute for Food and Veterinary Research (Denmark), the University of Prague (Czech
Republic), the Food and Environmental Research Agency (UK), and the University of Bristol (UK).
He was a Leverhulme visiting professor in the Institute of Advanced Sciences in the University of
Bristol during the years 2004 and 2005 and Marie Curie research fellow in the faculty of medical
and veterinary sciences in the University of Bristol (UK) until 2007.
His research interest is focused on the establishment of reliable, quantitative molecular strategies
for detection of important food-borne pathogens from environmental sources and various types of
foodstuffs, the characterization of the prevalence of the main foodborne pathogens in food and
food-related environments, and the development of emergent food preservation processes and
their effects in the microbial virulence. He has participated in a number of coordinated EU-funded
projects such as PROMISE, BASELINE, VITAL, FOOD-PCR, SACROHN, and MONI-QA, having
established active links with the leading European research groups working in food safety.
He has published more than 100 international scientific papers and book or book chapters
regarding food safety. He is currently a member of the editorial board of Applied and Environmental
Microbiology, International Journal of Food Microbiology, Food and Environmental Virology, and
International Journal of Food Contamination and the editor-in-chief of the journal Food Analytical
Methods. He was awarded with the XV Jaime Ferrán Award in 2013 by the Spanish Society for
Microbiology for his promising scientific career in microbiology.
Turid Rustad is a professor and the head of the food science group at the Department of
Biotechnology, Norwegian University of Science and Technology. The main research focuses on
the biochemistry of marine raw materials, the relationship between biochemistry and quality, and
changes in raw material properties during processing. Studies of enzymatic activities in different
raw materials have been linked to studies of changes in the biochemistry of these raw materials. She
has worked with characterization of composition and enzymatic processes in a wide range of
different raw materials, such as fish, fish by-products, and zooplankton in relation to different
storage and processing methods such as chilling, heating, superchilling, and frozen storage.
xiv Editorial Advisory Board

Noel W. Solomons has lived and worked in Guatemala for 40 years. He was born and educated in
Massachusetts in the United States. As a young child, he became an amateur naturalist and was a
nature counselor at various summer camps; this would guide him to a career in science. In his
young adulthood, he would participate in the civil rights and anti-war movements, only to become
disillusioned by the intractable nature of the injustice elements in the fabric of American society. As
a physician by graduate training, he performed his university studies at Harvard College and
Harvard Medical School; it was during overseas electives in his medical training that he visited
Peru and Colombia and committed to an expatriate life trajectory outside of his homeland. Clinical
training included a residency in internal medicine and infectious diseases at the Hospital of the
University of Pennsylvania and specialization in gastroenterology and clinical nutrition at the
University of Chicago. He became a resident of Guatemala in 1974 as an affiliated investigator at
the Institute of Nutrition of Central America and Panama. He would later commute for eight years
to a faculty position in the Department of Nutrition and Food Science of the Massachusetts
Institute of Technology. Assuming a full-time Guatemala commitment in 1985, he co-founded
the Center for Studies of Sensory Impairment, Aging and Metabolism (CeSSIAM) where he remains
its scientific director. Over 40 local university theses have been completed by Central American students in that institution as well as an
equal number of master’s degree research projects from international students from Europe, and North and South America. He has
supervised doctoral dissertations for 12 PhD candidates from the United States, Canada, Germany, Spain, and the Netherlands through
CeSSIAM.
Dr. Solomons has 332 publications indexed on Medline. In addition, he has edited two books and contributed over 100 articles, reviews,
editorials, and commentaries in nonindexed venues and over 50 book chapters. These are dedicated to the scientific and academic interests
of his career including: clinical nutrition; human growth and body composition; lactose maldigestion; dietary intake, nutritional status,
intestinal absorption, and food fortification related to various micronutrients (vitamins, trace elements, and essential fatty acids);
complementary feeding; nutrition in aging and chronic disease; and the interaction of malnutrition and infection.
Among the honors bestowed upon Dr. Solomons are the International Nutrition Prize of the International Union of Nutritional Sciences
and the Kellogg Prize of the Society for International Nutrition Research. He is a fellow of the American Society of Nutrition. He is an
academic member of the Guatemalan Academy of Medical, Physical and Natural Sciences and the Spanish Academy of Nutrition and Food
Science. He was the awardee of the 2010 National Medal for Science and Technology for Guatemala.
He has been a visiting professor in university courses in Mexico, Peru, Brazil, Indonesia, and Spain. He currently holds adjunct
professorial appointments at the Boston University School of Public Health, and the Friedman School of Nutrition Science and Policy
and the Department of Community Medicine and Public Health, both at Tufts University. He is a founding board of directors, member of
the Hildegard Grunow Foundation in Munich and the Essential Nutrient Foundation of Singapore. Finally, Dr. Solomons is a coordinator
for Central America of the Nevin Scrimshaw International Nutrition Foundation in Boston, and an associate editor for the Foundation’s
Food and Nutrition Bulletin. He serves on editorial boards for ten scientific journals.

Maria Tsimidou is a professor of food chemistry and the head of the Laboratory of Chemistry and
Technology in the School of Chemistry at the Aristotle University of Thessaloniki (AUTh), Greece.
Her teaching is food chemistry, analysis, quality control, and food legislation. Research interests are
related to virgin olive oil chemistry, quality and authenticity, saffron chemistry, authenticity and
quality, antioxidant activity of plant extracts and constituents, new sources of targeted bioactive
compounds (squalene, carotenoids, and phenols), and analytical procedures for their determina-
tion. She has published many research papers, review articles, and contributions to scientific books
and encyclopedias on the above-mentioned topics. Currently, she is an associate editor in the
European Journal of Lipid Science and Technology and chairs the COST ACTION FA1101
“Saffronomics.”
 Editorial Advisory Board xv

Jorge Welti-Chanes earned his degree in biochemical engineering (1976) and master of science in
food engineering (1978) at Tecnológico de Monterrey (ITESM, Mexico), later he moved to Spain to
perform his doctoral studies in chemistry, in the area of food technology, obtaining his degree at
the University of Valencia. He is currently the national director of graduate studies at School of
Engineering and Sciences at Tecnológico de Monterrey also is professor and researcher in the areas
of biotechnology and food at the same institution. He started his academic activity in 1976 as a
university professor of ITESM, has additionally been a full professor at the National Polytechnic
Institute (IPN, Mexico) and the University of the Americas, Puebla, Mexico (UDLA). He has an
experience of 37 years as a teacher and university researcher, 20 of which were spent in combina-
tion with the development of administration work in education, science and technology. In the
UDLA, he was teaching in the Departments of Chemistry and Biology and Chemical Engineering
and Food, in the latter was responsible for the leadership for a period of a year and subsequently
became dean of the School of Engineering (1986–1988). From January 1989 to June 2002, he was
an academic vice chancellor at UDLA. He has published 14 books and has over 200 scientific
publications in refereed journals and books, has given more than 250 presentations at international conferences. He is an associate editor of
the journals Food Engineering Reviews and Journal of Food Science and participates as a member of the editorial boards of Journal of Food
Engineering and Current Opinion in Food Science. In May 2011, he received the Life Achievement Award by the International Association for
Engineering and Food (IAEF), for his career as a researcher and academic worldwide, and in January 2014, the Romulo Garza Award from
the Tecnológico de Monterrey for the impact of their research work and as recognition for being one of the most productive researchers in
the life of Tecnológico de Monterrey. He has been the president of ISOPOW and IAEF and is the currently president of the International
Society of Food Engineering (ISFE).
Peter J. Wilde graduated in biophysics at the University of East Anglia in 1985 and has been
researching the colloidal and interfacial properties of food systems at the Institute of Food Research
(IFR) for over 25 years. IFR is the only publicly funded UK research institute that focuses on the
underlying science of food and health to address the global challenges of food security, diet, and
health, healthy aging, and food waste. IFR is the one of eight institutes that receives strategic
funding from the Biotechnology and Biological Sciences Research Council (BBSRC). It also receives
funding from government agencies and departments, the EU, charities, and industry, from the UK
and overseas.
Pete’s research expertise is the interfacial behavior of proteins and other surface active compo-
nents in food relevant systems. The aim is to determine how the molecular and interfacial processes
control the functionality of foams and emulsions. Currently, the functional aspects of his research
have focused on improving the dietary impact of emulsified foods. These include fundamental
studies on how interfacial layers control emulsion rheology to develop novel fat reduction strate-
gies; the design of interfacial structures to control lipid digestion to promote satiety or the delivery of fat-soluble nutrients and drugs; and to
determine the physico-chemical role played by the salivary film in perceiving fat content in emulsions. The impact of this research will be to
aid the rational design of foods with enhanced nutritional benefits to address the global challenges of obesity, type 2 diabetes, and other
major diet-related conditions.
This page intentionally left blank
HOW TO USE THE ENCYCLOPEDIA

All articles in the encyclopedia are arranged alphabet- See also: Anemia: Causes and Prevalence; Anemia: Prevention and
ically as a series of entries. Dietary Strategies; Iron: Biosynthesis and Significance of Heme;
Iron: Physiology of Iron.

1. Contents
3. Index
Your first point of reference will likely be the
contents. The complete contents list appears at the The index provides the volume and page num-
front of each volume providing volume and page ber for where the material is located, and the
numbers of the entry. We also display the article index entries differentiate between material that
title in the running headers on each page so you is a whole article; is part of an article, part of a
are able to identify your location and browse the table, or in a figure.
work in this manner.

4. Contributors
2. Cross-references
A full list of contributors appears at the end of
The majority of articles within the encyclope- volume 5.
dia have an extensive list of cross-references that
appear at the end of each article, for example:

xvii
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INTRODUCTION

Until a few decades ago, virtually all known health effects of foods were related to their content of essential
nutrients. The clinical description of most diet-related illnesses mirrored the signs of essential nutrient
deficiencies, such as pellagra, beriberi, and others. Consequently, the key public health concern regarding
diet was ensuring that everyone consumed enough food. It was only in the past 50 years that large-scale
epidemiological observations began to associate chronic diseases like diabetes and cardiovascular disease with
nonessential diet constituents such as saturated fat, fiber, and cholesterol. Taking advantage of the emergence of
digital informatics, these studies were able to manipulate increasingly large sets of data and provide, for the first
time, a picture of the secular changes in the health of large populations and its association with what they ate
regularly. These findings progressively shifted the concern from eating enough to avoiding excessive consump-
tion of certain foods. Eating enough was replaced by eating well.
But it turned out that defining how to eat well is far more complex than defining minimum needs of
essential nutrients. First, there is no single paradigm to study those relationships, given the wide variety of
biological mechanisms and the long exposures involved. Second, many of the experimental models used to
define essential nutrient needs are not applicable to the study of long-term effects of diets in free-living
populations. And it is now clear that experiments with isolated dietary compounds do not reflect the actual
effects of the complex food matrix we consume daily. Finally, while the discovery of essential nutrients and
their role in health was the domain of a few specialties speaking a common language (primarily biochemists
and physiologists), the study of the long-term effects of whole diets in humans must of necessity involve
epidemiologists, social and behavioral scientists, food scientists, clinicians, policy experts, etc., making far more
difficult the development of consensus and foundational concepts.
It is thus not surprising that today we have still not achieved a stable consensus on how to eat ‘well.’
Furthermore, while few nonscientists would care about the minimum requirement of a vitamin to sustain life,
there are plenty of opinions among nonscientists on how to eat ‘well.’
Our goal in preparing this encyclopedia has been to contribute to the understanding of that complex
diet–health relationship by providing a multidisciplinary, integrative and accurate source of information. We
aim to serve the needs not only of established and in-training scientists, but also of the increasingly important
group of professionals who are key to disseminate and sustain the practice of science: journalists, science
writers, science administrators, fund raisers, donors, and policymakers. In preparing this work, we had the
enormous advantage of working with one of the publishers with the most extensive expertise in major reference
works, Elsevier. This first edition builds on the impressive breadth of knowledge of over 922 authors and on the
tireless work of our editorial advisory board. We are very grateful to all of them.
Benjamin Caballero
Paul Finglas
Fidel Toldrá

xix
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VOLUME 2 TABLE OF CONTENTS

Editors-in-Chief v
Editorial Advisory Board vii
How to use the Encyclopedia xvii
Introduction xix

Chemometrics 1
F Marini

Cherries (Prunus Spp.): The Fruit and Its Importance 10

W Loescher

Chilled Foods: Effects on Shelf-life and Sensory Quality 14

D Bermúdez-Aguirre and J Welti-Chanes

Chilled Foods: Modified Atmosphere Packaging 19

LM Cunha and SC Fonseca

Chilled Foods: Packaging Under Vacuum 23

M Rossi

Chilled Foods: Principles 28

GG Amador-Espejo and ME Bárcenas Pozos

Chlorophyll 37
C Yilmaz and V Gökmen

Cholecalciferol: Properties and Determination 42

AK Hewavitharana and FP Gomes

Cholesterol: Absorption, Function and Metabolism 47

V Vučić and Z Cvetković

Cholesterol: Factors Determining Blood Cholesterol Levels 53

Z Rasic-Milutinovic, G Perunicic-Pekovic, D Jovanovic, N Simovic, Z Gluvic, D Ristic-Medic, and M Glibetic

Cholesterol: Properties, Processing Effects, and Determination 60

T Dinh and L Thompson

Choline: Physiology 70
SH Zeisel

Choline: Properties and Determination 73

MM Phillips

Chromatography: Combined Chromatography and Mass Spectrometry 79

Z Zhang, X Hu, and P Li

Chromatography: Focus on Multidimensional GC 85

C Cordero, C Cagliero, E Liberto, B Sgorbini, P Rubiolo, and C Bicchi

Chromatography: High-Performance Liquid Chromatography 93

H Gika, G Kaklamanos, P Manesiotis, and G Theodoridis

xxi
xxii Volume 2 Table of Contents

Chromatography: Supercritical Fluid Chromatography 100

C Galea, D Mangelings, and YV Heyden

Chromium: Physiology 108

JB Vincent

Chromium: Properties and Determination 114

JB Vincent

Cider (Cyder; Hard Cider): The Product and Its Manufacture 119
E Coton, M Coton, and H Guichard

Cirrhosis 129
S Honigbaum, J Lucas, and KB Schwarz

Citrus Fruits 136

AC Matheyambath, P Padmanabhan, and G Paliyath

Clostridium botulinum 141

A Harris

Clostridium: Occurrence and Detection of Clostridium perfringens 146

R Labbé and V Juneja

Clostridium: Food Poisoning by Clostridium perfringens 149

K Miyamoto and M Nagahama

Clostridium: Occurrence and Detection of Clostridium botulinum and Botulinum Neurotoxin 155
JW Austin

Cobalamin (Vitamin B12): Metabolism and Disorders 160

E Andrès and N Dali-Youcef

Cobalt: Properties and Determination 166

F Cámara-Martos and R Moreno-Rojas

Cobalt: Toxicology 172

F Cámara-Martos and R Moreno-Rojas

Cocoa: Composition and Health Effects 179

DD Mellor

Cocoa: Production, Chemistry, and Use 185

A Caligiani, A Marseglia, and G Palla

Codex Alimentarius 191

I Stankovic

Codex Alimentarius Commission: Role in International Food Standards Setting 197

V Kotwal

Coenzymes and Cofactors 206

RB Rucker and W Chowanadisai

Coffee: Analysis and Composition 225

MC Cid and M-P de Peña

Coffee: Decaffeination 232

AS Franca

Coffee: Health Effects 237

R Tofalo, G Renda, R De Caterina, and G Suzzi

Coffee: Types and Production 244

LR Batista, SM Chalfoun de Souza, CF Silva e Batista, and RF Schwan

Colon: Diseases and Disorders 252

R Arbizu and S Nurko

Colon: Structure and Function 259

R Arbizu and S Nurko
 Volume 2 Table of Contents xxiii

Colors: Health Effects 265

D Villaño, C Garcı´a-Viguera, and P Mena

Colors: Properties and Determination of Natural Pigments 273

A Giuliani, L Cerretani, and A Cichelli

Colors: Properties and Determination of Synthetic Pigments 284

E Diacu

Condensed Milk 291

SD Kalyankar, MA Deshmukh, CD Khedkar, SS Deosarkar, and AR Sarode

Consumer Protection Legislation 296

K Purnhagen and B van der Meulen

Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs 301

Z Escobedo-Avellaneda and J Welti-Chanes

Controlled Atmosphere Storage: Effect on Fruit and Vegetables 308

A Valdez Fragoso and H Mújica-Paz

Convenience Food 312

TA Brunner

Cooking: Domestic Techniques 316

AJ Rosenthal

Copper: Physiology 321

J Bertinato

Cream: Clotted Cream 327

RS Chavan, A Kumar, and S Bhatt

Cream: Types of Cream 331

SS Deosarkar, CD Khedkar, SD Kalyankar, and AR Sarode

Cured Foods: Health Effects 338

J Ruiz-Carrascal

Cystic Fibrosis, Nutrition in 343

S Sabharwal

D 345

Dahi 345
CD Khedkar, SD Kalyankar, SS Deosarkar, and AM Patil

Dairy Products: Dietary and Medical Importance 352

F Visioli

Date Palm: A Wealth of Healthy Food 356

KM Farag

Diarrheal Diseases 361

Z Bhutta and S Syed

Dietary Exposure Assessment 373

D Arcella, F Héraud, and M Gilsenan

Dietary Fiber: Bran 378

A Kamal-Eldin

Dietary Fiber: Determination 383

R Mongeau and SPJ Brooks

Dietary Fiber: Energy Value 392

SPJ Brooks and R Mongeau

Dietary Fiber: Physiological Effects 400

IT Johnson
xxiv Volume 2 Table of Contents

Dietary Fiber: Properties and Sources 404

R Mongeau and SPJ Brooks

Dietary Practices 413

AFG Cicero and T Stallone

Dietary References: US 418

J Dwyer and NJ Armstrong

Dietary Surveys: National Food Intake 432

MC Ocké, CTM van Rossum, and EJ de Boer

Drying: Effect on Nutrients, Composition and Health 439

SV Crowley and JA O’Mahony

Drying: Physical and Structural Changes 446

SV Jangam, AS Mujumdar, and B Adhikari

Drying: Principles and Types 456

JM Barat and R Grau

E 463

Eating Disorders 463

CC Schreyer, S Makhzoumi, JW Coughlin, and AS Guarda

Eggs: Composition and Health Effects 470

ML Fernandez and CJ Andersen

Eggs: Use in the Food Industry 476

CG Belyavin

Elderly: Nutrition Requirements 480

R Chernoff

Emerging Foodborne Enteric Bacterial Pathogens 487

SJ Forsythe

Emulsifiers: Types and Uses 498

R Miller

Energy Metabolism 503

JA Coss-Bu and NM Mehta

Energy: Intake and Energy Requirements 511

DJ Millward

Enteral Feeding 519

DL Waitzberg and RS Torrinhas

Enzymes: Analysis and Food Processing 524

T Haertlé

Enzymes: Functions and Characteristics 532

D Talens-Perales, J Marı́n-Navarro, and J Polaina

Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects 539
JL Smith and PM Fratamico

Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection 545

S Fanning, L Rogers, K Power, and PÓ Gaora

Essential Oils: Isolation, Production and Uses 552

CM Cook and T Lanaras

Essential Oils: Properties, Composition and Health Effects 558

G Buchbauer and IM Wallner

Ethnic Foods 563

OI Bermudez
 Volume 2 Table of Contents xxv

Extrusion Cooking: Chemical and Nutritional Changes 569

JAG Arêas, CM Rocha-Olivieri, and MR Marques

Extrusion Cooking: Principles and Practice 576

L Moscicki

F 581

Famine, Hunger, and Undernourishment 581

R Milà-Villarroel, C Homs, J Ngo, J Martı´n, and M Vidal

Fat Replacer 589

RS Chavan, CD Khedkar, and S Bhatt

Fats: Classification and Analysis 596

M Narváez-Rivas and M León-Camacho

Fats: Production and Uses of Animal Fats 604

SB Smith and DR Smith

Fatty Acids: Determination and Requirements 609

M Narváez-Rivas and M León-Camacho

Fatty Acids: Essential Fatty Acids 615

B Lands

Fatty Acids: Fatty Acids 623

S Petrovic and A Arsic

Fatty Acids: Metabolism 632

PC Calder

Fatty Acids: Trans Fatty Acids 645

AH Lichtenstein

Fermented Foods: Composition and Health effects 649

D Ansorena and I Astiasarán

Fermented Foods: Fermented Meat Products 656

F Leroy and L De Vuyst

Fermented Foods: Fermented Milks 661

CD Khedkar, SD Kalyankar, and SS Deosarkar

Fermented Foods: Fermented Vegetables and Other Products 668

R Di Cagno, P Filannino, and M Gobbetti

Fermented Foods: Origins and Applications 675

A Bevilacqua, M Sinigaglia, and MR Corbo

Fermented Foods: Use of Starter Cultures 681

PM Malo and EA Urquhart

Fish Oils: Composition and Health Effects 686

C Jacobsen

Fish Oils: Production and Properties 693

AK Carvajal and R Mozuraityte

Fish: Dietary Importance and Health Effects 699

HK Mæhre, I-J Jensen, and K-E Eilertsen

Fish: Fish in the Human Diet 706

B Blakistone, R Kleiner, and J McGuire

Fish: Processing 710

SP Aubourg

Flavor Enhancers: Characteristics and Uses 716

D Baines and M Brown
xxvi Volume 2 Table of Contents

Folic acid and Folates: Physiology and Health Effects 724

C Witthöft and M Hefni

Food Additives: Classification, Uses and Regulation 731

GA Blekas

Food Allergies 737

SL Taylor and JL Baumert

Food Allergies: Occurrence and Analysis 743

S Sforza and B Prandi

Food and Agriculture Organization of the United Nations 749

E Casadei and J Albert
 Chemometrics
F Marini, University of Rome “La Sapienza”, Rome, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Chemometrics can be defined as the chemical discipline,
which makes use of mathematical, statistical, and logical
methods to extract meaningful information from experimental
data and to optimize processes and/or products. It is evident
from this definition that it accompanies all stages of the chem-
ical measurement process, from sampling to interpretation
passing through the definition of the optimal experimental
conditions and data collection and processing. Even if it finds
its roots in analytical chemistry, chemometrics is highly inter-
disciplinary and its domain of application is becoming wider
and wider. However, since the very beginning, food-related
issues have constituted an important field of application of
chemometric techniques. Indeed, foodstuffs are rather com-
plex matrices and their authentication often relies on a holistic
characterization through the measurement of different chemi-
cal indexes or through the recording of whole instrumental
fingerprints. The construction of traceability models for the
verification of labeling compliance of products with a desig-
nated origin, the monitoring and control of food production
processes, the correlation of volatile compound profiles with
the sensory evaluation of a trained panel, and the indirect
quantification of one or multiple analytes based on cheap
and rapid spectroscopic techniques are only a few emblematic
examples of application where the use of chemometrics is not
only advisable but also necessary.
Chemical Data: Types and Representation
As anticipated, the main goal of chemometrics is to extract
useful and meaningful information from chemical data. There-
fore, prior to illustrating the chemometric toolbox with all its
plethora of methods, it is essential to understand the data,
which constitute the basis for modeling. A chemical system is
usually characterized and described by a set of measured or
calculated variables, which can be either quantitative or qual-
itative in nature. In the former case, variables are defined on a
numerical (interval or ratio) scale, can undergo any kind of
algebraic operation, and can assume an infinite (continuous)
set of values; examples of these types of indices are pH, con-
centration, and temperature. On the other hand, qualitative
variables may assume only a discrete set of values, which are
often categorical or expressed in the form of attributes: the
presence or absence of a constituent, genuine/adulterated,
and sensory panel evaluation on a 1–5 scale are all examples
of such kind. Usually, one variable only is not enough to
characterize a chemical system, so that multiple descriptors
are recorded on the same sample: these may come from differ-
ent instruments or analytic procedures (e.g., total acidity, per-
oxide number, and concentration of iron or calcium) or be
acquired with the same platform (e.g., absorbances at different
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00779-0
wavelengths or detector intensities at different retention times,
in the case of spectroscopic or chromatographic profiles,
respectively). The resulting data are then multivariate and each
sample may be described by a numerical row vector xi, collect-
ing the results of all the measurements performed:


xi ¼ xi1 xi2 xi3 . . . xip [1]
p being the total number of variables. Accordingly, when more
than a single sample is analyzed, the data can be arranged into
a matrix X, having as many rows as the number of samples (n)
and, as already stated in eqn [1], as many columns as the
number of variables (p):
0 1
x11 . . . x1p
B C
X¼B @ ⋮ O ⋮A
C [2]
xn1 ... xnp
Each row of the matrix X, then, contains the results of the
experimental measurement performed on a particular sample,
while each column is made of the values of a single variable
along all the samples.
One of the advantages of the matrix representation in eqn
[2] is that it has a geometric counterpart: Each variable can be
thought as an axis in a (hyper-)space having p dimensions so
that the value of the matrix element xij represents the coordi-
nate of the ith object along the jth direction. Accordingly, each
sample can be described as a point in the p-dimensional space
(see Figure 1).
Experimental Design
Although this aspect is often still neglected by many chemists,
chemometrics enters the analytical process long before the data
processing and interpretation stage, on one hand as it is nec-
essary to sketch the most appropriate sampling strategies in
order to have representativeness and to meet specific require-
ments (e.g., a target accuracy or precision) and, on the other, as
the quality of the data obtained and the possibility of retrieving
the information sought strongly rely on a careful experimental
planning. Indeed, as already pointed out by Fisher, “Statistical
procedure and experimental design are only two different
aspects of the same whole, and that whole comprises all the
logical requirements of the complete process of adding to
natural knowledge by experimentation.”
By the term experimental design, one indicates the family of
techniques whose aim is to identify a parsimonious set of
experimental conditions. The aim of experimental design tech-
niques is (a) to understand the effect of controlled variables
(factors) on one or more responses (often with the final intent
of optimization) and (b) to define an empirical model
(response surface) for the relation between the responses
(dependent variables) and the factors (independent variables),
which may be used either to support the optimization process
1
2 Chemometrics

x2 = [1 3 7]
6

Variable 3
x1 = [4 5 2]
4

0
6
4 4
3
2 2
Variable 2
1 Variable 1
0 0

Figure 1 Geometric representation of data vectors as points in the multidimensional space of the variables.

Selection of factors and responses

Definition of experimental domain

Screening designs
Discard unimportant factors
Selection of the strategy

Simultaneous designs Sequential designs

Two level factorial designs Selection of optimum

Interactions No interactions

Multilevel factorial designs Multilevel OVAT

Selection of optimum Selection of optimum

Figure 2 Flowchart of experimental design.

defined in point (a) or for prediction purposes. In both cases, a
systematic and efficient mapping of the experimental domain,
at the same time looking for the minimum number of exper-
iments to be performed, is sought.
Given the scopes summarized earlier, the family of experi-
mental designs encompasses a wide range of techniques of
increasing complexity, as evidenced by the pipeline shown in
Figure 2.
Screening designs are the most parsimonious ones, as they
include only the experiments needed to estimate in a
semiquantitative fashion the main effect of the investigated
factors, not taking into account the possible interactions
among variables. A linear relation is assumed between the
response(s) y and each of the f controlled factors:
y ¼ b0 þ b1 x1 þ b2 x2 þ . . . þ bf xf
where b0 is an offset term, corresponding to the predicted
response in the center of the experimental domain, while
b1. . .bf are estimates of the effect of the factors 1. . .f on y. A
direct consequence of the assumption of linearity is that only
two levels are investigated for each factor and the resulting
strategies are on one hand the so-called saturated or supersat-
urated factorial and on the other the Plackett–Burman designs.
Screening designs are normally used to have information on
which of possible factors may be discarded as very likely unaf-
fecting the response. After screening, it is necessary to choose
the strategy that may be more appropriate for the specific
problem: Indeed, there are two options available, the factorial
approach and the sequential approach. The sequential strategy
is most suited when there are only a few factors, the goal is
optimization of (possibly) a single response, and there is no
interest in calculating the response surface. It operates by
selecting a small number of initial experiments and then iter-
[3] atively choosing the coordinates for the successive experiment
based on the coordinates and the responses of the previous
ones, up to when a stopping/optimality criterion is met. On
the other hand, in the factorial approach, the whole set of

experiments to be conducted is defined a priori, before any of
those is actually carried out: This approach is in general to be
preferred, but it is for sure the one to be adopted when the
effect of controlled factors on multiple responses has to be
investigated and also when modeling is the purpose. In this
context, two level factorial designs, that is, factorial designs in
which each factor is controlled only at two values, allow to
assess the main effect of the factors and at the same time to
verify the presence of interactions. Indeed, they assume a linear
model with mixed terms, which, in the case of two factors,
takes the form
y ¼ b0 þ b1 x1 þ b2 x2 þ b12 x1 x2 [4]
where b12 is the term that accounts for the interaction between
factor 1 and factor 2, while all other terms have the same
meaning as in eqn [3]. Although such mathematical assump-
tion is often too approximate to lead to reliable estimation of
the main effects and interaction terms or to accurate predic-
tions of the response values, still, two level factorial designs
allow to have a semiquantitative understanding of the relation
between dependent and independent variables and to assess
the presence or absence of interactions.
When the linearity assumption is released, so that non-
linear (almost always second- or third-order polynomial) rela-
tionships are modeled, factors cannot be controlled at two
levels only anymore, so that multilevel designs are needed.
The use of multilevel designs, the most famous of which are
the central composite, the Box-Behnken, and the Doehlert
ones, together with the family of optimal designs (D-optimal,
A-optimal, and so on), allows to better approximate the
response surface, which, in the case of three factors, usually
takes the form
y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b12 x1 x2 þ b23 x2 x3
þ b13 x1 x3 þ b11 x21 þ b22 x22 þ b33 x23 [5]
where b11, b22, and b33 account for the effect of the squared
factors and all the other terms have the same meaning as in
eqns [2] and [3]. Specific designs may also be used when the
factors to be investigated are the constituents of a mixture or
when they are qualitative variables.
Exploratory Analysis
Exploratory analysis is a strategy/philosophy that makes use of
a variety of techniques to summarize the main characteristics
of a data matrix in an easy-to-understand form, often with
visual graphs, without using a statistical model or having for-
mulated a hypothesis. Its main purposes are to maximize
insight into a data set, to uncover underlying structure, to
extract important variables, to detect outliers and anomalies,
to test underlying assumptions, and to develop parsimonious
models. All these characteristics suggest that exploration is and
should always be the first step in data analysis, even when the
final goal is some kind of predictive modeling.
According to the definitions reported earlier, exploratory
data analysis is normally carried out by means of two families
of techniques: projection methods and clustering algorithms.
The need for the projection method is evident when thinking
that, in the situations when many variables are measured on
Chemometrics 3
the samples, which is what normally occurs with chemical
systems, graphical inspection of the data in the original
p-dimensional space described in the section ‘Chemical Data:
Types and Representation’ may result difficult or even unfea-
sible. Indeed, as the name suggests, projection methods oper-
ate by projecting the data on a relevant subspace, which can be
used to summarize the data conveniently and explore the data
set using plots and figures, in order, for instance, to find
patterns, relation, differences, and similarities between objects
and variables. The most famous and used multivariate projec-
tion method is for sure principal component analysis (PCA),
whose aim is to find the best low-rank approximation of the
original data in a least squares sense. This is equivalent to state
that PCA identifies those directions in the multivariate space,
which in turn account for the maximum explained variance
and which are mutually orthogonal, and projects the original
data onto this reduced subspace. Mathematically, the PCA
model can be expressed by the following equation:
T ¼ X P [6]
nF np pF
where T is the matrix collecting the coordinates (scores) of the
sample onto the new reduced (F usually  p) set of variables
(called principal components), while the matrix P contains the
coefficients of the projection (loadings). The same relation can
be rearranged as in eqn [7], to show the so-called bilinear
nature of the PCA decomposition:
^ þ E ¼ TPT þ E
X¼X [7]
where the matrix E (residuals) accounts for the portion of the
variability originally present in the data, which is not explained
by the PCA model and the hat (^) indicates that X is approxi-
mated by the model. Indeed, bilinear modeling, that is, the
approach that assumes that a data matrix X may be approxi-
mated by the product of two low-rank matrices, one represent-
ing the coordinates of the samples onto a reduced subspace
and the other expressing the variable contribution to the def-
inition of the subspace, is one of the core elements of chemo-
metrics. Due to its characteristics, PCA represents the optimal
tool to perform exploratory data analysis, as it can provide
information about the relationship among samples and
among variables, together with the possibility of noise filtering
and outlier detection. Graphical representation of the samples
in the principal component space (scores plot) allows to identify
the presence of clusters or groups in the data or to evidence
some trends; on the other hand, correlation between the exper-
imental variables or irrelevant descriptors may be assessed by
visually inspecting the elements of the loadings matrix (load-
ings plot). An example of the application of PCA in the context
of food analysis may be observed in Figure 3, where the scores
and the loadings plots for a problem involving the authentica-
tion of honey samples are shown. In particular, the data set was
made on the results of 15 chemical analyses on 73 honey
samples coming from different botanical origin (sulla, heather,
eucalyptus, chestnut, honeydew, and wildflower).
Inspection of the scores plot in Figure 3(a) evidences the
presence of different clusters in the data: If no information
about the origin of samples were available, one would have
stated that there were seven different groups of honey; how-
ever, integrating the scores plot with the information available,
4 Chemometrics

PC 2 (26.97%)
1

−1 honeydew
eucalyptus
−2 chestnut
sulla
−3 heather
wildflower
−4
−4 −3 −2 −1 0 1 2 3 4
(a) PC 1 (39.08%)
0.5
Conductivity
Color
0.4
pH Specific rotation

0.3

0.2 Free acidity

Total acidity
PC 2 (26.97%)

0.1
% DP2 HMF
0
13C/12C(proteins)
Moisture %dextrose
−0.1 Lactones
Diastase
−0.2

13C/12C(whole)
−0.3
%fructose
−0.4
−0.6 −0.4 −0.2 0 0.2 0.4 0.6
(b) PC 1 (39.08%)
Figure 3 Principal component analysis of honey data set. (a) Scores and (b) loadings plots.

it is possible to affirm that there are six clusters, one for each of
the different floral origin of the samples and that the group
corresponding to honeydew is further split in two subclusters.
Moreover, in general, it is also possible to see how the clusters
corresponding to multifloral origin (honeydew and wild-
flower), which are separated from the unifloral ones along
PC2, are less homogeneous, as it could be expected. Informa-
tion about the variables can, instead, be obtained by looking at
the loadings plot reported in Figure 3(b): At first, one can
observe that dextrose and lactones are correlated, as well as
color and specific rotation or free and total acidity, as they fall
very close to one another in the PC space; on the other hand,
the carbon isotope ratio in the proteic fraction does not seem
to contribute much to the model as its loadings are almost
zero. Lastly, comparison of the scores and the loadings plot
allows to interpret the differences observed among samples in
terms of the original variables. As stated, unifloral honeys are
separated from multifloral ones along the second principal
component: looking at the loadings plot, this means that the
former are characterized by a higher fructose content and
carbon isotope ratio, at the same time showing a lower pH,
conductivity, color, and specific rotation. On the other hand,
wildflower is separated from honeydew along PC1, and the
same component differentiates also among the various uni-
floral honeys: this means that in going from honeydew to
wildflower or from chestnut to heather, there are a systematic
increase of dextrose, lactones, free and total acidity, and hydro-
xymethylfurfural (HMF) and a corresponding decrease of dia-
stase and DP2.
The second family of techniques normally used for explor-
atory data analysis are clustering algorithms. As the name
suggests, the aim of clustering techniques is to look for the
presence of groups (usually of samples, but it is possible to
apply the same concepts to variables or to both) in the data,
based on the calculation of some kind of similarity or dissim-
ilarity index. Indeed, the underlying idea is that object belong-
ing to the same group will be similar to one another and
dissimilar to the object belonging to other clusters, so that

the definition of a proper measure to quantify (dis-)similarity
plays a key role for this family of methods. In many applica-
tions, dissimilarity is expressed in terms of a distance measure
in the multivariate space, but other indexes may also be used.
Once the proper way of quantifying (dis-)similarity is chosen,
clustering may be carried out according to two different
approaches: hierarchical and partitional. In hierarchical clus-
tering, as the name suggests, objects are ranked from the most
similar to the most dissimilar in a hierarchical fashion, and the
main result is a graphical representation called a dendrogram. In
particular, at the beginning of the hierarchical clustering pro-
cedure, each sample is considered a cluster per se; at each step,
the two most similar clusters are merged together and the
procedure is continued until all objects are joined into a single
group. This approach is called agglomerative (bottom up) and
it is the most widely used: hierarchical clustering may also be
carried out in a divisive way (top down), by starting from a
single cluster and iteratively arriving to as many groups as the
number of objects, but this approach is more computationally
intensive and, therefore, it is rarely adopted. Here, it must be
stressed that, whatever the approach, hierarchical clustering
does not provide a unique grouping: partitioning of object
into clusters may be achieved only by cutting the dendrogram
at a specific level, which usually corresponds to a large distance
among the merged groups. As an example, the dendrogram
corresponding to agglomerative hierarchical clustering for the
honey data set already presented in the case of PCA is reported
in Figure 4.
The dendrogram in Figure 4 shows very clearly the presence
of clusters among the data, and also, as already evidenced by
PCA, the groups of honeydew and wildflower samples are less
homogenous than the other ones (their final merging distance
is higher). Moreover, it can be also seen how the identified
number of clusters would vary, depending on the level at
25
20
15
Distance
10
0
DDDDDDDDDDDWWWWWWWWWWWWWHHHHHHHHHHHHE EEEE EEEEE EECCCCCCCCCCCCCSSSSSSSSSSSS
Sample Index
Figure 4 Dendrogram resulting from hierarchical clustering of honey data. Dashed and dotted lines (corresponding to distance values of 8 and 10,
respectively) indicate two possible threshold values, which may be used to cut the dendrogram in order to define sample grouping.
Chemometrics 5
which the dendrogram be cut: If one chooses a threshold
distance of 8 (dashed line), then honeydew samples would
be seen as two different groups and a total number of seven
clusters would be identified; instead, if a threshold distance of
10 (continuous line) be adopted, then the number of detected
groups would be six. On the other hand, partitional clustering
attempts to directly decompose the data into a predefined
number of groups by minimizing some criterion, which
should reflect the local structure. The most famous member
of this family of algorithm is k-means where the criterion
function is the sum of the squared distances of each sample
to its nearest cluster centroid:
XN 2
E¼ i¼1
jjx i  mCðxi Þ jj [8]
where mCðxi Þ is the vector collecting the coordinates of the
centroid of the cluster closer to xi.
Calibration
When dealing with food analysis or characterization, explor-
atory data analysis, although necessary, may not be sufficient,
as many problems may require the prediction of one or more
quantitative or qualitative properties of the samples. Calibra-
tion is the use of empirical data and prior knowledge for
determining how to predict quantitative information Y from
available measurement X via some mathematical transfer func-
tion f:
^ þ EY ¼ f ðX Þ þ EY
Y¼Y [9]
where the matrix of residuals EY collects the variance in Y not
accounted for by the regression model, that is, the difference
between the actual values of Y and those predicted by the
6 Chemometrics
model (Ŷ). In food analysis, calibration plays a key role as quite
often, the direct measurements of the properties of interest may
not be feasible or it may be too expensive, time-consuming, or
inaccurate, so that quantification is usually based on secondary
measures (e.g., chromatographic peak area, absorption or emis-
sion intensity, current, and voltage). In this context, the sim-
plest relation between the experimental measurements
collected in X and the properties to be predicted is a linear one:
^ ¼ XB
Y [10]
where B is a matrix of the so-called regression coefficients,
which constitute the model parameters and uniquely define
the mathematical relation. In particular, each column of the
regression coefficient matrix collects the weights associated
with the independent variables for the prediction of the corre-
sponding column of the Y matrix, and this information can be
used also for the sake of interpretation. Indeed, in principle,
the larger the absolute value of the coefficient bij (the ith row
and jth column element of B), the higher the contribution of
the ith X-variable to the prediction of the jth response. How-
ever, particular care should be taken when inspecting the
values of B, as their magnitude directly reflects the scales of
both X and Y. Moreover, when dealing with instrumental
fingerprints, interpretation can be further hindered by the
presence of overlapping signals: As an example, one may con-
sider the case when a spectroscopic measurement is used to
predict the concentration of an analyte in a solution. If no
interferent is present, the regression coefficients would resem-
ble the spectrum of the pure analyte; however, if the solution
contains also an interferent, whose spectrum partially overlaps
with that of the analyte, the regression vector no longer looks
like the pure spectrum because negative parts and shifts in
position of peak maximum are introduced. Accordingly, there
may be cases when a negative regression coefficient is correctly
obtained for a variable that is positively correlated with the
response. Operationally, the most straightforward way of esti-
mating the regression coefficients in eqn [10] is provided by
multiple linear regression (MLR). MLR is the multivariate gen-
eralization of the univariate least squares method; the values of
the coefficient are calculated as the ones, which minimize the
sum of squared residuals:
   
^ 2
min jjEY jj2 ¼ min jjY  Yjj [11]
B B
Accordingly, the matrix B is estimated as
 1
BMLR ¼ X T X X T Y [12]
Unfortunately, even if MLR is the simplest method for
linear regression, it is quite often inapplicable to the matrices
resulting from food analysis and characterization. Indeed, in
order for the term (XTX)1 in eqn [12] to be estimated accu-
rately, the matrix X should meet some mathematical
requirements, which are rarely fulfilled in problems involving
instrumental fingerprinting of real-world samples: the number
of samples should be lower than the number of predictors and
the variables should be as uncorrelated as possible from one
another. In this context, the bilinear approach introduced in
the section ‘Exploratory Analysis’ for PCA, by involving the
projection of the samples onto a low-dimensional space of
orthogonal variables, represents a way of overcoming the
previously mentioned limitations. In particular, principal com-
ponent regression (PCR) is a calibration method that origi-
nated by directly combining a preliminary dimensionality
reduction step by means of PCA with the calculation of a
MLR model on the resulting scores. In mathematical terms, at
first, the matrix X is decomposed into the product of scores and
loadings according to eqn [7], and successively, the scores are
used as predictors to build the MLR model:
 
^ ¼ TV ) V ¼ TT T 1 TT Y
Y [13]
V being the matrix of regression coefficients relating Y to T.
By combining eqns [13] and [7], it is possible to obtain the
model coefficients directly in terms of the original independent
matrix X:
^ ¼ TV ¼ XPV ¼ XBPCR ) BPCR ¼ PV
Y [14]
Due to the nature of PCA decomposition, PCR provides a
reliable answer to the drawback discussed earlier for MLR, even
if it may not provide the best low-rank representation of the
data to be used for calibration purposes. Indeed, principal
components are calculated as those directions in space that
account for the maximum variance in the data set; however,
when multiple sources of spurious (irrelevant) variation are
present in the data set, the directions of maximum variance
may not account for the correlation with the responses to be
predicted. On the other hand, partial least squares regression
(PLS) makes active use of the information in Y to define the
low-rank subspace onto which the data should be projected. In
PLS, both the X- and the Y-blocks are decomposed in a bilinear
fashion, and the axes of the low-dimensional subspace (called
latent vectors) are chosen so that the scores of Y (U) have
maximum covariance with those of X (T) and are linearly
dependent, through what is called the inner relation (last one
of the following equations):
T ¼ XR
Y ¼ UQT þ EY [15]
U ¼ TC
where R is a matrix of weights, governing the projection of the
X-block, Q are the loadings for the Y-block, and C is a diagonal
matrix of coefficients. Also, in the case of PLS, it is possible to
combine the equations in eqn [15] to obtain a matrix of
regression coefficients directly relating Y to X:
Y ¼ UQT þ EY ¼ TCQT þ EY ¼ XRCQT þ EY ¼ XBPLS þ EY
BPLS ¼ RCQT
[16]
However, inspection of regression coefficients is not the
only tool for interpretation, when dealing with PLS: the bilin-
ear nature of the relations reported in eqn [15] suggests that
scores and loadings plots also constitute a valid support to
model understanding and diagnostics.
Although, for all the methods described so far, a linear
relationship between the property (or properties) to be pre-
dicted and the secondary measurements is assumed, this need
not always be the case: the important is to have a defined
calibration equation in order to be able to make predictions
on future samples, so that several nonlinear algorithms have

been proposed in the literature to tackle with more complex
functional dependencies. In this framework, since a detailed
discussion would be far beyond the scope of the present article,
it is just worth mentioning, among the various possibilities,
kernel- or dissimilarity-based approaches, neural networks, or
locally weighted regression.
Classification
Food-related issues may not always call for a quantitative
prediction, and rather, problems such as the authentication
of a good, its quality control, and traceability (just to cite a
few) involve the assessment of one or more qualitative prop-
erties. For instance, one may be interested in assessing whether
a product is organically grown or not, or if a wine was pro-
duced in Italy, Spain, South Africa, or Chile, or, again, if a food
will be good, acceptable, or bad according to consumer prefer-
ences. From a chemometric standpoint, all those methods,
which deal with the possibility of predicting one or more
qualitative responses on a set of samples, belong to the family
of classification tools. Indeed, classification techniques aim at
building models, which, based on the values of the measured
variables, assign a sample to a category or class, the latter being a
group of objects sharing similar characteristics. Accordingly, in
the language of classification techniques, the wine authentica-
tion problem, cited earlier as an example, would involve four
categories (‘Spain,’ ‘Italy,’ ‘South Africa,’ and ‘Chile’), while the
three classes ‘good,’ ‘bad,’ and ‘acceptable’ would be consid-
ered for the food preference one. Since samples can be repre-
sented as points in the multivariate space of the variables,
classification may be seen under a geometric perspective as
the search for surfaces identifying regions of space where it is
more likely to find objects belonging to a particular category.
In this context, it is particularly useful to operate a distinction
between two possible approaches: discrimination and class
modeling. Discriminant techniques partition the space in as
many regions as the number of categories in the data set, so
that if an object falls in the region corresponding to a particular
class, it is univocally assigned to it; as a consequence, each
sample is predicted to belong to one and only one of the
categories postulated by the problem. On the other hand,
modeling techniques, as the name suggest, try to model each
category independently on the others and operate by identify-
ing a region of the multivariate space where it is likely to find
samples from that particular class (the model space): If a
sample falls within that region, it is accepted by the class
model; otherwise, it is rejected. Accordingly, when more than
one category is modeled, a sample can be accepted by only one
class (and then be univocally assigned to it), by more than one
(i.e., confused), or by none (and be considered an outlier). In
the remainder of the section, discriminant and modeling
approaches will be further discussed through the illustration
of two widely used methods, respectively, partial least squares
discriminant analysis (PLS-DA) and soft independent model-
ing of class analogies (SIMCA).
PLS-DA is a discriminant classification technique based on
the PLS algorithm already described in the section ‘Calibration,’
and it was introduced to overcome the limitations suffered by
traditional methods such as linear (LDA) and quadratic (QDA)
Chemometrics 7
discriminant analysis, in the presence of ill-conditioned X matri-
ces. Indeed, the same kind of problems (high number of highly
correlated variables), which make MLR unsuitable to build
regression models, hinders the applicability of LDA and QDA
for classification. Accordingly, after finding a suitable coding that
allows to transform a classification problem into a regression
one, the use of the PLS algorithm represents a solution to these
drawbacks. In particular, PLS-DA is based on coding the infor-
mation about class belonging into a binary dummy matrix Y
having as many rows as the number of samples and as many
columns as the number of classes: the matrix element yij will be
equal to 1 if the ith sample belongs to class j or to zero if it does
not. A PLS model is then built between the experimental matrix X
and the dummy matrix Y, and classification is achieved on the
basis of the predicted values Ŷ, usually via the introduction of a
suitable threshold. An example of application is reported in
Figure 5, where the use of PLS-DA on chromatographic data to
discriminate olive oils from the PDO Sabina from other extra
virgin oils is shown. In this case, since there are only two catego-
ries, due to the symmetry of the problem, the dummy matrix Y
boils down to a vector y in which 1 indicates ‘Sabina’ and 0 ‘other
oils.’ Classification is then achieved by setting a threshold of 0.5
to the predictions: all the samples for which the predicted
response is higher than the threshold are classified as Sabina
oils, while all the others are recognized as from other origins.
Differently than what happens for discriminant methods,
modeling techniques focus on capturing the similarity among
samples belonging to the same category, rather than the differ-
ences between individuals from competing classes. Under
many respects, they can be considered as outlier detection
techniques, as their aim is to verify whether a sample fits the
model of a particular category or not. In particular, SIMCA
operates by describing the class-related variability in the exper-
imental fingerprint using a PCA model of appropriate
dimensionality:
XG ¼ TG PTG þ EG [17]
where the subscript G indicates that only the samples from
category G are used to define the projection. Then, for each
sample, an overall distance to the class model, measuring the
extent of outlyingness, is defined as the combination of the
distance to the model space (which is a function of the resid-
uals) and the distance within the model space (which accounts
for the distance of the sample scores to the origin of the PC
space). Accordingly, if the distance to the model is below a
prespecified threshold, the sample is accepted by the category;
otherwise, it is rejected. When more than a single category is
modeled, a straightforward way of representing the results of
SIMCA is the so-called Coomans plot, which is shown in
Figure 6 for the same data set used to exemplify PLS-DA.
The axes of the Coomans plot represent the sample dis-
tances to the two investigated categories, and the thresholds
used to define acceptance/rejection by the class models divide
the plot in four different regions: Objects falling in the upper-
most left region of the plot are univocally accepted by the class
‘Sabina,’ while those mapped onto the rightmost lower part are
uniquely accepted by ‘other oils’; the samples falling in the
lowermost left part of the plot are confused between the two
categories, while those in the uppermost right regions are
considered as outliers by both classes.
8 Chemometrics

1.2
Other origins
1 Sabina

0.8

0.6

Y predicted
0.4

0.2

−0.2

−0.4
0 10 20 30 40 50
Sample Index
Figure 5 Illustration of how classification is accomplished in PLS-DA: samples are assigned to one or the other class based on the predicted y values
and the green dashed line indicates the classification threshold. Accordingly, all samples except the two Sabina oils that fall below the threshold
are correctly classified.

8
Other origins
Distance to the model “other origins”

7 Sabina

0
0 1 2 3 4 5 6 7 8
Distance to the model “Sabina”
Figure 6 SIMCA modeling of the olive oil data set: Coomans plot. The dashed lines indicate the acceptance thresholds used by the two class models.

A Continually Increasing Toolbox
Although the topics presented in the previous sections cover
most of the fields of application of chemometrics to food
analysis and characterization, the chemometric toolbox is con-
tinuously evolving to match the increase in the complexity of
the problems to be tackled and, at the same time, in the
availability of high-throughput instrumentation. For instance,
multiway and multiset resolution techniques may be used to
extract chemically relevant profiles from data collected by
means of hyphenated techniques or, in general, resulting
from experiments where signals are recorded as a function of
different sources of variability. On the other hand, hyperspec-
tral image analysis techniques allow to extract both spatial
information (e.g., texture and homogeneity) and spectral
information from the samples, while data fusion approaches
combine information from multiple sources into a holistic
characterization of the sample for both exploratory and pre-
dictive purposes.
In general, one may affirm that chemometric techniques
constitute an essential and valid tool for all of those who are
involved at different levels in the characterization and analysis
of foodstuff.
See also: Authenticity of Food; Food Fraud; Infrared Spectroscopy:
Applications.

Further Reading
Bevilacqua M, Marini F, Biasioli F, and Gasperi F (2013) Advances in analysis of
instrumental food sensory quality data. In: Kilcast D (ed.) Instrumental assessment
of food sensory quality, pp. 313–352. London: Woodhead Publishing.
Bevilacqua M, Nescatelli R, Bucci R, Magrı̀ AD, Magrı̀ AL, and Marini F (2014)
Chemometric classification techniques as a tool for solving problems in analytical
chemistry. Journal of AOAC International 97: 19–28.
Brereton R (2009) Chemometrics for pattern recognition. New York, NY: Wiley.
Bro R, van den Berg F, Thybo A, Andersen CM, Jørgensen BM, and Andersen H (2002)
Multivariate data analysis as a tool in advanced quality monitoring in the food
production chain. Trends in Food Science and Technology 13: 235–244.
Brown SD, Tauler R, and Walczak B (eds.) (2009) Comprehensive chemometrics.
Chemical and biochemical data analysis. Oxford: Elsevier.
Forina M, Lanteri S, and Armanino C (1987) Chemometrics in food chemistry. Topics in
Current Chemistry 141: 91–143.
Chemometrics 9
Leardi R (2003) Chemometrics in data analysis. In: Lees M (ed.) Food authenticity and
traceability, pp. 299–320. London: Woodhead Publishing.
Leardi R (2008) Chemometric methods in food authentication. In: Sun D-W (ed.)
Modern techniques for food authentication, pp. 585–616. New York, NY: Academic
Press.
Marini F (2013) Chemometrics in food chemistry. Oxford: Elsevier.
Martens H and Martens M (2001) Multivariate analysis of quality: an introduction.
New York, NY: Wiley.
Martens H and Næs T (1991) Multivariate calibration, 2nd ed. New York, NY: Wiley.
Munck L, Nørgaard L, Engelsen SB, Bro R, and Andersson CA (1998) Chemometrics in
food science – a demonstration of the feasibility of a highly exploratory, inductive
evaluation strategy of fundamental scientific significance. Chemometrics and
Intelligent Laboratory Systems 44: 31–60.
Oliveri P and Downey G (2012) Multivariate class modeling for the verification of food-
authenticity claims. Trends in Analytical Chemistry 35: 74–86.
 Cherries (Prunus spp.): The Fruit and Its Importance
W Loescher, Michigan State University, East Lansing, MI, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction: Cherry Taxonomy and Types
Commercially, the two most important cherry species are sweet
cherry (Prunus avium, L.) and tart cherry (Prunus cerasus L.),
both tree fruits native to Southeastern Europe and Western
Asia. They are closely related and graft-compatible and will
hybridize to form interspecific (Duke) cultivars. Sweet cherry
(diploid, with a base chromosome number of 8 and a somatic
number of 16) probably originated between the Black Sea and
the Caspian Sea, but it spread into Europe in ancient times.
Tart cherry (tetraploid, with a base chromosome number of 16
and somatic chromosome number of 32) is native to the same
areas as sweet cherry, and there is good evidence that crosses
between Prunus avium and the ground cherry (Prunus fruticosa
Pall) gave rise to tart cherry. There are other cherry species, but
most, for example, Nanking cherry (Prunus tomentosa), have
limited commercial value as fruits.
Sweet cherries can be divided into two major types based on
fruit characteristics. Heart-type cherries are ovoid or heart-
shaped with relatively soft flesh, often ripening early. Most of
the commercially important cultivars, however, are of the
Bigarreau type with firmer, crisp-fleshed fruit, ripening mid to
late season. Fruit flesh may be red or yellow, and the skin may
be dark (red to nearly black) or light (yellow-red to yellow-
white).
Many sweet cherry cultivars grown throughout the world
originated in Europe, but a number of important ones were
selected or bred in local cherry districts. European cultivars
grown in the United States are Napoleon (Royal Ann), Black
Tartarian, Eagle, Early Purple, Early Rivers, Elkhorn, Hedelfin-
gen, Knight’s Early Black, Lyon, and Schmidt. The cultivars
Windsor, Van, Sam, Vista, Victor, Sue, Vega, Summit, and
Stella were developed in Canada. Chinook and Rainier were
developed in Washington. Bing, Lambert, Black Republican,
Corum, and Hoskins were selected and developed in Oregon.
Chapman, Burbank, Bush Tartarian, and the new cultivars
Mona, Larian, Jubilee, Berryessa, and Bada originated in Cali-
fornia. Recent introductions include Ulster and Hudson from
New York and Angela from Utah.
The most important sweet cherry cultivars in the Western
United States, where over 80% of the US crop is produced,
have been dark-fruited, crisp-fleshed cultivars: Bing (the lead-
ing cultivar in North America), Van, and Lambert. But others
may be available because of their use as pollenizers or as the
result of recent fresh market demand for large, light-colored,
and crisp-fleshed fruits from cultivars like Rainier. Firmness,
size, color, and soluble solids are all important market consid-
erations, and growers in regions where summer rains are prev-
alent, for example, the eastern United States and Eastern
Europe, are at a disadvantage because the main cultivars are
often the softer-fleshed, rain cracking-resistant types, for exam-
ple, Emperor Francis, Hedelfingen, and Schmidt. In these
regions, light-fleshed cultivars, Rainier, Napoleon (Royal
10 Encyclopedia of Food and Health
Ann), Corum, and Emperor Francis, are best for making into
maraschino cherries (because pigment is undesirable), but a
few are nonetheless grown for the fresh market. Napoleon is
also used for canning. Bing is mainly a fresh market cultivar,
and Lambert is used both for canning and fresh market. Black
Republican and other very firm, dark cherries are good for
freezing.
Tart cherry fruit are generally soft, juicy, and depressed-
globose in shape, but colors may range from the Morello
types with red to dark red flesh and juice to the Amarelle
types with nearly colorless juice and flesh.
Although new cultivars are being tested, there are only a few
tart cherry cultivars commonly grown in North America, rang-
ing from the light red Early Richmond, to the medium red-
skinned Montmorency, to the late dark red English Morello,
but Montmorency is still the standard. In Western Europe,
Schattenmorelle and Sternsbaer are common, but many others
are grown in Russia, Slovenia, Romania, and Hungary. Most,
unlike sweet cherry, are more or less self-fertile and generally
do not require pollenizers. Almost all of those grown in the
United States and Western Europe are harvested mechanically
and sold for processing, primarily as a frozen or canned ingre-
dient for use in manufactured food products such as pies, but
more recently as a dried fruit product, and in Europe and other
areas, there have been uses developing for juice, liqueur, and
marmalade production and combinations with yogurt.
Production Areas
Turkey, the United States, Iran, and Russia are large producers
of both sweet and tart cherries (FAO Statistics, Table 1). In
some areas of northern Europe, tart cherries are, after apples,
the second most important fruit grown. Otherwise, within
Europe, tart cherry production is concentrated in Eastern
Europe, Slovenia, Hungary, and Romania, while sweet cherry
production is more common in Western Europe, Italy,
Switzerland, France, and Spain. Sweet cherry production is
increasing in the Southern Hemisphere, New Zealand,
Australia, and Chile, for fresh shipments to northern markets
in their winter season. In the United States, sweet cherry culti-
vation has also been increasing, with production mostly in the
west, not only in Washington but also in Oregon and
California. Most US tart cherry production occurs near the
Great Lakes, primarily in Michigan with 70–75% of the US
crop, which with New York, Wisconsin, and Pennsylvania
totals 90–95% of the US crop.
Average world production values for sweet and tart cherries
are now approximately 1 200 000 and 2 200 000 metric tons,
respectively. Wide annual supply fluctuations, especially
regionally, in both sweet and tart cherries characterize produc-
tion and create high risks in product availability and price
change for producers, processors, and marketers. Annual US
http://dx.doi.org/10.1016/B978-0-12-384947-2.00138-0
 Cherries (Prunus spp.): The Fruit and Its Importance 11

Table 1 Average values and yields of tart (sour) and sweet cherries (top 20 producers) over the years 2010–12

Sour cherries average Sour cherries Sweet cherries average Sweet cherries Total average yield Total average value
Country annual yield (MT) value ($1000) annual yield (MT) value ($1000) (MT) ($1000)

Turkey 188 388 115 033 445 734 566 649 634 122 681 682
The United 179 667 109 708 324 057 411 964 503 723 521 672
States
Iran 166 733 101 811 200 864 255 353 367 598 357 163
Italy 165 893 101 297 111 006 141 118 276 898 242 415
Spain 102 744 62 737 95 179 120 998 197 923 183 735
Chile 77 159 47 114 78 716 100 069 155 875 147 183
Uzbekistan 76 687 46 826 80 333 102 125 157 021 148 951
Syria 55 677 33 997 67 540 85 861 123 217 119 858
Ukraine 32 267 19 702 72 800 92 548 105 067 112 250
Russia 29 575 18 059 71 567 90 980 101 142 109 039
Romania 17 833 10 889 74 225 94 359 92 058 105 249
Greece 16 333 9973 47 567 60 470 63 900 70 443
Poland 13 821 8439 39 727 50 504 53 548 58 943
Austria 12 238 7472 53 954 68 590 66 192 76 063
China 9096 5554 32 000 40 680 41 096 46 234
France 7308 4462 41 135 52 293 48 443 56 755
Germany 6778 4138 30 290 38 507 37 069 42 645
Lebanon 6616 4039 22 833 29 027 29 449 33 066
Serbia 6494 3965 24 322 30 919 30 816 34 884
Bulgaria 5338 3259 24 842 31 581 30 180 34 840

Source: United Nations FAO statistics.

tart cherry production ranged, for example, from 38 000 to
133 000 metric tons in the last several years, but there has
been a gradual downward trend in average production since
the mid-1960s to about 120 000 tons (in 2014), with a farm
value of about $50 million, but the processed value is at least
thrice that. Sweet cherry production, however, has been
increasing, especially recently as markets develop in Japan
and the Far East of the Pacific Rim for fresh cherries grown in
the Western United States and elsewhere.
Between 2010 and 2012, the world’s cherry acreage
increased by 4.2%, reaching over 400 000 ha, according to
the United Nations Food and Agriculture Organization
(FAO). Turkey has the largest cherry acreage worldwide
(increasing its share from 11% to 12% in 2012), with the
United States being the second with 8.7%. Italy is third with
Syria (at 7.4%) and then Iran and Spain, with shares of 7.2%
and 6%, respectively. Chile’s share increased (from 3.4% to
3.8%). Turkey also increased its share in the world’s produc-
tion, with 21.3%. The United States was second with 17%,
while Iran, Syria, and Italy reduced their shares to 8.9%,
4.6%, and 3.6%, respectively. Chile ranked sixth in world
production, with a 4% share of the total in 2012, a sharp
increase compared with the 2.8% in 2010.
Growth and Management
Flowering and fruit set – Sweet cherry flowers are in clusters of
two to four usually borne laterally on short spurs on 2-year-old
twigs or near the base of longer 1-year-old shoots. Floral initi-
ation takes place in July, after the crop is harvested, and only
on buds where the subtending leaves opened relatively early in
summer. Flower buds are of the unmixed type and do not give
rise to a lateral or bourse shoot. As a result, flowering spurs,
unlike those on apples and pears, do not remain productive.
Flowers generally have a single pistil but in very hot summers
may form two pistils that result in undesirable double fruits.
With few exceptions, for example, ‘Stella’ (and its progeny) and
the new ‘Lapins’ and ‘Sweetheart’ cultivars, commercial sweet
cherries are self-sterile (self-incompatible) and therefore
require another cultivar for pollination. There are, however,
intrasterile groups, where none of the group will cross-
pollinate any other member of the group. Bing, Lambert, and
Napoleon are one such group.
Tart cherry flowers develop much like sweet cherry, with
buds of two to four flowers on either spurs or lateral buds. Tart
cherry cultivars range from compatible to self-incompatible.
Montmorency, for example, is only partially compatible but is
always grown without a pollinator. Fruit set, however, clearly
limits yield on the fully incompatible cultivars, but overcrop-
ping may occur in other cultivars with excessive flowering or
fruit set resulting in too few leaves or leaf buds to develop fruit
of adequate size and quality.
In both sweet and tart cherries, flower development and
fruit set may frequently be harmed by late frosts, although tart
cherry is hardier and generally blooms later than sweet cherry.
Wide annual supply fluctuations are consequently common in
major growing areas for both species due to spring frosts or to
low midwinter temperatures where lack of wood hardiness is a
contributing factor. Sweet cherries are less hardy than apples,
but some tart cherry cultivars may be as hardy as the apple
cultivars McIntosh or Northern Spy. In addition, the best-
quality sweet cherry cultivars tend to be more susceptible to
rain cracking and do best in regions with dry summer growing
conditions. Fruit also develops good quality in regions often
too cool for peaches or apricots. Climate also dictates that
12 Cherries (Prunus spp.): The Fruit and Its Importance
cherries be grown where winter chilling temperatures (about
1000 h for most sweet cherries, longer for tart cherry) are
adequate to break rest; thus, cherry culture is generally limited
to cooler temperate regions.
Tree size and rootstocks – Tree size plays a central role in
production of quality fruit. Dwarf trees have many advantages:
Light penetrates better, favoring photosynthesis; the tree pro-
duces more and better fruit; spraying can be done more
efficiently, usually with reduced use of chemicals; and dwarf
trees are easier to harvest. Cherries are no exception, but dwarf-
ing rootstocks have not until recently been available for
either sweet or tart cherry. The common rootstocks, ‘Mazzard’
and ‘Mahaleb,’ only slightly affect tree size, if at all. ‘Colt’ is
similar and may be somewhat drought- and cold-susceptible.
Recently, however, dwarfing rootstocks have been developed
in several breeding programs, and is being tested. For example,
of 17 cherry rootstocks developed in Giessen, Germany, most
produce relatively large trees, but two of these rootstocks give
trees about 25% of the standard, and several rootstocks devel-
oped in Belgium may also be promising.
Harvesting and handling – Sweet cherries are almost all
hand-harvested, particularly those intended for the fresh mar-
ket. Avoiding pitting and bruising throughout harvest, sorting,
and packing is a major problem in delivering high-quality fruit
to the fresh market. Bruise susceptibility of some white- or
yellow-fleshed cultivars may even require field packing to min-
imize loss. Mechanical harvesting of tart cherries for proces-
sing, however, has been a major technological development,
which substantially reduces grower costs. A grower and his
family plus some high school students (a crew of 6–8) can
mechanically harvest as much as 200–300 hand pickers for-
merly did. The results include huge savings in direct labor
costs, large reductions in housing costs, and substantial savings
in labor fringe costs. The US tart cherry industry used essen-
tially completely mechanical harvesting during the 1970s,
although there have since been additional improvements in
equipment and techniques.
Some aspects of cherry processing have also substantially
changed, which has led to greater efficiencies and product
quality in the cherry industry. Almost all tart cherry processors
have adopted electric-eye sorting equipment, which substan-
tially reduces in-plant sorting labor, and destemming equip-
ment efficiently removes the stems from mechanically
harvested cherries. Although picking of sweet cherries for the
fresh market is still by hand, subsequent handling has been
improved dramatically very recently with the substitution of
hydraulic flumes for conveyor belts to reduce bruising and
pitting throughout sorting and packing.
Quality Factors
Soluble solids (primarily hexose sugars and sorbitol) and fruit
color (depending on the type) are the best indicators of quality
for both sweet and tart cherries, although fruit acid level may
be important in tart cherry. Except for soluble carbohydrates,
vitamins A and C, and certain flavonoids that may be impor-
tant as antioxidants in some cultivars, cherries are relatively
low in nutrients, but calcium, iron, magnesium, phosphorus,
and copper contents are high compared to apple, peach, grape,
and strawberry. High-quality tart cherry fruit typically has at
least 15% soluble solids, while sweet cherries should have
nearly 20% (or higher). Standards for harvesting and market-
ing may, however, often be lower. Optimum conditions also
often vary with use. To facilitate brining (bleaching in sulfur
dioxide solutions for maraschino cherries), fruit may be picked
prematurely before color and soluble solids are adequate for
the fresh market. Stem fruit removal force is carefully moni-
tored for tart cherries that will be mechanically harvested, and
abscission may be brought on by treatment with ethephon,
which releases ethylene, expediting abscission and fruit drop in
response to mechanical shaking.
Disorders, Diseases, and Pests
Disorders – In processing brined sweet and tart cherries, the
solution pocket problem involves subepidermal splits in the
flesh, which fill with brine solution and with ruptured cell
contents. Time of harvest, degree of turgidity at brining, tem-
perature, or any procedures that reduce either the sugar or
water content of the fruit will tend to decrease the problem.
Rain cracking (swelling followed by rupture of the epider-
mis) of sweet cherries occurs mostly during the harvest period
when the fruit is mature or nearly so and has been wet with
rain for some time. Primary cause is absorption of water
directly through the skin of the fruit and not through the root
system. Cultivar cracking susceptibility has been tested exten-
sively. In testing, Bing, one of the best-quality cultivars, was
worst, followed by Napoleon, Lambert, Emperor Francis,
Giant, Schmidt, Yellow Spanish, and Montmorency, which
did not crack. In another ranking, Bing invariably cracked
very badly and was followed by Lambert, Giant, Gil Peck,
and Hedelfingen. In still another test, Van, Merton Glory,
Vega, and Vista were very susceptible, while Emperor Francis,
Schmidt, and Sam were less, and Sue, Kristin, Ulster, and Early
Rivers were the least susceptible. From the long-range view-
point, breeding programs under way ultimately may produce
desirable crack-resistant cherries. Cracking may be reduced by
some chemical treatments, for example, rain-activated calcium
sprayers, but results with hormone (auxin (NAA) and gibber-
ellin (GA3)) applications are equivocal. In some parts of the
world, for example, Europe, covering trees with plastic film has
been widely used to avoid cracking. Elsewhere, this approach
or a high tunnel method has also been used to promote earlier
flowering and fruiting, thus capturing high early market
returns that justify the extra costs.
Pitting of sweet cherry is a condition in which areas near the
surface of the fruit become sunken, forming dimples or pits,
and may occur before or after harvest, and there are at least
three different sources: usually from bruising during handling,
from feeding by sucking insects such as the soldier bug, and
perhaps from physiological injuries, for example, adverse low-
temperature stress during postharvest cooling or growing
conditions.
Diseases – Bacterial canker, one of the most important sweet
and tart cherry pathogens, is caused by two different patho-
gens, Pseudomonas syringae and Pseudomonas morsprunorum, and
is characterized by oozing of gum (gummosis) at infection
sites. Disease development is most prevalent during the

cool, wet periods of early spring. Crown gall, caused by
Agrobacterium tumefaciens, can affect sweet and tart cherry root-
stocks and is characterized by galls forming usually near infec-
tion sites caused by wounds, sometimes man-made, for
example, cultivation injuries, or due to damage from subterra-
nean chewing insects or rodents. Some rootstocks, however,
are only moderately susceptible, and some hybrids may be
tolerant. Brown rot, caused by the fungi Monilinia fructicola or
M. laxa, affects both sweet and tart cherries and reduces yield in
infected and decaying blossoms, twigs, and fruit. Fruit decay
after harvest is also a problem. The fungi persist in mummified
fruit on the tree and the ground and infection continues from
these inocula the following spring. Growing regions with
cooler and drier summers provide some relief. Cherry leaf
spot, Blumeriella jaapii (Coccomyces hiemalis), is the most seri-
ous disease affecting tart cherry and most ground cherries.
Infection occurs in the spring on expanding leaves and con-
tinues throughout the season under favorable conditions, for
example, high humidity. Severely infected leaves become chlo-
rotic and abscise, and if defoliation is severe, fruit may not
ripen properly and tree vigor and hardiness are reduced. Pow-
dery mildew (Podosphaera oxyacanthae) is similar. Other fungal
pathogens may sometimes be important, causing blights,
crown or root rots, and replant (orchard reestablishment)
problems. Cherry dieback is thought to be a complex of several
disorders, one of which may be mycoplasma disease.
X-disease, leafhopper-transmitted and often devastating, is
also due to mycoplasma.
Several viruses cause poor vegetative growth, reduce yields,
and may even result in tree death, but others are symptomless.
Among the most severe is prunus necrotic ringspot virus, which
is pollen-transmitted and present in all cherry-growing areas of
the world. Little cherry (prune dwarf) disease is another pollen-
transmitted virus and very destructive. Prunus stem pitting
disease is caused by the tomato ringspot virus and is spread
by nematodes.
Pests – Bird damage can be very serious, and some areas may
require protective netting to reduce predation. The cherry fruit
fly passes the winter in the soil as a pupa, adult flies emerge in
late spring, and females feed on surfaces of leaves and fruit and
lay eggs in the nearly ripe fruit. On hatching, the larvae (mag-
gots) feed on the fruit flesh. The larvae are easily killed by
holding fruit near 0  C, but fumigation, until recently most
often with methyl bromide, may be required to meet quaran-
tine restrictions for shipping overseas. Other pests include
black cherry aphid, plum curculio, European red mite, peach
tree borer, and two-spotted mite.
Economic Problems and Future Developments
Although the tart cherry industry is facing a serious problem of
excessive productive capacity in some years and persistent
Cherries (Prunus spp.): The Fruit and Its Importance 13
oversupplies, this industry has adopted new technologies and
practices in the last 10 years, which substantially improve its
cost efficiency and productivity. Much of the newly planted
acreage uses efficient trickle irrigation and closely planted
orchard systems that also involve hedging techniques. These
recent new techniques, especially in combination, provide
large increases in yields per hectare and hence substantial
reductions in costs.
Considerable genetic diversity still exists in Eastern Europe
and Russia, the center of origin for both tart and sweet cherries.
Although breeding programs have been limited, exploiting that
diversity should do much to overcome the growing, handling,
and processing problems that face growers using the industry
standards, the sweet ‘Bing’ and the tart ‘Montmorency’ in the
United States. Sweet cherry growers especially need dwarfing
rootstocks and spur types for growth control, and all growers
need cultivars with disease and pest resistance, less self-sterility,
and a range of maturities so that there are longer seasons for
fresh markets. Sweet cherry growers also need cultivars with
rain-cracking resistance and, for postharvest fresh market qual-
ity, resistance to bruising. Tart cherry growers need new culti-
vars for diversifying and strengthening marketing options, for
example, fresh and frozen juice products, dyes for cosmetics
and the food processing industry, and dry stem scars and small
freestone pits to facilitate handling and processing. A combi-
nation of new marketing strategies and products for tart
cherries and advances in breeding of both sweet and tart
cherries would clearly benefit the economic potential of the
entire cherry industry.
See also: Apples; Berries and Related Fruits; Drying: Effect on
Nutrients, Composition and Health; Fruit Juices; Peaches and
Nectarines; Plums and Related Fruits; Strawberries.
Further Reading
Ayala M, Zoffoli JP, and Lang G (2014) Proceedings of the sixth international cherry
symposium (2009). Acta Horticulturae 1020: 1–536.
Brettin TS, Karle R, Crowe EL, and Iezzoni AF (2000) Chloroplast inheritance and DNA
variation in sweet, tart, and ground cherry. Journal of Heredity 91: 75–79.
Brown SK, Iezzoni AF, and Fogle HW (1996) Cherries. In: Janick J and Moore JN (eds.)
Fruit breeding, vol. I: tree and tropical fruits, pp. 213–255. New York: Wiley.
Iezzoni A, Schmidt H, and Albertini A (1990) Cherries (Prunus spp.). In: Moore JN and
Ballington Jr. JR Jr. (eds.) Genetic resources of temperate fruit and nut crops,
pp. 110–173. Wageningen: International Society for Horticultural Science.
Kappel F, Lang G, Azarenko A, et al. (2013) Performance of sweet cherry rootstocks in
the 1998 NC-140 regional trial in western North America. Journal of the American
Pomological Society 67: 186–195.
Lang GA (2000) Precocious, dwarfing, and productive – how will new cherry rootstocks
impact the sweet cherry industry? HortTechnology 10: 719–725.
Lang GA (2013) Tree fruit production in high tunnels: current status and case study of
sweet cherries. Acta Horticulturae 987: 73–81.
Webster AD and Looney NE (eds.) (1996) Cherries: crop physiology, production and
uses. New York: Oxford University Press 464 pp.
 Chilled Foods: Effects on Shelf-life and Sensory Quality
D Bermúdez-Aguirre and J Welti-Chanes, Tecnológico de Monterrey, Monterrey, Nuevo Leon, Mexico
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The commercial cold chain has been evolved together with the
progress on Food Science and Technology. In the past, chilled
food was a term used for those items that need to be kept under
refrigeration because of quick microbial spoilage. Sometimes,
these products were seafood and fish caught in remote areas of
the world and needed to be transported to specific markets.
However, nowadays, the chilled food chain also includes some
novel products that must be kept under refrigerated condi-
tions; temperature abuse is not an option because of the
microbial risk inherent in the product. These novel products
are called cooked-chilled foods or ready-to-eat meals.
During the storage of chilled foods, temperature must be
kept at specific values and recorded to ensure the microbial
safety of the product. Consumers must be aware of the impor-
tance of controlling the temperature of these products and make
sure their responsibility to handle the product from the store to
the final consumption. Even if the product is kept with ice on a
boat or kept on a supermarket on the fridge, temperature must
be carefully monitored to ensure that spoilage microorganisms
are growing slowly or the microbial growth is delayed.
Several microbial species have been identified in specific
products and these are the ones that should be monitored
during the storage; microorganisms such as Bacillus spp. are
frequently found in chilled goods and it is recognized as one of
the foodborne organisms, and high counts of this sporeformer
microorganism can promote gastrointestinal diseases. Patho-
gens such as Listeria monocytogenes, or even spores of Clostrid-
ium botulinum, can be identified in some chilled foods, because
of a poor pasteurization process, lack of hygienic measures,
cross contamination, or underprocessing of the product.
Furthermore, sensory quality of chilled foods can be drastically
compromised because of some chemical reactions taking place
during storage, such as rancidity, changes in color and flavor, or
changes in texture. Refrigerated temperatures delay some of these
chemical reactions but do not eliminate them completely. Even if
the reaction rate is slow, the chemical processes are taking place,
and if the product is stored for several weeks, noticeable changes
on sensory properties could be observed.
This article presents a brief discussion about some microbial
and sensory changes in chilled foods. The specific microbial
groups of representative food products are included as well as
some of the novel approaches to minimize microbial risks and
to improve the current technologies used to preserve chilled
foods. Sensory changes in some refrigerated products and some
novel strategies used by food technologists are also included.
Chilled Foods
The term chilled foods includes a number of products: some of
them are ready-to-eat, others require some quick preparation
14 Encyclopedia of Food and Health
by the consumer, and another category includes chilled prod-
ucts that will be used as ingredients for other products. In
general, chilled foods must be kept at a temperature 5  C to
ensure the microbial quality of the product through the chilled
chain until they reach the consumer. Chilled foods include
entrées, pasta, vegetables, fresh soups, salad dressing, desserts,
deli products, dips, ready-to-eat meals, different kinds of meat,
fish and seafood, and poultry products. All of them, regardless
of the product, must follow the food safety regulations in
each country for processing, handling, transportation, storage,
and final consumption. Often, a hazard analysis and critical
control point (HACCP) program is followed to process, pre-
serve, handle, prepare, transport, and package chilled foods.
Some of the main concerns of chilled foods are related with
chemical, sensory, and microbial changes of the product by the
end of the shelf life when the consumer is eating the product.
Chemical changes on the product can seriously compromise
the nutritional quality of the product leading to a food item
without the original nutrient content. Sensory changes are
related more with the acceptability of the product by the con-
sumer; even though these changes do not put at risk the health
of the user, they might affect the perception and consumption
of the product, making it unsuitable for eating. Finally, one of
the most important characteristics of the chilled foods is the
microbial quality; even though the growth of the microorgan-
isms is delayed during storage of these products because of low
temperature, there are some species such as psychrophilic
microorganisms that can grow and represent a risk for the
consumption. Besides, some of the emerging pathogens might
stay alive on the product and grow if the temperature is not
controlled, producing a high risk of foodborne illnesses.
Shelf Life
The shelf life of the product depends on several factors such as
initial microbial counts, the quality of the raw ingredients, the
processing technology, the addition of antimicrobials, and the
use of preservation factors such as decrease of pH or aw and
storage temperature. The chemical composition of the product
will also be an important factor to consider during the shelf life
studies, as the richer in nutrients the product is, the faster its
microbial growth. Furthermore, aw is an important fact to
consider during the shelf life, especially for chilled foods that
have high moisture content. Water is important not only for
microbial growth but also for the promotion of chemical reac-
tions on the product.
Regardless of the chemical composition of the product and
the initial microbial population, an additional aspect to con-
sider during the shelf life is the packaging material and the
packaging conditions of the product. Some packaging mate-
rials represent a strong barrier against moisture, oxygen, and
temperature, which together will extend considerably the shelf
http://dx.doi.org/10.1016/B978-0-12-384947-2.00144-6
 Chilled Foods: Effects on Shelf-life and Sensory Quality 15

life of the product. However, on the chilled food chain, some product has been considerably extended when comparing
products are not packaged because they are sold ‘fresh and raw’ with control samples kept only on ice; microbial loads
such as fish or produce or some products have a very weak reported for muscles are considerably lower for those treated
barrier with very basic packaging materials. Some products products compared with control samples. Counts of aerobes,
might be packaged using some specific conditions such as anaerobes, psychrotrophs, Enterobacteriaceae, and lipolytic
vacuum and modified or controlled atmospheres, which also and proteolytic microorganisms are affected because of the
contribute to the extension of the shelf life. Also, several pres- presence of acids and free radicals.
ervation factors have been tested together with low tempera- This traditional method to preserve fish, using ice, is com-
tures during food processing to extend the shelf life of different monly used for transportation from the origin to the final
products; physical and chemical hurdles have been used as market of the fish. Boxes with  30% of ice are used to keep
shown in Table 1. the fish with low microbial growth and minimize the chemical
reactions during transportation.
In the last few years, a new approach has been studied to
Microbial Shelf Life reduce the amount of ice required for transportation of fish.
Superchilled fish has 10–15% of ice since the temperature of
Fish and seafood
the product is reduced to about 1–2  C below its freezing
Several marine products such as seafood and fish have a short
point. The ice is surrounding the product, protecting it from
shelf life because of the chemical reactions taking place
microbial spoilage and enzymatic reactions, creating an ice
together with the postmortem changes. All of these chemical
shell within the product. Other studies on keeping chilling
changes can promote faster microbial growth of the product.
temperatures on fish include the use of ice with different
Spoilage microorganisms on fish include aerobic and proteo-
shapes such as the traditional flake ice or the use of small
lytic bacteria, coliforms, and lactic acid species. Pathogens on
spherical ice crystals known as slurry, flow, or fluid ice. The
fish include Vibrio cholera, L. monocytogenes, Escherichia coli, and
latest provides an extended shelf life of the fish rather than the
Salmonella spp., among others (Table 2). Numerous studies
flake ice (up to two times longer) because of the direct contact
have been conducted to incorporate some compounds on the
of the crystals within a bigger superficial area of the fish delay-
ice that keeps the temperature of chilled foods low. Some of
ing microbial growth and preserving the texture of the product.
the compounds have antimicrobial properties to delay the
Other approaches that have been researched to extend the
microbial growth and extend the shelf life of the product.
shelf life of fish include the use of chilling slurry, edible films,
Studies have been done with plants and herbal extracts, for
example, thyme, oregano, clove, basil, and rosemary, among
others. Other compounds that have been tested together with Table 2 List of microorganisms found in chilled food according
ice include some organic acids, alone and in combination. to the product
Some examples are citric, ascorbic, and lactic acids. The food
Product Microorganisms Product Microorganisms
products tested include marine species such as seafood (ancho-
vies) and different kinds of fish (sardines and mackerel, Fish and Lactic acid bacteria Fresh Coliforms
among others). In most of the cases, the shelf life of the seafood (LAB) produce
Vibrio spp. Yeast and molds
Listeria Lactic acid bacteria
Table 1 Examples of preservation factors used together with
monocytogenes (LAB)
refrigerated conditions to extend the shelf life of chilled foods
Escherichia coli Salmonellaa
Food item Physical factors Chemical factors Pseudomonas E. coli O157:H7a
spp. L. monocytogenesa
Fish and seafood Herbal extracts H2S-producing
Ready-to-eat meals Irradiation bacteria
(meats and Rabbit meat Pseudomonas Lamb E. coli
vegetables) spp.
Ready-to-eat meals High hydrostatic Lactic acid bacteria Lactic acid bacteria
(beef) pressure (LAB) (LAB)
Fish Essential oils Brochothrix Pseudomonas spp.
Shrimp Vacuum packing thermosphacta
Seafood Gas flushing Enterobacteria Yersinia
Fish Organic acids enterocolitica
Fish Vacuum packing Modified atmosphere Cooked- L. monocytogenes
packaging (MAP) chilled
Ready-to-cook meats Chitosan foodsa
and fish Clostridium
Fish Bacteriocins (nisin) botulinum
Fish Different shapes Bacillus cereus
of ice crystals Clostridium
Sausages Olive oil perfringens
Fish Super chilling a
(partly freezing) No common microorganisms but they might be present if the food has not been
properly handled.
16 Chilled Foods: Effects on Shelf-life and Sensory Quality
and modified and/or controlled atmosphere. The use of chill-
ing slurry has been tested in cod, basically using seawater slurry
( 2  C) that can reduce the temperature of the product faster
than regular ice. During the shelf life, chilling slurry has shown
better results on the fish because of the low microbial growth
and the freshness of the product. However, some of the prob-
lems associated with the use of chilling slurry are associated
with the weight gain and salt intake of the fish, both consid-
ered drawbacks for the product.
Meat products
One of the meat that are highly valued is lamb; some of the
most important markets are away from the highest consumers,
for example, the lamb from New Zealand needs to travel to
foreign markets that sometimes takes several weeks to reach its
final destination. The vacuum-packed lamb needs to keep a
temperature below 1.5  C to ensure a shelf life between 60
and 70 days. Some of the bacteria that can be found in lamb
meat include E. coli, Pseudomonas spp., lactic acid bacteria, and
even Yersinia enterocolitica (Table 2). It is essential to keep the
product under strict temperature conditions to delay the micro-
bial growth; besides, such as in other meat products, cross
contamination can occur during the handling of the animal.
Miscellaneous foods
As part of this category, there is a group of very complex foods
that include all the cooked-chilled foods that have meat, poul-
try, fish, and vegetables as part of the list of ingredients. Sales
on these products have been drastically increased in the last
few years because of the variety of products, innovative
concepts, and developed products that fit several lifestyles.
The average shelf life of these products is about 5 days when
the temperature has been 3  C. However, if the temperature
is not well controlled, the products might represent a risk for
consumption because of the kind of bacteria that can grow on
them. These products are generally pasteurized and packaged
such as mashed potatoes, pork chops, ratatouille, minced fish,
spaghetti, and mac and cheese. A comprehensive list of these
products is presented in Table 3. However, one of the main
risks of these products is associated with the presence of some
pathogenic microorganisms such as L. monocytogenes and
spores of C. botulinum that can survive the pasteurization and
be latent on the product. These cooked-chilled foods are also
known as refrigerated processed foods of extended durability
(REPFED) ready-to-eat meals.
Depending on the thermal treatment applied to this kind of
products, there are three categories of REPFEDs:
(a) Mild thermal treatment at 70  C for 2 min. Basically, this
treatment is applied to inactivate L. monocytogenes in at
Table 3 Examples of cooked-chilled foods (REPFEDs) available on the market; classification is made based on the main ingredient
Meat Poultry
Pork chops Chicken and rice
Meatballs with tomato paste Curry chicken
Veal stew Chicken with vegetables
Beef burgers Grilled chicken
Sliced ham Turkey meat
Roast beef Poultry sausages
least 6 log reduction. These products are pasteurized
in the package used to sell them. However, this mild
thermal treatment cannot inactivate spores in the product
and represents a risk for the consumer if the food is not
adequately handled by the consumer until the final
consumption.
(b) Medium thermal treatment at 90  C for 10 min. This
treatment has the goal to inactivate the strains of non-
proteolytic vegetative cells of C. botulinum, Bacillus cereus,
and L. monocytogenes. The product is pasteurized in the
package used to sell it, and because the thermal treatment
is not really strong, some spores can resist the process and
survive. However, the temperature used for this process
can produce some thermal damage on the spores reducing
the possibility of producing a foodborne problem.
(c) Repackaged chilled foods. This kind of products is pasteur-
ized in some packaging material and after is repackaged in
the intended final package. Sometimes, these products are
pasteurized in opened containers and after packaged to be
sold. The big risks in these products are related with cross
contamination after the pasteurization with pathogenic
strains.
Cold storage is one of the hurdles used on these products to
extend the shelf life and delay the microbial growth; however,
most of the time, a previous thermal treatment and some other
preservation factors (such as reduction of pH, aw, and antimi-
crobials) are used together.
One of the microorganisms that have been also associated
with chilled foods is B. cereus. The spores of this microorgan-
ism can survive high temperature during conventional pasteur-
ization but it can also survive refrigeration temperatures and
grow during the storage of food, representing a microbiological
concern. B. cereus is frequently associated with foodborne gas-
troenteritis. Several foodborne outbreaks have been reported
in the food industry in the last decades because of the presence
of spore-forming bacteria, mainly from the genera Bacillus. The
vehicle of these microorganisms has been mainly associated
with the vegetables that are part of most of these cooked-
chilled foods.
Chemical Shelf Life
One of the main chemical reactions taking place on fish is
oxidation of the lipids; these reactions known as rancidity are
responsible of the generation of off-flavors and odors on the
product. Marine products have a characteristic odor that can be
easily noticeable. However, when there is lipid oxidation
Fish Vegetable Pasta
Salmon and rice Mashed potatoes Spaghetti
Fish in sauce Spinach mash Cannelloni
Minced fish Ratatouille Penne with vegetables
Crab cakes Carrots Mac and cheese
Lobster cakes Leak and potato mash Lasagna
Surimi Rice with vegetables Fresh pasta salad

because of the changes in pH during the shelf life, this odor
changes to a very unpleasant and putrid smell because of the
several biochemical reactions taking place, in addition to
microbial growth. Also, there are chemical changes associated
with proteins and microbial growth together with enzymatic
activity, releasing some nitrogen that is associated with fish
deterioration. Some metabolites coming from the microbial
activity such as amino acids are related with the protein hydro-
lysis taking place on fish and are also responsible for changes
on the texture of the product.
On the other side, in some meat products, the chemical
reactions taking place during the chilled storage are part of the
natural process of tenderization of the product. Changes in pH,
water-soluble compounds, and lipids on meat muscle during
the storage will promote some desirable chemical reactions that
provide the product with the characteristic flavor during the
cooking process. For example, on beef, the biochemical changes
during the postmortem period involving the adenosine triphos-
phate (ATP) degradation will produce specific flavor precursors
on the product once it is cooked. These changes involve the
reaction between sugars and amino acids producing specific
volatile compounds. Nevertheless, the degree of production of
these compounds is also influenced by other factors such as the
diet and race of the animal, the season, the animal age, and the
conditions of slaughtering, among others.
Because the quality of the product depends on the control
on the chemical reactions taking place during the storage,
several efforts focus on how to optimize these chemical
changes. Some examples are the reduction of temperature
keeping the product in special chambers, the use of special
packaging materials and conditions, the incorporation of
some chemicals on the ice, and the use of antioxidants and
radical scavengers, among others. The use of antioxidants has
been extensively documented; several compounds have been
tested in different food products to delay microbial growth, to
inhibit enzymes or reduce the chemical reactions catalyzed by
them, and to scavenge the free radicals in those products
having rancidity problems during storage.
Sensory Quality
The basic sensory evaluation of food products includes the
assessment of odor, flavor, taste, texture, and appearance. Dur-
ing chilling of foods, some physicochemical changes take
place; in some cases, these changes are part of the conditioning
process of the product, for example, the postmortem changes
mentioned earlier (such as ATP degradation) on fish, beef,
pork, and poultry products. The flavor and aroma compounds
in different kinds of meat are present on the water-soluble and
lipid fractions of the product. Furthermore, these biochemical
changes will impact the texture, general acceptability, and
overall sensory quality of the product. The chemical reactions
associated with the sensory characteristics of the food involve
sugars, lipids, and amino acids.
Several studies have shown that the conditioning process of
beef and pork meat provides better products in terms of sen-
sory characteristics when the product is kept at chilled condi-
tions as long as 21 days. Important and significant changes are
observed in aroma, flavor, taste, and texture characteristics
Chilled Foods: Effects on Shelf-life and Sensory Quality 17
such as tenderness, juiciness, and chewiness when the meat is
allowed for a long conditioning process. Another example is
the use of CO2 snow and brine chilled ( 2  C) in ground beef;
both techniques are able to delay the microbial growth on the
meat up to 21 days. The use of CO2 snow produced also a
better texture on the product having a more tender meat;
meanwhile, the chilled option showed cooking losses on the
product.
Some specific products from beef, such as the heart and
liver, which are used as by-products for other industries, are
preserved as chilled foods. However, when the product is
removed from the animal and immediately packaged under
vacuum conditions, the meat is better preserved in terms of
weight loss, off-flavor generation, and minimum microbial
growth. Then, it is highly recommended for this kind of prod-
ucts to package the product under vacuum just as the post-
mortem period starts.
The use of herbs has also been used in the meat industry to
improve the sensory quality of some products such as chilled
lamb. Some extracts from rosemary have antioxidant effects,
and once applied to chilled lamb, the product can have a
longer shelf life not only with minimum lipid oxidation but
also with excellent sensory properties in terms of flavor, tex-
ture, color, and aroma.
The main sensory issues observed on fish during the shelf
life are related to the loss of freshness observed on the flesh, the
changes on pigmentation, the presence of off-flavors, and the
appearance of the skin. Regarding some of the novel applica-
tions in chilled foods, some of the previously mentioned stud-
ies using herbal extracts to extend the shelf life of fish have
shown positive results regarding sensory characteristics. When
the ice contains some herbs such as oregano, thyme, and
rosemary, the product acquires a similar taste and flavor, mak-
ing it more appealing to the consumer.
Furthermore, when fish is stored under specific preserva-
tion factors, the shelf life can be extended and the quality
improved. For example, studying the use of superchilled
( 2  C) and chilled (4  C) salmon and having the same fish
under modified atmosphere, it is possible to extend the shelf
life considerably. Products under superchilled conditions are
able to show good quality and lower microbial counts
(<1000 cfu g 1) after 24 days of storage (total aerobic plate,
H2S-producing bacteria, and psychrotrophic counts). The
superchilled salmon shows also acceptable sensory properties
after 21 days of storage. The salmon kept under modified
atmosphere and chilled was spoiled after 10 and 7 days,
respectively.
Fruits and Vegetables
In this group, there are not only some products highly con-
sumed such as the ready-to-eat salad blends because of the
practical approach for the consumer but also some other prod-
ucts with lower demand such as some fruit cuts sold as chilled
food or just regular fresh produce. Chilled salads represent a
very good example of vegetables that have a reduced shelf life
because of the natural characteristics of the ingredients. Some
brands are sold using modified atmospheres to provide addi-
tional hurdles for the microorganisms. In most of the cases, the
18 Chilled Foods: Effects on Shelf-life and Sensory Quality
microbial growth is delayed long enough to provide a safe
product for the consumer but all the biochemical reactions
affect considerably the sensory characteristics of the product,
mainly the texture. Although chilled fruits and vegetables can
be considered a safe product, it is well known that some
foodborne outbreaks come from these products such as the
salad blends involving pathogens such as Salmonella. Also,
some consumers prefer to purchase fresh produce and disinfect
and prepare them at home rather than use commercial chilled
vegetables.
Some common products kept under chilling conditions
and sold by piece include leafy greens, mushrooms, cucum-
bers, green and red peppers, carrots, herbs, squash, green
beans, turnips, and celery, among others. These products are
kept under chilling conditions to preserve their freshness and
delay the microbial growth. However, when these products
remain on cold storage for longer periods of time, the known
chilling injury is observed. Some of these products kept at
4–5  C and 95% of relative humidity can start to show some
signs of injury such as tissue electrolyte leakage, stress because
of ethylene production, and changes observed on appearance.
Conclusions
The chilled food chain represents a high percentage of the food
production around the world. Hundreds of food items are
currently sold as final products or ingredients and hundreds
are new in the market each year. However, one of the main
concerns related with these goods is still the microbial quality
because of the constant foodborne outbreaks and the possibil-
ity of microbial growth during storage. It is really important to
monitor the storage temperature of chilled foods throughout
the production chain until the product reaches the consumer.
Research has been done and is ongoing in the food technology
field trying to identify new preservation factors to be used
together with low temperatures not only to extend the shelf
life of the product but also to ensure that pathogenic microor-
ganisms are completely eliminated from the product after pro-
cessing. The main challenge is to find these preservation factors
strong against microorganisms but gentle with the nutritional
and sensory quality of the product.
Further Reading
Brown M (ed.) (2008) Chilled foods: a comprehensive guide, 3rd ed. Cambridge:
Woodhead Publishing Ltd.
CFA (2015). Chilled Food Association. http://www.chilledfood.org/.
Daelman J, Jacxsens L, Lahou E, Devlieghere F, and Uyttendaele M (2013) Assessment
of the microbial safety and quality of cooked chilled foods and their production
process. International Journal of Food Microbiology 160: 193–200.
ECFF (2015). European Chilled Food Federation. http://www.ecff.net/.
ICFMS (International Commission on Microbiological Specifications for Foods) (2011)
Microorganisms in foods 8: use of data for assessing process control and product
acceptance. New York: Springer.
Man CMD and Jones AA (eds.) (2000) Shelf-life evaluation of foods Gaithersburg, MD:
Aspen Publishers.
Mascheroni RH (ed.) (2012) Operations in food refrigeration Boca Raton, FL: CRC
Press.
Mills J, Donnison A, and Brightwell G (2014) Factors affecting microbial spoilage and
shelf-life of chilled vacuum-packed lamb transported to distant markets: a review.
Meat Science 98: 71–80.
Peck MW and Stringer SC (2005) The safety of pasteurized in pack-chilled meat
products with respect to the foodborne botulism hazard. Meat Science 70: 461–475.
Sofos J (ed.) (2013) Advances in microbial food safety Cambridge: Woodhead
Publishing Ltd.
 Chilled Foods: Modified Atmosphere Packaging
LM Cunha and SC Fonseca, Universidade do Porto, Vairão, Portugal
ã 2016 Elsevier Ltd. All rights reserved.
Rationale
Preservation of food products, by quality optimization and
shelf life extension, is an important challenge of the food
industry that allows the valorization of the final food product.
Because food products are very sensitive to temperature, the
reduction and maintenance of low temperatures are a crucial
technology for food preservation. The technological develop-
ment of chilling equipment in the last century allowed a wider
range of food items in the market and enlarged the number of
consumers. With the benefits of preservation by chilling, foods
do not need to be so severely processed, such as in the tradi-
tional operations of canning, drying, smoking, and salting, and
could be presented in the fresh state, minimally processed, or
mildly processed as in pasteurization. Therefore, chilled foods,
submitted to low-impact processing, result in higher quality
and nutritional retention, however, typically with a relative
short shelf life that reduces product marketability. Thus, this
type of products may take advantage of the hurdle concept,
associating chilling with the modified atmosphere packaging
(MAP) technology in order to enlarge product shelf life from
50% to 400%. One success example of this hurdle technology
concept is the application of MAP in chilling-sensitive produce,
allowing to overcome the impact of low-temperature injury.
Modified Atmosphere Packaging
MAP technology relies on modification of the atmosphere
inside the package and surrounding the food product that
leads to an increased product shelf life, by acting at the micro-
biological, metabolic, and physicochemical levels in the
product.
The modified atmosphere inside the package is maintained
over storage time due to the control of gas transport through
the packaging material that should have different barrier prop-
erties depending on whether it is nonrespiring or respiring
products. Packaging of nonrespiring products needs a high-
barrier material for gases in order to maintain the atmosphere
injected, since no relevant metabolic activity exists in the prod-
uct. Packaging of respiring products, such as fresh fruits and
vegetables, is more complex than nonrespiring products
because it involves the interplay between the natural respira-
tion process of the fresh products and the gas exchange
through the package containing the product, to generate and
maintain the adequate atmospheric composition to product
preservation. Thus, the packaging material needs a selective
permeability to the gases involved.
MAP consists in the generation of an atmosphere that is
different than normal atmospheric air. The typical composi-
tion of atmospheric dry air is 78.08% (v/v) N2 (nitrogen),
20.96% O2 (oxygen), 0.93% Ar (argon), and 0.04% CO2
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00145-8
(carbon dioxide) and other trace gases. Normally, nitrogen,
oxygen, and carbon dioxide are the three most common gases
used in MAP. In very limited cases, it is also used carbon
monoxide, ozone, ethylene oxide, nitrous oxide, helium,
neon, argon, propylene oxide, ethanol vapor, hydrogen, sulfur
dioxide, and chlorine. The presence of O2 inhibits the growth
of anaerobic microorganisms but promotes the growth of aer-
obic ones and some undesired reactions of oxidation and
vitamin loss. The increased level of CO2, compared with air,
leads to a bacteriostatic and fungistatic effect, in part due to the
acidic effect of dissolved CO2 in the product, and also
contributes to respiration rate reduction of many respiring
products. The lower level of O2, compared with air, has a
positive effect on respiration rate reduction. Superatmospheric
O2 (30–80%) may have an identical effect on reducing the
metabolic processes. The inert and tasteless N2 gas, due to its
low solubility in food products and inhibition of aerobic
spoilage microorganisms growth, is normally used as filling
gas to maintain the atmospheric pressure and avoid package
collapse.
Even though vacuum packaging (VP) may be interpreted as
a modification of the original package atmosphere and in that
sense could be considered a type of MAP, normally, it is not
regarded in literature as a MAP system due to the large differ-
ences between both packaging systems; in fact, VP has no
atmosphere inside the package.
The term scientifically used as modified atmosphere is
commercially substituted by protective atmosphere in food
labeling, in order to be easily understood and not raise suspi-
cions from consumers. Another term close, which sometimes is
wrongly applied as synonymous, is controlled atmosphere that
should not be confused with modified atmosphere. In con-
trolled atmosphere, the gas composition surrounding the
product is continually monitored and regulated to maintain
the desired gas concentrations, normally applied not in a pack-
age but in a container or a storage room. Thus, the control of
the atmosphere in modified atmosphere is less precise than in
controlled atmosphere.
MAP is a technology that despite its wide commercial appli-
cation in Europe dates back to the 1970–80s of last century
meets the demands of today’s consumer and market trends for
safer and healthier products and convenience products, prod-
ucts already sliced and ready to eat or to prepare. This preser-
vation technology, combined with temperature control, does
not need to use chemical preservatives and based on the con-
cept of barriers (‘hurdles’) allows the reduction of salt and
additives in the product, maintaining its nutritional value.
Moreover, the increase in the shelf life of the product has the
effect of reducing product losses, thus reducing distribution
costs and widening the market for such products. Thus, with
the use of chilling and MAP technologies for preservation,
many food products may be available out of season and/or
shipped to distant consumer markets with a high standard
19
20 Chilled Foods: Modified Atmosphere Packaging
quality. However, the following limitations should also be
pointed out: (i) increased product cost, (ii) loss of benefits
once the pack is opened or leaked, (iii) restricted temperature
control, (iv) specific recommended atmosphere for each type
of product, and (v) consequently development of a MAP solu-
tion for each type of product.
The two most common types of retail MA packages are
ellipsoid–cylindrical polymeric bags with sealed ends and par-
allelepipedic trays heat-sealed across the top with polymeric
films. Polymeric films are the most popular among available
barriers to create modified atmospheres. These materials pre-
sent different properties depending on the chemical structure,
production process, and additives. The plastic material may be
composed of only a film (monolayer) or a set of layers of
different polymers (multilayer). Since there is no monolayer
plastic material having properties that make it suitable for all
applications in the food industry, it is necessary to combine the
properties of various plastics in a multilayer system suitable for
each case.
The main desired factors to consider in the selection of the
polymeric material for MAP are
• type of packaging (e.g., flexible or rigid package or tray with
a semirigid cover);
• permeability properties of CO2 and O2;
• permeability to water vapor;
• physical properties (e.g., strength, transparency, and
durability);
• effectiveness and strength of heat sealing;
• resistance to degradation by chemicals;
• nontoxic and chemically inert;
• easy printing on the outer surface.
The most common plastic polymers used for packaging
foods are EVOH (ethylene vinyl alcohol), PVC (polyvinyl
chloride), PVDC (polyvinylidene chloride), PET (polyethylene
terephthalate), PP (polypropylene), PE (polyethylene), amor-
phous polyester, and nylon. Typically, these polymers are
coated on the inside face with a chemical agent for dispersing
droplets of condensed water during storage, ensuring good
visibility of the product. LDPE (low-density polyethylene)
and PVC have permeability characteristics that make them
most suitable for the packaging of respiring products and
PVDC and polyester in the case of products with low respira-
tion rates.
MAP for Nonrespiring Chilled Foods
Definition
MAP of nonrespiring products consists of complete removal of
the original atmosphere inside the package, replacing it with
the desired atmosphere by injection at the time of closing or
sealing of the package, without additional manipulation of the
atmosphere inside the package. Therefore, the package has a
crucial role in maintaining the initial atmosphere throughout
storage time of the product.
The main reason of food preservation in nonrespiring
products is the control of microbial growth, and the most
common atmospheres used are the ones rich in CO2 and
poor or absent in O2. There are many studies in literature
studying different atmospheres in specific products to con-
clude the most appropriate atmosphere for the preservation
of that specific product.
Design
The successful application of MAP technology is dependent on
a correct design of the packaging system, taking in attention of
the gas transfer phenomena between outside and inside pack-
aging material and between inside atmosphere and product
itself. If the desired atmosphere composition is initially
injected, the purpose of the packaging material will be to
maintain over time, as close as possible, that initial atmo-
sphere. The packaging material should therefore be
– CO2 barrier (after the initial injection of CO2 into the atmo-
sphere within the packaging, the packaging is intended to
prevent the outflow of this gas over time since CO2 has a
beneficial antimicrobial activity),
– O2 barrier (after the initial removal of O2 from the atmo-
sphere within the packaging, the packaging is intended to
prevent the entrance of O2 over time that would lead to color
changes and reduce product acceptability by the consumer,
with the exception of red color fresh meat that is favorable in
the presence of O2),
– barrier to aromatic compounds (in order to prevent loss of
natural flavor of the product and prevent entry of off-flavors
to the product),
– barrier to water vapor (normally, food products have a
higher water content that facilitates its loss; thus, the pack-
aging should prevent the exit of water that decreases product
sensorial quality and commercial value).
However, temperature fluctuations in the distribution chain
occur frequently, and the presence of condensed water inside
the package is depreciated; water condensed could be a good
medium for microbial growth and consequently limits the
shelf life of the product. Therefore, it would be desirable that
the packaging would be capable of absorbing the water
released from the product.
The volume of gas should usually be between two and three
times the volume of the food product due to the high solubility
of CO2 in the product.
Examples of MAP Applications
The main MAP applications in nonrespiring products are in
cheeses, fresh meat, cured and smoked meat-based products,
snacks (potato chips), fresh fish, fresh pasta, coffee, bakery
products, and ready-to-eat foods.
For food products where the main spoilage parameter is
oxidative rancidity, the gas mixture should be O2-free. In
opposition, the presence of O2 in the MA composition is
used in fresh red meat in order to avoid the red color loss.
Usually, a mixture of around 20–60% CO2 and 40–80% N2 is
adequate if the main objective is the microbial control. The
use of concentrations of 20–30% (v/v) CO2 and nonuse of O2
for cooked and smoked meat-based products are common
nowadays.

MAP for Respiring Chilled Foods
Definition
MAP of fresh produce is an atmospheric modification that
relies on the interplay between the natural process of product
respiration and gas exchange through the package. The main
goal is the control of the metabolic activity, using low-O2 and
high-CO2 atmospheric compositions that interfere in many
metabolic processes. The potential effects of low levels of O2
and high levels of CO2 are related with reduction of (i) respi-
ration rate, (ii) ethylene production and sensitivity to ethylene
action, (iii) developmental alterations, (iv) incidence and
severity of certain physiological disorders, and (v)
susceptibility to decay, with the resultant benefit of retarding
senescence and extending the shelf life of the fresh produce.
Each different product and in some cases different cultivars
have different responses to low O2 and high CO2. Exposure
of fresh fruits and vegetables to O2 levels below their tolerance
limits or to CO2 levels above their tolerance limits may hazard
the product and decrease their storage life. The beneficial and
injurious effects of low O2 and high CO2 specifically for fresh
fruits, fresh vegetables, and fresh-cut fruits and vegetables were
thoroughly studied and presented in scientific literature. The
normal gas mixtures are 1–5% O2 and 0–20% CO2.
Types of MAP
The modified atmosphere inside a package can be achieved by
either passive or active procedures. In passive procedure, the
package is closed with the atmospheric air, and due to prod-
uct respiration process, there are increase in carbon dioxide
(CO2) and a depletion of oxygen (O2) concentration, changes
that are regulated by the gas exchange through the package so
that, at equilibrium, adequate low O2 and high CO2 concen-
trations are reached. The O2 concentration decreases and the
CO2 concentration increases until the product respiration rate
equals the gas exchange rate through the package and steady
state values are attained. At steady state, the O2 flow entering
the package is equal to the O2 consumed by respiration, and
the CO2 flow leaving the package is equal to the CO2 pro-
duced by respiration. Throughout the transient period, the O2
and CO2 concentrations are not the most adequate to product
preservation. In order to achieve the desired atmosphere more
rapidly, the modification of the atmosphere in the package
can be accelerated by the active procedure, although an
increasing cost is inconvenient. In the active one, the proce-
dure is similar with the nonrespiring products: removal and
replacement of the atmosphere before closing the package. In
this procedure, there is no lag time to achieve the optimal gas
concentrations, and the interplay between the product respi-
ration and the gas exchange through the package will allow
to maintain the desired gas concentrations over product
shelf life.
Design
An MAP system not properly designed may be ineffective or
even shorten the storage life of a product. If respiration is
much slower than the gas exchange through the package,
Chilled Foods: Modified Atmosphere Packaging 21
the initial concentrations will almost not be altered. In
opposition, if respiration is much faster than the gas
exchange, O2 concentrations will rapidly deplete and anaer-
obic respiration will be induced, thus the importance of a
correct design of the packaging system. MAP design can be
improved by the development of mathematical models
used to predict the gas concentrations inside a package
instead of the ‘trial and error’ approach. Modeling a MAP
for a particular food product requires the analysis of differ-
ent components of the system package–environment–
commodity: (i) the gas exchange through package, (ii) the
recommended gas concentrations, and (iii) the respiration
rate for a specific product and storage conditions. Several
models have been developed to predict the gas concentra-
tions inside an MAP system with different levels of mathe-
matical complexity.
Examples
MAP has a special interest in high added value and highly
perishable products, achieving an increase in shelf life of up
to 800%. For example, the export of highly perishable products
such as strawberries and other red fruits needs an increased
product shelf life. The recommended storage conditions under
MAP for strawberry are 4–10% (v/v) O2 and 15–20% (v/v)
CO2, at 0–5  C combined with high relative humidity
(90–95%). The use of MAP to extend the shelf life of mush-
rooms has been extensively reported, and the recommended
MAP conditions for mushrooms at optimum storage tempera-
ture (0–2  C) combined with high relative humidity (95%) are
3–5% (v/v) O2 and <12% (v/v) CO2, depending on each
mushroom species.
Another example of high added value and highly perishable
products are the fresh-cut ones. Fresh-cut products have shorter
shelf life than intact products, owing to cell damage. Thus,
chilling and MAP technologies for extending fresh-cut product
shelf life may have a major impact on the fresh-cut market. The
higher respiration rates of fresh-cut products, as well as their
higher tolerance to CO2 in general, require the use of packag-
ing materials with a high O2 transfer rate. Recent advances in
MAP have been driven by the requirements of minimally pro-
cessed vegetables. An important commercial application of
MAP is cut lettuce. The success is attributed to retardation of
browning, coupled with maintaining a fresh appearance. The
recommended MAP conditions for cut lettuce at optimum
storage temperature (0–5  C) combined with high relative
humidity (90–95%) are 0.5–3% (v/v) O2 and 5–10% (v/v)
CO2. Other common fresh-cut products taking advantage of
MAP are shredded carrots (2–5% (v/v) O2 and 15–20% (v/v)
CO2 at 0–5  C).
See also: Apples; Brassica: Characteristics and Properties; Cheese:
Processing and Sensory Properties; Chilled Foods: Packaging Under
Vacuum; Chilled Foods: Principles; Controlled Atmosphere Storage:
Applications for Bulk Storage of Foodstuffs; Controlled Atmosphere
Storage: Effect on Fruit and Vegetables; Convenience Food; Cured
Foods: Health Effects; Meat: Eating Quality and Preservation;
Pasteurization: Principles and Applications; Preservation of Foods;
Storage Stability: Mechanisms of Degradation.
22 Chilled Foods: Modified Atmosphere Packaging
Further Reading
Caleb OJ, Mahajan PV, Al-Said FA-J, and Opara UL (2013) Modified atmosphere
packaging technology of fresh and fresh-cut produce and the microbial
consequences – a review. Food and Bioprocess Technology 6: 303–329.
Fonseca SC, Oliveira FAR, and Brecht JK (2002) Modelling respiration rate of fresh
fruits and vegetables for modified atmosphere packaging: a review. Journal of Food
Engineering 52(2): 99–119.
Hotchkiss JH and Al-Ati T (2002) Application of packaging and modified atmosphere to
fresh-cut fruits and vegetables. In: Lamikanra O (ed.) Fresh-cut fruits and vegetables
– science, technology and market. Boca Raton, FL: CRC Press, Chapter 10.
Lencki RW (2005) Modified atmosphere packaging for minimally processed foods.
In: Sun D (ed.) Emerging technologies for food processing, pp. 733–756.
Amsterdam: Elsevier.
Mahajan PV, Oliveira FAR, Sousa MJ, Fonseca SC, and Cunha LM (2006) An interactive
design of ma-packaging for fresh produce. In: Hui YH (ed.) Handbook of food
science, technology and engineering, vol. 3. Boca Raton, FL: CRC Press, pp. 119-1
to 119-16.
Mullan M and McDowell D (2003) Modified atmosphere packaging. In: Coles R,
McDowell D, and Kirwam MJ (eds.) Food packaging technology, pp. 303–331.
Oxford: Blackwell Publishing/CRC Press.
Ooraikul B (2003) Modified atmosphere packaging (MAP). In: Zeuthen P and Bogh-
Soresen L (eds.) Food preservation techniques. Boca Raton, FL: Woodhead
Publishing Limited/CRC Press, Chapter 17.
Rodriguez-Aguilera R and Oliveira JC (2009) Review of design engineering methods
and applications of active and modified atmosphere packaging systems. Food
Engineering Reviews 1: 66–83.
Rojas-Graü MA, Oms-Oliu G, Soliva-Fortuny R, and Martı́n-Belloso O (2009) The use of
packaging techniques to maintain freshness in fresh-cut fruits and vegetables: a
review. International Journal of Food Science and Technology 44: 875–889.
Sandhya (2010) Modified atmosphere packaging of fresh produce: current status and
future needs. LWT - Food Science and Technology 43: 381–392.
Simpson R, Acevedo C, and Almonacid S (2008) Mass transfer of CO2 in MAP systems:
advances for non-respiring foods. Journal of Food Engineering 92: 233–239.
Sivertsvik M, Rosnes JT, and Bergslien H (2002) Modified atmosphere packaging.
In: Ohlsson T and Bengtsson N (eds.) Minimal processing technologies in the food
industry. Boca Raton, FL: Woodhead Publishing Limited/CRC Press, Chapter 4.
Relevant Websites
http://www.aipia.info/ – Active & Intelligent Packaging Industry Association.
http://www.ba.ars.usda.gov/hb66/index.html – Commercial Storage of Fruits,
Vegetables and Florist and Nursery Stocks, draft version of the forthcoming revision
to USDA Agricultural Handbook 66.
http://www.eufic.org – European Food Information Council.
http://www.foodpackages.net/freepress/ – Book on MAP from Free Press.
http://www.foodproductiondaily.com/ – News on Food and Beverage Processing and
Packaging.
http://www.ishs.org/ – International Society for Horticultural Science.
http://modifiedatmospherepackaging.com/ – Modified Atmosphere Packaging.
http://www.poscosecha.com – International Directory of Postharvest Suppliers.
http://postharvest.ucdavis.edu/ – University of California Postharvest Technology
Center.
 Chilled Foods: Packaging Under Vacuum
M Rossi, Sealed Air s.r.l., Milan, Italy
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from the Encyclopedia of Food Sciences and Nutrition, volume 2, pp. 1191–1196, ã 2003, Elsevier Science Ltd.
The Role of Oxygen in Food Spoilage
The presence of oxygen is one of the major factors of spoilage
of foods and causes the following:
(1) Oxidation reactions, damaging vitamins, fatty substances,
pigments, and flavoring substances that are often catalyzed
by enzymes
(2) Growth and activity of aerobic microorganisms (aerobic
bacteria, yeasts, and molds)
It is therefore essential, in order to prolong the freshness of
foodstuffs, to eliminate the presence of oxygen in contact with
the foodstuff itself, and to prevent further access of oxygen
during storage. Vacuum packaging is one of the simplest and
most widely used systems to achieve this objective. This type of
packaging has enjoyed increasing success since the late 1950s,
in parallel with the development of the technology of plastics
owing also to the changes in the distribution chain of perish-
able foods requiring increased hygiene and storage life.
This article outlines the principles of vacuum-packaging
technology and the features of the packages utilized and
describes the main fields of application of vacuum packaging
to chilled foods.
Vacuum Packaging: A Definition
‘Vacuum packaging’ is a term improperly but commonly used
to define a packaging system that implies the reduction of the
partial pressure of atmospheric gases (oxygen being 20% of
them) inside a package. A vacuum inside a package can be
obtained mainly through two systems:
• Steam flushing of the headspace
• Sucking of air from the package headspace by means of
equipment (vacuum chamber and nozzle) based on a vac-
uum pump
The former system is mainly utilized in hot packaging of shelf-
stable products, which are generally packaged in rigid con-
tainers. The elimination of air is achieved by means of a
steam flush that replaces atmospheric gases inside the package;
steam condensation in the package headspace after chilling
reduces the inner gaseous pressure. The latter system is com-
monly utilized for packaging of perishable foodstuffs that have
to be stored in chilled conditions. In this case, packaging
equipment based on a vacuum pump is generally utilized in
combination with flexible packages, which, after evacuation,
are closed hermetically (by means of a seal or sometimes a tight
clip) and collapse on the packaged product once the package is
exposed to atmospheric pressure. Depending on the type of
equipment employed, a residual pressure of atmospheric gases
as low as 500 Pa can be obtained in the package.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00146-X
Figure 1 illustrates the main systems utilized for evacuating
flexible packages:
(1) Nozzle (Figure 1)
(2) Single vacuum chamber (Figure 1)
(3) Divided vacuum chamber (Figure 1)
Packaging Materials Utilized for Vacuum Packaging
Plastics are the main raw material utilized for manufacturing
the flexible materials employed in vacuum packaging of chilled
foods. These materials must have the following properties:
• Flexibility
• Mechanical resistance to various forms of abuse (abrasion,
puncture, and flex cracking)
• Gas-barrier properties adequate to the application require-
ments (generally expressed as gas permeation rates)
• Thermosealability or clippability
• Good optics (haze and gloss)
• Printability
• Suitable dimensional behavior (dimensional stability
to heat, formability after heating, and shrinkability after
heating)
To combine and balance to the required level of the earlier
mentioned properties, it is often necessary to mix different types
of individual materials. Different resins can be mixed together
(resin blends), resin additives (such as plasticizers, pigments,
stabilizers, and slip agents) can be used, and, more commonly,
a multilayer material is produced, each layer being composed of
a distinct individual material that imparts its properties to the
overall structure.
Individual components most commonly used in
manufacturing flexible materials utilized for vacuum packag-
ing are listed in the succeeding text, together with their abbre-
viations and main properties:
• Polyethylene (PE): sealability, formability, moisture bar-
rier, and low cost
• Polypropylene (PP): moisture barrier, thermal resistance,
and dimensional stability
• Ethylene–vinyl acetate (EVA) copolymer: easy sealability
and thermal shrink properties
• Ionomers: mechanical strength, easy sealability, grease
resistance, and formability
• Polyamides (PA): mechanical strength, gas barrier, and
formability
• Polyesters (PET): mechanical resistance, heat resistance,
and gas barrier
• Ethylene–vinyl alcohol (EVOH) copolymer: gas barrier and
easy processability in coextrusion
• Polyvinylidene chloride (PVDC): gas barrier and grease barrier
23
24 Chilled Foods: Packaging Under Vacuum

Atmospheric pressure

Product

(a)
Vacuum pump

Ballooning Closing device open Ballooning

Step a Step a Product

Product

Vacuum pump Vacuum pump

Package is closed

Step b Step b
Product Product

Vacuum pump

Step c Product Product

Step c

(b) Atmospheric pressure (c) Atmospheric pressure

Figure 1 Main systems utilized for obtaining vacuum packages. (a) Nozzle system. Air is extracted from the package (a bag or a pouch) through a
nozzle, and then, the package is closed. This is the simplest way of extracting the air, but it does not allow high levels of vacuum in the package.
(b) Most common system utilized for evacuating bags, pouches, and thermoformed packages. (c) Divided vacuum chamber. This allows a
better evacuation of the package headspace by using two separate chambers where a vacuum is applied sequentially. Reproduced from Chilled storage:
packaging under vacuum. Macrae, R., Robinson, R. K. and Sadler, M. J. (eds.) (1993). Encyclopedia of food science, food technology and
nutrition. Academic Press.

PE, PP, EVA, and ionomers are classified in the wide family of
resins called polyolefins. Aluminum foils and vacuum-
metallized plastic films are also utilized because of the excel-
lent gas-barrier properties of aluminum.
Multilayer materials are manufactured using various
techniques:
(1) Coextrusion: the molten resins are combined in the final
structure by extruding them through a round or flat extru-
sion die, which keeps them separate in discrete layers.
(2) Lamination: previously extruded plastic films and, some-
times, aluminum foil are joined together by means of
glues (glue lamination) or with resins that have adhesive
properties (extrusion lamination).
(3) Coating: preextruded films are coated with a layer of mol-
ten or dissolved resin (a latex).
Package Configurations Used for Vacuum Packaging
of Chilled Foods
These can be classified into four main categories: shrink bags
such as Cryovac, pouches, thermoformed packages, and skin
packages such as Darfresh (Cryovac and Darfresh are registered
trademarks of Cryovac Inc., a subsidiary of Sealed Air
Corporation).
Shrink Bags
Shrink bags are available in the form of premade bags that can
be prepared in different packages (e.g., taped bags) to allow
their utilization on automatic equipment. The main feature of
these bags is their ability to shrink when exposed for a short
time to heat (for instance, a few seconds at 90
C). This behav-
ior is the consequence of treatment imparted to the packaging
material during its production (oriented polymeric chains that
retain a built-in tension, making them able to shrink when
relaxed by heating).
Shrinking increases the packaging material thickness
(which varies between 40 and 120 mm), improving mechanical
resistance and gas-barrier properties; determines a tighter pack-
age around the product, limiting dripping out of juices in
moist products such as meat; and improves the appearance
by eliminating excess of packaging material around the prod-
uct. Shrink bags, at the onset of their introduction on the
market in the 1950s, were monolayer PVDC materials, but
subsequent technological evolution gave rise to complex coex-
truded multilayer structures having polyolefins as the main
components and gas-barrier layers made of PVDC or EVOH.
Some materials are electronically cross-linked to improve
mechanical properties.
Shrink bags are used for vacuum packaging of industrial
units of fresh meat, processed meat, and cheese and for con-
sumer units of processed meat and cheese.
Pouches
Pouches can be utilized in two forms:
• Premade plastic envelopes of different sizes that are loaded
with the product, then evacuated, and sealed in vacuum
chambers
Chilled Foods: Packaging Under Vacuum 25
• In-line prepared pouches from machines using rollstock
material (horizontal form fill seal and vertical form fill
seal machines)
The earlier mentioned machines, starting from a flat web,
produce a film tubing that can be either horizontal or vertical
(hence the different definitions), which is filled with the prod-
uct, sealed transversely, and cut into final packages.
The machines can be equipped with a vacuum chamber to
evacuate the pouch before final sealing. Form fill seal machines
are widely employed in food packaging; however, their utiliza-
tion for vacuum packaging of perishable foods is rather limited
as thermoforming is preferred whenever a high packaging
output and automation of packaging operation are required.
Premade pouches are commonly utilized for vacuum pack-
aging of industrial units of fresh red meat such as whole primal
cuts, processed meat (ham, bacon, salami, and bologna), and
cheese. Typical structures of materials for pouches, obtained by
means of glue or extrusion lamination, are based on bioriented
PA6 or PET (biorientation plus heat setting enhances dimen-
sional stability and mechanical resistance), which can be
coated with PVDC to increase the gas-barrier properties and
are laminated to suitable sealing layers (PE, EVA, or PP and
ionomers when heat resistance for pasteurization or cooking in
the package is requested). For long storage-life applications
(pasteurized cooked ham or sausages) where maximum gas-
barrier properties are needed, aluminum foil is also used.
The total thickness of the materials mentioned earlier varies
between 70 and 250 mm, the higher thickness being employed
for heavy and hard products.
Thermoformed Packages
These are obtained on continuous thermoforming machines,
which use rollstock materials. Two rolls are used, one for the
bottom web, which is unwound, heated by a warm plate, and
formed into cavities where the product to be packaged is sub-
sequently loaded, and one for the top web, which is sealed onto
the bottom web inside a vacuum chamber from which the air
has been removed. Thermoforming is widely applied to the
packaging of perishable foodstuffs because of the flexibility of
the process, the packaging speed, and the ease of automation.
A wide variety of packaging materials are utilized. Bottom
webs are generally based on PA laminated or coextruded with
all types of polyolefins. PVDC and EVOH are used when high
gas-barrier properties are needed. Some bottom webs are also
heat-shrinkable after thermoforming.
Top webs are generally laminated structures similar to those
used for pouches, the thickness of which seldom exceeds
100 mm. Metallized materials are often used in the top web
formulation.
Thermoforming is utilized for packaging many kinds of
perishable products, both consumer and industrial units,
including whole ham and fresh meat primal cuts, with the
exception of the biggest units of cheese and processed meat.
Skin Packages
Skin packaging represents an evolution of thermoforming
where the top web is softened by means of heat and subse-
quently formed onto the packaged product.
26 Chilled Foods: Packaging Under Vacuum
The bottom web can be either flexible or rigid and can be
preformed into a tray or a product-sized cavity into which the
product to be packaged is loaded.
In skin packaging, the top film conforms tightly to the
product shape with advantages in terms of appearance, limited
product crushing due to vacuum, and the possibility of contour
sealing around the product, limiting the exudation of liquids
from the product. Because of its appealing appearance and the
high cost of the packaging materials, skin-packaging utilization
is limited to consumer or family units of meat (both fresh and
processed), fish, prepared meals, and cheese.
Top webs are usually based on ionomeric resins or on
coextruded structures of polyolefins. PVDC or EVOH provides
the necessary gas barrier.
Bottom webs are generally either laminated or coextruded
structures similar to those used for thermoforming.
When a rigid tray is required, polyvinyl chloride (PVC),
polystyrene (PS), or PET is employed.
Influence of Vacuum Packaging on the Storage
Behavior of Chilled Foods
Fresh Meats and Poultry
Meats contain an abundance of nutrients necessary for the
growth of microorganisms; they are particularly rich in soluble
organic substances such as carbohydrates, amino acids, and
nucleotides. Therefore, spoilage of fresh meat and poultry
during chilled storage is mainly due to the growth of aerobic
psychrophilic bacteria belonging to the Pseudomonadaceae
family and to the Moraxella–Acinetobacter group. The growth
of these bacteria results in development of off-odors (due to
hydrogen sulfide, ammonia, amines, and indole) and bacterial
slimes, which contribute to meat discoloration.
Vacuum packaging in oxygen-impermeable materials limits
oxygen supply to the typical aerobic spoilage microflora, pro-
viding conditions suitable only for the slower-growing lactic
acid bacteria, which, in chilled conditions, require several
weeks to produce off-odors.
In addition, vacuum packaging has an impact on meat
color, which is mainly due to the presence in the muscle tissue
of myoglobin, a conjugated protein where the protein moiety
(globin) is bonded to a heme group.
The iron atom of the hematin nucleus can form complexes
with different ligands and can be in either the ferrous (Fe2þ) or
ferric (Fe3þ) oxidation state. The globin can be in either
the native or the denatured state. Among the different com-
plexes of heme, globin, and ligands, three are important in
fresh meat:
• Oxymyoglobin, with oxygen as the ligand and iron in the
ferrous state, characterized by a bright red color
• Myoglobin, with water as the ligand and iron in the ferrous
state, characterized by a purplish-red color
• Metmyoglobin, with water as the ligand and iron in the
ferric state, characterized by a brown color
Oxymyoglobin is the pigment normally present on the surface
of meat exposed to air and gives the meat its bright and
attractive color.
During storage, as a consequence of the growth of aerobic
bacteria that reduce the availability of oxygen on the meat
surface and of the reduction in the capability of the meat to
reduce its own metmyoglobin level, metmyoglobin tends
to predominate, imparting its brown color to the meat surface,
which contributes to consumer rejection of the product.
In vacuum-packaged meat, as a consequence of preventing
oxygen access to the meat surface, the pigment is of the myo-
globin color, and the meat appears darker than a sample
exposed to air or packaged in modified atmospheres. Display-
ing of vacuum-packaged consumer units of fresh meat can
create problems of consumer acceptance because of the pur-
plish color of the meat surface. In this case, proper consumer
warning has to be given to explain the origin of the color and
the advantages of vacuum packaging in terms of prolonged
storage life.
The storage life at 0–2
C of fresh primal meat vacuum-
packaged in bags or pouches is 4–8 weeks, allowing full aging
of meat types such as beef that require maturation. Consumer
units in the form of meat slices have a storage life of 2–3 weeks.
Processed Meats
It is useful to classify the many existing types of processed
meats into two main categories of products:
• Products having high water activity, the production pro-
cesses of which often imply cooking (frankfurters, patties,
bologna, fresh sausages, cooked ham, and luncheon meats)
• Cured products with low water activity (raw ham, salami,
and bacon)
All the products mentioned earlier can benefit from vacuum
packaging, which avoids surface drying and mold growth
and greatly slows down the oxidation of fat and pigments.
Pasteurizable packages are also commonly used for microbial
stabilization of some products (cooked ham, patties, and frank-
furters). Furthermore, cook-in-the-package technology has been
developed for the production of cooked ham, allowing optimi-
zation of the production process, hygienic quality, and yield.
The chilled storage life of vacuum-packaged processed
meats is very variable, depending primarily upon product
composition and packaging material characteristics, and can
vary from a couple of weeks (certain types of fresh sausages) to
several months (cured products and pasteurized or cook-in-
the-package products).
Cheese
Vacuum packaging is mainly applied to hard and semihard
types of cheese, for both industrial and consumer units, and
its effect in storage-life extension is due mainly to
• elimination of surface drying,
• slowing down of fungal growth,
• limitation of oxidation of fatty substances.
An interesting application of vacuum packaging is the curing
in the package technology that is utilized for certain types
of cheeses (Emmentaler, Gouda, and Edam). The cheese is
vacuum-packaged at an early curing stage and cured inside

the package. This technique improves yield (limiting rind for-
mation and water loss) and allows a certain product standard-
ization. Curing in the package is particularly demanding in
terms of gas transmission properties of the packaging material;
a good compromise is necessary between a sufficiently high
carbon dioxide permeability to allow the escape of gas formed
inside the cheese during curing and a sufficiently low oxygen
transmission rate to avoid mold growth. The gas transmission
rate requirements can vary from cheese type to cheese type and
also depend upon production conditions, which are often
variable among different dairies.
Fish
Wet fish is probably the most perishable type of foodstuff
because of the high content of soluble substances in the flesh,
many of which contain nitrogen and triglycerides characterized
by polyunsaturated fatty acids (in fatty types of fish such as
clupeids, salmonids, and scombroids). In addition, fish flesh is
characterized by a high level of enzymatic activity, which plays
a major role in the early stages of spoilage.
Vacuum packaging retards the growth of aerobic spoilage
bacteria and limits the oxidation of fat. However, vacuum-
packaged fish is very perishable and has to be carefully stored
at temperatures below 2
C, the normal storage life being 5–7
days. Prepackaged consumer units of wet fish, utilizing skin
packages, have recently been introduced at a commercial level
due to the need for a distribution system capable of handling
in a relatively easy way a product characterized by bad smells,
dripping out, and hygienic and preparation problems at the
consumer level. For processed fish (smoked, salted, and pick-
led), vacuum packaging is commonly utilized because it limits
the spoilage factors of this kind of product (mainly fat oxida-
tion and mold growth). The storage life of vacuum-packaged
processed fish depends upon the water activity level, ranging
between 2 and 3 weeks for slightly salted fish and a few months
for highly salted fish.
Prepared Foods
Vacuum packaging is also utilized for a wide range of prepared
foods (delicatessen products, cooked meats and poultry, pre-
pared meals, and salads).
The spoilage mechanism and perishability of these prod-
ucts are related to their chemical composition (pH and addi-
tives) and heat treatment (pasteurization), and therefore, their
storage life is very variable, ranging from 2 weeks to several
months.
An interesting application of vacuum packaging to prepared
foods is the cook-in-the-package technique, which implies
packaging under vacuum of raw or partially cooked foods
that are cooked inside the package, pasteurized (when
Chilled Foods: Packaging Under Vacuum 27
required), chilled and stored, and reheated and unpackaged
at the time of consumption. This technology allows the ratio-
nalization of meal preparation in central units (e.g., in institu-
tional kitchens and restaurant chains) because of the storage
life of the prepared meal (1 week or more) and it has also been
introduced to food-processing plants to prepare meals to be
distributed chilled at the retail level.
See also: Controlled Atmosphere Storage: Applications for Bulk
Storage of Foodstuffs; Lactic Acid Bacteria; Meat: Structure; Oxidation
of Food Components; Spoilage: Bacterial Spoilage.
Further Reading
Brody AL (ed.) (1989) Controlled/modified atmosphere/vacuum packaging of foods.
Trumball, CT: Food and Nutrition Press.
Choi S-J and Burgess G (2007) Practical mathematical model to predict the
performance of insulating packages. Packaging Technology and Science 20(6):
369–380.
Farber JM and Dodds KL (eds.) (1995) Principles of modified atmosphere and sous
vide product packaging. Lancaster, PA: Technomic.
Kadoya T (ed.) (1990) Food packaging. San Diego, CA: Academic Press.
Mathlouthi M (1994) Food packaging and preservation. London: Blackie Academic &
Professional.
Mills J, Donnison A, and Brightwell G (2014) Factors affecting microbial spoilage and
shelf-life of chilled vacuum-packed lamb transported to distant markets: a review.
Meat Science 98: 71–80.
Renerre M and Labadie J (1993) Fresh meat packaging and meat quality. Proceedings
of the International Congress on Meat Science and Technology 39: 361–387.
Robertson GL (1992) Food packaging – principles and practice. New York: Marcel
Dekker.
Robertson GL (2013) Food packaging: principles and practice, 3rd ed. Boca Raton, FL:
CRC.
Schneider Y, Kluge C, Weiß U, and Rohm H (2010) Packaging materials and equipment.
In: Law BA and Tamime AY (eds.) Technology of cheese making, 2nd ed.
Wiley-Blackwell, p. 413. Chichester, United Kingdom.
Sun D-W (2011) Handbook of frozen food processing and packaging, 2nd ed. Boca
Raton, FL: CRC Press.
Yam KL (2009) Encyclopedia of packaging technology. USA: Wiley.
Relevant Websites
www.fda.gov/Food/GuidanceRegulation/RetailFoodProtection/FoodCode/ucm188201.
htm – FDA - U.S. Food and Drug Administration.
http://www.food.gov.uk/business-industry/manufacturers/shelf-life-storage/vacpac –
Food Standards Agency.
http://www.mda.state.mn.us/global/mdadocs/food/foodsafety/mod-vacpack/vacpack-
speaker_notes.aspx – Minnesota Department of Agriculture - Speaker notes from
Vacuum Packaging Food Safety Principals speech.
http://www.ngdc.noaa.gov/mgg/curator/meetings/2007/presentations/IODP/
Core_preservation_complete_CuratorsMeeting_200709.pdf – NOAA National
Centers for Environmental Information - Presentation from Curators Meeting.
http://www.uvm.edu/extension/food/pdfs/vacuum_packaging_factsheet_2013aug.pdf
– University of Vermont - Reduced Oxygen Packaging.
 Chilled Foods: Principles
GG Amador-Espejo and ME Bárcenas Pozos, Universidad de las Américas Puebla, Cholula, Mexico
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Nowadays, the population exodus to urban areas has required
some improvements in food preservation techniques and
greater distribution services of perishable products. Due
to modern consumer preferences of quality, the most impor-
tant method of preservation and distribution to the main
world market is by chilling and freezing. Today’s lifestyles
have also changed consumer behavior, product preferences,
and eating trends resulting in new requirements for food
producers.
Chilling is one of the preservation techniques that is most
applied with food products, having been established in the
nineteen century. Because of the complexity of the refrigeration
process, it needed more time to be developed than the heating
process. People learned to produce heat about 500 000 years
ago, but the chilling process started about 150 years ago—
although some reports from the second century (BC) estab-
lished the use of 4
of coldness for medical purposes. By
definition, the refrigeration process can be described as a sim-
ple reduction of the ambient temperature (i.e., if the ambient
temperature is around 40
C the product transport can operate
with a refrigeration system to reduce the temperature to 30
C),
even though some authors have considered the refrigeration
temperatures to only be in the range of 0–10
C, based on the
preservation principle involved.
The refrigeration principle consists of a reduction in the
product temperature which reduces the kinetic energy, ulti-
mately minimizing the rate of motion of the molecules inside
the product. This rate determines the speed at which many
important reactions take place. In the case of food products,
many spoiling reactions can be controlled by temperature,
such as: microbial growth, physiological processes (ripening
of fruits and vegetables), and chemical and enzymatic
reactions (enzymatic and non-enzymatic browning, vitamin
degradation, lipid oxidation, and biocompound degradation).
Basically, all food products can be preserved by refrigera-
tion for some time, depending on the final temperature
reached and the chilling rate. However, the final storage tem-
perature and the amount of time that the product is subjected
to refrigeration have different effects that can compromise the
product quality. In this sense, in rapidly cooled peaches, a
serious defect can be observed, known as ‘wooly texture,’ in
which a lack of juiciness without variation of the tissue water
content is shown. Another typical case of the effect of the
refrigeration process on food products is the phenomenon
called ‘cold shortening,’ which is an irreversible reduction of
the muscle tissue – that can be detected in beef, lamb, and pork
meat – when the temperature is reduce rapidly.
This contribution establishes the principles related to the
chilled process, equations related, new techniques, and also,
improvements in the refrigerant compounds employed in food
processing.
28 Encyclopedia of Food and Health
Chilling
The chilling process can be described as the method to remove
heat from a product. It is the most benign process applied to
food, presenting only a few changes in flavor, texture, and
nutritious value. In general, refrigeration temperatures for food
product preservation are below 15
C and above the freezing
point. Although human beings have been using cold storage
since ancient times – by placing products in ice or at low
temperatures outside – the refrigeration process, or mechanical
refrigeration, has no more 150 years of application.
Due to their nature, food products are vulnerable to spoil-
ing, reducing their commercial life by three mechanisms:
• The growth of live organisms can contaminate and deteri-
orate food products. Microorganisms, such as bacteria,
parasites, and molds can be in the air and soil. Insects and
rodent can contaminate food products with their own
microbial fauna.
• Chemical reactions, such as browning or oxidation, can
produce different compounds causing changes in flavor or
texture of the product.
• Biochemical reactions, like respiration, ripening, and brow-
ning (in vegetables and animal meat after slaughter) are
basically carried out by enzymes within the product. Also,
these reactions may reduce the product quality and other
physical phenomena such as gain or loss of moisture that
reflects on moisturize or dehydration.
On the other hand, the term ‘shelf life’ refers to the time-
frame that the product (stored under the recommended con-
ditions) will:
• remain safe for consumption;
• be certain to retain desired sensory, chemical, physical, and
microbiological characteristics; and
• fulfill any label declaration of nutritional information.
This definition of shelf life implies the storing conditions,
which, when referring to refrigerated conditions, may vary
enormously due to temperature changes during the transpor-
tation and in-home handling before their consumption.
The main effect of chilling is the decrease of storage tem-
peratures, which also implies a reduction in the rate of the
reactions mentioned above, presenting an increase in the
shelf life of the product. The deterioration rate can lessen as
the temperature decreases to the minimum in the range of the
refrigeration temperatures. Although, even below freezing tem-
peratures, some reactions still take place in some products.
The most important effect of the chilling process on food
products is the reduction of the microbial growth, which
increases the shelf life of the product. The most common
temperature for microorganism growth is around 36
C; but
microbial growth has been detected in extremely low
(34
C), and extremely high (100
C) temperatures as well.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00143-4
 Chilled Foods: Principles 29

The microbial grow division in this large range of temperature
refers to three main groups. The first group of microorganisms
that grow well at or below 7
C, and have their optimum
between 20 and 30
C, are referred to as psychrotrophs. Further,
the group that can grow well between 20 and 45
C, with their
optimum range between 30 and 40
C, are referred to as
mesophiles. While the group that grows well at and above
45
C, with their optimum range between 55 and 65
C, are
referred to as thermophiles.
Psychrotroph microorganisms that can grow under refriger-
ated conditions are: a. bacteria Shewanella, Alcaligenes, Flavobacter-
ium, Corynebacterium, Lactobacillus, Micrococcus, Pseudomonas,
Psychrobacter, Brochothrix Enterococcus, and others, being Pseudo-
monas and Enterococcus the most commonly detected and c. molds
c strains of Aspergillus, Cladosporium, and Thamnidium. Under
refrigerated conditions, the growth of some yeast has been
detected, but in less numbers than bacteria and molds.
Although shelf life is enhanced by chilling, it only increases
by a small amount of time compared to other preserving
techniques, such as heat treatment, freezing, or drying.
Further, it is important to point out that chilling can be
applied not only to enhance the product shelf life, but to
control the growth reaction rate of specific microorganisms
desirable in food products. This is the case of the chilled
ripened process in some cheeses, meats, and wines.
It can be assumed that the lower the storage temperature,
the higher the shelf life; but this is not always true. Some
products, mainly fruits and vegetables, may suffer when they
are stored at temperatures close to 0
C. For instance, peaches
can suffer undesirable internal and core browning (in a process
called woolliness) if they are stored at temperatures close to
0
C. In the case of potatoes, when they are stored below 3
C
quality deterioration occurs as a consequence of changes in the
starch–sugar system that produces the accumulation of sugars.
Table 1 presents the most common deterioration problems
related to cold storage. Further, much research has been carried
out to find the best temperature storage conditions for different
food products, with the intention to reduce undesirable reac-
tions and optimize their shelf life. Table 2 presents the opti-
mum storage conditions for some food products.
Moreover, another parameter that must be considered dur-
ing food refrigeration storage is the relative humidity. If the
water activity in the product is higher than the relative humidity
inside the refrigeration room, there will be loss of moisture in
the product. On the contrary, if the relative humidity in the
refrigeration room is higher than the water activity, water con-
densation may occur, which, in many cases, produces microbial
growth and spoilage of the product. In this sense, high relative
humidity conditions (between 90% and 95%) are preferred for
fruit and vegetable products—based on their high moisture
content. On the contrary, products with low moisture content,
such as cheese or nuts, should be stored under reduced relative
humidity conditions (< 85%). To prevent microbial growth in
the refrigeration chamber with high relative humidity, it is
necessary to avoid water condensation on the chamber walls.
The Refrigeration Process
Refrigerants
Refrigerants are chemical substances that can take the heat out
from the product and release it outside the refrigeration sys-
tem. The substances employed in vapor compression systems,
such as NH3, CO2, and R134 are named primary refrigerants

Table 1 Deterioration of fruits and vegetables under refrigeration storage conditions

Product Critical storage temperature (
C) Type of deterioration between the critical and freezing temperature

Mango 10–13 Gray discoloration of peel

Unequal ripeness
Melon 7–10 Spot, peel putrefaction
No ripeness
Apple 2–3 Internal and core browning, scald, moist breaking
Orange 3 Spot, brown marks
Sweet potatoes 13 Internal discoloration, spot
Rotting
Eggplant 7 Scald on surface
Rotting
Olive 7 Internal browning
Avocado 4–13 Brown discoloration in pulp
Squash 10 Rotting
Green beans 7 Spot and redness
Lime 7–9 Spot
Lemmon 14 Spot, red mark
Papaya 7 Spot, rotting
No ripeness
Cucumber 7 Spot, rotting
Pineapple 7–10 Green color upon ripening
Banana 12–13 Opaque color upon ripening
Watermelon 4 Spot, unpleasant odor
Tomato (green) 13 Light color upon ripening, rotting
Tomato (ripe) 7–10 Rotting, softening, soaked water
30 Chilled Foods: Principles

Table 2 Optimum storage conditions different foods

Food product Storage temperature (
C) Relative humidity (%) Storage life (days)

Banana 11–15.5 85–95 7–10

Apricot 0.5 to 0 90 7–14
Bean (snap) 7 90–95 7–10
Broccoli 0 95 10–14
Carrot 0 98–100 28–42
Celery 0 95 30–60
Cherry 1 90–95 14–20
Chicken Just above the freezing point 85–90 20–30
Cucumber 10–15 90–95 10–14
Eggplant 7–10 90–95 7–10
Fish Just above the freezing point 90–95 7–10
Lemon 10–14 85–90 30–180
Lime 9–10 85–90 40–140
Lettuce 0–1 95–100 14–20
Meat Just above the freezing point 85–90 10–15
Milk (pasteurized) Just above the freezing point <85 10–15
Mushroom 0 90 3–4
Peach 0.5 to 0 90 14–30
Plum 1 to 0 90–95 14–30
Potato 3–10 90–95 150–240
Spinach 0 95 10–14
Strawberry 0.5 to 0 90–95 5–7
Tomato 4–10 85–90 4–7
Watermelon 4–10 80–90 14–20

Table 3 Application of different refrigerant compounds used in the industry

Refrigerant

Formula Number Latent heat (kJ kg1) Boiling point 100 kPa (
C) Environmental classification Toxicity Flammability

CCl3F R-11 194.20 23.8 CFC Low Low

CCl2F2 R-12 163.54 29.8 CFC Low Low
CHCl2F R-21 254.20 44.5 HCFC Low Low
CHClF2 R-22 220.94 40.8 HCFC Low Low
R-123 170.47 27.83 HCFC Low Low
C2H2F4 R-134 217.28 26.05 HFC Low Low
NH3 R-717 1328.48 33.3 High High
CO2 R-744 352.00 78.5 Low Low

and are used to chill the product. Substances employed for the
transportation of the chilled product from one cooled place to
another are named secondary refrigerants. In this sense, solu-
tions with a freezing point below 0
C are used as secondary
refrigerants; the most commonly employed of these solutions
being ethylene glycol, propylene glycol, and calcium chloride.
Their properties are similar; however, propylene glycol has the
advantage of being safe if it comes into contact with food.
The first refrigerant applied in the industrial vapor com-
pression systems was ethyl ether, around 1850. After, other
refrigerant products were employed, such as carbon dioxide,
methyl chloride, butane, ammonia, ethane, and gasoline—
among others. During the decade of the 1920s, after many
factory workers were injured and or killed due to leaks of
these first refrigerants, their use was limited and finally prohib-
ited for use in the industry.
Later, during the first part of the twentieth century, the
company DuPont developed the refrigerant R-21; the first
member of the chlorofluorocarbon family. Sometime later,
the company developed R-12 (Freon), naming it as the most
adequate refrigerant for commercial use. However, during the
1970s, it was discovered that Freon had a negative impact on
two worldwide problems: the thinning of the protective ozone
layer at the stratosphere (protecting the ground from ultra-
violet radiation from the sun); and the increase in global
warming by the greenhouse effect on the atmosphere. For
these reasons, the refrigerants R-12 and R-22 have been
replaced by other kinds of refrigerants, such as R-134a and
R-123. Table 3 presents the main characteristics of the primary
refrigerants used in the industry and in domestic equipment.
From this, the most important characteristics are the vaporiza-
tion heat and low boiling point, followed by the toxicity and

flammability. Even though the ammonia (R-717) has good
thermodynamic properties, the high toxicity and flammability
make it extremely difficult to use in chilling industry.
Refrigerants can be classified by the halocarbon numbering
system. This system was first adopted by the DuPont
Company. But also, it has been adopted by some professional
associations, such as the American Association American Soci-
ety of Heating, Refrigerating and Air-Conditioning Engineers
(ASHRAE). The general model to name a refrigerant is R-xyzC,
where R stands for refrigerant, x stands for the number of
carbon atoms, y stands for the number of hydrogen atoms
and z stands for the number of fluorine, chlorine atoms.
Table 4 presents the classification convention for this system.
Further, there is a classification systems presented by the
International Union of Pure and Applied Chemistry (IUPAC).
In this case, the refrigerants are classified by chemical compo-
sition, being:
• CFC. Chlorine-Fluor-Carbon compounds. They are pres-
ently banned because of their ozone depletion potential
(ODP). Production stopped world-wide in 1995 and
usage in 2000 (e.g., R12, R13, R500).
• HCFC. Hydro-Chlorine-Fluor-Carbon. The use of these
refrigerants is not allowed because of their zero ODP,
their production is going to stop in 2015, and usage in
2030, worldwide, but in the European Union, in 2001 for
new equipment, in 2010 for old equipment, and in 2015
for any use (e.g., R22, R409).
• HFC. Hydro-Fluor-Carbons. Their use is allowed because of
their zero ODP, but they contribute to the global warming
potential (GWP). Fluorocarbons, where all hydrogen atoms
are replaced by fluorine atoms, are often called perfluoro-
carbons (PFC) (e.g., R134a, R410A, R404A, and R407C).
Table 4 Refrigerants nomenclature by halocarbon numbering system
Code Significance
Rxyz Halocarbons
x ¼ number of carbon atoms 1
y ¼ number of hydrogen atoms þ1
z ¼ number of fluor atoms
R4xx Zeotropic mixtures
xx ¼ chronological order
A,B, for different compositions, as they become
accepted
R5xx Azeotropic mixtures
xx ¼ chronological order
R-7xx Inorganic compounds
xx ¼ molar mass (rounded)
a,b,. . . for different isomers, as they become
accepted
RxyzBt halons
xyz as for CFCs and t ¼ bromine
Halons Halons
Xyzt xyzt for C, F, Cl, and Br atoms
Chilled Foods: Principles 31
• HFO. Hydro-Fluor-Olefin. It almost has equal thermal
behavior to that of R132a, but with a much smaller GWP.
The most common R1234yf (2,3,3,3-Tetrafluoropropene,
C3H2F4) is being used as a replacement for R134a in auto-
mobile air conditioners.
• Natural refrigerants (no ODP or GWP): such as air, water,
ammonia (R717, NH3), carbon dioxide (R744), and pro-
pane (R290, C3H8), are flammable, or toxic, or have very
large vapor pressures.
• Halons. Containing bromine which replaces some chlorine
compounds. This refrigerants were developed in the 1960s,
and were employed more as a fire-fighting agents. However,
because of the ODP, their production stopped in the 1990s.
There are signs used to detect a leakage of the refrigerants, such
as odor in natural (ammonia) and artificial (hydrocarbons)
refrigerants, some physical effects (bubbling), chemical effects
(catalytic reaction in platinum wires) and fluorescence excita-
tion with ultra-violet lighting to detect CFC, HCFC, and HFC
refrigerants.
Cryogenic chilling is the term applied to refrigerants that
change their phase by absorbing latent heat to cool the food.
There are only three kinds of cryogen refrigerants: solid carbon
dioxide, liquid carbon dioxide, and liquid nitrogen.
The phase change mechanism in solid carbon dioxide is by
sublimation (solid to gas), removing latent heat of around
352 kJ kg1 at 78
C. Liquid cryogens remove latent heat by
vaporization, obtaining around 358 kJ kg1 at 196
C for
liquid nitrogen. In the case of liquid carbon dioxide, the latent
heat is similar to the solid. Further, the gas obtained also
absorbs sensible heat as it warms from 196
C (liquid nitro-
gen) or from 78
C (CO2) to give a total refrigerant effect (the
total amount of heat that can be removed by the refrigerant) of
Examples
R12: (R012) 1C ! x ¼ 0, 0H ! y ¼ 1, 2F ! z ¼ 2, plus 2Cl, that is, dichloro-difluoro-
methane, CCl2F2
The number of Cl is calculated by diminishing the number of flourine
R22: (R022) 1C ! x ¼ 0, 1H ! y ¼ 2, 2F ! z ¼ 2,
R134a: 2C ! x ¼ 1, 2H ! y ¼ 3, 4F ! z ¼ 4, C2H2F4
Plain hydrocarbons were also included:
R50 ¼ CH4, R170 ¼ C2H6, R290 ¼ C3H8
R404A: R125 þ R143a þ R134a, 44/52/4 in %wt
R410A: R32 (CH2F2) þ R125 (C2HF5), 50/50 in %wt*
R407C: R32 þ R125 þ R134a, 23/25/52 in %wt
R500: 50% R125 þ 50% R134a wt was the first on the market
R502: 48.8% R22 þ 51.2% R115 was the third on the market
R507: R125 þ R143a, 50/50 in %wt
R717: ammonia (MNH3 ¼ 17 g mol1)
R718: water (MH2O ¼ 18 g mol1)
R744: carbon dioxide (MCO2 ¼ 44 g mol1)
R764: sulfur dioxide (MSO2 ¼ 64 g mol1)
R12B1: 1C, 0H, 2F, 1Br, 1Cl
Halon 1211 ¼ CF2ClBr, bromochlorodifluoromethane, liquid
Halon 1301 ¼ CF3Br, bromotrifluoromethane, gas
Halon 2402 ¼ C2F4Br2, dibromotetrafluoroethane, liquid
32 Chilled Foods: Principles
690 and 565 kJ kg1, respectively. This effect can be used to
chill diced meat or vegetables at up to 3 ton h1, making the
plant operation and storage processes faster.
The main disadvantages of these substances are their cost
(because most of the time the refrigerant is not reusable), and
their ability to cause asphyxia, which is higher for carbon
dioxide, and to a lesser extent with nitrogen. Regulations are
set for a maximum safe limit for operators of 0.5% CO2 by
volume, and that excess carbon dioxide must be removed from
the processing area by an exhaust system to ensure operator
safety, which also incurs additional setup costs. Other hazards
associated with liquefied gasses include cold burns, frostbite,
and hypothermia after exposure to intense cold.
A refrigerant’s safety classification is based on the combina-
tion of toxicity and flammability. According to the ANSI/ASH-
RAE Standard 34-1997, safety groups are classified as follows:
• A1 lower toxicity and no flame propagation
• A2 lower toxicity and lower flammability
• A3 lower toxicity and higher flammability
• B1 higher toxicity and no flame propagation
• B2 higher toxicity and lower flammability
• B3 higher toxicity and higher flammability
The Refrigeration Cycle by Vapor Compression
The refrigeration process taking place in household and
warehouse fridges includes a mechanism that removes energy
from a place with low temperature and transfers it to a place
with high temperature. In the first place, the second law of the
thermodynamics indicates that heat flows from a place with
higher temperature to a place with lower temperature to equil-
ibrate the temperature in the system. However, according to the
Clausius statement, heat can flow from a place with low tem-
perature to a place with higher temperature by applying some
effort. In the refrigeration process, the objective is to transfer
heat from a place with low temperature to a place with higher
temperature. To achieve this, the refrigerant uses the transfor-
mation enthalpies presented when it changes from the liquid to
vapor. A scheme of this process is presented in Figure 1. The
refrigeration process starts in point 1, called the compression
point, in which the fluid in a vapor state receives energy from the
compressor and circulates to the point 2. At point 2, the fluid
increases its pressure, temperature, and enthalpy. Then, the
refrigerant enters into the condenser, where the fluid liberates
Condenser
Expansion
Compressor
devise
Evaporator
Figure 1 Chilling cycle system (Qi is the heat remove from the food
product; Qo is the heat released to the environment).
heat to the system surroundings—due to the temperature differ-
ences between them. As a consequence of this heat transfer, the
refrigerant decreases its temperature and condenses, presenting a
phase change from the vapor to the liquid phase at constant
pressure (point 3). Afterward, the refrigerant flows through an
expansion valve, with a decrease in pressure in an isoentropic
process, to obtain a vapor–liquid mixture (point 4). Finally, the
mixture enters into the evaporator (which corresponds to the
inside part of domestic fridges) where the refrigerant receives
heat from the food products, until it reaches the conditions
presented in point 1 to restart the cycle.
Further, the refrigeration cycle can be described in a pressure-
enthalpy diagram (Figure 2). First, the refrigerant increases the
pressure during the compression process (points 1–2). Later,
there is a decrease in the enthalpy during the condensation
that takes place at constant pressures (point 2–3). After, the
fluid reduces its pressure in the expansion valve at isoenthalpic
conditions (points 3–4). Finally, the refrigerant increases its
enthalpy (by the increase in the temperature) at isobaric condi-
tions (points 4–1) to restart the process.
In this diagram, the amount of energy released by the
refrigerant in the condenser can be determined by the
enthalpies in points 2 and 3. In this sense, the heat flow
released by the refrigerant is:


Q_ s ¼ w H
^3  H ^2 [1]
In the eqn [1], w represents the flow mass of the refrigerant
and H represents the enthalpy per unit mass of refrigerant. In
this case, the heat value obtained is negative because the
amount of energy in point 2 that represents the enthalpy of
the refrigerant when the fluid leaves the compressor is higher
than in point 3, when the refrigerant leaves the condenser.
To know the amount of energy removed by the refrigeration
system, it is necessary to apply the eqn [2]. In this equation,
w represents the flow mass of the refrigerant, H1 represents the
enthalpy in point 1 after the evaporation process in the chilling
chamber, and H4 (point 4) represents the energy of the refrig-
erant before flowing to the evaporator.


Q_ E ¼ w H
^1  H^4 [2]
P
(MPa)
QH
Pcod H3 H2
Pevap Wcomp
H4 Re H1
H
(kJ/kg)
Figure 2 Chilling cycle system in an enthalpy–pressure diagram.

The value of QE represents the most important part of the
refrigeration system, a parameter known as refrigerant capacity.
Another important parameter in the refrigeration process is
the coefficient of performance (COP), which is defined as the
quotient of the refrigerant capacity and the work externally pro-
vided to the system (in the compression operation) (eqn [3]).


H^1  H^4
COP ¼ [3]
H^2  H^1
Further, the refrigeration facilities used for these applica-
tions must be designed to overcome heat gain through the
walls of the system, and also to perform the descent of the
temperature. To calculate the energy required for chilling food
products, the following equations must be applied (eqn [4]).

compressor, while the liquid is fed into an expansion valve to
Q ¼ mCp Ti  Tf [4]
• Q is the total energy removed from the food product
• m: mass of the product being chilled (kg)
• Cp: Specific Heat value (kJ kg1 K) of the food product
• Ti  Tf: Difference between the initial and final chilling
temperature (K)
There is abundant information regarding the Cp of different
food products above the freezing point and below the freezing
point. However, the Siebel equation for above freezing prod-
ucts (Tk < 274 K) can be applied to predict the Cp in kJ kg1 K;
in which, ‘a’ is the mass fraction of water in the food product
(eqn [5]).
Cp ¼ 3:3494a þ 0:8374 [5]
Moreover, the size of the refrigeration plant and the proces-
sing time required to chill different products are calculated
using unsteady-state heat transfer methods. Many assumptions
are made to simplify the estimation, that is, the initial temper-
ature of a food is constant and uniform throughout the prod-
uct and the temperature of the cooling medium, respiratory
activity, and all thermal properties of the food are constant
during cooling.
Total heat load ¼ Heat from respiration
þ Sensible heat of containers
þ Heat evolved from operators and lights
þ Heat loss through roofs and walls
þ Heat loss through floor
A British thermal unit is defined as the amount of heat energy
required to raise the temperature of one pound of water 1
F
from 59 to 60
F; and 1 Btu h1 ¼ 0.293 watt (W).
Another unit of refrigeration widely used in the industry of
ventilation and air conditioned is ton of refrigeration, or sim-
ply ton, being 1 ton ¼ 12 000 BTU h1 of heat removed. This
equals the heat absorbed by 1 ton (2000 lb) of ice melting at a
temperature of 32
F over 24 h, and because the heat of fusion
of ice at 32
F is 144 Btu lb1 (eqn [6]).
1  2000  144
1 ton ¼ ¼ 12000 BTU h1 [6]
24
The conversion factor of 1 ton in the SI system is 1
ton ¼ 3.516 kW
Chilled Foods: Principles 33
Refrigeration System Derivations
With the intention of reducing the power needed in the com-
pression stage or achieving higher evaporation values than in
the regular vapor compression cycle, many derivations of this
arrangement have been developed, and they are presented in
the following sections.
Chilling system with gas separation (flash economizer)
In systems with a gas separation arrangement, the fraction of
vapor is separated from the saturated liquid in a manner that,
conditions of a saturated liquid that, when expanded produces
a mixture (point 3), which can be separated into a liquid
(point 4) and a vapor (point 6). This vapor is send into the
the evaporator; for this reason, the system needs two compres-
sors. The power savings in the compressors by employing this
system can be up to 16% (Figure 3).
Systems with two compressors and one evaporator
A useful option to achieve an important reduction in the power
needed to compress the refrigerant is the application of a two-
stage compression systems. In this system, a two-stage com-
pression and intermediate refrigeration section are employed.
This system requires higher initial investments, but the savings
in power in the long term, and the low temperature achieved in
the evaporator section justify its use.
Systems with two compressors and two evaporators
Sometimes different refrigeration temperatures are required in
diverse processes in a food industry in the operation plants. Then
it is required to develop a chilling system with more than one
evaporator. Evaporators operating at two different temperatures
can work in an efficient way in a two-stage system, employing an
intermediate refrigerator and vapor separator. In this system, the
point 3 corresponds to the saturation temperature T3 of the
second evaporator, and is set by this temperature (Figure 4).
To evaluate this system, it is necessary to carry out a mass and
energy balance in the different sections of the arrangement.
The next assumption is taken within the system, where w is
the flow mass in the system (eqn [7]):
w1 ¼ w2 ¼ w7 ¼ w8 [7]
Balances in the evaporators:


QE1 ¼ w1 H ^7
^1  H [8]
3 2
Condenser
4 5
Liquid-vapor
separator 1
6 Two-stage
compressor
Evaporator
Figure 3 Expansion process with gas separation.
34 Chilled Foods: Principles

5
Condenser
4
High
pressure
compressor
3

6
Evaporator
QE2

6
Heat exchanger and
evaporation tank
1

7 Low pressure
compressor

8
Evaporator
QE1

Pressure

5 4

6
7 3
2

1
8

Enthalpy
Figure 4 Operation with two compressors and two evaporators.



^ 2 ¼ w3 H
Second compressor : ðPowÞ2 ¼ w3 W ^3
^4  H [14]
QE2 ¼ w6 H ^6
^3  H [9]

Balances in the second evaporator and intermediate Chilling system with heat exchanger
refrigerator: In this arrangement, the liquid refrigerant, before passing
^ 2 þ w5 H
w2 H ^ 5 þ QE2 ¼ w3 H
^ 3 þ w7 H
^7 [10] through the expansion valve, is sent to a heat exchanger with
the intention of increasing the heat in the system (and it
Also, in this subsystem, the flow mass can be determine by facilitates the state change to vapor). The vapor flowing after
the eqn [11]. the evaporator, before entering the compressor, is providing
the heat in the heat exchanger (Figure 5).
w2 þ w5 ¼ w3 þ w7 [11]

Since w2 ¼ w7, it is also true that w3 ¼ w5; therefore: Magnetic refrigeration

As an emerging technology, magnetic refrigeration has the
w3 H^3  H ^ 5 ¼ w2 H ^2  H^ 7 þ QE2 [12] potential to achieve high energy efficiency using
Power of the compressors: environmentally friendly refrigerants. These devices use the

magnetocaloric effect (MCE), defined as the temperature
^ 1 ¼ w1 H
First compressor : ðPowÞ1 ¼ w1 W ^1
^2  H [13] change that most of the magnetic materials present when
 Chilled Foods: Principles 35

they are subjected to a changing magnetic field. Since the MCE
in the best magnetocaloric materials available presents a max-
imum temperature change of 4 K in a magnetic field of 1 T, the
magnetic refrigeration device must use a regenerative process to
obtain a temperature span high enough to be applied for
refrigeration purposes. The only disadvantage of this technol-
ogy is the high price of the magnets.
Condenser
One of the most recent devices with interesting results is the
one developed with a rotating mechanism in which the magnet
rotates with magnetocaloric material. The magnet’s (which has
a high price, one of the disadvantages of this technology)
design consists of a complex arrangement of permanent mag-
nets and soft magnetic materials, which are assembled in the
shape of an inner rotor. This device seems to be the prototype
with better characteristics for being successfully applied in
refrigeration systems. It also can help to obtain the higher
temperature span in the system.

Refrigerated transport
The refrigerated transport of chilled foods must be seen as a
global operation, taking into account the movement of chilled
Expansion
device products from one cold storage area to another without varia-
HX Compressor
tions in the temperature. For this, the actual movement of food
products is carried out by way of refrigerated vehicles, ships,
Evaporator and aircrafts, the latter being the shipment mode least used. In
the movement of products, temperature preservation is crucial
Figure 5 Chilling system with heat exchanger. to delivering products of the highest quality. Use of the finest

Refrigeration unit Cargo space

(a)

Condenser fan
Receiver

Suction line heat

exchanger
Condenser

Compressor
Thermostatic
expansion
valve
Evaporator

Accumulator

(b) Evaporator fan

Figure 6 (a) A refrigerated truck system and (b) schematic of the refrigeration unit.
36 Chilled Foods: Principles
facilities cannot compensate bad manufacturing or handling
practices, such as mistakes in the loading, packaging, or fluc-
tuations in the storage temperatures. Furthermore, it is impor-
tant that the temperature of the refrigeration equipment be
adequate; but it is even more important to make sure that the
food product is stored at the correct temperature.
The amount of refrigerated containers around the world is
getting bigger every day. In 2002, at least a million refrigerated
road vehicles, and about 400 000 refrigerated containers were
reported in use all over the world. The temperature range of the
refrigeration equipment is wide, from an insulated box con-
taining ice, to a complex container equipped with computer
systems to maintain the temperature, depending on the prod-
uct being transported, within a range of 25 to 30
C.
A typical refrigeration transport system is presented in
Figure 6. In this, the refrigeration unit interacts with the truck
cargo space to regulate the space to a certain temperature.
Figure 6 shows the scheme of the refrigeration unit, in which
all the components are interconnected, forming a complete
vapor compression system. As in the common chilling system,
the system employed in the vehicle uses energy to extract heat
from the refrigeration chamber and transfer it to the external
environment to maintain the product temperature.
See also: Chilled Foods: Effects on Shelf-life and Sensory Quality;
Chilled Foods: Modified Atmosphere Packaging; Chilled Foods:
Packaging Under Vacuum; Freezing Theory.
Further Reading
American Society of Heating, Refrigerating and Air Conditioning Engineers (1985)
ASHRAE handbook fundamentals. Atlanta, GA: ASHRAE.
Bjørk R, Bahl CRH, Smith A, and Pryds N (2010) Review and comparison of magnet
designs for magnetic refrigeration: a review. International Journal of Refrigeration
33: 437–448.
Dalkilic AS and Wongwises S (2010) A performance comparison of vapor-compression
refrigeration system using various alternative refrigerants. International
Communications in Heat and Mass Transfer 37: 1340–1349.
Domanski PA, Brown JS, Heo J, Wojtusiak J, and McLinden MO (2014) A
thermodynamic analysis of refrigerants: performance limits of the vapor
compression cycle. International Journal of Refrigeration 38: 71–79.
Dupont Refrigerants. (2013). Understanding the Refrigerant "R" Nomenclature.
Dupont™. http://www2.dupont.com/Refrigerants/en_CA/products/understanding.
html.
Fellows P (2000) Food processing technology, principles and practice. Boca Raton, FL:
CRC.
Gonçalves AA and Blaha F (2011) Cold chain in seafood industry. In: Larsen ME (ed.)
Refrigeration: theory, technology and applications. Hauppauge, NY: Nova Science
Publishers, Inc.
Ibarz A and Barbosa-Canovas G (2003) Unit operations in food engineering. Boca
Raton, FL: CRC.
Jay JM, Loessner MJ, and Golden DA (2005) Modern food microbiology, 7th ed.
New York, NY: Springer.
Li B, Otten R, Chandan V, Mohs WF, Berge J, and Alleyne AG (2010) Optimal on–off
control of refrigerated transport systems. Control Engineering Practice
18: 1406–1417.
National Refrigerants Inc. (2004) Refrigerant reference guide, 4th ed. USA: National
Refrigerants Inc.
United States Department of Agriculture (2013) Refrigeration and food safety. USA:
United States Department of Agriculture. Food Safety and Inspection Service.
Wang S (2003) Handbook of air conditioning and refrigeration, 2nd ed. New York:
McGraw-Hill.
Relevant Websites
http://www.chilledfood.org/ – Web site of the Association for chilled food manufacturers
in the United Kingdom.
http://www.danfoss.com/North_America/BusinessAreas/
RefrigerationþandþAirþConditioning/ProductþSelectionþToolsþDetails/
DIRcalc.htm – DIRCalc™ is a program that helps you size and calculate Industrial
Refrigeration plants.
http://www2.dupont.com/Refrigerants/en_US/products/DUPREX/DUPREX.html –
DuPont Refrigerant Expert™ (DUPREX) DUPREX Version 3.2 is a software tool from
DuPont specifically developed to allow users to easily and quickly generate data for
DuPont refrigerants.
http://www.ecff.net/ – Web site of the European Chilled Food Federation. An
Organization for chilled food manufacturers.
http://www.engineeringtoolbox.com/specific-heat-capacity-food-d_295.html –
Information of the Cp for different food products.
http://www.rdandt.co.uk/data/rdandt/downloads/Case%20study-Adande.pdf –
Refrigeration Developing and Testing LTD. 2011. RD&T A novel multi-temperature
refrigerator.
 Chlorophyll
C Yilmaz and V Gökmen, Hacettepe University, Ankara, Turkey
ã 2016 Elsevier Ltd. All rights reserved.
Sources and Production
Chlorophyll is a natural green pigment found in photosyn-
thetic organisms such as plants and algaes. In higher plants
and algaes, except the blue-greens, chlorophyll is located in
chloroplasts. Chlorophyll takes an important role in human
diet as it is consumed as a part of vegetables and fruits. To
date, there have been no standardized and certain informa-
tion about the chlorophyll content of plants in literature. The
change in chlorophyll contents depends on cultivar, harvest
time, ripening stage, and parts of the plant. Storage condi-
tions and processing of plants may also play an important
role. Besides, various extraction and quantification methods
cause incompatible chlorophyll contents. Chlorophyll con-
tents of some raw vegetables, obtained from different studies,
are given in Table 1.
Due to having green color and widespread occurrence in
many plants, chlorophyll might be thought of as a natural
coloring agent in foods. However, it is not a suitable coloring
agent due to its unstable nature against processing conditions.
Therefore, to have stable green coloring agents, metal-substituted
chlorophylls are commercially produced by chemical modifica-
tion of natural chlorophylls. The most popular and industrially
used metal-substituted chlorophyll is water-soluble copper chlor-
ophyllin. It is produced from crude chlorophyll extract as a result
of hydrolysis of phytyl and methyl esters, removal of cyclopenta-
none ring, and replacement of magnesium by copper. It is gen-
erally present as its sodium or potassium salt.
Chemical and Physical Characterization of Chlorophyll
The basic chemical structure of chlorophyll is a cyclic
tetrapyrrole. Tetrapyrrole is not only found in the structure of
chlorophyll but also in some other biologically important mol-
ecules such as heme and vitamin B12. It has four pyrrole rings
linked together at their carbon atoms via a one-carbon bridge. It
is generally present bound with a metal ion, which provides
different biological functions to the molecule. In chlorophyll,
four pyrrole rings are linked with methine (dC]) bridges, and
the four nitrogen atoms are coordinated with a central magne-
sium atom (Figure 1). Beside four pyrrole rings (A, B, C, and D),
chlorophyll contains an additional fifth isocyclic ring (ring E). A
monounsaturated isoprenoid alcohol (phytol) esterified to pro-
pionic acid located at C-17 gives chlorophyll a hydrophobic
character.
There are five types of well-known chlorophylls named as
chlorophyll a, b, c, d, and e. Apart from them, chlorophyll f has
been identified in recent years. Chlorophyll a and chlorophyll b
are the most abundant types in higher plants. They are generally
present in an average ratio of 3–1. However, it has been stated
that this ratio varies depending on genetic and environmental
factors. Chlorophyll a and chlorophyll b differ from each other
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00147-1
due to a chemical compound on the second pyrrole ring (ring B)
at the C-7 position. Chlorophyll a has a methyl group (
CH3)
at the C-7 position, whereas chlorophyll b has a formyl group
(
CHO). Chlorophyll d has a very similar structure with chlo-
rophyll a, except that it contains formyl group instead of vinyl
group (dHC]CH2) at the C-3 position. Although chlorophyll
a, b, and d are formed from the dihydroporphrin macrocycle
named as chlorin, chlorophyll c has a fully unsaturated porphy-
rin macrocycle. It also has a propenoic acid (a trans acrylic acid)
at the C-17 position instead of propionic acid. This propenoic
acid is not esterified with a long-chain alcohol like phytol as in
the other chlorophylls. Molecular structures of major chloro-
phylls are shown in Figure 1.
Chlorophylls have many conjugated double bonds with the
ability to absorb visible light. However, all chlorophylls have
specific wavelength absorptions due to their differences in
structures causing different green hues among plants. The
absorbance spectrums of chlorophylls show two dominant
bands as Q-band and Soret band. Chlorophyll a has character-
istic bands in the 432 and 669 nm regions that correspond to
the Soret band and Q-band, respectively. The corresponding
bands for chlorophyll b are located at 455 and 644 nm, respec-
tively. Chlorophyll c has characteristic bands between 578 and
630 nm and between 443 and 450 nm. Due to being structural
similarity, absorbance spectrum of chlorophyll d is very close
to that of chlorophyll a.
In chloroplasts, chlorophylls are found bound to caroten-
oids, lipids, and lipoproteins via noncovalent bonds. For that
reason, chlorophylls are easily extracted with organic solvents,
especially polar solvents such as acetone, methanol, and
ethanol.
Effect of Processing and Storage Conditions
on Chlorophyll
Stability of chlorophyll depends on storage and processing
conditions such as pH, temperature, oxygen, metals, and
enzymes. Depending on the conditions, there could be
changes in molecular structure of chlorophyll causing discol-
oration that is an indicator of low quality. Thus, several
studies have been carried out to prevent chlorophyll
degradation.
pH
Chlorophyll is susceptible to low pH conditions. Chlorophyll
degradation with the effect of acidic conditions is the result of
degradation of chlorophyll to pheophytin (Figure 2). This
reaction is called pheophytinization, and two hydrogen ions
replace the magnesium ion found in the center of the porphy-
rin ring. As a result, the bright green color of chlorophyll turns
to olive-brown, which causes negative consumer perception. In
37
38 Chlorophyll

the presence of chlorophyllase enzyme, pheophytin losses its the process. For instance, fermentation of vegetables lowers the
phytyl group, resulting in pheophorbide formation. Pheophor- pH in brine causing chlorophyll degradation. Besides, higher
bide also has an olive-brown color like pheophytin. process temperatures trigger the formation of pheophytin in
Although acidity is not enough for the formation of pheo- processed vegetables. Natural organic acids found in food
phytin at the beginning of the process, pH may decrease during matrix and fatty acids arising from lipid hydrolysis might be
released as a result of heat treatment.
To prevent pheophytin formation and accordingly discol-
Table 1 Chlorophyll content of some vegetables oration, alkalizing agents such as magnesium carbonate might
be used. However, alkalizing agents cannot maintain alkaline
Chlorophyll content (mg kg
1 fresh weight)
conditions permanently. It is especially important during long
Spinach 791a, 1083b, 1270c period of storage that results in color losses.
Green beans 71–133d, 75a
Brussels sprouts 32e, 60c
Broccoli 21a, 79c, 128b
Parsley 632a, 995f Heating
Cucumbers 36a Heat treatment is widely applied in food processing, espe-
Green peas 50a cially for pasteurization, sterilization, blanching, and drying
Leeks 87a
purposes. Chlorophyll is susceptible to heat treatments,
Green paprika 38a
which causes some structural changes. Mild heat treatments
Zucchini 68a
Celery 23a, 34g result with the formation of chlorophyll isomers (Chloro-
phyll a0 and Chlorophyll b0 ). They differ from chlorophyll
a
Bohn, T., et al. (2004). Chlorophyll-bound magnesium in commonly consumed
vegetables and fruits: relevance to magnesium nutrition. Journal of Food Science 69,
347–350.
b
Sanchez, H., et al. (2007). The effect of high pressure and high temperature processing - phytol
on carotenoids and chlorophylls content in some vegetables. Food Chemistry 163, Chlorophyll Chlorophyllide
Chlorophyllase
37–45.
c
Khachik, F., et al. (1986). Separation, identification and quantification of the major
carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid - Mg+2 Acid/heat - Mg+2 Acid/heat

chromatography. Journal of Agricultural and Food Chemistry 34, 603–1986.

d
Cubas, C., et al. (2008). Optimization of the extraction of chlorophylls in green beans
(Phaseolus vulgaris L.) by N,N-dimethylformamide using response surface - phytol
Pheophytin Pheophorbide
methodology. Journal of Food Composition and Analysis 21, 125–133. Chlorophyllase
e
Olivera, D. F., et al. (2008). Effect of blanching on the quality of Brussels sprouts
(Brassica oleracea L. gemmifera DC) after frozen storage. Journal of Food Engineering -COOCH3 Heat -COOCH3 Heat
84, 148–155.
f
Arnold, C., et al. (2014). Carotenoids and chlorophylls in processed xanthophyll-rich
food. LWT – Food Science and Technology 57, 442–445.
g
Pyropheophytin Pyropheophorbide
Vina, S. Z., et al. (2007). Quality changes in fresh-cut celery as affected by heat
treatment and storage. Journal of the Science of Food and Agriculture 87, 1400–1407. Figure 2 Formation of chlorophyll derivatives and factors affecting.

H2C HC CH3
3 5 7
2 4 6 8
H 3C CH2 CH3
A B
1 N 21 22 N 9

20 Mg 10

19 N 23 24 N 11
H3C
18 D 14 C
17 16 12 CH3
H 15 E 13
H
132 131
171 CH2
H O
172 CH2
COOCH3
173 C O

H CH3 H CH3

Figure 1 Molecular structure of chlorophylls.


with carbomethoxy group (
COOCH3) at the C-132 posi-
tion. Heat treatments may also cause loss of Mg2þ ion and
formation of pheophytin. Chlorophyll a leads to formation
of pheophytin more rapidly than chlorophyll b, as its heat
stability is lower. Increased temperatures or long periods of
heat treatments result in the formation of pyroderivatives
of chlorophylls (pyropheophytin and pyropheoporbide);
those have no carbometoxy group at the C-132 position
(Figure 2).
Blanching is a heat treatment implemented prior to
freezing or canning of vegetables to inactivate enzymes.
This treatment provides protection of nutritional and sen-
sorial quality. However, conditions that require inactivating
enzymes are different from each other. It has been reported
that blanching conditions required to inactivate lipoxygen-
ase (LOX) causes more chlorophyll loss than peroxidase
(POD). Thus, it could be said that blanching conditions
required to inactivate LOX are not proper for protection of
chlorophyll.
To preserve the green color of fruits and vegetables, control
of the thermal treatment is important. It has been shown that
high temperature in a short time might be an effective
approach to preserve chlorophyll.
Enzymes
Enzymes such as chlorophyllase, LOX, POD, and polyphenol
oxidase induce loss of quality in fruits and vegetables. Chlor-
ophyllase, a thylakoid membrane glycoprotein, is found in
green plants and causes the destruction of chlorophyll. Its
activity continues during the processing and storage period of
fruits and vegetables. Chlorophyllase cause the conversion of
chlorophyll to chlorophyllide by catalyzing cleavage of the
phytol group. Chlorophyllide is known as a green chlorophyll
derivative. Formation of chlorophyllide is desirable to preserve
the green color in canned vegetables. However, when acid is
present in the medium, chlorophyllide losses its magnesium
ion and causes pheophorbide formation. Olive fermentation
could be a good example for formation of chlorophyllide and
then pheophorbide. Besides, chlorophyllase hydrolyzes pheo-
phytins, which gives rise to the formation of pheophorbide
(Figure 2).
The formation of chlorophyllide depends on temperature
conditions as chlorophyllase is activated in a temperature
range (60–82.2  C) and degraded at higher temperatures
(>100  C). Studies have shown that degradation of chloro-
phylls to pheophytins and degradation of chlorophyllides to
pheophorbides follow first-order kinetics.
Besides chlorophyllase, POD and LOX enzymes in fruits
and vegetables play a crucial role in chlorophyll degradation.
LOX oxidizes fatty acids containing cis,cis-1,4-pentadiene
structure. LOX is responsible for formation of hydroperoxides
and free radicals that cause khaki/yellow or colorless prod-
ucts. Oxidative degradation of chlorophyll is named
‘chlorophyll bleaching.’ LOX could be inactivated by heat
treatment. Therefore, blanching is generally performed for
canned or frozen vegetables to inactivate LOX and protect
color.
POD is one of the oxidoreductase enzymes and is found in
many cell organelles including chloroplast. Although POD plays
Chlorophyll 39
a significant role in ripening of fruits and vegetables, it is also
responsible for chlorophyll degradation. In the degradation
mechanism of chlorophyll via POD, phenolic compounds are
oxidized with hydrogen peroxide. As a result, formed phenoxy
radicals oxidize chlorophyll to colorless compounds.
Metals
Chlorophyll forms complex with some metal ions, which
causes some changes in color. In canned vegetable foods, the
magnesium ion at the center of chlorophyll might be replaced
with iron. This replacement results in an undesirable brown-
gray color. If zinc and copper salts are present in the medium
during processing, some greener areas on the surface of vege-
tables could appear. This color change is called as ‘regreening.’
From this point of view, green vegetables are blanched in water
containing certain amounts of Zn2þ and Cu2þ salts, and they
become greener than their natural forms. Formation of com-
plexes between chlorophyll and Zn2þ and Cu2þ are named the
‘Veri-Green process.’ These metal complexes are more stable
than natural chlorophyll. The complex formation rate could
change depending on types of chlorophylls, metals, and envi-
ronmental conditions such as pH. Although complexes pro-
vide stable green color, their usage might cause detrimental
health problems. Zn-chlorophyll complexes are preferably
used in the industry, as Cu-chlorophyll complexes are not
innocent.
In canned foods, after pheophytin is formed with the effect
of heat, it is complexed with zinc and resulted with formation
of zinc-pheophytin. Decarboxymethylation of zinc pheophy-
tin causes the formation of zinc pyropheophytin. Another
suggested pathway for zinc pyropheophytin formation is direct
complexation of zinc with pyropheophytin.
Surface-active anionic compounds affect the formation of
metal complexes. These compounds adsorb onto the chloro-
plast membranes, which results in increasing the negative sur-
face charge and thereby increasing complex formation.
Oxidation
Chlorophyll is found in many vegetable oils such as virgin
olive oil and rapeseed oil. During bleaching of edible oils,
chlorophyll and its derivatives are generally removed or
their concentration is reduced as much as possible. Due
to being sensitizers, chlorophyll and its derivatives might
cause photosensitized oxidation in edible oils. Photo-
sensitized oxidation should be taken into account, as it is
as important as autoxidation by means of oxidation prod-
ucts. Low-molecular-weight off-flavor compounds, degrada-
tion products of fatty acids, and oxidized polymers are
some of the products that cause unacceptable consumer
perceptions.
Photosensitizers such as chlorophyll, riboflavin, and myo-
globin absorb light energy and turn into an excited single-
state sensitizer. Following that, an excited triplet-state sensi-
tizer is generated from the excited single-state sensitizer via
intersystem crossing. An excited triplet-state sensitizer might
follow two pathways. In the type I pathway, sensitizers play a
role as free-radical initiators. An excited triplet sensitizer
reacts with a substrate such as linoleic acid by donating an
40 Chlorophyll
electron or accepting hydrogen. Accordingly, free radicals are
generated, and they react with triplet oxygen via a free radical
oxidation mechanism. In type II pathways, an excited triplet
sensitizer reacts with triplet oxygen, which results in forma-
tion of a singlet sensitizer and singlet oxygen. After that, the
singlet oxygen interacts with unsaturated fatty acids, and
hydroperoxides are produced. The amount of hydroperoxides
depends on the number of double bonds of unsaturated
fatty acids.
When chlorophyll is dissolved in alcoholic solutions or
exposed to air, oxidation at the C-132 position might occur.
Oxygen or oxygen-containing compound are located in the
C-132 position of the chlorophyll.
Free radical scavengers such as carotenoids and tocopherols
prevent chlorophyll-sensitized oxidation of fatty acids.
b-carotene, a singlet oxygen quencher, prevents oxidation in
edible oils. Moreover, storage in a controlled atmosphere
might be used for the same purpose.
Refining
Chlorophyll and pheophytins are critical compounds and
impart a greenish color to edible oils. Chlorophyll is especially
responsible for color of olive oil that depends on the types and
ripeness degrees of olives. However, excess chlorophyll causes
an unacceptable consumer perception. Chlorophyll and its
derivatives could be partly removed by decolorization step of
refining process. However, removing chlorophyll completely is
a difficult and not economical process.
Bioavailability and Health Effects
Knowledge about potential health-related benefits of chloro-
phyll has drawn attention in recent years. Therefore, many
studies about the bioavailability of chlorophyll have been
carried out to expose the effectiveness of chlorophyll on
human health. However, investigations about the bioavailabil-
ity of chlorophyll generally focus on in vitro studies.
Absorption of natural chlorophylls might change
depending on such factors as type, solubility, digestive
stability, and food matrix. During digestion, natural chloro-
phylls are converted to pheophytins because of acidity in
the stomach. As a result, Mg-free derivatives might be pre-
dominantly found in human feces. According to studies, the
absorption of Mg-free chlorophyll derivatives by intestinal
cells is easier than that of natural chlorophyll. It has been
also found that specific metallo-chlorophyll derivatives like
Zn-pheophytin are stable in the gastric conditions and
available for absorption.
Commercial copper chlorophyllin is a complex mixture
containing different chlorin-based components such as copper
chlorin e6 and copper chlorin e4. These chlorin-based compo-
nents are absorbed at different rates. The absorption rate of
copper chlorophyllin components might be changeable
depending on the food matrix that may protect them from
ingestion conditions.
Although chlorophyll causes photosensitized oxidation,
it might show an antioxidative effect based on some
conditions. If chlorophyll is exposed to light, the pro-
oxidant effect of chlorophyll increases. However, it plays
an important role as an antioxidant in dark medium.
Change of chlorophyll structure also affects antioxidant
activity. It has been reported that the antiradical capacity
of metallo-derivatives such as Mg-chlorophylls and Zn-
pheophytins is much more than that of metal-free deriva-
tives such as pheophytins and pyropheophytins. In addition
to natural chlorophyll derivatives, it has been remarked that
copper chlorophyllin displays antioxidant activity against
oxygen species.
Chlorophyll and its derivatives have antimutagenic and
anticarcinogenic effects. These effects are against many muta-
gens and carcinogens such as polycyclic aromatic hydrocar-
bons, heterocyclic amines, and aflatoxin B1. According to a
common opinion, the protective mechanism of chlorophyll
is formation of complex between porphyrin ring of chloro-
phyll and carcinogens–mutagens. However, the chemical
structure of mutagens–carcinogens is an effective factor for
formation of a complex. As a result, DNA-adduct formation is
prevented and bioavailability of dietary carcinogens or muta-
gens is reduced. It has been shown that green vegetables might
decrease the risk of colon cancer in humans. Moreover, it has
been stated that chlorophyll and its derivatives prevent skin
cancer when experimental animals are used. Studies have also
shown that chlorophyllin has a significant antimutagenic effect
against antitumor drugs that might have detrimental effects on
healthy cells.
The amount of chlorophyll in the diet is significant to
determine contribution of chlorophyll consumption to
human health. Because of its high concentration in vegetable-
rich diets, health benefits of chlorophyll might increase. As can
be seen from Table 1, the average chlorophyll content of green
beans (90 mg chlorophyll per kg vegetable) is less than that of
parsley (800 mg chlorophyll per kg vegetable). Therefore, the
health effect of chlorophyll in green bean could be provided by
a lower amount of parsley.
See also: Carotenoids: Occurrence, Properties and Determination;
Carotenoids: Physiology; Colors: Health Effects; Colors: Properties and
Determination of Natural Pigments; Colors: Properties and
Determination of Synthetic Pigments; Cooking: Domestic Techniques;
Storage Stability: Shelf Life Testing.
Further Reading
Daood HG (2003) Chlorophylls. In: Caballero B, Trugo L, and Finglas PM (eds.)
Encyclopedia of food sciences and nutrition, 2rd ed., pp. 1196–1205. Oxford:
Academic Press.
Fennema OR (ed.) (1985) Food chemistry. New York: Marcel Dekker.
Ferruzzi MG and Blakeslee J (2007) Digestion, absorption, and cancer
preventative activity of dietary chlorophyll derivatives. Nutrition Research
27: 1–12.
Heaton JW, Lencki RW, and Marangoni AG (1996) Kinetic model for chlorophyll
degradation in green tissue. Journal of Agricultural and Food Chemistry
44: 399–402.
Lanfer Marquez UM and Sinnecker P (2008a) Chlorophylls: properties, biosynthesis,
degradation and functions. In: Socaciu C (ed.) Food colorants: chemical and
functional properties, pp. 25–49. Boca Raton, FL: CRC Press.
 Chlorophyll 41

Lanfer Marquez UM and Sinnecker P (2008b) Chlorophylls in foods: sources and
stability. In: Socaciu C (ed.) Food colorants: chemical and functional properties,
pp. 195–211. Boca Raton, FL: CRC Press.
Mı́nguez-Mosquera MI, Gandul-Rojas B, Gallardo-Guerrero L, Roca M, and Jarén-
Galán M (2008) Chlorophylls. In: Hurst WJ (ed.) Methods of analysis for functional
foods and nutraceuticals, 2rd ed., pp. 337–387. Boca Raton, FL: CRC Press.
Tumolo T and Lanfer-Marquez UM (2012) Copper chlorophyllin: a food colorant with
bioactive properties? Food Research International 46: 451–459.
Relevant Websites
http://www.fao.org/ag/agn/jecfa-additives/specs/monograph5/additive-127-m5.pdf –
Food and Agriculture Organization, 2008, Chlorophyllins, copper complexes
sodium and potassium salts.
http://www.fao.org/ag/agn/jecfa-additives/specs/Monograph1/Additive-128.pdf – Food
and Agriculture Organization, 2002, Chlorophylls.
 Cholecalciferol: Properties and Determination
AK Hewavitharana and FP Gomes, University of Queensland, Brisbane, QLD, Australia
ã 2016 Elsevier Ltd. All rights reserved.
Background
Cholecalciferol (vitamin D3) is the common form of vitamin D
synthesized in animals. Ergocalciferol (vitamin D2) is the form
that is synthesized mainly in plant sources such as mushrooms.
The term vitamin D usually covers both forms, and it plays a
crucial role in the regulation of intestinal absorption and
metabolism of calcium and phosphate, both of which are
vital for the healthy homeostasis of bones. Despite being
known as a vitamin, vitamin D is a prohormone that is con-
verted to its hormonal active form (1,25-dihydroxyvitamin D
(1,25(OH)2D3 or 1,25(OH)2D2)). Vitamin D was discovered
in the early twentieth century from research on the treatment of
rickets, a bone disorder caused by vitamin D deficiency in
children. The original vitamin D, which was named D1, was a
mixture of compounds that was prepared by ultraviolet (UV)
radiation of ergosterol that was found in plants and fungi. In
1931, vitamin D1 was purified by Askew and the compound
ergocalciferol or vitamin D2 was isolated. At that time, synthe-
sis of vitamin D in human was not understood, as ergosterol
could not be found in human tissues. Few years later, Windaus
synthesized 7-dehydrocholesterol (ergosterol equivalent in
human skin) from cholesterol, and its UV-radiated product
was named cholecalciferol or vitamin D3.
Physical and Chemical Properties
Cholecalciferol is a white crystalline powder with the chemical
name 9,10-seco(5Z,7E)-5,7,10(19)-cholestatriene-3b-ol. As
shown in Figure 1, its chemical structure differs from that of
steroids by the bond cleavage at 9–10 followed by ring open-
ing, which is responsible for antirachitic activity. This ring
opening reaction occurs in human skin on exposure to UV-B
radiation (290–315 nm) of 7-dehydrocholesterol (provitamin
D3). As shown in Figure 1, the ring opening is followed by the
formation of a double bond to produce previtamin D3. Subse-
quent temperature-dependent rearrangement of this double
bond produces cholecalciferol with a characteristic conjugated
triene structure. Once formed, it is carried into circulation by
vitamin D-binding protein. Cholecalciferol itself is biologically
inactive, and its conversion to function biologically comprises
two enzymatic hydroxylation reactions, producing 25-
hydroxyvitamin D3(25(OH)D3) followed by 1,25(OH)2D3.
Ergocalciferol, which is ingested through plant food sources,
also undergoes similar process to produce the biologically
active form 1,25(OH)2D2. The structures of the vitamin D
analogues commonly found in human plasma are shown in
Figure 2, and these and many other analogues found in plasma
and other biological fluids are collectively called vitamin D.
Although vitamin D is traditionally implicated in bone
health, its deficiency was found to have effects on many other
diseases such as hypertension, diabetes mellitus, cancer, and
autoimmune and cardiovascular diseases. As a result, there has
42 Encyclopedia of Food and Health
been a proliferation of research on vitamin D in recent times.
Some research indicates that different forms of the vitamin D
may be responsible for this variety of physiological effects. In
general, the activities of vitamins are expressed in international
units (IU). Expression in IU is useful in expressing the total
activity when several forms of the vitamin with different bio-
potencies are present in one food source. With vitamin D, both
cholecalciferol and ergocalciferol are considered to have a
similar potency: 1 IU ¼ 0.025 mg. However, the potencies of
other forms of vitamin D are not available in IU; therefore,
this concept is not as widely used as with other vitamins such
as vitamin A. Evaluation of potencies of different forms of
vitamin D is made more challenging due to the fact that each
form may have different physiological effects. As shown in
Figure 2, in terms of chemical structure, cholecalciferol differs
from ergocalciferol at the C17 side chain where ergocalciferol
has an additional methyl group at C24 and an extra double
bond between C22 and C23. It is worth mentioning that
cholecalciferol and ergocalciferol possess similar physical and
chemical properties. Table 1 lists the physical and chemical
properties of cholecalciferol and ergocalciferol. While their
potencies in humans are considered equivalent by pharmaco-
poeias, some studies have demonstrated that cholecalciferol is
more potent than ergocalciferol and better absorbed by the
intestine. It has also been found that its major circulating
metabolite 25(OH)D3 has higher affinity than that of ergocal-
ciferol (25-hydroxyvitamin D2 (25(OH)D2)) to vitamin
D-binding protein, resulting in a longer half-life.
Cholecalciferol is the common form that is consumed as
vitamin D. It has been extracted from fish liver oil, but
nowadays, it is mostly synthesized for use in nutritional sup-
plements and in fortified foods. Despite its insolubility in
water due to high lipophilicity, cholecalciferol is readily solu-
ble in a wide range of organic solvents including alcohols and
both chlorinated and nonchlorinated hydrocarbons such as
chloroform and hexane. Cholecalciferol undergoes a reversible
thermal isomerization to previtamin D3 and/or degradation
when stored in alcoholic solutions and exposed to oxygen and
light. However, it is stable for long periods when stored in
absolute ethanol without the presence of oxygen and light at
low temperature (20  C). It is also stable in oils, fats, and
fortified food, when stored in dark at low temperatures. It has
been found that cholecalciferol is protected against degrada-
tion in food matrices and once released from them can be
vulnerable to degradation by light and oxygen. Cholecalciferol
is stable in alkaline solutions but not in acidic conditions. In
acidic conditions, it undergoes isomerization to inactive forms
such as isotachysterol and 5,6 trans isomers.
Occurrence and Forms in Food
It is well known that cholecalciferol is primarily obtained via
exposure of the skin to sunlight. Some critical factors including
skin pigmentation, time of day, sunscreen use, altitude, skin
http://dx.doi.org/10.1016/B978-0-12-384947-2.00148-3
 Cholecalciferol: Properties and Determination 43

21 hence, dietary intake has become the main source as vitamin D

Provitamin D3 deficiency has become common.
20 22
18 23 24 Vitamin D can be naturally found in a limited number of
12
17 26 foods. Cholecalciferol is usually present in animal foods, while
19 11 13 25 ergocalciferol can be found in fungi (mushrooms) and plants.
D 16 Provitamins (7-dehydrocholesterol and ergosterol) are present
C
8 27 in animals and plants (including the ones used as food sources
1 9 14
2
10 15
A B by humans); therefore, exposure to sunlight produces vitamin
3 5 D in them. Mushrooms dried in sunlight are considered a good
4 7
HO
6 plant source of vitamin D2. Fish liver oils are regarded as the
best source of cholecalciferol. Fish, such as tuna and salmon,
are generally accepted as relatively good sources. Both chole-
calciferol and ergocalciferol can be found in milk, cholecalcif-
erol being the predominant form. Seasonal variations can
affect the concentration of cholecalciferol in cow’s milk due
UV-B radiation
to changes in sunlight exposure of its skin. Although alimen-
tary forms are mainly composed of ergocalciferol and chole-
calciferol, small portions of calcidiol and calcitriol can also be
found in some dietary products. Table 2 provides a list of
nonfortified foods containing vitamin D with their respective
concentrations. It should be noted that the analytical methods
available for measuring vitamin D in food were usually devel-
oped to measure only the two calciferols; therefore, the
Previtamin D3
reported values may not reflect the total amounts that include
the contributions by other forms.

Fortification of Foods
HO
The serious concern of the adverse outcomes (wrinkling and
skin cancer) that can occur with high exposure to sunlight has
decreased obtaining vitamin D through sunlight exposure. In
addition, the general trend of low-fat diets causes vitamin D
Heat deficiency because this fat-soluble vitamin is removed along
with fat during the production of low-fat food. Although an
optimal cholecalciferol daily intake level has not yet been
established, current recommended daily intake is in the
range 400–600 IU. This is difficult to obtain through dietary
sources alone without exposure to sunlight. As a result, vita-
21 min D supplementation is commonly prescribed. In order to
22 address the vitamin D deficiency in a large fraction of the
Vitamin D3 18 24
12 20 population, a common practice used in many countries is to
23 26 fortify certain foods with vitamin D. However, fortification of
11
13 17 25 food with vitamin D must be monitored because of toxico-
C D 16
9 logical concerns such as hypercalcemia with fatal conse-
8 27
14
15 quences at very high levels. Either cholecalciferol or
ergocalciferol is used in the fortification of food, although
7
6
cholecalciferol is the preferred form. There is no consensus
about the extent to which food fortification should be per-
19 formed and the prevalent action varies according to the legis-
5
4 10 lation of each country.
3
A 1 Common vehicles of vitamin D fortification are milk, milk-
HO
2 based beverages, milk powder, margarine, and cereal products.
Cholecalciferol is usually added to the milk in oily form due to
Figure 1 Photochemical conversion of provitamin D3 to vitamin D3. its easier solubilization in oil-based foods. Milk powder is
fortified by mixing cholecalciferol powder with milk powder
into the bulk. In this process, it is important to make sure
type, age, obesity, latitude, clothing, and season directly influ- particle sizes of milk powder and vitamin powder are compa-
ence the vitamin D intake from the sun. Due to concerns of the tible in order to avoid separation of the components, leading
depleted ozone layer and skin cancer, the significance of sun- to a heterogeneous mix. It must be stored in a cool and dry
light in vitamin D production has decreased in recent years; space to ensure minimal cholecalciferol loss. In the case of
44 Cholecalciferol: Properties and Determination

HO HO
Vitamin D2 (Ergocalciferol) Vitamin D3 (Cholecalciferol)

OH OH

HO HO
25-Hydroxyvitamin D2 (Ercalcidiol) 25-Hydroxyvitamin D3 (Calcidiol)

OH
OH

HO OH
HO OH
1,25-Dihydroxyvitamin D2 (Ercalcitriol) 1,25-Dihydroxyvitamin D3 (Calcitriol)

Figure 2 Chemical structures of ergocalciferol, ercalcidiol, ercalcitriol, cholecalciferol, calcidiol, and calcitriol.

margarine, the fortification is performed by mixing oily chole-
calciferol with a fraction of warmed oil, followed by homo-
genization with the fat blend prior to the emulsification
procedure. In addition to fortified foods, cholecalciferol can
be obtained from vitamin D supplements that generally con-
tain 1000 IU in each tablet.
Analytical Methods
Unlike with photosynthesized vitamin from skin, with dietary
intake, vitamin D levels can rise to over 400 ng ml1 (about
2 mg or 80 000 IU in total), leading to vitamin D toxicity. The
natural levels of vitamin D in common foods are very low and
the fortified levels are also relatively low unlike with most
other vitamins. The determination of cholecalciferol content
in foods is a challenging analytical task as, in natural foods and
even in fortified foods, only small amounts are present and a
wide range of compounds are extracted along with cholecalcif-
erol that cause difficulties in sample preparation and instru-
mental analysis.
Vitamin D molecules in foodstuffs and biological fluids are
bound to lipids and/or proteins; therefore, chemical bonds
need to be broken to release the vitamin before analysis. In
general, saponification is used to release vitamin D and to
remove other lipids from foodstuff, and solvent-based protein
 Cholecalciferol: Properties and Determination 45

Table 1 Properties of ergocalciferol and cholecalciferol

Properties Ergocalciferol (vitamin D2) Cholecalciferol (vitamin D3)

Molecular formula C28H44O C27H44O

Molar mass (g mol1) 396.63 384.62
Molar volume 406.9 cm3 mol1 396.9 cm3 mol1
Melting point ( C) 115–118 84–85
pKa 14.74 14.74
log P 9.148 9.085
lmax (nm) 264.5 264.5
Extinction coefficient (E1%
cm (hexane)) 459 485
Extinction coefficient (E1%
cm (ethanol)) 462 485
Optical rotation (chloroform) þ52 þ52
Mass solubility (water) (Sparingly soluble (5.9E5 g l1)) (Sparingly soluble (6.5E 5 g l1))
Molar solubility (water) (Sparingly soluble (1.5E7 mol l1)) (Sparingly soluble (1.7E 7 mol l1))

Table 2 Dietary sources of vitamin D using techniques such as ELISA, radio immunoassay, or
high-performance liquid chromatography (HPLC) with UV
Food source Vitamin D content (IU/100 g)
spectrophotometric or mass spectrometric detection (HPLC-
Beef (meat) 13 UV or HPLC–mass spectrometry (MS)). HPLC–MS is the
Beef (liver) 8–40 most common chromatographic technique used nowadays
Pork (meat) 84 due to its superior selectivity and sensitivity relative to HPLC-
Pork (liver) 40 UV. However, vitamin D compounds produce weak signals in
Chicken 50–65 MS detector because of their poor ionizability. Recently
Poultry 80 reported methods have overcome this problem by derivatiza-
Poultry skin 900
tion with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to pro-
Butter 35
Eggs 28
duce Diels–Alder adducts that have better ionization efficiency
Cheese 12 in MS detection.
Cream 50 Chromatographic separation is usually carried out on a
Cabbage 0.2 reversed-phase analytical column, and the detection wave-
Spinach 0.2 length commonly used with UV detection is 265 or 280 nm.
Corn oil 9 Electrospray ionization and atmospheric pressure chemical
Cod liver oil 10 000 ionization are the most frequently used mass spectrometric
Cod 85 ionization modes, and most methods used tandem mass spec-
Shrimp 150 trometric detection (HPLC–MS/MS) rather than HPLC–MS to
Salmon 220–440
achieve better specificity and sensitivity.
Sardines 1500
Mackerel 120
There are many different immunoassay kits commercially
Herring liver oil 140 000 available for vitamin D assay. With immunoassays, the vitamin
Herring 330 extract is subjected to an antibody-specific vitamin D assay.
The detection step of immunoassays is based on different
spectroscopic techniques such as UV–visible, chemilumines-
precipitation is used to release it from biological fluids. In cence, Raman scattering, and radioactivity measurements. Two
saponification, vitamin D compounds are released from lipop- major limitations of immunoassays for vitamin D analysis are
rotein complexes. In protein precipitation, vitamin is released matrix effects and the inability to distinguish between 25(OH)
from the proteins by denaturing the protein. Commonly used D3 and 25(OH)D2. Raman spectroscopy for immunoassay has
saponification procedure consists of mixing the sample shown high sensitivity, enabling the detection of vitamin D
with ethanolic potassium hydroxide and heating to approxi- metabolites at very low concentrations; however, this method
mately 80  C for about 1 h. The unsaponifiable portion still cannot distinguish between the different vitamin D
containing the vitamin is then extracted using a nonpolar metabolites.
solvent such as hexane. Subsequently, the organic phase is Despite being the best method for vitamin D analysis, one of
removed by evaporation and the residue reconstituted in a the major drawbacks of HPLC–MS or HPLC–MS/MS is that the
suitable solvent depending on the analytical method used. MS detection is susceptible to matrix effects. Matrix effects in MS
Commonly used protein precipitation method consists of occur when the compounds that coelute with the analyte inter-
mixing the sample with acetonitrile to precipitate out the pro- fere with the ionization process causing ionization suppression
teins by denaturation. The precipitated proteins are removed or enhancement. As there is no way to ensure complete elimi-
after centrifugation, the supernatant is dried under nitrogen nation of the interfering compounds that coelute with the
or by evaporation, and the residue is reconstituted in a suit- analyte, the only way to obtain accurate data is to remove the
able solvent depending on the analytical method used. The contribution from the interferences at the quantification step.
extracted vitamin is subsequently detected and quantified The inclusion of stable isotope-labeled (SIL) internal standards
46 Cholecalciferol: Properties and Determination
(IS) in both the samples and standards followed by quantifica-
tion using internal standard calibration has become the most
common method used to correct for matrix effects in MS detec-
tion. As the chemical properties and ionization process of SIL-IS
are almost identical to those of the vitamin, and as it elutes at
the same retention time as the vitamin and experiences the
same extent of matrix effects, it provides the best option to
correct for matrix effects when IS calibration method is used.
As a separate SIL-IS is needed for each form of the vitamin
quantified, and some of them are not commercially available,
many published methods for vitamin D had no corrections
applied for matrix effects of MS detection. Some methods
used a single SIL-IS for all vitamin forms although this does
not remove matrix effects from all vitamin D analogues. Quan-
tification with HPLC-UV methods has used either IS calibration
using noncoeluting compounds such as laurophenone or reti-
nyl acetate or external standard calibration.
Eleven analytical methods for the analysis of vitamin D in
foodstuffs have been validated by the Association of Official
Analytical Chemists International (AOACI); however, only four
of them have been used in laboratories. In summary, these
methods are similar, in which saponification is used to extract
the vitamin D content and HPLC-UV is used for quantification.
The scope of these methodologies is only vitamin D3, limiting
their applications to food matrices containing other forms of
vitamin D. In addition, these approaches were validated for a
limited number of food matrices, containing high levels of fat,
which make them inappropriate to the application in matrices
with low-fat content such as cereals and juices.
See also: Calcium: Physiology; Calcium: Properties and
Determination; Cereals: Dietary Importance; Chromatography: High-
Performance Liquid Chromatography; Dairy Products: Dietary and
Medical Importance; Fish: Fish in the Human Diet; Fish Oils:
Composition and Health Effects; Fish Oils: Production and Properties;
Immunoassays: Principles; Milk: Role in the Diet; Milk Powder.
Further Reading
Ball GFM (2006) Vitamin D. In: Ball GFM (ed.) Vitamins in foods: Analysis,
bioavailability, and stability, pp. 107–116. Boca Raton, FL: CRC Press.
Ball GFM (2004) Vitamin D. In: Ball GFM (ed.) Vitamins: Their role in the human body,
pp. 188–224. Oxford: Blackwell Publishing Ltd.
Ball GFM (1998) Vitamin D. In: Ball GFM (ed.) Bioavailability and analysis of vitamins
in foods, pp. 163–189. London: Chapman & Hall.
Byrdwell WC, Devries J, Exler J, et al. (2008) Analyzing vitamin D in foods and
supplements: Methodologic challenges. The American Journal of Clinical Nutrition
88(2): 554s–557s.
Combs GF (2012) Vitamin D. In: Combs GF (ed.) The vitamins, 4th ed., pp. 139–180.
San Diego: Academic Press.
Eitenmiller RR, Ye L, and Landen WO (2008) Vitamin D. In: Eitenmiller RR, Ye L, and
Landen WO (eds.) Vitamin analysis for the health and food sciences, 2nd ed.,
pp. 83–112. Boca Raton, FL: CRC Press.
Gomes FP, Shaw PN, Whitfield K, Koorts P, and Hewavitharana AK (2013) Recent trends
in the determination of vitamin D. Bioanalysis 5(24): 3063–3078.
Holick MF (2007) Vitamin D deficiency. New England Journal of Medicine 357(3):
266–281.
Houghton LA and Vieth R (2006) The case against ergocalciferol (vitamin D2)
as a vitamin supplement. The American Journal of Clinical Nutrition 84(4):
694–697.
IUPAC-IUB Joint Commission on Biochemical Nomenclature (1982) Nomenclature of
vitamin D. Molecular and Cellular Biochemistry 49(3): 177–181.
Ottaway PB (ed.) (1993) The technology of vitamins in food. Cornwall: Springer.
Perales S, Alegrı́a A, Barberá R, and Farré R (2005) Review: Determination of vitamin D
in dairy products by high performance liquid chromatography. Food Science and
Technology International 11(6): 451–462.
Schmid A and Walther B (2013) Natural vitamin D content in animal products. Advances
in Nutrition 4(4): 453–462.
Wolf G (2004) The discovery of vitamin D: The contribution of adolf windaus. The
Journal of Nutrition 134(6): 1299–1302.
Woollard D and Indyk H (2003) Cholecalciferol properties and determination.
In: Caballero B, Finglas P, and Trugo L (eds.) Encyclopedia of food sciences and
nutrition, 2nd ed., pp. 1205–1213. Amsterdam: Academic Press.
Relevant Websites
www.stanford.edu/group/hopes/cgi-bin/wordpress/2013/04/vitamin-d3/ – Stanford.
www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/SCOGS/ucm261118.htm –
FDA.
 Cholesterol: Absorption, Function and Metabolism
V Vučić, University of Belgrade, Belgrade, Serbia
Z Cvetković, Clinical Hospital Center Zemun, Belgrade, Serbia
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Cholesterol plays an essential role in maintaining membrane
integrity and proper fluidity. It is an abundant component of
the plasma membranes of eukaryotic cells, which makes up
around 25–30% of the total lipids present in cell membranes.
The cholesterol contents of cell membranes are tightly regulated
and have an impact on cell permeability. The large hydrophobic
domain of cholesterol fits into spaces between phospholipids,
thereby preventing diffusion of water-soluble molecules through
the membrane. Furthermore, cholesterol reduces the fluidity of
cell membranes with its rigid structure and stabilizes membranes
under different conditions. Alterations in membrane fluidity
further influence membrane transport processes, including
Naþ/Kþ-ATPase. Although around 70% of plasma cholesterol is
esterified, cholesterol in cellular membranes is in free form.
In addition to the role in the stability and architecture of the
plasma membrane, cholesterol is a precursor for the biosynthe-
sis of bile acids, vitamin D, and steroid hormones in mammals.
Fluctuations of circulating levels of cholesterol under physiolog-
ical conditions are controlled by a series of mechanisms that
balance between de novo synthesis, absorption of dietary
cholesterol, and removal from peripheral tissues. Therefore,
cholesterol levels in circulation depend not only on the amount
of cholesterol supplied from the diet (exogenous) and synthe-
sized in the body (endogenous) but also on the removal, uptake
from cells, and the conversion of cholesterol into other mole-
cules (bile acids and steroid hormones). Concentration of intra-
cellular cholesterol is coordinated by the same mechanisms, in
line with the phases in the cell cycle. All these processes are
tightly regulated and maintain concentration of cholesterol
within a normal range. However, many chronic non-
communicable diseases are associated with alterations in these
mechanisms, including cardiovascular diseases and cancer.
Cholesterol Biosynthesis
Most animal cells are able to produce cholesterol, but the
production rates vary depending on the cell types and organ
function. Cholesterol de novo synthesis is a highly regulated,
multistep process known as the mevalonate pathway. Primary
place of cholesterol production is the liver, which contributes
20–25% of total cholesterol synthesis, while cholesterol is also
synthesized in the intestines, adrenal glands, and reproductive
organs. The only precursors for the biosynthesis of cholesterol
are two-carbon metabolite of acetate, acetyl coenzyme A (acetyl
CoA). Two acetyl-CoA molecules form acetoacetyl CoA, which
is combined with another acetyl CoA and then hydrated to 3-
hydroxy-3-methylglutaryl CoA (HMG-CoA) in the cytoplasm.
This reaction is catalyzed by the enzyme HMG-CoA synthase.
The next step is the reduction of HMG-CoA to mevalonate (the
key intermediate in cholesterol biosynthesis) by the enzyme
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00151-3
HMG-CoA reductase in microsomes, which requires NADPH.
This is an irreversible and rate-limiting step in the cholesterol
biosynthesis, which is commonly used as therapeutic target in
hyperlipidemic disorders using statin drugs (HMG-CoA
reductase-competitive inhibitors).
HMG-CoA reductase is placed in the membrane of the endo-
plasmic reticulum (ER). This enzyme contains multiple trans-
membrane domains and a catalytic domain that converts
HMG-CoA into mevalonate. The catalytic domain (C-terminal
domain) faces the cytosol, while the membrane domains play
an important regulatory role. Namely, these domains are sterol-
sensitive, and they change the conformation of the enzyme
when interact with sterols. Hence, when cholesterol level in
the cell is high, conformation changes downregulate the activity
of HMG-CoA reductase, and less mevalonate is produced. Lower
synthesis is compensated by increased rate of uptake of choles-
terol from blood to cells, thereby lowering the level of choles-
terol in the circulation. On the contrary, when levels of
cholesterol in cells decline, they respond by increasing the
gene expression of not only proteins included in cholesterol
biosynthesis, in particular HMG-CoA reductase, but also pro-
teins responsible for the uptake of cholesterol from the external
environment, such as low-density lipoprotein (LDL) receptor. In
this way, cholesterol concentration in cells increases.
Further, conversion of mevalonate to 3-isopentenyl pyro-
phosphate includes three reactions, which all require ATP.
These reactions, as well as those that follow, are carried out in
the ER. The next steps involve the synthesis of farnesyl pyro-
phosphate, which is used for the synthesis of squalene. Sq-
ualene is converted to lanosterol through series of cyclization
and migrations of hydrogen and methyl groups. Finally, cho-
lesterol is synthesized from lanosterol in several reactions
including oxidation, reduction, and demethylation. The
whole process consists of 37 reactions.
Cholesterol Transport
Although cholesterol in synthesized in the ER, its concentration
in the ER is very low. This is because the largest part of synthe-
sized cholesterol is transported to other sites. Most of the cho-
lesterol is carried to the plasma membrane, where it modulates
membrane fluidity and maintains barrier between cell and envi-
ronment. However, cholesterol is also sent to other organelles.
Some of them are a part of the secretory pathway (e.g., the Golgi
complex and lysosomes), and they receive cholesterol by vesic-
ular transport. For the other intracellular destinations, such as
the mitochondria, lipid transport proteins deliver cholesterol.
These proteins interact with the ER membrane, bind cholesterol,
then diffuse through the cytoplasm to target organelles, and
release cholesterol into their membranes.
Furthermore, cholesterol is delivered to circulation. Given that
cholesterol is a highly insoluble molecule, it is transported
47
48 Cholesterol: Absorption, Function and Metabolism
through circulation by endogenous transporters called lipopro-
teins. The five major classes of lipoproteins are chylomicrons,
very low-density lipoprotein (VLDL), intermediate-density lipo-
protein (IDL), LDL, and high-density lipoprotein (HDL), and
their characteristics are presented in Table 1. Lipoproteins have
a common structural configuration, although the relative lipid
and protein composition of each lipoprotein varies. All of them
contain phospholipid monolayer with anchored proteins, which
protect inner hydrophobic core of neutral lipids, for example,
cholesterol esters and triglycerides. On the surface of lipoproteins,
there is some free cholesterol as well. Based on this architecture,
lipoproteins are ideal carriers for lipophilic compounds. In addi-
tion to other lipids, lipoproteins not only transport and deliver
dietary cholesterol to peripheral tissues but also remove the excess
cholesterol from peripheral tissues to the liver, thereby maintain-
ing the homeostatic balance. However, each class of lipoproteins
has different structures, specific functions, and thus different
effects on health. From the clinical point of view, LDL
cholesterol and HDL cholesterol are particularly attractive, even
though other lipoproteins have clinical significance as well.
Cholesterol Absorption
Absorption of dietary cholesterol is closely related to plasma
concentrations of cholesterol. In human diet, the major
sources of cholesterol are egg yolk, meat, and dairy products.
A variable proportion of dietary cholesterol is esterified to fatty
acids, while the rest remains free. However, only free choles-
terol appears to be absorbed, so cholesterol esters have to be
hydrolyzed prior to absorption, mostly by the action of pan-
creatic enzymes. Additional and often overlooked source of
cholesterol originates from the bile, and all biliary cholesterol
is nonesterified and therefore suitable for absorption. More-
over, among 60–80% of cholesterol that is absorbed from the
diet and the bile, greater part originates from the bile since it is
already prepared to be absorbed.
The main sites of cholesterol absorption are the duodenum
and proximal jejunum. The process occurs through the intesti-
nal mucosal cells on the surface of the intestinal villi. Digestion
of lipids starts in the stomach when the food is mixed with
gastric and lingual enzymes, forming a crude emulsion. This
emulsion is mixed in the duodenum with pancreatic enzymes
and bile salts. Pancreatic enzymes, lipase, cholesterol esterase,
Table 1 Composition and physical properties of lipoprotein classes
Lipoprotein Chylomicrons VLDL
Source Intestine Liver, intestine
Diameter (nm) 90–1000 30–90
Density (g ml1) <0.95 0.95–1.006
Total lipids (%) 98–99 90–93
Triglyceride 85–88 50–55
Free cholesterol 1 8–10
Cholesteryl esters 3 12–15
Phospholipids 8 18–20
Protein (%) 1–2 7–10
Apoprotein A-I, A-II, B-48, C-I, C-II, C-III B-100, C-I, C-II, C-III, E
and phospholipase A all have hydrolytic functions and liberate
cholesterol from the esters. Since many products of pancreatic
enzymes are poorly soluble, the role of bile salts is to emulsify
lipids, acting as a surfactant. The obtained mixture of lipids,
triglycerides, and cholesterol is readily soluble and is able to
pass through the thin water layer around luminal cells. In this
way, the mixture comes up to absorptive enterocytes. Although
it has been thought for years that lipids passively diffuse
through the plasma membrane of enterocytes, recent studies
have shown that several proteins are involved in intestinal
transport of cholesterol. The cholesterol carriers that are iden-
tified so far are NPC1L1, SR-B1, CD13, CD36, P-glycoprotein,
and annexin 2–caveolin 1 complex. Some of them affect intra-
cellular concentration of cholesterol and are controlled by
transcription factors. However, the list of protein mediators
of intestinal cholesterol transport is likely still incomplete.
Inside the intestinal cell, cholesterol is shuttled to the ER,
where it is packaged into chylomicrons. Chylomicrons are large
particles (around 1 um), with lipid-containing core (similar to
the other lipoproteins) and the surface with anchored apopro-
teins, mostly apoB-48, apoE, A-1, A-2, and A-4. Amphipathic
nature of apoproteins makes chylomicrons soluble in blood and
lymph fluid. Because of the size of chylomicrons, they cannot
pass through the blood vessels, but instead enter the lymph. This
secretion into the lymph fluid requires apoB-48 on the surface of
chylomicrons. From the lymph, chylomicrons enter the blood-
stream via the thoracic duct and the right subclavian vein.
Thence, they are sent to the adipose and muscle tissue that
utilize a part of the triglycerides in chylomicrons, before arriving
at the liver. The triglycerides from chylomicrons are extracted by
the enzyme lipoprotein lipase (LPL) while passing through the
capillaries of the muscle and adipose tissue and used by muscles
or stored in adipocytes. The remaining particle is called chylo-
micron remnant. The remnant is denser than chylomicron; it
contains less triglycerides and thus is relatively rich in esterified
cholesterol. It is rapidly removed from the circulation by the
liver, via LDL receptors or the LDL receptor-like protein (LRP).
Chylomicron receptor LRP is placed on the surface of hepato-
cytes, interacts with apoE in the remnants, and thus facilitates
the uptake to the liver.
In the liver, lipids are repackaged into VLDL particles,
which necessarily contain apoB-100, synthesized mainly in
hepatocytes. The amounts of apoB-100 are controlled by the
amount of lipids: When more lipids are available, the
IDL LDL HDL
VLDL VLDL Liver, intestine, VLDL,
chylomicrons
25–35 20–25 5–25
1.006–1.019 1.019–1.063 1.063–1.210
88–90 78–80 43–67
25–30 10–15 3–15
8–10 8–10 2–10
32–35 37–48 15–30
25–27 20–28 26–46
10–12 20–22 33–57
B-100, C-I, C-II, C-III, E B-100 A-I, A-II, C-I, C-II, C-III,
D, E

proteolysis of apoB-100 is reduced and the production of
VLDL increases. In contrast, when the concentration of lipids
falls down, more apoB-100 is proteolyzed and the generation
of VLDL decreases as well. Therefore, the concentration of
VLDL is regulated by the proteolysis of apoB-100. VLDL are
secreted from the liver, and they are the main carriers of endog-
enously produced triglycerides. As with chylomicrons, VLDL is
also subjected to LPL-mediated hydrolysis of triglycerides in
capillary walls, and then, surface proteins (with the exception
of apoB-100) are transferred to HDL. One important protein
involved in the transfer of cholesteryl esters and triglycerides
between VLDL, LDL, and HDL is facilitated by cholesterol ester
transfer protein (CETP), which is also an important target for
cholesterol-lowering drugs. Therefore, both chylomicrons and
VLDL deliver triglycerides to tissues for storage or as a source of
energy. Although a part of the remnants of VLDL is removed by
the liver, most are converted to LDL via IDL. In addition, there
is another pathway for lipid transfer by chylomicrons and
VLDL, heparan sulfate proteoglycan-mediated pathway.
After hydrolysis of triglycerides from VLDL, IDL particles
are produced. Some of IDL are taken up by the liver, through
the binding of apoE to the LDL receptors. Nevertheless, the
majority of IDL is modified by hepatic lipase forming LDL.
LDL Cholesterol
In LDL particles, cholesterol makes  50% of the weight, while
around 25% are proteins. The crucial protein component is
apolipoprotein B-100 (apoB-100), along with 80–100 addi-
tional ancillary proteins that increase LDL solubility. Core of
the LDL particles is highly hydrophobic and contains mostly
triglycerides and cholesterol esterified by various fatty acids.
The basic function of LDL is delivering cholesterol to cells for
storage or further biogenesis, by receptor-mediated endocyto-
sis involving the uptake of the whole lipoprotein particle.
Especially during periods of rapid growth and development,
LDL particles provide cholesterol to most peripheral tissues via
the LDL receptor. In spite of this important function, high
levels of LDL cholesterol in serum strongly correlate with
increased risk of cardiovascular events. Thus, LDL cholesterol
is widely called ‘bad cholesterol.’ Furthermore, recent evidence
showed possible relationship between oxidized form of LDL
and atherosclerosis. The oxidation of LDL is a complex process,
which affects both the protein and the lipids: they undergo
oxidative changes and form complex products. Primarily, non-
enzymatic oxidative changes in amino acids result in extensive
alteration in the apoB-100 composition and structure. In addi-
tion, lipid peroxidation causes generation of aldehydes and
ketones that covalently modify amino acid side-chain residues
of apoB-100 and the other proteins. Modified LDL is then
scavenged and degraded by macrophages. They bind oxidized
LDL by the scavenger receptor, which is expressed primarily on
macrophages. The receptor–LDL complex is taken up and then
targeted to the lysosome for degradation. If macrophages
phagocyte a high amount of LDL, cholesterol accumulates in
their cytoplasm, which becomes filled with lipid droplets. The
lipid droplets in the cytoplasm give the macrophages a foamy
appearance, because these cells are named foam cells. They are
not harmful as such, but they can accumulate in the wall of
large blood vessels, leading to the formation of an atheroma,
Cholesterol: Absorption, Function and Metabolism 49
which are known as precursors for atherosclerosis. The unrest-
ricted generation of foam cells and its ability to promote
inflammatory responses and differentiation of monocytes in
the arterial wall imply that oxidized LDL is a key factor in the
development of atherosclerosis.
In order to be packaged into lipoproteins, a large amount of
cholesterol is esterified with long-chain fatty acids. In this way,
the problem of transport of insoluble cholesterol through
circulation is solved. However, cholesterol esters cannot pass
through membrane and delivery is enabled by lipoprotein
receptors. A prototype of these receptors is LDL receptor,
which mediates the uptake and lysosomal degradation of
plasma LDL, thereby providing cholesterol to cells. This is a
cell surface transmembrane protein that binds LDL on apopro-
tein sites and carries it into the cell by receptor-mediated
endocytosis. After binding LDL, the receptor–LDL complex is
taken up, clustered, and then endocytosed by clathrin. Then,
the vesicle is fused with an acidic endosome. The decrease in
pH induces a conformational change in the LDL receptor,
which releases the LDL cholesterol, and the receptors either
are then destroyed or can be sorted into vesicles and returned
to the surface of the cell. The remaining endosome is delivered
to the lysosome, which is more acidic than endosome. Lyso-
somal enzymes degrade the protein component and hydrolyze
the cholesterol esters. The liberated cholesterol can be used by
the cell for the synthesis of plasma membranes, vitamin D, and
steroid hormones or stored in the cytoplasm in the form of
cholesterol ester droplets.
This mechanism is crucial for efficiently removing the
excess LDL and maintaining desirable levels of LDL in blood.
Therefore, it is important that cells express an adequate
amount of LDL receptors on their surfaces and that their func-
tion is appropriate. Several genetic mutations in the LDL recep-
tor diminish its function or reduce the number of receptors on
the cell surface, leading to increased levels of serum LDL and
hypercholesterolemia.
LDL Receptor Mutations
Mutations in the LDL receptor gene cause inherited disorder
characterized by high serum cholesterol level, called familial
hypercholesterolemia (FH). FH is principally an autosomal
dominant disorder. LDL receptor is located on chromosome
19 at 19p13.1–p13.3, and more than 1000 mutations have
been identified in this gene so far. In addition, mutations in
two other genes also produce the FH phenotype: the apolipo-
protein B-100 gene that codes the most important protein
component of LDL and proprotein convertase subtilisin/kexin
type 9 (PCSK9). Nevertheless, mutations in LDL receptor gene
are more common and affect about 1 in 500 individuals. These
genetic changes are classified in five broad classes according to
the effect on LDL receptor function. Class 1 mutations affect
the synthesis of the receptor in the ER, and these mutations are
alternatively referred to as ‘receptor-negative’ mutations. In
class 2 mutations, the LDL receptor is not transported from
the ER to the Golgi that leads to the degradation of the recep-
tor. In class 3 mutations, the binding of LDL to the receptor is
improper. In class 4 mutations, the internalization of the
receptor–ligand complex is inhibited, and in class 5 mutations,
the internalized receptors cannot recycle properly. The classes
50 Cholesterol: Absorption, Function and Metabolism
2–3 are often termed ‘receptor-defective.’ Class 5 mutations
cause relatively mild phenotype of FH, since LDL receptors
are still present on the cell surface, although they have to be
newly synthesized. Classes 2 and 3 are identified as the most
common.
Most people with FH are heterozygotes, having one normal
copy of the LDL receptor gene and one mutated copy of the
gene. These people have an increased risk of cardiovascular
disease that typically begins in their forties or fifties. However,
homozygotes have two mutated genes for the LDL receptor,
inherited from both affected parents. In these people, clinical
picture is more severe and their FH clinical onset of symptoms
usually appears in childhood or adolescence.
HDL Cholesterol and Reverse Cholesterol Transport
Unlike LDL, HDL cholesterol is widely known as ‘good cho-
lesterol’ because of its strong inverse association with the risk
of cardiovascular disease. HDL particles play the largest role in
removing cholesterol from the peripheral tissues to the liver,
suppressing cholesterol accumulation in the peripheral tissues.
The essential function of HDL cholesterol is the initiation of a
multistep process, reverse cholesterol transport, via plasma to
the liver, which processes the excretion of excess cholesterol
from the body. Furthermore, many other atheroprotective
activities have also been attributed to HDL, such as antioxi-
dant, anti-inflammatory, hemostasis, and endothelial cell
maintenance functions. It prevents oxidative modification of
LDL, thereby reducing the generation of macrophage foam
cells and atheroma plaque formation within the arteries.
HDL also decreases the activity of macrophage chemotactic
factor 1 that participates in low-grade systemic inflammation
and signals the infiltration of monocytes to arterial walls. In
addition, HDL acts opposite to LDL inhibiting platelet aggre-
gation and enabling activity of nitric oxide synthase, which is
also inhibited by oxidized LDL. Nevertheless, the most impor-
tant function of HDL is the promotion of reverse cholesterol
transport, especially removing cholesterol from atherosclerotic
plaques. A number of epidemiological studies have suggested
that low level of this lipoprotein induces the development of
coronary artery diseases. On the contrary, high HDL choles-
terol level is associated with the reduction of incidence of
atherosclerosis and cardiovascular disease.
HDL constitutes a heterogeneous group of lipoproteins that
differ in density, size, lipid composition, and apolipoprotein
content, as well as electrophoretic mobility. Based on the
electromobility determined by electrophoresis, HDL particles
could be separated into two major subfractions, HDL2 and
HDL3. Although the proteomics of HDL is very complex, the
majority of HDL particles contain apolipoprotein A-I (apoA-I),
which is the most abundant apolipoprotein in human plasma
in healthy population. The second most abundant protein in
HDL is apoA-II, which is also included in many HDL particles.
In addition, over 85 other proteins have been identified so far,
showing proteomic complexity and diversity of HDL that is
related to numerous functions of HDL.
ApoA-I synthesis in the liver and intestine is the first step in
HDL metabolism. The next step is the interaction of ApoA-I
with cells expressing ABCA1, a member of the ABC transporter
superfamily (ATP-binding cassette transporters) that transports
the specific substrates across cell membranes utilizing the
energy of ATP binding and hydrolysis. ABCA1 is situated at
the plasma membrane and intracellular organelles, where it
can facilitate transport of various molecules across extra- and
intracellular membranes. Although studies in animal models
of tissue-specific ABCA1 deficiency confirmed that hepatic
ABCA1 was crucial for HDL biogenesis, nonhepatic tissues
are also very important. The ABCA1 pathway not only is
involved in lipid efflux from both peripheral and hepatic
tissues but also is important for the formation of nascent
HDL and maintenance of plasma HDL levels. At first, lipid-
poor apoA-I removes free cholesterol from peripheral cells
through ABCA1 transporter to generate nascent pre-bHDL.
This particle, consisting of a phospholipid bilayer surrounded
by a protein coat, has a great affinity for free cholesterol and
collects cholesterol, which is then stored in the lipid bilayer.
This process is enhanced by the activation of the lecithin–
cholesterol acyltransferase enzyme, which esterifies free cho-
lesterol stored in the bilayer with fatty acyl groups from lecithin
(phosphatidylcholine), forming hydrophobic cholesterol
esters. The obtained esters are moved to the hydrophobic
core, which is growing up. During circulation, nascent HDL
increases binding more cholesterol and phospholipids. The
shape of HDL is then changing from the disk to a sphere in a
complex process mediated by ABCG1, hepatic lipase, endothe-
lial lipase, CETP, and phospholipid transfer protein. At this
step, further cholesterol collection is ceased; mature spherical
HDL particle is formed and recognized by the liver. Mature
HDL interacts with other apoB-containing lipoproteins, such
as IDL and VLDL. Thus, ABCA1 and its transporter are impor-
tant for HDL metabolism, and the defective ABCA1 transporter
due to mutations leads to severe reduction of HDL, a rare
inherited disorder called the Tangier disease.
In humans, HDL cholesteryl esters are cleared from plasma
via two major pathways. In the first one, CETP facilitates an
exchange for triglyceride in HDL cholesteryl ester and transfer
to LDL, which is returned to the liver through the LDL receptor-
mediated pathway. The other way is nonendocytic hepatic
uptake of HDL by scavenger receptor B1, multiligand receptor
that binds not only HDL but also VLDL and LDL. The final step
for the elimination of cholesterol from the liver is secretion
into bile, either directly or after conversion to bile salts. Several
transporters are involved in biliary cholesterol secretion, such
as ABC transporters (ABCB11, ABCB4, and ABCG5/G8), the
Niemann–Pick C1-like protein 1, the phosphatidylserine flip-
pase, ATPase class I type 8B member 1, and the HDL choles-
terol uptake receptor – SR.
Cholesterol-Lowering Drugs
Regarding the established role of the total and LDL cholesterol in
atherosclerosis and CVD, the American College of Cardiology
(ACC) and the American Heart Association (AHA) proposed in
2013 the guideline on the treatment of blood cholesterol to
reduce atherosclerotic cardiovascular risk in adults. Atheroscle-
rotic cardiovascular disease (ASCVD) includes coronary heart
disease, stroke, and peripheral arterial disease. Healthy diet,
maintenance of healthy weight, and lifestyle modification (reg-
ular exercise and avoidance of tobacco) are proposed as crucial

components in ASCVD risk reduction, both prior and with the
use of cholesterol-lowering drugs.
The most used cholesterol-lowering drugs are statins, a large
group of inhibitors of HMG-CoA reductase. A comprehensive
meta-analysis from the Cholesterol Treatment Trialists’ Collab-
oration of 27 randomized trials revealed that they reduced the
risk of major coronary events by 24% for each 1 mmol l1
reduction of LDL concentration, stroke by 15%, and coronary
revascularization by 24%. It has been assumed that fully imple-
mented public policies would result that more than a third of
all middle-aged and older adults in the United States and
United Kingdom will be recommended for statin therapy.
Statins act on crucial step of cholesterol biosynthesis, the
mevalonate synthesis, which is regulated to ensure sufficient
production of isoprenoids. The inhibition of cholesterol bio-
synthesis leads to raised expression of LDL receptors in liver cell
membranes, enhancing clearance of the circulating LDL parti-
cles from blood, and to raised expression of PCSK9, an enzyme
responsible for LDL receptor catabolism. Additional ‘pleiotro-
pic’ effects of statins (such as reduced vascular and systemic
inflammation, including sepsis, and reduced cancer risk) may
result from the inhibition of the biosynthesis of isoprenoid
intermediates and of small GTP-binding protein, thus influenc-
ing the signal transduction in membrane receptors involved in
processes of cell proliferation, differentiation, and apoptosis.
Statins downregulate antigen presentation and decrease T-cell
activation and reduce antigen expression on endothelial cell
and the levels of inflammatory cytokines TNF-a, IL-1b, IL-6, IL-
8, NF-kB, and C-reactive protein. Furthermore, they reduce the
production of reactive oxygen species, inhibit the production of
thrombin and thrombomodulin, decrease plasminogen activa-
tor inhibitor-1 expression, and increase tissue plasminogen
activator levels and tissue factor, thus leading to increased
degradation of fibrin. The increased anticoagulation and fibri-
nolysis prevent endothelial cell disruption.
Statins are strongly recommended as secondary prevention
in individuals with clinical ASCVD and as primary prevention
in individuals with primary elevation of LDL above
4.9 mmol l1, in individuals with diabetes, and in those
with estimated 10-year ASCVD risk  7.5% with LDL concen-
tration 1.8–4.9 mmol l1. High-intensity statin therapy on
average lowers LDL by  50%, moderate-intensity statin
therapy lowers LDL by 30–50%, and lower-intensity statin
therapy lowers LDL by approximately <30%. In people over
75 years of age, high-intensity statin therapy in secondary
prevention is not recommended. These drugs are not rou-
tinely recommended for individuals with NYHA class II–IV
heart failure or who are receiving maintenance hemodialysis.
Statins are metabolized by cytochrome P450 pathway and
thereby occasionally may cause hepatotoxicity (<3% of
patients) and myopathy (<0.2% of patients), and in < 0.05%
of statin-treated patients, muscle injury leads to rhabdomyol-
ysis, myoglobinuria, and renal failure. After over 25 years of
wide use of statins, it is proved that benefits of statin therapy
are of far greater significance than the risk of adverse effects
of statins such as muscle weakness, impairment of hepatic
function, the increased incidence of type 2 diabetes, and
cataract.
Approved nonstatin agents for LDL reduction include bile
acid-binding resins (colestipol, cholestyramine, and colesevelam),
Cholesterol: Absorption, Function and Metabolism 51
which have the advantage of not being absorbed systemically
and can be used in pregnancy; fibrates (fenofibrate, bezafibrate,
and gemfibrozil), which mainly decrease triglycerides and
increase HDL; niacin, which slightly decreases LDL and triglycer-
ides while increasing HDL; cholesterol absorption inhibitor ez-
etimibe, which acts on epithelial Niemann–Pick C1-like
protein; and omega-3 fatty acid supplements. Although these
agents can clearly improve lipid profiles in many patients, con-
temporary event reduction trials have shown little evidence to
support their use either as monotherapy or as an adjunct to
statins in general population.
The new agents – mipomersen and lomitapide – were
approved in 2013 by the US Food and Drug Administration
to treat patients with homozygous FH. Mipomersen is an anti-
sense oligonucleotide that binds to a specific 20-base sequence
on messenger RNA coding for apolipoprotein B-100. Lomita-
pide is an inhibitor of microsomal triglyceride transport protein
assisting in the transfer of triglyceride to apolipoprotein B and
also reduces circulating LDL cholesterol by targeting VLDL
production.
CETP inhibitors such as anacetrapib and evacetrapib are very
effective in raising HDL (>100% in phase 3 trials), but their
effects on HDL function are not fully understood, although
some data suggest that they have the potential to promote
cholesterol efflux capacity. They are also effective in reducing
LDL levels (>30%) and concentrations of Lp(a).
The most promising novel agents effective in LDL reduction
are monoclonal antibodies targeting PCKK9, a protein secreted by
hepatocytes that binds to the LDL receptors leading to its
cellular internalization and lysosomal degradation. The inhi-
bition of PCSK9 also reduces Lp(a). Three large randomized
control human trials on safety and efficacy of alirocumab,
bococizumab, and evolocumab are currently ongoing.
See also: Adipose Tissue: Structure and Function of Brown Adipose
Tissue; Adipose Tissue: White Adipose Tissue Structure and Function;
Cholesterol: Factors Determining Blood Cholesterol Levels;
Cholesterol: Properties, Processing Effects, and Determination; Elderly:
Nutrition Requirements; Fat Replacer; Fatty Acids: Essential Fatty Acids;
Fatty Acids: Metabolism; Fatty Acids: Trans Fatty Acids; Hypertension
and Diet; Obesity: Causes and Prevalence; Obesity: The Role of Diet;
Phospholipids: Physiology; Phospholipids: Properties and Occurrence;
Triacylglycerols: Characterization and Determination; Triacylglycerols:
Structures and Properties; World Health Organization.
Further Reading
Cortes VA, Busso D, Mardones P, Maiz A, Arteaga A, Nervi F, and Rigotti A (2013)
Advances in the physiological and pathological implications of cholesterol.
Biological Reviews 88(4): 825–843.
Fisher EA, Feig JE, Hewing B, Hazen SL, and Smith JD (2012) High-density lipoprotein
function, dysfunction, and reverse cholesterol transport. Arteriosclerosis,
Thrombosis, and Vascular Biology 32(12): 2813–2820.
Gylling H (2014) Clinical utility of serum markers of cholesterol absorption and
synthesis. Current Opinion in Lipidology 25(3): 207–212.
Levy E, Spahis S, Sinnett D, et al. (2007) Intestinal cholesterol transport proteins: an
update and beyond. Current Opinion in Lipidology 18(3): 310–318.
Ridker PM (2014) LDL cholesterol: controversies and future directions. Lancet
384(9943): 607–617.
52 Cholesterol: Absorption, Function and Metabolism

Stone NJ, Robinson JG, Lichtenstein AH, et al. (2014) 2013 ACC/AHA guideline on the
treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in
adults: a report of the American College of Cardiology/American Heart Association
Task Force on Practice Guidelines. Journal of the American College of Cardiology
63(25 Pt B): 2889–2934.
Tarling EJ, Vallim TQ, and Edwards PA (2013) Role of ABC transporters in lipid
transport and human disease. Trends in Endocrinology and Metabolism 24(7):
342–350.
Uehara Y and Saku K (2014) High-density lipoprotein and atherosclerosis: roles of lipid
transporters. World Journal of Cardiology 6(10): 1049.
Varghese MJ (2014) Familial hypercholesterolemia: a review. Annals of Pediatric
Cardiology 7: 107–117.
Relevant Websites
http://lipidlibrary.aocs.org/Lipids/lipoprot/index.htm – AOCS Lipid Library.
http://themedicalbiochemistrypage.org/cholesterol.php – The Medical Biochemistry
Page.
 Cholesterol: Factors Determining Blood Cholesterol Levels
Z Rasic-Milutinovic and G Perunicic-Pekovic, University of Belgrade, Belgrade, Serbia
D Jovanovic, Institute of Public Health ‘Milan Jovanovic-Batut’, Belgrade, Serbia
N Simovic, Z Gluvic, D Ristic-Medic, and M Glibetic, University of Belgrade, Belgrade, Serbia
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Whole-body cholesterol balance is regulated by the net effects
of dietary cholesterol absorption, de novo cholesterol biosyn-
thesis, and whole-body cholesterol clearance, mostly by biliary
excretion from the liver.
In the intestinal tract, cholesterol originates from two
sources: food intake and biliary secretion into the duodenum.
Several proteins in the brush border membrane of enterocytes
are involved in mediating intestinal cholesterol absorption.
Whereas various transporters, including fatty acid translocase/
cluster determinant 36, scavenger receptor class B type I (SR-BI),
and Niemann-Pick C1-like 1 (NPC1L1), may influence choles-
terol uptake, the ATP binding cassette (ABC) transporter family,
including several cholesterol carriers (ABCA1, ABCB1, and
ABCG5/G8), act as efflux pumps favoring cholesterol export
out of absorptive cells into the lumen or basolateral compart-
ment. Among all the cholesterol transporters, the enriched
NPC1L1 protein in the apical membrane of enterocytes is con-
sidered essential for intestinal cholesterol absorption, and
genetic modifications of NPC1L1 in cultured intestinal cells
alter cholesterol uptake.
Although all tissues in the body are capable of synthesizing
cholesterol from acetyl coenzyme A (CoA), the liver is the main
site for de novo cholesterol synthesis and stores it as cholesterol
ester after esterification by acetyl-CoA acetyltransferase. The
major rate-controlling enzyme in hepatic cholesterol synthesis
is 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase,
which is used as a pharmacological target of statin treatment.
Cholesterol, as a water-insoluble molecule, needs to be
transported in the plasma associated with various lipoprotein
particles, such as chylomicrons, very low-density lipoproteins
(VLDLs), intermediate-density lipoproteins (IDLs), low-
density lipoproteins (LDLs), and high-density lipoproteins
(HDLs). Approximately 60–80% of cholesterol is transported
through the bloodstream in the core of LDL particle. On the
other hand, crucial molecules in cholesterol transport are apo-
lipoproteins located on the surface of LDL particles. Each LDL
particle contains one molecule of apoB, a lipoprotein respon-
sible for carrying cholesterol to peripheral tissues and binding
to LDL receptors. Since more than 90% of the removal of LDL
takes place in the liver, the liver determines the rates of LDL
clearance from plasma. Hence, the liver is the only organ
capable of eliminating excess cholesterol from the body, by
either secretion into bile or conversion into bile acids. The
movement of excess cholesterol from peripheral tissues to the
liver is a result of reverse cholesterol transport (RCT), which is
promoted by HDL particles.
The body maintains a stable cholesterol pool by regulating
mechanisms of absorption, synthesis, and elimination. The
dominant factor that determines cholesterol absorption in
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00152-5
the small intestine is intake. Hence, when dietary cholesterol
intake is very low, its absorption is upregulated. Conversely, if
dietary intake is too high, absorption is decreased and biliary
excretion is intensified. The percent of cholesterol absorbed in
healthy subjects vary significantly from person to person from
29% to 80%. However, about 25% of the population has
exaggerated response to an overload of dietary cholesterol.
Additionally, among dietary and genetic factors, many physio-
logical and pathological conditions can influence plasma cho-
lesterol levels.
Aging
Numerous studies have demonstrated that regardless of phys-
ical activity levels and nutritional status, levels of plasma total
cholesterol rise progressively with age. Plasma LDL cholesterol
(LDL-C) levels increase progressively from young adulthood to
approximately age 60 in men and to age 70 in women. Pro-
longed intestinal transit time may be a factor for increasing
cholesterol absorption with aging. Slow intestinal transit is
associated with an increased rate of bacterial biotransforma-
tion of bile acids, with enhanced enterohepatic recirculation of
deoxycholic acid. At the same time, diet rich in cholesterol and
saturated fatty acids (SFA), usually presented in elderly sub-
jects, can be the reason for increased cholesterol absorption.
Another possible explanation could be a gradual reduction in
the rate of LDL clearance from the circulation, presumably as
the result of a reduced activity of LDL receptors, diminished
number of functioning hepatic LDL receptors, and prolonged
turnover time (‘downregulation’) of the recirculating LDL
receptors. In response to cholestyramine, a bile acid-binding
agent, the elderly can reach the same values of LDL clearance as
younger individuals, which explains that they still have the
capacity for ‘upregulation’ of hepatic LDL receptors. Another
explanation for reduced LDL receptor expression may be a
consequence of a reduced hepatic demand for cholesterol,
due to a reduced bile acid synthesis occurring with age. In
mice, aging increases biliary cholesterol secretion and reduces
level of hepatic bile acid synthesis, which hence increases the
susceptibility of developing cholesterol gallstones in elderly
animals. This finding may be explained with the decreased
activity of hepatic 7-a hydroxylase, an enzyme that metabolizes
cholesterol to biliary salts. Some of the rise in LDL-C levels
with age could be related to the sedentary lifestyle and increase
of body weight, which is typical in older population. In addi-
tion, various concomitant diseases (diabetes mellitus, hypo-
thyroidism, and nephropathy) and commonly used drugs
more frequently present in the elderly population are associ-
ated with hypercholesterolemia. Also, it has been shown that
growth hormone (GH) has key roles in cholesterol metabolism
53
54 Cholesterol: Factors Determining Blood Cholesterol Levels
and that its secretion is reduced with aging. Experiments per-
formed in rodents have demonstrated that the administration
of GH is able to completely reverse age-dependent increase in
plasma cholesterol and the reduced levels of bile acid synthesis
to the same level as seen in young animals.
Gender
Compared with men, women have lower levels of LDL-C and
VLDL cholesterol and higher HDL cholesterol (HDL-C) levels.
There is no difference in cholesterol absorption fraction
between men and women. Lower concentrations of LDL-C
are associated with accelerated LDL production and enhanced
LDL clearance, which may be explained with higher levels of
estrogens in women. Studies in rats have shown that estrogen
treatment is followed by an increase of hepatic LDL receptors
and a faster clearance of LDL particles. The sex difference in
HDL concentrations is associated with greater synthesis rate of
apolipoproteins A-I and A-II, major proteins of HDL particle
responsible for fat efflux from peripheral tissues to the liver. It
has been shown that postmenopausal women have higher
plasma cholesterol levels than premenopausal women of the
same age. In postmenopausal women, the decrease in plasma
estrogen levels after menopause may play a significant role in
the reduction of the clearance of LDL particles and subsequent
increase of LDL-C. Estrogen replacement treatment has been
shown to markedly decrease LDL-C in dyslipidemic postmen-
opausal women. Sexual dimorphism in cholesterol metabo-
lism cannot be explained only by the different levels of sex
hormones. There are studies that showed that surgically
induced menopause without hormone replacement therapy
has no effect on plasma cholesterol concentrations when com-
pared with surgical control group (hysterectomy with conser-
vation of the ovaries). There are seemingly many factors that
need to be explored in the future, and one of them is certainly
the difference in insulin action between men and women, with
higher rate of circulating insulin and therefore greater suppres-
sion of lipolysis in women. It is unlikely that differences in
body composition are responsible for this phenomenon,
because sex differences in cholesterol metabolism exist even
they are matched for percentage of body fat. Androgen action is
most likely not responsible for the sex differences in plasma
LDL-C concentration. Testosterone administration is associ-
ated with only modest reduction of LDL-C concentration
when given to hypogonadal men in replacement doses and
has no effect on LDL-C concentration in eugonadal men.
Genetic Factors
Serum lipid levels are associated with genetic factors. It has been
estimated that genotype participates with around 40–60% in the
serum total cholesterol variability. There is growing evidence
about association between 95 genetic loci and LDL-C, HDL-C,
and triglycerides, discovered in genome-wide association
studies. In the Framingham Heart Study (FHS), offspring and
third-generation cohorts (3110 participants) of middle-aged to
elderly adults with available genomic DNA were genotyped for
lipid single-nucleotide polymorphisms (SNPs), and genetic risk
scores (GRS) were calculated for each individual and lipid frac-
tion. The results showed modest association between lipid GRS
and corresponding lipid, which was the strongest with the long-
term average lipid measure, and between HDL-C GRS and TG
GRS. Lu et al. investigated whether common genetic variants in
genes involved in cholesterol metabolism could predict the
plasma cholesterol levels. They found that out of 361 SNPs in
243 genes, 23 SNPs were associated with plasma total choles-
terol levels. The results of the study with the multiple gene
approach reported that 10 out of 17 candidate genes were
associated with lipid levels in Caribbean Hispanic subjects,
where the genetic variants on three genes, APOA5, APOB, and
CYP7A1, accounted for the largest proportion of lipids varia-
tion. The longitudinal cohort of black and white siblings,
enrolled in the Bogalusa Heart Study, showed association of
long-term levels and trends of LDL-C with chromosomes 1
and 19. There are several evidences of strong connections
between ten common variants in the genes for LPL, CEPT, and
APO and plasma lipid concentrations in children according to
the Gene–Diet Attica Investigation on childhood obesity (GEN-
DAI). Significantly higher total cholesterol and LDL-C were
observed in APOE E4 carriers compared to E3/E3 homozygotes
and E2 carriers. The association of APOE genotype with total
cholesterol/HDL-C ratio was further modulated by body mass
index (BMI). Carriers of the cholesteryl ester transfer protein
(CETP) TaqIB B2 allele had significantly higher HDL-C and
lower total cholesterol/HDL-C ratio compared to B1/B1 indi-
viduals. Suggested potential prediction of lifelong exposure to
an adverse lipid profile in children is very important for apply-
ing precautionary principle and preventive measures.
Dietary Factors
Modern diet is characterized by the high intake of SFA, refined
starches and sugars, both known to have adverse effect on
serum lipids levels. In addition, the human diet contains a
large portion of oxidized fatty acids and oxidized cholesterol
because of the food processing (e.g., frying and heating).
Lipid Intake
It is clear that plasma cholesterol levels correlate to the quality
and quantity of dietary lipid intake. Tarahumara and Guate-
malan Indians who are consuming a diet low in fat exhibit low
serum total cholesterol and LDL-C. However, when these peo-
ple are placed on typical Western diet, their total cholesterol
and LDL-C increase and synthesis of VLDL increased, mainly
due to increased flow of free fatty acids (FFA) to the liver. In
subjects with increased BMI, it has been demonstrated that
excess body weight correlates with increased synthesis and
turnover of cholesterol in the adipose tissue.
SFA and Cholesterol
Effects of dietary fats and cholesterol on circulating cholesterol
levels, among others, depend on the type of fats and individual
characteristics. A high intake of dietary cholesterol increased
fasting LDL levels by
10% in a dose-dependent manner,
while 12% reduction of fasting LDL levels reduced coronary
 Cholesterol: Factors Determining Blood Cholesterol Levels
risk by 19%. It was estimated that each 100 mg increase in dietary
cholesterol intake resulted in an increase of 0.05 mmol l1 in
serum LDL-C concentration. On the other hand, very-low-fat
diets caused dyslipidemia (high triglycerides and low HDL-C
concentrations). In the ‘Great Fat Debate,’ scientists emphasized
that the adequate replacement of SFA in diet is crucial in terms of
reduction of LDL-C and cardiovascular risk. The general recom-
mendation is to minimize dietary saturated fat by displacing it
with unsaturated fat, primarily polyunsaturated. However, in
practice, people usually replace it with refined carbohydrates,
which also increase cardiovascular risk.
Polyunsaturated and Monounsaturated Fatty Acids
The beneficial effect of unsaturated fatty acids on lipid metab-
olism in man is well documented. Supplementation of diet
by linoleic acid (n6) leads to a decrease in total cholesterol,
LDL-C, and HDL-C levels and in LDL/HDL ratio. Lowering
effect on serum total cholesterol and LDL-C could come from
polyunsaturated and monounsaturated fatty acids from the
corn oil. Favorable fat content from the corn oil positively
influence HDL-C, rising its level and also increasing ratio
of HDL-C to total cholesterol and decreasing ratio LDL-C to
HDL-C. The (n3) fatty acids have an effect different from that
of the (n6) fatty acids, in that they enhance HDL-C levels. The
effects of n3 PUFA on circulating levels of plasma lipopro-
teins have been shown to be variable. No consistent changes
have been observed in LDL-C or HDL-C concentrations with
n3 PUFA consumption. Greater part of this variability is
attributed to a lack of control of confounding factors such as
dietary cholesterol, fat level, and fatty acid composition. The
most comprehensive study evaluating benefits of fish oil (i.e.,
EPA þ DHA) supplementation represents meta-analysis
comprising 47 placebo-controlled randomized trials, which
found clinically significant reduction of triglycerides (by
0.34 mmol l1), no change in total cholesterol, and slight
increases in HDL-C and LDL-C (by 0.01 and 0.06 mmol l1,
respectively) in hyperlipidemic subjects. Another meta-analysis
of 21 trials evaluating lipid outcomes after fish oil consump-
tion found net change in triglycerides (0.30 mmol l1), HDL-C
(0.04 mmol l1), and LDL-C (0.15 mmol l1) and no effect on
total cholesterol. According to previous reports, the mecha-
nisms by which dietary n3 PUFA may induce changes
in plasma LDL-C levels by decreasing absolute rates of LDL
synthesis and catabolism without any effect on the fractional
catabolic rate. Studies using rats have shown that consumption
of fish oils alters hepatic LDL receptor expression. Now, it is
known that the effect of EPA/DHA on blood lipids (including
cholesterol) is based on the action of these n3 PUFAs as
ligands of the various PPAR isoforms and on the modulation
of the signaling pathway of the transcription factor SREBP.
Principal transcription factor that binds the promoter region
of the genes coding for proteins controlling cholesterol
homeostasis is SREBP-2. However, the fact that since SREBP-2
is not directly ligated by DHA (EPA), a relationship between
PPARa ligation and SREBP-2 activation is presumed and is still
not unequivocally explained. The last relationship is possibly
species-dependent. Furthermore, the more efficient absorption
of dietary cholesterol by rodents compared to humans may
account for the differences in the extent of observed increases
55
in LDL-C. The limited but rather consistent increases reported
in LDL-C in humans consuming fish oils may be due to the
relatively small proportion of dietary cholesterol being deliv-
ered to the liver compared to the amount delivered to the livers
of hamsters. Improvement of HDL-C together with insulin
resistance index HOMA (homeostasis model assessment) was
noted in hemodialyzed patients, after EPA þ DHA supplemen-
tation. That was explained by probably activation of PPAR
isoforms and consequently improving insulin signaling on
postreceptor level. It was shown that consumption of fatty
seafood can modulate fasting insulin, ghrelin, and leptin dur-
ing an 8-week intervention in obese young adults. Effects were
partly gender-specific, and the most consistent effect on circu-
lating hormones was mediated by weight loss. PPARa agonists
are the center of a regulatory hub impacting fatty acid uptake,
fatty acid activation, intracellular fatty acid binding, mitochon-
drial and peroxisomal fatty acid oxidation, ketogenesis, triglyc-
eride turnover, lipid droplet biology, gluconeogenesis, and bile
synthesis/secretion.
Therefore, it is important to understand the effects of die-
tary n3 PUFA on cholesterol metabolism and circulating lipid
levels and their interrelation with other dietary components, so
that the beneficial effects of n3 PUFA are not compromised.
As to the influence of oleic acid (n9), controversial results
have been published. Some authors did not see modification
of HDL-C levels, but others showed an increase of HDL-C
especially after an olive oil regime. Significant increase of
HDL-C and apo A1 levels among walnut consumers without
modified regular diet was established. The increase of HDL-C
may be due to the fatty acid composition of walnuts, as
it provides mostly linoleic acid (n6), alpha-linolenic acid
(n3), and oleic acid (n9), or might be related to the nature
of the protein amino acids.
Dietary Fiber, Soy Protein, and Plant Sterols
The serum levels of total cholesterol, LDL-C, Apo A1, and Apo B
were significantly lower in one Chinese ethnic group, who
consume diet primarily based on corn and then on rice, soy,
buckwheat, sweet potato, and pumpkin products, compared
with another Chinese ethnic group, who primary consume rice
and then corn, broomcorn, potato, and taro products, which are
more harmful for lipid profile. Corn-based diet of the first group
is rich with dietary fiber that reduces serum total cholesterol
level in healthy and hyperlipidemic subjects and with plant
high-quality protein that might raise serum HDL-C levels and
promote the transportation and excretion of free cholesterol.
Consumption of soy protein significantly decreases concen-
trations of total cholesterol, LDL-C, and triglycerides in circula-
tion. It was shown that after 24 weeks of treatment based on
soybean, patients with combined hyperlipidemia, isolated
hypercholesterolemia, and isolated hypertriglyceridemia have
decreased LDL-C level by 38%, 32%, and 8%, respectively.
Results of the meta-analysis showed association of soy protein
with significant decreases in serum total cholesterol by 77%,
LDL-C by 5.25%, and triglycerides by 7.27% after short initial
period and significant increases in serum HDL-C by 3.03% after
more than 12 weeks of consumption. A better effect on the lipid
profile was found after intake > 80 mg. Dietary fiber from fruits,
vegetables, and whole-grain products lowers levels of total
56 Cholesterol: Factors Determining Blood Cholesterol Levels
cholesterol and LDL-C possibly through the bile acid metabo-
lism and alteration in serum sex hormone concentrations.
Plant sterols (around 2 g day1) have been shown to block
intestinal absorption of cholesterol and lower total plasma
LDL-C.
In nutritional epidemiology, examining the relation between
diet and its effect should not be focussed on the intake of single
nutrients, food items, or food groups, but should be focussed on
the overall diet and food preparation methods and eating pat-
terns. If we want to achieve healthy serum lipid levels and
prevent chronic diseases, we should follow general diet recom-
mendations to consume great amount of fruits, vegetables, nuts,
and fish; moderate amount of dairy and vegetable oils; and
whole-grain foods in place of refined starches and sugars and
to avoid sugar-sweetened beverages, processed meats, and foods
that contain partially hydrogenated vegetable oils.
Alcohol Consumption
Serum lipid level is associated with alcohol consumption.
Effects depend in part on the amount of consumed alcohol,
so that moderate intake protects individuals against cardiovas-
cular diseases. Alcohol consumption was the independent neg-
ative risk factor for insulin resistance and improved lipid
profiles that are known to be worsened by insulin resistance.
The increase in HDL-C and decrease in LDL-C in drinkers result
from the inhibition of CETP that promotes transfer of choles-
teryl ester from HDL to VLDL (a precursor of LDL) and LDL.
Inconsistent results among earlier studies may partly result
from variability in the prevalence of obesity among subgroups
and according to the amount or frequency of alcohol con-
sumption. Harmful effect of heavy alcohol consumption may
be attributable to increased triglyceride synthesis.
Coffee Consumption
Coffee is consumed as a beverage worldwide; however, its effect
as a cardiovascular risk factor is still controversial. Roasted
coffee contains naturally present antioxidants and others that
are formed during the roasting process. Chlorogenic acids and
caffeine may play a role in the inhibition of lipid peroxidation,
free radical scavenging, and anti-inflammatory activity. How-
ever, coffee also contains diterpenes, cafestol, and kahweol.
High consumption of these compounds can raise serum levels
of total cholesterol and LDL-C. Most of them are retained by the
paper filter, which substantially reduces the cholesterol-raising
effects. It should be mentioned that instant coffee was associ-
ated with lower serum concentrations of total cholesterol and
higher serum LDL-C level. Average change in total cholesterol
for each cup ranged from 0.007 to 0.026 mmol l1. When
examining the effects of coffee beverage on serum lipoprotein
levels, we should take into account coffee preparation such as
the use of milk, sugar, ice cream, and alcohol.
Physical Activity
The sedentary lifestyle is one of the principal risk factors of
highly prevalent illnesses such as type 2 diabetes,
cardiovascular disease, osteoporosis, and some cancers. Asso-
ciation between sedentary lifestyle and the current pandemic of
obesity and metabolic syndrome is clear. Attempt to exactly
measure the effect of sedentary lifestyle on metabolic syn-
drome and other cardiovascular risk factors, based on duration
of leisure-time physical activity, have shown that even 25 min
day1 produces benefits, better HDL-C function or higher level
of paraoxonase 1 activity. The difference for glycemia, total
cholesterol, and HDL-C level disappears after adjustment for
age, gender, and cigarette smoking. However, low physical
fitness among young adults has been shown to longitudinally
predict hypercholesterolemia and, among middle-aged adults,
hypertension. This association was attenuated when adjusted
for obesity.
Seasonal variation in physical activity has been reported to
coincide with seasonal changes in blood lipid levels, particu-
larly total cholesterol. Environmental changes in ambient tem-
perature, daylight, and monthly precipitation are thought to
induce seasonal changes in physical activity, particularly by
extreme environmental factors (e.g., hot or cold temperatures).
Significant increase in nonoccupational activity due to yard
work and exercise or recreational activities was noted during
the warmer months. Estimates of the amplitude of seasonal
variation in activity energy expenditure in this report were
consistent with those in the Framingham Offspring Study.
The FHS is a population-based prospective family study that
began in Framingham, MA, in 1948 with the recruitment of the
Original Cohort. In 1971, children of the Original Cohort,
called the Offspring Cohort, were enrolled, and finally, in
2002, the grandchildren of the Original Cohort were enrolled
making the FHS the longest-running family-based study in
history. For the past 62 years, investigators at the FHS have
collected data related to CVD and its risk factors. Recent public
health recommendations have noted the importance of envi-
ronmental factors as potential barriers to regular participation
in healthful levels of such activity.
Environmental Contaminants
There are increasing evidences of the role of environmental
contaminants (e.g., heavy metals and persistent pollutants) in
serum lipid level variation. Nonoccupational exposure to var-
ious chemicals can occur through contaminated drinking
water, foods, air, and cigarette smoking, and even low-level
exposure may be harmful to health. Hereafter, examples of the
influences of the most common contaminants on lipid metab-
olism will be described.
Arsenic is widely distributed in the environment and usually
contaminates drinking water sources. Experimental studies on
rats have shown an increase in total cholesterol level after
10–20 weeks of exposure to arsenic, added as arsenite or arse-
nate in drinking water. This effect becomes more significant
under high-cholesterol diet and when the exposure occurs
earlier in life. Mechanism by which arsenic modifies choles-
terol metabolism has not been yet elucidated. Possible way is
modulation of RCT that transfers cholesterol from the periph-
ery back to the liver by modifying the densities of cholesterols.
Altered lipid metabolism (low HDL-C, hypertriglyceride-
mia, and high total cholesterol and LDL-C level) may be a
 Cholesterol: Factors Determining Blood Cholesterol Levels
consequence of chronic cadmium exposure, possibly due to
decreased plasma lipoprotein lipase activity and increased
activity of HMG-CoA reductase.
The results of relationship between blood lead level and
serum cholesterol and lipoprotein levels are conflicting. Occu-
pational exposure to lead was positively associated with levels
of total cholesterol and HDL-C. Lead-exposed patients had
decreased total cholesterol and LDL-C and increased HDL-C
levels. Recently, an association between blood lead and total
cholesterol based on an age-adjusted model has been
confirmed.
Fat-soluble chlorinated organics, such as dioxins, furans,
polychlorinated biphenyls (PCBs), and chlorinated pesticides,
were related with unfavorable serum lipid levels. Correlation
between serum PCBs levels and plasma lipid levels was firstly
showed in occupational studies. The strong relationship
between PCBs levels and cholesterol and triglycerides was
reported with average value for the sum of PCB congeners of
4.2 mg. The proposed mechanisms by which PCBs affect serum
lipids are the activation of certain cytochrome P450 enzymes
and increased synthesis of lipids.
Perfluorooctanesulfonate (PFOS) used as surfactant in a wide
variety of commercial products has been recently recognized to
disrupt serum lipid level. Positive association between plasma
concentration of PFOS and serum concentration of HDL-C and
negative association with total cholesterol/HDL-C ratio and
triglycerides level were reported. The possible pathway is per-
oxisome proliferation, leading in hepatotoxicity and alteration
of lipids homeostasis.
It was postulated in the systematic association study about
broad environmental correlation to lipid levels. There was
favorable association of HDL-C (3–4% higher HDL-C) with
iron and mercury exposure and unfavorable association with
PCBs, dibenzofurans, organochlorine pesticides, and all per-
sistent organic pollutants. Multiple environmental factor anal-
ysis enabled better understanding of their relationship with
characteristics in the general population.
Cigarette Smoking
Cigarette smoking is a well-known risk factor for atherosclero-
sis and may influence serum lipid levels. Significant increase in
serum total cholesterol, triglycerides, VLDL, and LDL-C and
decrease in protective HDL were reported among chronic
smokers, hypertensives, and chronic smokers with high
blood pressure. However, confounding factors such as diet,
BMI, stress, alcohol consumption, and physical activity have
not been controlled in some studies. The effect of smoking on
the serum lipid profiles may be influenced by age, gender, and
different smoking statuses. It was found that smoking was
associated with lower total cholesterol and LDL-C levels in
elderly men and in middle-aged women and also with
decreased HDL-C levels in 65–74-year-old men and 55–64-
year-old women when compared with nonsmokers. The data
from meta-analysis were in contrast due to differences in study
populations and their dietary habits, physical activities, life-
style, or public health awareness. Positive association between
current smoking and dyslipidemia was reported in women but
not in men. Further, current female smokers in the age groups
57
between 20 and 34 years and of 50 years or older have had the
highest risk of dyslipidemia. The possible reason for gender
difference in the association between cigarette smoking and
unfavorable serum lipid levels could be an interaction of some
hormonal factors with components of the inhaled smoke. The
nicotine provokes the secretion of catecholamines and other
hormones (e.g., cortisol and GH) leading to an increased
serum concentration of FFA, which stimulates hepatic secre-
tion of VLDL and triglycerides. Cigarette smoke has great oxi-
dative potential and can promote oxidative modifications in
LDL and other biomolecules and may contribute to additional
endogenous oxidant formation, through its effects on the
inflammatory-immune response. Some natural antioxidants
such as dietary polyunsaturated fats and vitamin E are connect-
ing with lower risk for atherosclerosis, although they may
contribute to lipid oxidation in smokers. Further studies are
needed to explain the mechanism by which cigarette smoke
changes serum lipid levels and what are the most responsible
substances for these changes, since cigarette smoke besides
nicotine contains multiple toxic compounds.
Stress
Serum cholesterol levels are changing under emotionally
stressful situations. Numerous studies have demonstrated
that acute and chronic stressors are related in alterations in
cholesterol concentrations. Investigators have established a
negative link between cholesterol and psychological and
physical aggressions. However, there is a positive correlation
between cholesterol and psychological stress. This correlation
can be explained with increased peripheral fat tissue lipolysis
caused by heightened sympathetic nervous system activity and
increased levels of catecholamines, glucocorticoids, and gluca-
gon. The final result of these processes is increased release of
fatty acids into the circulation. In contrary, deficit of serotonin
and lower cholesterol levels have been implicated with physi-
cal aggression and increased incidence of accident, suicide, and
homicide. The possible explanation can be that in primitive
man, cholesterol served as a sentinel compound for survival.
Hence, when primitive man was experiencing lack of food, low
blood cholesterol was leading and preparing him for food
seeking and increased risk in hunting. Chronic stress induces
both functional and structural adaptations within the hypot-
halamo–pituitary–adrenal axis that are suggestive of long-term
alterations in neuroendocrine reactivity to subsequent
stressors. Experimental evidence of chronic stress-induced
hyperlipidemia in animal models has been documented by
significant increases of blood total cholesterol, LDLs, and tri-
glycerides and decrease in HDLs concomitant with increased
oxidative stress.
Diseases
Obesity
It is hard to interpret the influence of obesity itself on choles-
terol metabolism, due to the confounding of metabolic disor-
ders accompanied with this condition. Abnormalities, such as
hypertriglyceridemia, hyperglycemia, and insulin resistance,
58 Cholesterol: Factors Determining Blood Cholesterol Levels
commonly seen in obese population, may affect the distribu-
tion and size of lipoprotein particles independently of adipos-
ity. The typical dyslipidemia observed in obesity does not
include total cholesterol level disturbances. Obesity is charac-
terized by the higher development of small dense, more ath-
erogenic LDL particles. Viscerally, obese men were found to
have significantly reduced LDL receptor binding of lipopro-
teins compared with lean healthy controls. Although hyper-
cholesterolemia is not a concomitant feature of insulin
resistance and obesity, both of them are characterized by
decreased expression of hepatic LDL receptors due to higher
rates of hepatic cholesterol synthesis and diminished choles-
terol absorption.
Many studies show that total cholesterol and LDL-C con-
centrations respond more weakly to diets low in saturated fat
and cholesterol in obese than in lean subjects, due to large
amount of cholesterol in the enterohepatic pool in obese
people. Additional amount taken in with the diet would not
be recognized as small to activate hepatic LDL receptors, and
hepatic LDL receptors are most probably suppressed by this
large stream of endogenous cholesterol from enterohepatic
circulation. Therefore, the most effective way for the obese to
normalize their blood cholesterol is to lose weight. This shows
us that cholesterol metabolism is tightly regulated so that if
cholesterol synthesis is upregulated, cholesterol absorption is
diminished and vice versa.
Diabetes Mellitus and Metabolic Syndrome
Considerable attention has been focussed on dyslipidemia
accompanying diabetes and metabolic syndrome. Metabolic
syndrome is generally characterized by abdominal obesity,
insulin resistance, hypertension, and blood lipid disorders
including high TG and low HDL-C, high apoB, and small
dense LDL-C. Metabolic syndrome is associated with increased
endogenous cholesterol synthesis and reduced intestinal cho-
lesterol absorption predominantly as a consequence of visceral
obesity, independently of overall obesity. It has been proposed
that hyperinsulinemia in insulin resistance may upregulate the
expression of SREBP-1c, a transcription factor that stimulates
the synthesis of fatty acids and the production of VLDL
particles. However, SREBP-2, another transcription factor that
upregulates de novo cholesterol synthesis, does not appear to
be affected by hyperinsulinemia or hyperglycemia. Besides
elevated liver VLDL production, decreased postprandial
triglyceride metabolism represents a major pathway of the
hypertriglyceridemia that characterizes diabetic condition.
Increased cholesterol absorption has also been reported in
type 2 diabetes with slightly increased concentrations of total
cholesterol and LDL-C. It has been shown that changing car-
bohydrate content of a mixed meal altered the postprandial
accumulation of chylomicrons. Furthermore, a high glucose
level alters the genetic expression of various genes involved in
HDL metabolism in HepG2 cells, including human ABCA1,
SR-BI, and hepatic lipase.
Cushing Syndrome
It is well known that chronic overt hypercortisolism, as in
Cushing syndrome, is characterized by systemic alterations
leading to increased risk of metabolic and cardiovascular diseases.
Cortisol excess could inhibit insulin secretion, glucose uptake,
and glycogen synthesis; worsen insulin sensitivity; and increase
gluconeogenesis. However, it was hypothesized that adrenal inci-
dentaloma (the presence of adrenal mass and patient has no signs
of hormonal excess or obvious underlying malignancy) may be
itself an unrecognized manifestation of the metabolic syndrome.
The pattern of dyslipidemia in Cushing syndrome is the same as
in metabolic syndrome or in type 2 diabetes.
Hypothyroidism
Hypothyroidism is associated with increased TC and LDL-C
due to limited synthesis of the LDL hepatic receptors. Also, the
activity of HMG-CoA reductase is significantly lowered, which
may explain the poor response to hypolipemic treatment.
Acromegaly
Patients with acromegaly have a relative risk to present glucose
alterations, with a 2.6 times and 2.1 times higher risk of
impaired glucose tolerance and diabetes, respectively, than
the general population, and show a higher prevalence of hyper-
triglyceridemia and low HDL-C. Active acromegaly in women
is strongly associated with higher visceral adiposity dysfunc-
tion, insulin resistance, and the features of MetS; therefore,
more careful metabolic management is suggested in acrome-
galic women.
Chronic Renal Failure
The typical dyslipidemia in chronic renal failure is characterized
by hypertriglyceridemia and low levels of HDL-C, as the result
from decreased lipoprotein lipase activity. The decreased level
of hepatic lipase observed in renal failure may account for the
presence of IDLs and the high HDL2 subfraction. The pattern
of dyslipidemia depends on the degree of proteinuria and the
kind of terminal renal failure treatment. Total and LDL-C can
also be increased, with a predominance of the small dense LDL
in the event of proteinuria or peritoneal dialysis.
Drugs
Many drugs, besides lipid-lowering drugs, can affect serum cho-
lesterol levels. It has been reported that thiazide diuretics increase
total and LDL-C levels by 5–10% in a dose-dependent way. These
side effects are short term and not contraindication for their
use. Loop diuretics have a similar effect. In contrary, the use of
potassium-sparing diuretics and indapamide shows no changes
in cholesterol levels. The effects of beta-blockers on total
cholesterol and LDL-C are negligible. Furthermore, they could
decrease HDL-C by
5–20%. However, Celiprolol, a selective
beta1 blocker with weak beta2 sympathomimetic activity even
improves the lipid pattern. The only antihypertensive agents that
lower total and LDL-C levels are alpha1-blocking agents.
Oral estrogen preparations given to postmenopausal
women, premenopausal women, women with polycystic ovary
syndrome, men with prostatic carcinoma, and male-to-female
transsexuals reduce total and LDL-C and increase HDL-C levels.
 Cholesterol: Factors Determining Blood Cholesterol Levels
Combined (estrogen/progestogen) hormone replacement
therapy has similar effect. Tamoxifen, a selective receptor estro-
gen modulator, beneficially affects both total cholesterol and
LDL-C, by decreasing them, due to its partial estrogenic activity.
Danazol, a drug that is used in treatment of endometriosis,
induces an increase of LDL and decrease of HDL-C level. Iso-
tretinoin treatment increases LDL and total cholesterol levels
by
15% compared to pretreatment levels. Patients treated
with immunosuppressive therapy (cyclosporine, azathioprine,
and corticosteroids) show increased level of total cholesterol,
LDL-C, and HDL-C in spite of the combination of drugs and
gender of the patient. The largest effect on cholesterol metabo-
lism has cyclosporine in female population, and it is associated
with small dense, more atherogenic LDL particles. Tacrolimus
does not affect total cholesterol and LDL-C levels.
Protease inhibitors, especially ritonavir, increases total cho-
lesterol level by 30–40%.
Considering anticonvulsive therapy, carbamazepine seems
to have most effect on TC levels, which are more pronounced
in women and independent of the dosage. Contrary, valproic
acid is the only anticonvulsant with a more favorable effect on
the lipid profile.
Conclusions
In this article, we try to summarize all factors that determine
cholesterol balance in the body, from absorption, to synthesis,
to clearance. Some of them, as age, gender, and unbalanced
food with more saturated fats and refined carbohydrates, are
well known. However, there is growing amount of evidence
about the benefit effect of diet, which contains more fatty
seafood, corn, soy protein, olive oil, and dietary fiber from
fruits, vegetables, and whole grain. Maybe, it is possible to
improve, by epigenetic impact with regular diet, known spe-
cific genetic variants of genes involved in cholesterol metabo-
lism in childhood. We also emphasize that improvement of
low physical fitness could reduce hypercholesterolemia. In the
future, we have to pay attention to environmental contami-
nants (e.g., heavy metals and persistent pollutants) that are
able to influence lipid metabolism.
See also: Cereals: Dietary Importance; Cholesterol: Absorption,
Function and Metabolism; Fish: Dietary Importance and Health Effects;
Soy Beans: Dietary Importance.
59
Further Reading
Altmann SW, Davis Jr. HR Jr., Zhu LJ, Yao X, Hoos LM, and Tetzloff G (2004) Niemann-
Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science
303(5661): 1201–1204.
Berrougui H and Khalil A (2009) Age-associated decrease of high-density lipoprotein-
mediated reverse cholesterol transport activity. Rejuvenation Research 12(2):
117–126.
Delgado M, Gutierrez A, Cano MD, and Castillo MJ (1996) Elimination of meat, fish,
and derived products from the Spanish– Mediterranean diet: effect of the plasma
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between posttraumatic stress disorder and dyslipidemia. Journal of Psychosomatic
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supplementation in hyperlipidemia: a systematic review and meta-analysis.
International Journal of Cardiology 136: 4–16.
Hu FB (2002) Dietary pattern analysis: a new direction in nutritional epidemiology.
Current Opinion in Lipidology 13: 3–9.
Hunter RF, Tullya MA, Donnelly P, Stevenson M, and Kee F (2014) Knowledge of UK
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Preventive Medicine 65: 33–39.
Jump DB (2008) N-3 polyunsaturated fatty acid regulation of hepatic gene transcription.
Current Opinion in Lipidology 19: 242–247.
Kuller LH (2011) The great fat debate: reducing cholesterol. Journal of the American
Dietetic Association 111: 663–664.
Kikkawa K, Nakajima K, Shimomura Y, et al. (2015) Small dense LDL cholesterol
measured by homogeneous assay in Japanese healthy controls, metabolic
syndrome and diabetes patients with or without a fatty liver. Clinica Chimica Acta
438: 70–77.
Petel CJ, Cullen MR, Ioannidis JPA, and Butte AJ (2012) Systematic evaluation of
environmental factors: persistent pollutants and nutrients correlated with serum lipid
levels. International Journal of Epidemiology 41: 828–843.
Rasic-Milutinovic Z, Popovic T, Perunicic-Pekovic G, Arsic A, Borozan S, and
Glibetic M (2012) Lower serum paraoxonase-1 activity is related with linoleic and
docosahexanoic fatty acids in type 2 diabetic patients. Archives of Medical Research
43: 75–82.
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Relevant Websites
www.aace.com – AACE is American Association of Clinical Endocrinologists.
www.nice.org.uk – NICE is National Institute for Health and Care Excellence from UK.
www.ama-assn.org – AMA (American Medical Association) and AMA publications.
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www.oldwayspt.org/programs/mediterranean-food-alliance – Oldway mediterranean
diet pyramid.
 Cholesterol: Properties, Processing Effects, and Determination
T Dinh, Mississippi State University, Mississippi State, MS, USA
L Thompson, Texas Tech University, Lubbock, TX, USA
ã 2016 Elsevier Ltd. All rights reserved.
Properties
Introduction
Cholesterol is the most highly regarded small molecule in biol-
ogy because of the great research effort by multidisciplinary
scientists and the number of highly decorated research awards,
especially the 13 Nobel Prizes, bestowed upon those scientists
dedicating their work to study the structure, biosynthesis, biolog-
ical functions, absorption and metabolism, and quantification of
this fascinating molecule. Cholesterol, a complex four-ring mol-
ecule with essential biological functions, is surprisingly synthe-
sized from the most basic two-carbon substrate, acetate
(Figure 1). Although being an essential cellular component of
animal tissues and the sole precursor of many steroid hormones,
cholesterol creates various health complications by being oxi-
dized or accumulating elsewhere excessively, such as in the artery
wall, which are preconditions to the formation of atherosclerosis.
Cholesterol was first discovered in 1815 as a component of
human gallstones by the French chemist M.E. Chevreul. The
structure of cholesterol was correctly identified in 1932; however,
the process amazingly had begun decades before without mod-
ern technologies such as nuclear magnetic resonance. From then
on, using isotopic tracers, the discovery of various biosynthetic
and metabolic pathways of cholesterol began.
21 22 24 26
18
20 25
12 H 23
11
19 17 27
1
13
H14 16
9
2 sisting of triglycerides and cholesterol esters, surrounded by a
10 8 15
5
H H
3
HO
7 teins. Chylomicrons (synthesized in the intestine) and very
4 6
Figure 1 Cholesterol: the most regarded small molecule in biology.
ACD/ChemSketch Freeware 2012, Advanced Chemistry Development,
Inc., Ontario, Canada.
Chemical Properties
Naturally occurring sterols, including cholesterol, have 1,2-
cyclopentano-phenan-threne skeleton with 27–30 carbons, a
hydroxyl group at C-3, and a minimal 7-carbon side chain
at C-17 of the ring structure (Figure 2). The variation in
the structures of the ring skeleton and the side chain differen-
tiates mammalian and plant sterols. Cholesterol is the most
abundant sterol in animals and the vital structural component
of animal plasma membranes and is essentially absent in
plants.
Cholesterol can be found free or esterified to long-chained
fatty acids in animal tissues. Hepatocytes need cholesterol to
synthesize lipoproteins and bile acids, whereas other cell types
incorporate cholesterol into their cell membranes. Cholesterol
60
required for cellular functionality can be taken exogenously
through low-density lipoprotein (LDL) from the bloodstream
or synthesized endogenously from acyl-coenzyme A (CoA).
Cholesterol is hydrophobic, which is enhanced by the loss of
the hydroxyl group through esterification. Cholesterol is trans-
ported in the bloodstream by lipoproteins, primarily in the
form of cholesterol esters. Because of their hydrophobicity,
cholesterol esters are more suitable for cellular storage, whereas
free cholesterol acquired by cells through hydrolysis of choles-
terol esters accumulates in the cell membranes as a structural
component.
Uptake and Use
Isotopic labeling techniques confirmed that a two-carbon
metabolite of acetate in acetyl-CoA was the sole building
block for the biosynthesis of cholesterol in the endoplasmic
reticulum. In addition to endogenous biosynthesis, cholesterol
is also available to enterocytes from dietary sources, bile, and
turnover of intestinal mucosal epithelium. Cholesterol enters
the intestine in the free-form by the action of pancreatic cho-
lesterol esterase. It is packed into more hydrophilic micelles
containing conjugated bile acids, monoglycerides, and lysolec-
ithin to approach the absorptive cells. Approximately 60–80%
of dietary and biliary cholesterol is absorbed; however, biliary
cholesterol is to a greater extent absorbed because it is already
in the form of micelles. Cholesterol is transported in the circu-
latory system and to the body tissues by plasma lipoproteins,
which share a common structure of a neutral lipid core con-
monolayer of phospholipids, free cholesterol, and alipopro-
low-density lipoproteins (VLDL, largely synthesized in the
liver) deliver triglycerides to tissues for storage or energy
purposes, and they are converted to LDL in the process. The
LDL can also be synthesized in the liver. The LDL primarily
transports cholesterol to tissues and glands for storage or fur-
ther synthesis. Both endogenously synthesized and exoge-
nously absorbed cholesterol must be returned to the liver for
excretion or degradation. The removal of cholesterol at the
extrahepatic tissues and in the circulatory system is facilitated
by the high-density lipoprotein (HDL). The HDL is removed
by the liver or steroidogenic tissues through a selective uptake
process of cholesterol and other lipid components.
Cholesterol, if not stored as cholesterol esters in the cyto-
plasm, is used for structural purposes in the cell membrane or
the synthesis of bile acids, steroid hormones, and vitamin
D. Cholesterol contributes up to 30% of lipid mass in the cell
membrane and is vital to the less fluid and more structured
lipid phase. The oxidation of cholesterol in tissues, governed
by enzymatic pathways, yields important steroid precursors;
however, the oxidation caused by food processing through
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00150-1
 Cholesterol: Properties, Processing Effects, and Determination 61

nonenzymatic pathways usually results in biologically toxic
oxysterols.
Cholesterol Content in Foods
Cholesterol in the human diet primarily comes from meat,
poultry, eggs, dairy products, and seafood (Tables 1–4). There-
fore, it is expected that, of all the food groups presented in the
USDA National Nutrient Database for Standard Reference
(SR), only those without the previously mentioned protein
sources such as vegetables, spices and herbs, cereals, and
vegetable cooking oils have an undetectable amount of choles-
terol. The database provides the cholesterol content of various
types of protein sources in various forms of raw, cooked, and
further processed products, which makes it possible to extrap-
olate the approximate cholesterol content of similar foods.
Cholesterol is an integral component of the animal cell mem-
brane, and it is also deposited in fat droplets; therefore,
cholesterol content of fresh ingredients does not change
much unless there is a remarkable change in the structure
and metabolism of muscle or fat cells. Factors influencing
cholesterol content across protein sources are species, diet,
muscle oxidative pattern, muscle fiber type and structure, and
fat content.
In general, raw and cooked meat and poultry have approxi-
mately 40–100 mg of cholesterol per 100 g of meat (Table 1).
Few exceptions, such as nonenhanced, cooked chicken dark
meat, can contain up to 150 mg/100 g. Chicken skin, both raw
and cooked, also has more than 100 mg/100 g. Mechanically
deboned meat and poultry have an approximately 10–20%
greater amount of cholesterol because of bone marrow contam-
ination. Variety meats have much greater cholesterol content,
from 300 mg/100 g of raw liver to 3000 mg/100 g of braised
brain. Egg and dairy products are other sources of cholesterol
in the human diet (Table 2). With an exception of more than
200 mg/100 g in few salted butter products, cholesterol content

21 22 24 26 21 22 24 26
25
18 25 18
20 20
12 H 23 12 H 23 OH
11 27 11
19 13 17 19 13 17 27
1 9
H 16 1 9
H 16
14 14
2 2
10 8 15 10 8 15
3 5
H H H H
5
7 7
HO HO 3
4 6 4 6
cholesterol 25-hydroxycholesterol
21 22 24 26 21 22 24 26
18 18 25
20 25 20
12 H 23 12 H 23
11 11 27
19 13 17 27 19 13 17
1 H 16 1 9
H 16
9 14 14
2 2
10 8 15 10 8 15
H H 3 5
H 7
H
5 7
HO 3 OH HO OH
4 6 4 6
7α-hydroxycholesterol 7β-hydroxycholesterol
21 22 24 26 21 22 24 26
18 25 18 25
20 20
12 H 23 12 H 23
11 27 11 27
19 13 17 19 13 17
1 9
H 16 1 9
H 16
14 14
2 2
10 8 15 10 8 15
5
H H 3 5
H H
7 7
HO 3 HO
4 O 6 4 O 6
5,6α-epoxycholesterol 5,6β-cholesterol
21 22 24 26 21 22 24 26
18 25 18 25
20 20
12 H 23 12 H 23
11 27 11
19 13 17 19 13 17 27
1 9
H 16 1 9
H 16
14 14
2 2
10 8 15 10 8 15
5
H 7
H H H
5
6 7
HO 3 O HO 3
4 6 4 OH
7-ketocholesterol OH
cholestane-3β,5α,6β-triol

Figure 2 Structures of cholesterol and the most commonly found cholesterol oxides. ACD/ChemSketch Freeware 2012, Advanced Chemistry
Development, Inc., Ontario, Canada.
62 Cholesterol: Properties, Processing Effects, and Determination

Table 1 Cholesterol content of meat and poultry Table 1 (Continued)

Cholesterol Cholesterol
content, content,
Meat and poultry mg/100 g Meat and poultry mg/100 g

Chicken Beef, variety meats and by-products, brain, cooked, 3100

Chicken, broiler or fryers, breast, skinless, boneless, 73 simmered
meat only, raw Beef, variety meats and by-products, brain, raw 3010
Chicken, broilers or fryers, breast, meat only, cooked, 85 Beef, variety meats and by-products, liver, cooked, 396
roasted braised
Chicken, broilers or fryers, dark meat, drumstick, 89 Beef, variety meats and by-products, liver, raw 275
meat only, raw Chicken, gizzard, all classes, cooked, simmered 370
Chicken, broilers or fryers, drumstick, meat and skin, 130 Chicken, gizzard, all classes, raw 240
cooked, roasted Chicken, liver, all classes, cooked, simmered 563
Chicken, broilers or fryers, skin only, cooked, roasted 83 Chicken, liver, all classes, raw 345
Chicken, broilers or fryers, skin only, raw 109 Pork, fresh, variety meats and by-products, brain, 2552
Pork cooked, braised
Pork, fresh, leg (ham), whole, separable lean only, 94 Pork, fresh, variety meats and by-products, brain, raw 2195
cooked, roasted Pork, fresh, variety meats and by-products, heart, 221
Pork, fresh, leg (ham), whole, separable lean only, 68 cooked, braised
raw Pork, fresh, variety meats and by-products, heart, raw 131
Pork, fresh, loin, whole, separable lean and fat, raw 63
Pork, fresh, loin, whole, separable lean only, cooked, 81 Source: US Department of Agriculture, Agricultural Research Service. (2014). USDA
roasted National Nutrient Database for Standard Reference, Release 27; http://www.ars.usda.
Pork, fresh, loin, whole, separable lean only, raw 59 gov/ba/bhnrc/ndl.
Pork, fresh, spareribs, separable lean and fat, cooked, 121
braised of dairy products is less than 100 mg/100 g. Cholesterol in dairy
Pork, fresh, spareribs, separable lean and fat, raw 80
products is accumulated for storage purposes in the form of
Beef
cholesterol esters within the fat globules; therefore, more con-
Beef, grass-fed, strip steaks, lean only, raw 55
Beef, ground, 70% lean meat/30% fat, patty, cooked, 82 centrated and more fatty dairy products have more cholesterol,
broiled for example, most milk products with approximately 4 mg/
Beef, ground, 70% lean meat/30% fat, raw 78 100 g, regular cream cheese with 110 mg/100 g, and fat-free
Beef, rib, whole (ribs 6–12), separable lean and fat, 82 cream cheese with 12 mg/100 g. Most cottage cheeses are low
trimmed to 1/8" fat, all grades, cooked, broiled in cholesterol, around 4–10 mg/100 g. Similarly, egg products
Beef, rib, whole (ribs 6–12), separable lean and fat, 70 containing more yolk have much more cholesterol than those
trimmed to 1/8" fat, all grades, raw containing more egg white because majority of the lipid compo-
Beef, round, top round, separable lean and fat, 90 nents of eggs are located in egg yolk. Whole chicken eggs have
trimmed to 1/8" fat, all grades, cooked, braised
approximately 372 mg cholesterol/100 g, whereas cholesterol in
Beef, round, top round, steak, separable lean and fat, 69
most egg white powders is undetectable. Moreover, whole eggs
trimmed to 1/8" fat, all grades, raw
Beef, short loin, porterhouse steak, separable lean 82 from turkey or goose can have as much as 900 mg/100 g. Most
only, trimmed to 1/8" fat, all grades, cooked, grilled cholesterol in eggs occurs in free-form, whereas much of choles-
Beef, short loin, porterhouse steak, separable lean 58 terol in dairy products is esterified.
only, trimmed to 1/8" fat, all grades, raw Fish generally has similar or lower cholesterol content com-
Lamb pared with meat and poultry, for example, raw and cooked
Lamb, domestic, foreshank, separable lean and fat, 106 tuna with 38–49 mg/100 g. Some fish such as salmon or trout
trimmed to 1/4" fat, choice, cooked, braised can have up to 60 mg/100 g of raw meat and up to 100 mg/
Lamb, domestic, foreshank, separable lean and fat, 72 100 g of cooked meat. Shellfish and crustaceans have much
trimmed to 1/4" fat, choice, raw
greater cholesterol content than meat, poultry, and fish. Crus-
Lamb, domestic, loin, separable lean only, trimmed to 95
taceans such as shrimps, crabs, and lobsters have lower choles-
1/4" fat, choice, cooked, broiled
Lamb, domestic, loin, separable lean only, trimmed to 66 terol content than mollusks, in the range of 50–70 mg/100 g of
1/4" fat, choice, raw raw and 90–120 mg/100 g of cooked meat. Some canned and
Veal breaded shrimp products of mixed species can have up to
Veal, leg (top round), separable lean and fat, cooked, 134 138–252 mg/100 g of meat. Squid has been reported to have
braised the greatest cholesterol content among seafood, in the range of
Veal, leg (top round), separable lean and fat, raw 78 200–300 mg/100 g for both raw and cooked products. Mol-
Veal, loin, separable lean and fat, cooked, braised 118 lusks with shell such as oysters have similar cholesterol content
Veal, loin, separable lean and fat, raw 79 to that of crustaceans (Table 3).
Variety Meats
Processed meat products and other processed foods formu-
Beef, variety meats and by-products, brain, cooked, 1995
lated with cholesterol-containing protein sources vary greatly
pan-fried
in cholesterol content, from a few milligrams in 100 g of soups
 Cholesterol: Properties, Processing Effects, and Determination 63

Table 2 Cholesterol content of dairy foods and eggs Table 3 Cholesterol contents of seafood

Cholesterol Cholesterol
content, content,
Dairy foods and eggs mg/100 g Seafood mg/100 g

Butter, salted 215 Crustaceans, crab, Alaska king, cooked, moist heat 53
Butter, whipped, with salt 219 Crustaceans, crab, Alaska king, raw 42
Cheese, cottage, creamed, large or small curd 17 Crustaceans, shrimp, mixed species, canned 252
Cheese, cottage, low-fat, 1% milk fat 4 Crustaceans, shrimp, mixed species, raw 126
Cheese, cream 110 Fish oil, cod liver 570
Cheese, cream, fat-free 12 Fish, salmon, Atlantic, farmed, cooked, dry heat 63
Cheese, gouda 114 Fish, salmon, Atlantic, farmed, raw 55
Cheese, low-fat, cheddar, or colby 21 Fish, salmon, pink, canned, drained solids 83
Cheese, mozzarella, part skim milk 64 Fish, seatrout, mixed species, cooked, dry heat 106
Cheese, mozzarella, whole milk 79 Fish, trout, rainbow, farmed, raw 59
Cheese, pasteurized process, American, fortified with 100 Fish, tuna, fresh, bluefin, cooked, dry heat 49
vitamin D Fish, tuna, fresh, bluefin, raw 38
Egg, duck, whole, fresh, raw 884 Mollusks, cuttlefish, mixed species, raw 112
Egg, goose, whole, fresh, raw 852 Mollusks, octopus, common, raw 48
Egg, quail, whole, fresh, raw 844 Mollusks, oyster, eastern, cooked, breaded and fried 71
Egg, turkey, whole, fresh, raw 933 Mollusks, oyster, eastern, farmed, cooked, dry heat 38
Egg, white, raw, fresh 0 Mollusks, oyster, eastern, farmed, raw 25
Egg, whole, raw, fresh 372 Mollusks, oyster, eastern, wild, cooked, moist heat 79
Egg, yolk, raw, fresh 1085 Mollusks, oyster, eastern, wild, raw 40
Milk, fluid, 1% fat, without added vitamin A and vitamin D 5 Mollusks, squid, mixed species, cooked, fried 260
Milk, low-fat, fluid, 1% milk fat, with added vitamin A and 5 Mollusks, squid, mixed species, raw 233
vitamin D
Milk, nonfat, fluid, with added vitamin A and vitamin D 2 Source: US Department of Agriculture, Agricultural Research Service. (2014). USDA
(fat-free or skim) National Nutrient Database for Standard Reference, Release 27; http://www.ars.usda.
Milk, nonfat, fluid, without added vitamin A and vitamin D 2 gov/ba/bhnrc/ndl.
(fat-free or skim)
Milk, reduced fat, fluid, 2% milk fat, with added nonfat 8
dietary cholesterol may raise serum LDL cholesterol, thereby
milk solids, without added vitamin A
Milk, reduced fat, fluid, 2% milk fat, with added vitamin A 8 increasing the risk of cardiovascular disease. As such, in the
and vitamin D Guidelines for Americans 2010, the US Department of Agricul-
Milk, reduced fat, fluid, 2% milk fat, without added 8 ture and the US Department of Health and Human Services
vitamin A and vitamin D recommend that Americans consume less than 300 mg of cho-
Milk, whole, 3.25% milk fat, with added vitamin D 10 lesterol per day to help maintain normal levels of serum cho-
lesterol as well as a healthy ratio of HDL/LDL cholesterol.
Source: US Department of Agriculture, Agricultural Research Service. (2014). USDA
National Nutrient Database for Standard Reference, Release 27; http://www.ars.usda.
gov/ba/bhnrc/ndl.
Processing Effects
to more than 100 mg in 100 g of ready-to-eat sausages Effects on Cholesterol Content
(Table 4). The variation is a direct result of diverse formula- Most value-adding processes cause little chemical effect on
tions, ingredients, cooking processes, storage conditions, and cholesterol content of foods. Cholesterol is primarily diluted
the oxidation of cholesterol during processing. Cholesterol or concentrated through food processing because of changes in
content of meat, poultry, eggs, dairy products, and seafood protein, lipid, and moisture contents. Therefore, cholesterol-
provided by the SR and their labeled proportions can be used reducing strategies for foods normally involve protein and fat
to calculate the cholesterol content of processed foods. Many replacement by those from plant sources and the development
restaurant foods are also available in the database. It is of further processes that compensate for the loss of texture and
expected that restaurant foods containing large amounts of flavor, such as extrusion or restructuring of protein, lipid
animal fat and ingredients such as chicken dark meat, bacon, hydrogenation, and flavor addition (natural or synthetic). It
and shrimp are abundant in cholesterol. is also important to note that the replacement by plant sources
will dramatically increase the content of phytosterols, many of
which are estrogen analogs and have potentials to cause phys-
Dietary Recommendations
iological effects on human. An important aspect of food pro-
Despite the fact that cholesterol is a necessary physiological cessing that changes cholesterol concentration is the loss of
and structural component, the body synthesizes adequate moisture. Cooking or any heat application usually increases
levels of cholesterol to meet physiological needs. Evidence cholesterol content because moisture is lost, whereas choles-
exists that in certain individuals, excessive consumption of terol is retained. However, some cholesterol can be lost during
64 Cholesterol: Properties, Processing Effects, and Determination
Table 4 Cholesterol content of processed foods
Processed foods
Beef, bologna, reduced sodium
Blood sausage
Bockwurst, pork, veal, raw
Cooking oils, vegetable oils
Fast foods, biscuit, with egg and bacon
Fast foods, biscuit, with egg and ham
Fast foods, cheeseburger; double, large patty, with
condiments and vegetables
Fast foods, cheeseburger; single, regular patty; plain
Fast foods, cookies, animal crackers
Fast foods, cookies, chocolate chip
Fast foods, croissant, with egg and cheese
Fast foods, croissant, with egg, cheese, and bacon
Fast foods, croissant, with egg, cheese, and ham
Fast foods, croissant, with egg, cheese, and sausage
Fast foods, English muffin, with egg, cheese, and
Canadian bacon
Fast foods, hotdog, plain
Fast foods, onion rings, breaded and fried
Frankfurter, beef and pork
Frankfurter, chicken
Liver cheese, pork
Liver sausage, liverwurst, pork
Pork, oriental style, dehydrated
Salad dressing, Italian dressing, commercial, regular,
without salt
Salad dressing, Italian dressing, reduced fat, without salt
Salad dressing, ranch dressing, commercial, regular
Salad dressing, ranch dressing, reduced fat
Salami, cooked, beef and pork
Salami, cooked, turkey
Sausage, chicken and beef, smoked
Sausage, turkey and pork, fresh, bulk, patty or link,
cooked
Snacks, potato chips, cheese flavor
Snacks, potato chips, reduced fat
Soup, bean with bacon, condensed, single brand
Soup, bean with frankfurters, canned, condensed
Soup, bean with ham, canned, chunky, ready-to-serve
Soup, bean with pork, canned, condensed
Soup, beef noodle, canned, condensed
Soup, beef with vegetables and barley, canned,
condensed, single brand
Soup, cheese, canned, condensed
Soup, chicken with star-shaped pasta, canned,
condensed, single brand
Soup, cream of asparagus, canned, condensed
Soup, cream of celery, canned, condensed
Soup, cream of chicken, canned, condensed, single
brand
Spices
Source: US Department of Agriculture, Agricultural Research Service. (2014). USDA
National Nutrient Database for Standard Reference, Release 27; http://www.ars.usda.
gov/ba/bhnrc/ndl.
cooking because of oxidation or fat drip. Oxidation occurs
more easily to free cholesterol, for example, cholesterol in
eggs or lean tissues, whereas cholesterol esters in fat droplet
are more likely to be lost through cooking, for example,
chicken skin or fat tissues. Many studies have documented an
increase in cholesterol content in cooked meat and poultry
Cholesterol compared with raw and have explained the phenomenon, at
content,
least in part, through the migration of cholesterol from adipose
mg/100 g
tissues or poultry skin to cooked lean tissues. The USDA Nutri-
56 ent Data Laboratory, responsible for quality control of data
120 published in the SR, routinely uses dry matter basis (based on
93 solid content) to evaluate data. Occasionally, depending
0 on cooking methods, some chicken products cooked with
235 skin on showed an increase in solid percentage, more than
156 what was expected through moisture loss, and an increase in
55
dry matter-based concentration of cholesterol, which could
only be explained by the migration of lipid from the skin,
43
16 which has more cholesterol than cooked lean tissue. Interest-
21 ingly, such a phenomenon was not observed in chicken
170 cooked with skin side down or with added water because of
167 either fat drip or water dilution, respectively.
140
123
168 Oxidation of Cholesterol
Cholesterol can be oxidized during cooking, heat applications
45
such as extrusion or pasteurization, irradiation, and prolonged
0
processes such as fermentation or storage. The oxidation of
50
96 cholesterol has a similar mechanism to that of unsaturated
174 fatty acids. With a double bond between C5 and C6, the single
158 bonds C4–C5 and C6–C7 are most prone to the attack by free
67 radicals from lipid oxidation and reactive oxygen species. Cho-
67 lesterol esters, however, are more susceptible at C7. Choles-
terol oxidation products (COPs), cholesterol oxides or
6 oxysterols, have similar carbon backbone to that of cholesterol
26 but possess more oxygen-containing functional groups such as
16
hydroxyl, ketone, or hydroperoxide (Figure 2). Most of the
89
added functional groups are located at C7 and sometimes at
76
70 C5 and C6. Rarely does the oxidation happen on the side chain,
84 such as at C25 (25-hydroxycholesterol). To initiate the oxida-
tion, a free radical can subtract a hydrogen at the C7 next to the
4 C5¼ C6 double bond, creating a free radical in the cholesterol
0 structural rings. The migration of this free radical and subse-
3 quent formation of hydroperoxide, hydroxyl, or ketone deriva-
9 tive preferably occur at C7 rather than C4 because of the existing
9 hydroxyl group at C3. Reactive oxygen species such as triplet
2
oxygen may attack C5 ¼ C6 double bond in an addition mode
4
to form 5,6-epoxycholesterol. This epoxy can be hydrated to
6
form a very toxic triol (cholestane-3b,5a,6b-triol). The degra-
3 dation of hydroperoxide at C7, however, forms hydroxyl radical
4 and 7-alkoxyl radical, which becomes 7-hydroxycholesterol
through hydrogen abstraction or 7-ketocholesterol through
4 reaction with 7-peroxyl. In addition, further dehydration of
11 cholesterol and intermediate products by prolonged heating
7 can promote the formation of conjugated cholesta-3,5-dien,
7-ketocholesterol, cholesta-3, 5-dien-7-one, and cholesta-3, 5,
0
7-trien. Cholesterol 7-hydroperoxide and intermediate free rad-
icals such as 7-alkoxyl, 7-peroxyl, or hydroxyl propagate new
free radicals from other cholesterol or unsaturated fatty acid
molecules to start the chain reaction (Figure 3). Factors
influencing cholesterol oxidation are similar to those affecting
fatty acid oxidation, such as pH and the availability of free
radicals, reactive oxygen species, light, antioxidants, and metal
catalysts. The physical state of cholesterol, which influences the
exposure and the arrangement of the ring structure, the side
chain, the double bond, and the hydroxyl group, is also very
 H H O-O H O-OH H O OH H OH
or Fe3+
H H H H H
l
ero
H H H H H H H H H H lest
cho
oxy
HO HO HO HO HO hydr
25-
+H Δ, Fe2+ +H
25
H 5 hydrogen abstraction from cholesterol thermal degradation or hydrogen abstraction from cholesterol
C2 s + O2 transition metal catalysts
H d at ls n sp ecie or unsaturated fatty acids or unsaturated fatty acids
ize ica
l rad xyge +H Δ, Fe2+ +H
ica free tive o
H H rad c
3 5 rea
7 radicalized at ide
HO C7
rox

Cholesterol: Properties, Processing Effects, and Determination

6 H yl H
pe H xyl H H
e rox ro lko
lp hyd ro la
o ol H
H ter H
ter
H ste H
add iplet o

s ole
or t

ole ole
s ch
ch ch
ition xyge

H
r

+ O2 H H H H H H H H H H
of s

HO HO HO HO HO OH H
O-O pe O-OH O OH ol
ter

reaction
alkoxyl-peroxyl
wi rox les
ingle

or Fe3+
th yl- c ho
H H oxy
n

hydration ch pe
ydr
t

ole ro
st xyl 7-h

de

−
HO OH

H 2 ion
er

hy
+ H2O ol or p

O
dr
or er

at
H fa oxy
tty l
H ac -alk
H id ox H
ra yl
H l di re on
H trio
ca ac H ol enati
H H 6 - ls tio ter drog
, 5 , n
ole
s dehy
H ols H H -3 h
HO ter n e H H c
O les ta eto
ho HO les -k
H H xy
c OH ch
o HO O 7
po OH
-e
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O 5,6

Figure 3 Primary pathways of cholesterol oxidation by free radicals. ACD/ChemSketch Freeware 2012, Advanced Chemistry Development, Inc., Ontario, Canada.

65
66 Cholesterol: Properties, Processing Effects, and Determination
important to the oxidative degree and the production of oxy-
sterols. Cholesterol esters are less susceptible to oxidation than
free cholesterol, as evidenced by a much greater percentage of
cholesterol lost in dried egg powder (primarily containing
free cholesterol) than that in cooked separable fats (primarily
containing cholesterol esters) found in the SR and various
studies. In addition to heat applications, irradiation has been
reported to accelerate the autoxidation of lipids including
cholesterol. However, refrigeration, freezing, and vacuum
packaging provide a protective effect, as expected. Moreover, it
is important to recognize the photosensitized oxidation of
cholesterol during retail display under fluorescent light. Muscle
foods are especially susceptible because myoglobin, muscle
pigment, is a photosensitizer and significantly increases
the generation of the excited singlet oxygen. Singlet oxygen
can be added directly to the double bond of cholesterol mole-
cule. Initial products of photosensitized oxidation are primarily
5-hydroperoxycholesterol and 6-hydroperoxycholesterol, which
differ from those generated through free radical-mediated path-
ways. As previously discussed, stable oxidation products are
epoxycholesterols and cholestanetriol. Most commonly found
oxysterols in foods are 7-OH, 7-keto, 5,6-epoxy, 3,5,6-triol, and
the side-chain derivative 25-OH (Figures 2 and 3).
Cholesterol Oxides in Foods and Health Implications
Similar to cholesterol, cholesterol oxides are limited to foods of
animal origin and seafood. Irradiation, direct heating, and
processing methods with high heat or high degree of dehydra-
tion for a prolonged period have been reported to produce
more oxysterols than the milder processing methods. The
7-hydroxycholesterol derivative was most commonly found
in egg products at an average concentration of 0.2 to less
than 50 ppm. Spray-dried egg yolk, however, was reported to
have more than 100 ppm of cholesterol oxides, most were
epoxycholesterol. More cholesterol oxide derivatives, as previ-
ously mentioned, have been found in meat and poultry prod-
ucts. Using beef tallow for prolonged deep-frying was reported
to oxidize 25% cholesterol, most of which was converted to
7-hydroxycholesterol and conjugated dien and trien deriva-
tives. Moreover, prolonged storage of dried pork for 3 years
could oxidize more than 30% cholesterol, the products of
which also included 5,6-epoxycholesterol. Cholesterol oxides
have not been found in milk regardless of fat content even after
prolonged heating. However, dehydration, fermentation, and
prolonged storage seem to be the primary causes of cholesterol
oxidation in dairy products. Examples of cholesterol oxides
found skim milk powder are especially troubling, including
up to 2.5 ppm of toxic triol and 25 ppm of 7-ketocholesterol.
A level of up to 17 ppm of 5,6-epoxycholesterol, 7-hydroxy-
cholesterol, and other side-chain oxidation derivatives has also
been reported in butter and cheeses. Grated cheeses have much
more cholesterol oxides, possibly because of greater surface
area that promotes oxidation. Seafood has a low concentration
of cholesterol oxides, approximately 5–9 ppm, which is pri-
marily in products subject to smoking and prolonged storage.
Dry-heat cooking methods seem to increase cholesterol oxides
in fish more than moist-heat cookery.
The structures of cholesterol oxides resemble that of cho-
lesterol; therefore, they can be delivered to various tissues in
similar pathways and interfere with the biological functions of
cholesterol. Cholesterol oxides have an increased polarity com-
pared with cholesterol, which allows them to be absorbed
more efficiently into the bloodstream. Macrophages also
absorb LDL with oxidized cholesterol at a much greater rate.
COPs have been reported to have greater atherogenic and cyto-
toxic effects than cholesterol. The atherogenic effects of choles-
terol oxides were found in animal models at prolonged
ingestion rates of 1–40 mg kg1 body weight. Although most
oxidation derivatives cause cytotoxicity in culture of various cell
types, only 25-hydroxycholesterol and cholestanetriol have
been reported to cause severe necrosis in vivo, for example,
aortic muscles and endothelium of rabbits. Radioactive label-
ing indicates that VLDL and LDL, the preferred cholesterol
transporter to human vascular cells, are also the preferred
mode of transportation for cholesterol oxides. Therefore,
oxidized VLDL and LDL are suspected to cause damage to
the endothelial integrity and death of the vascular muscle
cells. In addition, cholestanetriol, 7-ketocholesterol, and
25-hydroxycholesterol interfere with cholesterol synthesis,
membrane formation and integrity, and cell growth of various
tissues in vitro and in vivo. Recently, evidence indicates that
cholesterol oxides, primarily epoxycholesterols, can also be
mutagenic and even carcinogenic. They were reported to cause
mutation in the lungs of hamsters and tumors in skin of mice.
Some cholesterol oxides, for example, 7a-hydroxycholesterol,
are physiological metabolites and have not been found to
exhibit adverse effects on human health.
Quantification
Cholesterol and cholesterol esters are usually determined
together as total cholesterol, although cholesterol esters are
usually hydrolyzed to the free-form. Cholesterol must be
extracted and separated from other interfering lipid com-
pounds, especially fatty acids before it can be quantified.
Although various means can be used to measure cholesterol,
gas chromatography (GC) with flame ionization detection
(FID) and high-pressure liquid chromatography (HPLC) with
ultraviolet (UV) detection or mass spectrometry (MS) have
become the predominant techniques. The majority of the
recent cholesterol data in foods, however, have been generated
by GC-FID, including those in the SR.
Extraction
Cholesterol is traditionally analyzed together with fatty acid
composition; therefore, lipid extraction has been the first step
of most cholesterol determination procedures found in the lit-
erature. The lipid extraction employs mixtures of midpolar and
nonpolar solvents, among which the 2:1 (v/v) chloroform/
methanol is most popular. The two most commonly used
methods of this mixture were proposed by Folch and coworkers
in 1957 and Bligh and Dyer in 1959, which have been modified
by many others. Other solvent mixtures of hexane, diethyl ether,
isopropanol, and butanol have also been examined; however,
none comes close to the recovery of the chloroform/methanol
mixture. The polarity of the extraction mixture is very important,
especially in a two-step procedure (a polar solvent followed by a
 Cholesterol: Properties, Processing Effects, and Determination
nonpolar one), because cholesterol exists in a slightly polar free-
form (with hydroxyl group) and a much less polar esterified
form. Cholesterol, as a structural component of the cell mem-
brane, is also bound by other polar compounds such as proteins
and phospholipids. After chloroform/methanol extraction, the
organic layer is separated from the aqueous layer. It is important
to maintain a chloroform/methanol/water ratio of 8:4:3 to pre-
vent the selective loss of lipid compounds into the aqueous
phase, including cholesterol.
Following lipid extraction, lipids are saponified primarily
using concentrated ethanolic or aqueous potassium hydroxide
(KOH) solutions to liberate cholesterol from the esterified
form with fatty acids. With cholesterol becoming more and
more important because of its health implications, analysts
have explored shortcuts to simplify the analytic procedures,
the most important of which has been the use of direct sapon-
ification when cholesterol is the only lipid compound of
interest. Direct saponification yields either similar or better
recovery compared with the traditional two-step extraction/
saponification procedure. In some cases of complex matrices,
especially those with high emulsifying capability such as eggs or
emulsion-type meat products, direct saponification is much
more accurate and precise. Samples of various natures are
directly hydrolyzed in either ethanolic or aqueous KOH solu-
tion, which is followed by an extraction of the unsaponifiable
compounds by a nonpolar solvent, similar to that of lipid
extraction. Toluene or a mixture of hexane and a miscible
alcohol is most commonly used. Hexane is sometimes used
alone; however, at least three extractions are needed to obtain
the exhausted recovery and maintain the ratio of cholesterol
and an internal standard. Toluene so far has been the best
solvent for cholesterol extraction, accommodating a wide
range of polarity with a single extraction. However, the use of
toluene is prone to the formation of an emulsion, especially
with the direct saponification because soap, an emulsification
agent, is formed during saponification. Extraction using tolu-
ene, therefore, is followed by a careful cleanup process. A few
cautions are needed for direct saponification technique such as
the strength of the KOH solution, saponification time, and type
of KOH solution (i.e., alcoholic or aqueous).
Derivatization
Cholesterol is usually converted to the suitable derivatives for
various means of measurement. GC requires volatility and
thermal stability, whereas liquid chromatography requires sen-
sitivity and specificity, that is, enhanced UV absorption or
more informative ion fragments if cholesterol is quantified by
UV absorption or MS, respectively. Recently, cholesterol has
been determined without derivatization because of dramatic
improvements in columns and detectors.
Trimethylsilyl (TMS) ether is the most common cholesterol
derivative. The ether provides great volatility and improves
peak shape because the hydroxyl group is converted to a
much less polar ether group, preventing the interaction with
the polar sites of the GC columns. Among various derivatizing
reagents and conditions, N,O-bis(TMS)trifluoroacetamide
(BSTFA) in 1% trimethylchlorosilane is recommended because
of the maximum ether conversion and the ability to produce
hydrofluoric acid, which volatizes silicon dioxide and helps
67
maintain the sensitivity of the FID detector. In addition, the
use of butyrate and acetate cholesterol esters has been
explored; however, the resultant conversion rates and peak
shapes were compared less favorably than those of the TMS
ether. The ether, on the other hand, is susceptible to moisture;
therefore, moisture-free environment and moisture trap for the
GC system are required.
Free cholesterol has been analyzed by HPLC-UV,
HPLC-photodiode array (PDA), and HPLC-MS. Liquid chro-
matography provides great resolution between cholesterol and
other interferences; however, the UV detection is much less
sensitive than the FID. MS is typically used for definitive pur-
poses rather than for routine analysis. Cholesterol, if not deri-
vatized, does not yield informative mass fragments because it
has only one functional group. Recently, the development of
high-temperature, low-bleed GC columns with fused silica and
bonded or cross-linked stationary phase has allowed for the
determination of free cholesterol by GC-FID. The temperature
program can be isothermal or gradient, depending on the
interferences that need to be separated from cholesterol. Free
cholesterol often interacts with silanol groups on the surface of
the fused silica, which causes the tailing of the cholesterol
peak. This phenomenon can affect the linearity of the calibra-
tion curve if a wide range of standard concentrations and a less
polar internal standard such as cholestane are used. A column
with a nonpolar stationary phase, for example, (5%-phenyl)-
methylpolysiloxane, and a thick stationary film, that is, more
than 0.25 mm and preferably 1 mm, can be used to improve
peak shape and linearity. However, a nonpolar column may
not provide the best resolution for a mixture of unsaponifiable
compounds, the majority of which are slightly more polar than
the saponified fatty acids. A thick stationary phase can increase
column bleeding and hinder the sensitivity; therefore, high-
temperature limits of the columns are important. The column
technology nowadays seems to provide adequate thermal sta-
bility through cross-linked and bonded phases. Longer condi-
tioning time during column installation can be helpful.
Separation
Chromatographic separation of cholesterol depends primarily
on columns; and in the case of HPLC, it also depends on the
separation mode, that is, normal-phase or reversed-phase.
Cholesterol has been most commonly determined by GC; there-
fore, many GC columns are available. Packed columns have been
mostly replaced by capillary columns because of much better
resolution and reproducibility. Most capillary columns used in
cholesterol analysis have typically 10 000–30 000 theoretical
plates, providing a much better resolution than the 3000–5000
theoretical plates of the packed columns. Cholesterol is classified
as a nonpolar compound with a slight polarity provided by one
hydroxyl group; therefore, both nonpolar and polar stationary
phases have been used. A nonpolar phase (100% dimethylpoly-
siloxane) such as HP-1, DB-1, SE-30, CP-Sil 5CB, or SPB-1; a
slightly polar phase (5%-phenyl-methylpolysiloxane) such as
HP-5, DB-5, SPB-5, RTX-5, or ZB-5; and a midpolar phase
(50%-phenyl-methylpolysiloxane) such as DB-17, DB-1701,
NB-17, or CP-Sil24CB are the most commonly used GC station-
ary phases for cholesterol determination. The low-polarity phase
with 5% phenyl group has been used much more than other two
68 Cholesterol: Properties, Processing Effects, and Determination
phases in analyzing both free cholesterol and derivatized choles-
terol. Cholesterol derivatives have been separated on a thin-film
column, that is, less than 0.25-mm film thickness, whereas a
thicker film has been used for free cholesterol. A thickness of
more than 0.5 mm will improve peak shape of free cholesterol as
previously discussed but will significantly prolong the retention
of cholesterol in the GC column, sometimes unnecessarily.
Most HPLC methods for cholesterol determination were
not developed for routine analysis. The advantage of HPLC is
that it can be used to separate cholesterol, cholesterol esters,
triglycerides, diglycerides, tocopherols, and other sterols with-
out derivatization, and sometimes in a single run. It has been
reported that the GC does not provide an adequate separation
of free cholesterol from phytosterols and tocopherols, whereas
such a separation can be accomplished by manipulating the
HPLC mobile phase polarity in both normal-phase and
reversed-phase modes. Normal-phase HPLC mostly employs
highly pure silica microparticles (5 mm) such as mPorasil, Inert-
Sil, or Spherisorb and a 1–3% isopropanol/hexane mobile
phase. Other polar columns of alcohol-bonded silica and cya-
nopropylsilica have also been used. Reversed-phase mode
employing a nonpolar column offers better selectivity because
it allows for more manipulation of mobile phase polarity.
Most compounds coexisting with cholesterol in a post-
preparation mixture are also retained better on a reversed-
phase column. Reversed stationary phase commonly used for
cholesterol determination consists of an octyl (C8) or octade-
cyl (C18) being bonded to a highly pure silica microparticle
(5 mm). The C18 columns provide excellent retention and
require mobile phases with lower polarity. Common organic
modifiers for reversed-phase HPLC are acetonitrile, ethanol,
methanol, and isopropanol. Recently, ultrahigh-pressure liq-
uid chromatography (UPLC) has been used more for choles-
terol determination because it uses much less solvent and
decreases the retention time significantly to approximately
5 min. UPLC can also increase the resolution substantially
through using submicron particle sizes.
Detection
Cholesterol derivatives have been quantified by FID or MS
although the latter is primarily used for research or definitive
purposes. A flame ionization detector provides great sensitivity
and linearity over a wide range of concentrations. MS, how-
ever, is the method of choice when cholesterol is analyzed in a
more complex mixture and expected to coelute with other
unsaponifiable compounds, such as phytosterols or tocoph-
erols. MS is better used for cholesterol derivatives than free
cholesterol because cholesterol derivatives respond better to
the ionization and produce more informative ion fragments
for identification and quantification. Free cholesterol, like
many sterols with very few functional groups, does not
respond well to atmospheric pressure ionization such as elec-
trospray; therefore, electron impact ionization in a vacuum
chamber is usually employed. The detection limit for FID and
MS is in the range 1 ng on-column. The MS can be more
sensitive, depending on the ion-monitoring mode.
In addition, cholesterol and cholesterol derivatives have been
successfully determined by UV, fluorescence (FD), evaporative
light-scattering, and electrochemical (ECD) detections, which
are usually coupled with an HPLC system. UV detection is
the most commonly used technique, which is probably a
matter of convenience more than technical considerations
because of the issues with specificity or sensitivity. Cholesterol
has been detected in the range of 202–210 nm with the
maximum absorption at 205 nm. A simple scan of spiked cho-
lesterol standard may be needed to confirm lmax in a specific
solution. A PDA detector improves specificity of UV detection
because of multiwavelength monitoring. Fluorescence-generating
or fluorescence-tagging reagents such as 3,4-dihydro-6,7-
dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl azide and
naproxen acyl chloride can be used to convert cholesterol and
other sterols to fluorescing derivatives, which will substantially
increase specificity and sensitivity. The limit of detection (LOD)
for the FD is within few picograms on-column. UV absorption
can be enhanced to a LOD of 0.1 ng at a much more favorable
lmax of 250 nm by converting cholesterol to cholest-4-en-3,6-
dione in the Jones oxidation reactions. Such a LOD is very
unusual for UV detection. Electrochemically active derivatives
of cholesterol such as carbamate ester can be quantified by ECD
at very sensitive levels of picograms on-column. Cholesterol
can also be determined on the ECD without precolumn deriv-
atization in an oxidation mode, however, with much less sen-
sitivity at nanogram levels. An evaporative light-scattering
detector is mostly suitable for cholesterol determination in
simple biological fluids and is a generic technique, having a
similar LOD but lower precision compared with those of UV
detection. Electrochemical and fluorescence detectors are most
specific and sensitive for the HPLC technique but may require
cumbersome precolumn preparation.
Determination of COPs
Similar to the determination of cholesterol, that of COPs fol-
lows a series of extraction (direct or postextraction saponifica-
tion), derivatization, separation, and detection processes. The
separation and detection can be achieved by both GC and HPLC
with various detection techniques, also similar to those used for
cholesterol. Cholesterol oxides, however, normally occur at very
low concentrations, approximately 1% of cholesterol content,
and in various forms, which makes the extraction process
extremely important. Direct saponification has been recom-
mended to separate COPs from the interferences because it
eliminates selective losses of COPs during lipid extraction. Hot
alkali conditions, however, can generate more cholesterol oxides
or alter the ketone derivatives. Some researchers have suggested
that the selection of saponification temperature depends on the
COPs of interest and the nature of food matrices and that the
cold saponification may be more suitable for the ketone deriv-
atives. Antioxidants such as butylated hydroxytoluene can be
added to prevent further oxidation of COPs. Preparative chro-
matography has been used to isolate COPs directly from lipid
extract or to remove cholesterol from the unsaponifiable
materials and, in some cases, is the most important step to
improve selectivity and sensitivity. Cholesterol oxides, similar
to cholesterol, can be determined in the free-form or derivatized
form. However, COPs are much more susceptible to further
degradation. Therefore, the GC determination usually requires
derivatization, primarily to TMS ethers, to improve the thermal
 Cholesterol: Properties, Processing Effects, and Determination
stability. Although GC, coupled with FID or MS, offers relatively
quick and sufficient separation of major COPs, the use of tem-
perature programs hinders the ability of this technique to ana-
lyze thermally sensitive COPs. As a result, HPLC is preferred for
COP determination. Most COPs are more polar than choles-
terol; therefore, normal-phase HPLC has been used more than
reversed-phase HPLC. However, reversed-phase HPLC methods,
especially those using C18 columns, are becoming more and
more popular for the same reason previously discussed.
Although the use of a UV detector has been the detection
method of choice, some COPs do not possess UV chromo-
phores. In addition, a large number of COPs occur in foods,
similar to the case of flavor volatiles, which makes an absolute
separation and identification almost impossible. Hence, HPLC-
MS will probably be the primary technique to study COPs in
foods in the future because MS is a much more specific tech-
nique and some COPs have been suggested to contribute to
Alzheimer’s disease, Parkinson’s disease, multiple sclerosis,
and inflammation. Recently, the isotope dilution MS has also
been employed to study the roles of COPs in various patholog-
ical pathways of these diseases.
See also: Adipose Tissue: Structure and Function of Brown Adipose
Tissue; Adipose Tissue: White Adipose Tissue Structure and Function;
Beef; Cholesterol: Absorption, Function and Metabolism; Cholesterol:
Factors Determining Blood Cholesterol Levels; Chromatography:
Combined Chromatography and Mass Spectrometry; Chromatography:
Focus on Multidimensional GC; Chromatography: High-Performance
Liquid Chromatography; Dairy Products: Dietary and Medical
Importance; Fats: Classification and Analysis; Fatty Acids:
Determination and Requirements; Fermented Foods: Fermented Meat
Products; Fish: Dietary Importance and Health Effects; Fish: Fish in the
Human Diet; Lipoproteins; Meat: Role in the Diet; Meat: Structure; Milk:
Role in the Diet; Milk: Sources and Composition; Phospholipids:
Physiology; Phospholipids: Properties and Occurrence; Phytic Acid:
Properties, Uses, and Determination; Pickling; Pine Kernels; Pineapple;
Pituitary Gland: Pituitary Hormones; Plums and Related Fruits;
Polycyclic Aromatic Hydrocarbons; Pork Meat Quality, Production and
Processing on; Poultry: Processing; Shellfish: Role in the diet.
Further Reading
Abidi SL (2001) Chromatographic analysis of plant sterols in foods and vegetable oils.
Journal of Chromatography. A 935: 173–201.
69
Albuquerque TG, Oliveira MBP, Sanches-Silva A, and Costa HS (2014) Cholesterol
determination in foods: comparison between high performance and ultra-high
performance liquid chromatography. Food Chemistry.
Bloch K (1965) The biological synthesis of cholesterol. Science 150(3692): 19–28.
Boselli E, Cardenia V, and Rodriguez-Estrada MT (2012) Cholesterol photosensitized
oxidation in muscle foods. European Journal of Lipid Science and Technology
114: 644–655.
Bragagnolo N (2009) Cholesterol and cholesterol oxides in meat and meat products.
In: Nollet LML and Toldra F (eds.) Handbook of muscle foods analysis,
pp. 187–219. Boca Raton, FL: CRC Press.
Brown MS and Goldstein JL (1980) Multivalent feedback regulation of HMG CoA
reductase, a control mechanism coordinating isoprenoid synthesis and cell growth.
Journal of Lipid Research 21: 505–517.
Brown MS and Goldstein JL (1986) A receptor-mediated pathway for cholesterol
homeostasis. Science 232(4746): 34–47.
Cardenia V, Rodriguez-Estrada MT, Boselli E, and Lercker G (2013)
Cholesterol photosensitized oxidation in food and biological systems. Biochimie
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Coelho FP and Alves FA (1946) Liebermann–Burchard reaction. Nature 157: 803.
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system. In: Vance DE and Vance JE (eds.) Biochemistry of lipids, lipoproteins, and
membranes, 5th ed., pp. 533–553. Oxford, UK: Elsevier Science B.V.
Grundy SM (1983) Absorption and metabolism of dietary cholesterol. Annual Review of
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of various methods for the extraction of total lipids, cholesterol, other sterols from
food products. Journal of the American Oil Chemists Society 54: 81–83.
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(COPs) in animal products. Food Control 18: 939–947.
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Kenny AP (1952) The determination of cholesterol by the Liebermann–Burchard
reaction. Biochemical Journal 52(4): 611–619.
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foods and methods for its determination. Critical Reviews in Food Science and
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 Choline: Physiology
SH Zeisel, University of North Carolina, Chapel Hill, NC, USA
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from the Encyclopedia of Food Sciences and Nutrition, volume 2, pp. 1251–1253, ã 2003, Elsevier Science Ltd., with an
updated Bibliography section supplied by the Editor.
Metabolism of Choline
There have been several comprehensive reviews of the metab-
olism and functions of choline that describe its role in the
synthesis of the phospholipids in cell membranes, methyl
metabolism, cholinergic neurotransmission, transmembrane
signaling, and lipid–cholesterol transport and metabolism.
Choline can be acetylated, phosphorylated, or oxidized.
Choline, methionine, and folate metabolisms interact at
the point that homocysteine is converted to methionine
(Figure 1). Betaine–homocysteine methyltransferase catalyzes
the methylation of homocysteine using the choline metabolite
betaine as the methyl donor. Elevated plasma homocysteine
is an independent risk factor for cardiovascular disease
and stroke in humans. Treatment with betaine or choline
also lowers elevated plasma homocysteine in humans. In an
alternative pathway, 5-methyltetrahydrofolate–homocysteine
methyltransferase regenerates methionine. In addition, tetra-
hydrofolate is needed to scavenge one-carbon groups when
betaine is metabolized. Perturbing metabolism of one of
the methyl donors results in compensatory changes in the
other methyl donors, owing to the intermingling of these
metabolic pathways. Rats ingesting a choline-deficient diet
have diminished tissue concentrations of methionine and
S-adenosylmethionine, doubled plasma homocysteine con-
centrations, and diminished hepatic methylfolate content.
These effects are reversible by refeeding choline. Rats fed with
diets deficient in both methionine and choline for 5 weeks
have hepatic folate concentrations that are half of those present
in controls. Rats treated with the antifolate, methotrexate, have
diminished pools of choline metabolites in the liver. Severe
folate deficiency induced in rats, by feeding an amino acid-
defined diet containing no folate and succinylsulfathiazole for
4 weeks, has resulted in hepatic choline and phosphocholine
concentrations that were 65% and 80% lower, respectively,
than in controls.
Dietary Requirements
Though there is a pathway (in all tissues, but most active in the
liver) for the de novo biosynthesis of the choline moiety via the
sequential methylation of phosphatidylethanolamine using
S-adenosylmethionine as the methyl donor (Figure 1), only
some of the demand for choline can be met by using methyl
groups derived from one-carbon metabolism. Animals and
humans fed with a choline-deficient diet deplete choline stores
and develop liver dysfunction. Healthy male humans with
normal folate and vitamin B12 statuses fed with a choline-
deficient diet have diminished plasma choline and phosphati-
dylcholine concentrations and develop liver damage (elevated
plasma alanine aminotransferase). Liver cell death occurs in
70 Encyclopedia of Food and Health
choline deficiency, because hepatocytes initiate programmed
cell death (apoptosis) when deprived of choline. Because
methyl supplementation with betaine, methionine, folate, or
vitamin B12 does not prevent apoptotic death induced by
choline deficiency in hepatocytes, it must be that depletion of
intracellular choline moieties, rather than depletion of methyl
groups, is the critical parameter involved in the induction of
apoptosis. Some humans (male and female) fed with total
parenteral nutrition solutions devoid of choline, but adequate
in methionine and folate, develop fatty liver and liver damage
that resolve when a source of dietary choline is provided. Fatty
liver occurs because choline is required to make the phospha-
tidylcholine portion of the very-low-density lipoprotein parti-
cle. Animals fed with a choline-deficient diet may also develop
growth retardation, renal dysfunction and hemorrhage, or
bone abnormalities. A metabolite of choline, betaine, is used
in the kidney glomerulus as an osmolyte, and for this reason,
choline-deficient animals have trouble excreting concentrated
urine.
Human studies of choline requirements in women, chil-
dren, or infants have not been conducted. Thus, we do not
know whether choline is needed in the diet of these groups.
Female rats are less sensitive to choline deficiency than are
male rats perhaps because estrogen enhances females’ capacity
to form the choline moiety de novo from S-adenosylmethio-
nine. Pregnancy may be a time when dietary supplies of cho-
line are especially limiting. Though female rats are resistant to
choline deficiency, pregnant rats are as vulnerable to deficiency
as males are. During pregnancy, large amounts of choline are
delivered to the fetus across the placenta, and this depletes
maternal stores of the various forms of this nutrient. At birth,
humans and other mammals have plasma choline concentra-
tions that are much higher than those in adults. Also, the need
for choline is increased during lactation, because so much must
be secreted into milk; lactating rats are more sensitive to cho-
line deficiency than are nonlactating rats.
The Institute of Medicine (IOM) recently made recommen-
dations for choline intake in the diet. There were insufficient
data with which to derive an estimated average requirement for
choline, and so, only an adequate intake can be estimated. The
IOM report cautioned, “this amount will be influenced by the
availability of methionine and methyl-folate in the diet. It may
be influenced by gender, and it may be influenced by preg-
nancy, lactation, and stage of development. Although AIs are
set for choline, it may be that the choline requirement can be
met by endogenous synthesis at some of these stages.” The
IOM recommendations are shown in Table 1.
The recent report by Jacob that folate deficiency in
humans exacerbates choline deficiency highlights why
studies of choline–folate–homocysteine interrelationships in
humans are important for the future refinement of these
recommendations.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00155-0
 Choline: Physiology 71

This period occurs during embryonic days 12–17 in the rat

Sphingomyelin
(rats give birth on day 21; mice give birth one day earlier and
ceramide probably have a slightly different period of susceptibility).
AdoHcy Phosphatidylcholine
Choline supplementation during these critical days elicits a
AdoMet PtdEtn major improvement in memory performance at all stages of
DNA methylation CDP-Choline training on a 12-arm radial maze. Choline deficiency during
other methylations
Methionine the critical time frame reduces memory performance. The
Tetrahydrofolate Phosphorylcholine choline-induced spatial memory facilitation correlates with
changes in the birth, death, and migration of cells in the
methylenetetra-
hydrofolate
hippocampus during fetal brain development, with altered
reductase defect
Vit. B12 Choline distribution and morphology of neurons, biochemical
changes, and electrophysiological changes in the hippocam-
methyl-THF Acetylcholine pus. Human and rat brains mature at different rates; the rat
Betaine
Homocysteine brain is comparatively more mature at birth than the human
methyl-groups brain, but in humans, hippocampal development may con-
for methyl-THF tinue for months or years after birth.
Sarcosine
Figure 1 Choline, folate, and homocysteine metabolisms are closely
interrelated. The pathways for the metabolism of these three
nutrients intersect at the formation of methionine from homocysteine.
Choline and Cancer
PtdEtn, phosphatidylethanolamine; AdoHcy, S-adenosylhomocysteine; and
AdoMet, S-adenosylmethionine.
Dietary deficiency of choline in rodents causes the develop-
ment of hepatocarcinomas in the absence of any known car-
Table 1 Institute of Medicine recommendations for choline intake in cinogen. Choline is the only single nutrient for which this is
the diet true. It is interesting that choline-deficient rats not only have a
higher incidence of spontaneous hepatocarcinoma but also are
AI for infants 0–6 months 125 mg per markedly sensitized to the effects of administered carcinogens.
day, Several mechanisms are suggested for the cancer-promoting
18 mg kg1
effect of a choline-devoid diet. A progressive increase in cell
6–12 months 150 mg per day
proliferation that is related to regeneration after parenchymal
AI for children 1–3 years 200 mg per day
4–8 years 250 mg per day cell death occurs in the choline-deficient liver. Cell prolifera-
9–13 years 375 mg per day tion and its associated increased rate of DNA synthesis could
AI for males 14–18 years 550 mg per day be the cause of the heightened sensitivity to chemical carcino-
19 years and older 550 mg per day gens. Methylation of DNA is essential to the regulation of
AI for females 14–18 years 400 mg per day expression of genetic information, and the undermethylation
19 years and older 425 mg per day of DNA observed during choline deficiency (despite adequate
AI for pregnant women All ages 450 mg per day dietary methionine) may be responsible for carcinogenesis.
AI for lactating women All ages 550 mg per day Choline-deficient rats experience increased lipid peroxidation
AI, adequate intake level. in the liver. Lipid peroxides in the nucleus are a possible source
of free radicals that could modify DNA and cause carcinogen-
esis. Choline deficiency activates protein kinase C signaling,
The plasma choline concentration varies in response to diet usually involved in growth factor signaling in hepatocytes.
and can rise as much as twofold after a two-egg meal. Fasting Finally, a defect in cell suicide (apoptosis) mechanisms may
plasma choline concentrations vary from 7 to 20 mM, with contribute to the carcinogenesis of choline deficiency.
most subjects having concentrations of 10 mM. Individuals
that have starved for up to 7 days have diminished plasma
choline, but levels never drop below 50% of the normal. The
plasma phosphatidylcholine concentration also decreases in Summary
choline deficiency, but these values are also influenced by
factors that change plasma lipoprotein levels. The fasting Choline in the diet is important for many reasons. As our
plasma phosphatidylcholine concentrations are
1–1.5 mM. understanding of the importance of folate and homocysteine
nutrition increases, there should be increased interest in how
choline interacts with folate and homocysteine metabolisms.
Recent findings about choline in brain development should
Choline and Brain Development stimulate comparable studies in humans. During the next few
years, it is likely that food composition data will be available
Choline availability during embryogenesis and perinatal devel- for choline, and this will make it possible to examine interac-
opment may be especially important. There is a sensitive tions between choline, folate, and methionine when consider-
period in rodent brain development during which treatment ing epidemiological data. In addition, we should learn more
with choline (about three times the dietary intake) produces about choline requirements in women. For these reasons, inter-
long-lasting enhancement of spatial memory that is lifelong. est in choline as a nutrient for humans should be sustained.
72 Choline: Physiology
See also: Cancer: Diet in Cancer Prevention; Phospholipids:
Properties and Occurrence; Riboflavin: Properties and Determination;
Vitamins: Overview.
Further Reading
Anonymous S (1997) Betaine for homocystinuria. Medical Letter on Drugs and
Therapeutics 39: 12.
Bapiro TE, Frese KK, Courtin A, et al. (2014) Gemcitabine diphosphate choline is a
major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo.
British Journal of Cancer 111: 318–325. http://dx.doi.org/10.1038/bjc.2014.288.
www.bjcancer.com.
Buchman AL, Dubin M, Jenden D, et al. (1992) Lecithin increases plasma free choline
and decreases hepatic steatosis in long-term total parenteral nutrition patients.
Gastroenterology 102: 1363–1370.
Buchman AL, Moukarzel A, Jenden DJ, et al. (1993) Low plasma free choline is
prevalent in patients receiving long term parenteral nutrition and is associated with
hepatic aminotransferase abnormalities. Clinical Nutrition 12: 33–37.
Buchman A, Dubin M, Moukarzel A, et al. (1995) Choline deficiency: a cause of hepatic
steatosis during parenteral nutrition that can be reversed with intravenous choline
supplementation. Hepatology 22: 1399–1403.
EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies) (2014)
Scientific Opinion on the substantiation of a health claim related to cytidine
5-diphosphocholine and maintenance of normal vision pursuant to Article 13(5) of
Regulation (EC) No 1924/2006. EFSA Journal 12(2): 3575. http://dx.doi.org/
10.2903/j.efsa.2014.3575 11 pp.
Goddijn-Wessel T, Wouters M, van de Molen E, et al. (1996) Hyperhomocysteinemia: a
risk factor for placental abruption or infarction. European Journal of Obstetrics,
Gynecology, and Reproductive Biology 66: 23–29.
Ilcol YO, Ozbek R, Hamurtekin E, and Ulus IH (2005) Choline status in newborns,
infants, children, breast-feeding women, breast-fed infants and human breast milk.
Journal of Nutritional Biochemistry 16(8): 489–499.
Institute of Medicine and National Academy of Sciences USA (1998) Dietary reference
intakes for folate, thiamin, riboflavin, niacin, vitamin B12, pantothenic acid, biotin,
and choline, vol. 1. Washington, DC: National Academy Press.
Jacob R, Jenden D, Okoji R, Allman M, and Swendseid M (1998) Choline status of men
and women is decreased by low dietary folate. FASEB Journal 12: A512.
Jiang X, Jones S, Andrew BY, et al. (2014) Choline inadequacy impairs trophoblast
function and vascularization in cultured human placental trophoblasts. Journal of
Cellular Physiology 229(8): 1016–1027.
Kang S (1996) Treatment of hyperhomocyst(e)inemia: physiological basis. Journal of
Nutrition 126: 1273S–1275S.
Katz-Brull R, Seger D, Rivenson-Segal D, Rushkin E, and Degani H (2002) Metabolic
markers of breast cancer: enhanced choline metabolism and reduced choline-ether-
phospholipid synthesis. Cancer Research 62: 1966–1970.
Kim Y-I, Miller JW, da Costa K-A, et al. (1995) Folate deficiency causes secondary
depletion of choline and phosphocholine in liver. Journal of Nutrition
124: 2197–2203.
Kraichely RE, Strege PR, Sarr MG, Kendrick ML, and Farrugia G (2009)
Lysophosphatidyl choline modulates mechanosensitive L-type Ca2 þ current in
circular smooth muscle cells from human jejunum. American Journal of Physiology.
Gastrointestinal and Liver Physiology 296(4): G833–G839. http://dx.doi.org/
10.1152/ajpgi.90610.2008.
Meck WH and Williams CL (1999) Choline supplementation during prenatal
development reduces proactive interference in spatial memory. Brain Research
Developmental Brain Research 118: 51–59.
Penry JT and Manore MM (2008) Choline: an important micronutrient for maximal
endurance-exercise performance? International Journal of Sport Nutrition and
Exercise Metabolism 18: 191–203.
Pyapali G, Turner D, Williams C, Meck W, and Swartzwelder HS (1998) Prenatal choline
supplementation decreases the threshold for induction of long-term potentiation in
young adult rats. Journal of Neurophysiology 79: 1790–1796.
Savendahl L, Mar M-H, Underwood L, and Zeisel S (1997) Prolonged fasting results in
diminished plasma choline concentration but does not cause liver dysfunction.
American Journal of Clinical Nutrition 66: 622–625.
Selhub J, Seyoum E, Pomfret EA, and Zeisel SH (1991) Effects of choline deficiency and
methotrexate treatment upon liver folate content and distribution. Cancer Research
51: 16–21.
Sheard NF, Tayek JA, Bistrian BR, Blackburn GL, and Zeisel SH (1986) Plasma choline
concentration in humans fed parenterally. American Journal of Clinical Nutrition
43: 219–224.
Shin OH, Mar MH, Albright CD, et al. (1997) Methyl-group donors cannot prevent
apoptotic death of rat hepatocytes induced by choline-deficiency. Journal of Cellular
Biochemistry 64: 196–208.
Tessitore L, Sesca E, Greco M, Pani P, and Dianzani M (1995) Sexually differentiated
response to choline in choline deficiency and ethionine intoxication. International
Journal of Experimental Pathology 76: 125–129.
Varela-Moreiras G, Selhub J, da Costa K, and Zeisel SH (1992) Effect of chronic choline
deficiency in rats on liver folate content and distribution. Journal of Nutritional
Biochemistry 3: 519–522.
Varela-Moreiras G, Ragel C, and Perez de Miguelsanz J (1995) Choline deficiency and
methotrexate treatment induces marked but reversible changes in hepatic folate
concentrations, serum homocysteine and DNA methylation rates in rats. Journal of
the American College of Nutrition 14: 480–485.
Yuan Z, Tie A, Tarnopolsky M, and Bakovic M (2006) Genomic organization,
promoter activity, and expression of the human choline transporter-like protein.
Physiological Genomics 26(1): 76–90. http://dx.doi.org/10.1152/
physiolgenomics.00107.2005.
Zeisel SH (2004) Nutritional importance of choline for brain development. Journal of the
American College of Nutrition 23(6): 621S–626S.
Zeisel SH and Blusztajn JK (1994) Choline and human nutrition. Annual Review of
Nutrition 14: 269–296.
Zeisel SH, Zola T, daCosta K, and Pomfret EA (1989) Effect of choline deficiency on
S-adenosylmethionine and methionine concentrations in rat liver. Biochemical
Journal 259: 725–729.
Zeisel SH, daCosta K-A, Franklin PD, et al. (1991) Choline, an essential nutrient for
humans. FASEB Journal 5: 2093–2098.
Zeisel SH, Mar M-H, Zhou Z-W, and da Costa K-A (1995) Pregnancy and lactation are
associated with diminished concentrations of choline and its metabolites in rat liver.
Journal of Nutrition 125: 3049–3054.
Zeisel SH, Albright CD, Shin O-K, et al. (1997) Choline deficiency selects for resistance
to p53-independent apoptosis and causes tumorigenic transformation of rat
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 Choline: Properties and Determination
MM Phillips, National Institute of Standards and Technology, Gaithersburg, MD, USA
Published by Elsevier Ltd.
Introduction
Choline is an essential nutrient that is required for normal cell
function, with involvement in lipid synthesis and transport,
cellular methylation reactions, neural tube development, stem
cell proliferation and apoptosis, and cholinergic neurotrans-
mission. Choline is a precursor for phosphatidylcholine,
which comprises over half of the phospholipid content in
mammalian cell membranes, and is critical in the transport
of excess triglycerides from the liver. As a result, choline defi-
ciency has been associated with the development of hepatos-
teatosis, or fatty liver, and is the only nutritional deficiency
found to cause cancer in the absence of known carcinogens.
One important cellular methylation reaction involving choline
is in the biosynthesis of methionine, one pathway for which in
mammals involves the choline metabolite betaine. The alter-
native pathway involves folate metabolism, linking the intakes
of these nutrients through a sensitive metabolic equilibrium.
This shared pathway, through methyl group donation, is
believed to be responsible for neural tube closure in utero,
explaining a fourfold increase in risk for neural tube defects
in the children of pregnant women with choline deficiency.
Rodent studies have similarly indicated that choline supple-
mentation during pregnancy altered the brain structure and
long-term potentiation of offspring through increased stem
cell proliferation and decreased stem cell apoptosis; the reverse
was observed for mothers with choline deficiency. Choline is
also the precursor for acetylcholine, one of the most common
neurotransmitters responsible for influencing the function of
the brain, heart, and numerous other organs and organ systems
(Figure 1).
Estimation of Choline Intake
Choline is produced in the body as a by-product of the
biosynthesis of phosphatidylcholine. In this process,
S-adenosylmethionine is converted to phosphatidylcholine by
the enzyme phosphatidylethanolamine-N-methyltransferase,
and a new choline molecule is formed. The production of
choline via this pathway, however, is not sufficient to support
proper function, and thus, dietary supplementation of choline
is necessary. The one exception may be in women of childbear-
ing age; high levels of circulating estrogen may be linked to
upregulated biosynthesis of choline. Adequate intakes (AIs) for
choline have been established by the US Institute of Medicine at
425 mg day
1 for women and 550 mg day
1 for men. The AI
increases to 450 or 550 mg day
1 for women who are pregnant
or lactating, respectively. In a monitored dietary intake study,
the average daily choline consumption was determined to be
630 mg day
1 for men and 440 mg day
1 for women, levels
comparable with the AIs. Tolerable upper limits were deter-
mined as the lowest level causing adverse effects (hypotension)
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00154-9
in humans to be 1000 mg day
1 in children 1–8 years of age,
2000 mg day
1 from 9 to 13 years of age, 3000 mg day
1 from
14 to 18 years of age, and 3500 mg day
1 over age 19. In
addition to hypotension, other adverse effects from high cho-
line intake include fishy body odor (from triethylamine pro-
duction) and cholinergic side effects such as sweating,
vomiting, and diarrhea.
Choline intakes can be attributed to a number of choline-
related compounds, including free choline and betaine,
and a number of esterified forms such as phosphocholine
(PCho), phosphatidylcholine (PtdCho), glycerophosphocho-
line (GPCho), and sphingomyelin (SM) (Figure 2). Choline is
a small quaternary amine containing two carbons and a
hydroxyl group. Betaine, also known as glycine betaine or tri-
methylglycine, is formed from choline by oxidation of the alco-
hol group to a carboxylic acid. Because the oxidation of choline
to betaine is irreversible, however, the intake of betaine is not
typically included in the considerations for choline intake. In
the phosphorylated forms of choline, the hydroxyl group is
replaced with a phosphate (PCho and SM) or glycerophosphate
group (GPCho and PtdCho). In the polar PCho and GPCho
molecules, the phosphate and glycerophosphate groups are the
respective molecular termini. In PtdCho, the phosphate group
joins the choline molecule to a glycerol moiety esterified with
two fatty acids, forming a phospholipid. A large number of
structures make up the group of phospholipids classified as
PtdCho, with fatty acids that vary from 16 to 22 carbons with
varying degrees of saturation. In SM, the phosphate group con-
nects the choline molecule with a ceramide, which contains a
sphingosine (an unsaturated 18-carbon amino alcohol) and a
fatty acid with 16–24 carbons and up to one degree of satura-
tion. These structural differences result in a group of compounds
with significantly different chemical characteristics.
In 2004, the US Department of Agriculture (USDA) released
a database outlining the choline content of 630 common food
items, and a selection of the entries from this database have
been summarized in Table 1 to represent foods high in choline
from a variety of categories. The USDA database includes indi-
vidual values for betaine, free choline, PCho, PtdCho, GPCho,
and SM and a summed total choline content determined in
each food product. Foods highest in total choline content
include egg yolks, liver (from beef, veal, chicken, and turkey),
and wheat germ. The total choline content, however, is a
mathematical sum of free choline, PCho, PtdCho, GPCho,
and SM and may not represent the bioavailable choline con-
tent upon consumption. Betaine content is not included in the
calculation of total choline because, as mentioned previously,
the conversion from choline to betaine is irreversible, and
therefore, the consumption of betaine does not directly trans-
late to choline intake. Studies indicate that in a rat model, the
bioavailabilities of choline, PCho, and GPCho are similar, but
studies have not been conducted to translate these findings to
human subjects; no data are available to elucidate the
73
 Acetylcholine synthesis
Choline Lipid synthesis, transport
Egg yolks Pcho Cellular methylation reactions Blood
Stem cell proliferation, apoptosis Choline
Liver PtdCho Serum
PtdCho
Wheat germ GPCho Plasma
SM

Male AI: 550 mg day –1 10 µmol l–1 choline

Female AI: 425 mg day –1 1.0 µmol l–1 PtdCho

Deficiency:
Choline fatty liver
Pcho cognitive disorders Aqueous Free Solvent
Multi-step Total PtdCho
PtdCho Hydrolysis Overconsumption: Extraction Choline Extraction
Extraction Choline
GPCho hypotension
SM fishy body odor
cholinergic effects
Figure 1 The interrelationship and analytical determination of choline-containing compounds in dietary intake, metabolism, and health status.

OH
N+
Betaine
O

OH
N+
Choline

O OH
Phosphocholine N+ P
(PCho) HO O

OH
Glycerophosphocholine O O OH
(GPCho) N+ P
HO O

O R2
Phosphatidylcholine O O
N+
(PtdCho) _ P
O O
O R1

HN R2
Sphingomyelin
(SM) O O OH
N+ _ P
O O

Figure 2 Chemical structures of choline-related compounds.

 Choline: Properties and Determination 75

Table 1 Foods high in choline content

Free choline (mg/ Total cholinea Major

Food description Category 100 g food) (mg/100 g food) form

Egg, yolk, raw, fresh Dairy, eggs 1.3 682.4 PtdCho

Beef, variety meats and by-products, liver, cooked, braised Beef 61.8 426.1 PtdCho
Beef, variety meats and by-products, liver, cooked, pan-fried Beef 56.7 418.3 PtdCho
Veal, variety meats and by-products, liver, cooked, pan-fried Lamb, veal, 92.9 411.0 PtdCho
game
Veal, variety meats and by-products, liver, cooked, braised Lamb, veal, 88.6 398.8 PtdCho
game
Beef, variety meats and by-products, liver, raw Beef 56.2 333.2 PtdCho
Veal, variety meats and by-products, liver, raw Lamb, veal, 85.3 309.9 PtdCho
game
Chicken, liver, all classes, cooked, pan-fried Chicken, turkey 69.1 308.5 PtdCho
Chicken, liver, all classes, cooked, simmered Chicken, turkey 47.9 290.1 PtdCho
Egg, whole, cooked, fried Dairy, eggs 0.7 272.6 PtdCho
Egg, whole, raw, fresh Dairy, eggs 0.6 251.0 PtdCho
Egg, whole, cooked, hard-boiled Dairy, eggs 0.6 225.2 PtdCho
Turkey, liver, all classes, raw Chicken, turkey 63.8 221.9 PtdCho
Turkey, liver, all classes, cooked, simmered Chicken, turkey 9.7 220.2 PtdCho
Chicken, liver, all classes, raw Chicken, turkey 49.2 194.5 PtdCho
Turkey, heart, all classes, cooked, simmered Chicken, turkey 3.9 172.5 PtdCho
Cereals ready-to-eat, wheat germ, toasted, plain Breakfast 69.2 152.1 Free
cereals choline
Pork, cured, bacon, cooked, pan-fried Pork 12.3 130.8 PtdCho
Cake, chocolate, dry mix, regular, prepared without frosting Baked products 5.4 128.4 PtdCho
Turkey, heart, all classes, raw Chicken, turkey 24.6 126.8 PtdCho
Pork, cured, bacon, cooked, microwaved Pork 11.6 124.7 PtdCho
Spices, mustard seed, yellow Spices, herbs 46.2 122.6 PtdCho
Pork, cured, bacon, cooked, baked Pork 12.3 119.3 PtdCho
Pork, fresh, loin, center loin (chops), bone-in, separable lean Pork 2.2 102.8 PtdCho
only, cooked, broiled
Coffee, instant, decaffeinated, powder Beverages 93.7 101.9 Free
choline
Mutton, roasted from mutton sandwich Lamb, veal, 1.7 100.6 PtdCho
game
Beans, navy, mature seeds, raw Legumes 49.9 87.4 Free
choline
Fish, cod, Atlantic, cooked, dry heat Finfish, 17.7 83.7 PtdCho
shellfish
Noodles, egg, dry, enriched Cereal grains, 50.4 78.7 Free
pastas choline
Nuts, pistachio nuts, dry roasted, with salt added Nuts, seeds 10.7 71.5 PtdCho
Candies, milk chocolate Sugars, sweets 9.1 46.1 GPCho
Salad dressing, mayonnaise, soybean oil, with salt Fats, oils 0.2 46.0 PtdCho
Brussels sprouts, cooked, boiled, drained, without salt Vegetables 23.4 40.1 Free
choline
Orange juice, frozen concentrate, unsweetened, undiluted Fruits 8.1 19.9 Free
choline
a
Total choline includes the sum of free choline, glycerophosphocholine (GPCho), phosphocholine (PCho), phosphatidylcholine (PtdCho), and sphingomyelin (SM).
Source: Howe, J. C., Williams, J. R., Holden, J. M., Zeisel, S. H. and Mar, M.-H. (2004). USDA database for the choline content of common foods. Beltsville, MD: US Department of
Agriculture.

differences in the human absorption of the various forms
of choline. The water- and lipid-soluble forms of choline are
known to have different absorption mechanisms in the human
body, which translates to differences in both the method and
rate of distribution to tissues. It is also interesting to note that
in animal products (meats, dairy, and eggs), the major choline-
containing compound identified is PtdCho, while in plant
products (fruits, vegetables, and grains), PtdCho is not the
major choline compound. In addition to endogenous choline
and choline-containing compounds, choline can be added to
foods and supplements as choline chloride, choline bitartrate,
or lecithin. The PtdCho content of lecithin, a natural emulsifier
of plant or animal origin, is roughly 25% by mass, correspond-
ing to approximately 3–4% choline by mass.
In order to establish whether AIs are met, accurate and
precise analytical methods must be utilized for determination
76 Choline: Properties and Determination
of choline and choline-related compounds in foods and food
products. In general, two approaches have been taken in the
characterization of foods for total choline content: extraction-
based methods in which free choline and each choline ester are
determined independently and hydrolysis-based methods in
which all choline esters are hydrolyzed to free choline and the
total free choline content is subsequently determined. The
extraction approach, while complicated and time-consuming,
is valuable because the concentration of each choline-related
compound can be determined independently, and therefore,
the bioavailability can be considered with respect to evaluation
of total choline intake. This approach requires multiple steps to
isolate the hydrophilic (free choline, betaine, PCho, and
GPCho) and hydrophobic (PtdCho and SM) components
and separate quantitative analyses of the two groups. Con-
versely, hydrolysis approaches utilize acid, base, and/or
enzymes to cleave the esters from the choline moiety, allowing
a single analysis to be performed to determine the total con-
centration of hydrolyzed free choline. The hydrolysis approach
is appealing because the overall methodology is often more
simple and rapid, but unfortunately, the efficacy of the chem-
ical hydrolysis of the choline esters does not reflect the bio-
availability of the various chemical forms.
Once choline intakes are established, appropriate methods
are needed to assess the choline status in the population
through measurement of choline or choline biomarkers in
plasma and/or serum. Experts believe that choline health status
is most accurately reflected in the concentration of choline and/
or PtdCho in plasma. Fasting choline concentrations in human
plasma have been reported in the range of 7–20 mmol l
1, with
a median concentration of 10 mmol l
1. Concentrations of
PtdCho in human plasma have been reported in the range of
1.0–1.5 mmol l
1, representing mostly the PtdCho that consti-
tutes plasma lipoproteins. The methods for determination of
choline and PtdCho in plasma and serum face the same chal-
lenges as those for determination in foods, as the extraction and
sample cleanup steps are different for the hydrophilic choline
and hydrophobic PtdCho. In determination of biomarkers,
however, the concentrations of choline and PtdCho are both
measured; hydrolysis-based approaches for determination of
total choline content are not routinely utilized.
Extraction-Based Approaches for Total Choline
Determination in Foods
Extraction-based approaches that separately quantify individ-
ual choline-containing compounds in foods and food prod-
ucts are desirable from a nutritional standpoint, as the
bioavailabilities of the various choline esters may not be equiv-
alent, leading to misrepresentation of total choline from a
simple summation. These approaches typically include both
aqueous and organic extraction steps (either simultaneously or
in parallel) followed by direct analysis to determine the con-
centration of each choline-containing compound in the sam-
ple. Most approaches rely on a classic liquid–liquid extraction
approach in which the sample is diluted to a level of approx-
imately 80% water and combined 1:3 with a mixture of chlo-
roform and methanol (2:1). After homogenizing, an
additional one part chloroform is added and one part water
in distinct steps separated by additional mixing. The mixture is
then allowed to settle; the upper water/methanol layer con-
tains the hydrophilic components, including free choline,
betaine, PCho, and GPCho, while the lower chloroform layer
contains the hydrophobic PtdCho and SM. This method has
been demonstrated to remove 94% of the lipid content into the
chloroform layer, with no significant cross extraction between
the two layers. Reprocessing of any remaining solid material
with an additional aliquot of chloroform has been found to
remove the remaining lipid-soluble components, and by com-
bination of the separate chloroform layers, quantitative extrac-
tion can be achieved. Other approaches for extraction of
choline-containing compounds from food and food products
have been described using a parallel approach in which the
sample is split, hydrophilic compounds are isolated using
dilute acid, and hydrophobic compounds are isolated using a
combination of chloroform and methanol.
Once choline-containing compounds have been isolated
from a food or food product, they must be isolated from one
another for accurate quantitation. Some approaches separate the
various compounds using preparative techniques such as liquid
chromatography (LC) and thin-layer chromatography (TLC);
isolated compounds are then hydrolyzed individually to release
choline, and the free choline is measured by a technique such as
gas chromatography-mass spectrometry (GC-MS). Performing
separate analyses for each compound is time- and resource-
intensive, however, which has led to improvements in the
analytical approach. Newer methods have converted the prepa-
ratory approach described in the preceding text into the overall
analytical approach, removing the need for compound collec-
tion and separate analysis. In these approaches, both the aque-
ous and organic extracts can be analyzed using the same LC
approach with different mobile phase compositions, leading to
a fivefold reduction in the analysis time. This approach has been
coupled with mass spectrometry (MS) using stable isotope-
labeled internal standards to assign the values reported in the
USDA database (Table 1). In addition, the use of an isotope
dilution approach to quantitation greatly improves the accuracy
and precision of an LC-MS method by reducing errors from
sample handling and variability in sample injection and ioniza-
tion within the mass spectrometer.
Improvements in column technology and better understand-
ing of fundamental chromatographic processes have also led to
improvements in the simultaneous determination of choline-
containing compounds. Utilization of hydrophilic interaction
liquid chromatography (HILIC) may be the future of total cho-
line analysis, as both hydrophilic and hydrophobic components
can be analyzed simultaneously, again reducing the analysis
time by a factor of 2. Coupled with isotope dilution-tandem
mass spectrometry (ID-MS/MS), this type of analysis can even
provide information about the distribution of various fatty acid
moieties of the choline esters PtdCho and SM. Although still in
its infancy, this approach may offer the full suite of information
needed to truly understand choline intake from foods in a more
straightforward and high-throughput technique.
Hydrolysis-Based Approaches for Total Choline
Determination in Foods
Despite concerns about equivalence in bioavailability of differ-
ent choline-containing compounds in foodstuffs, a majority of

historical approaches for determination of total choline rely on
hydrolysis by acid, base, and/or enzymes, which removes all
information about the distribution of choline esters. The ben-
efit of hydrolysis, although the sample preparation can be
labor- and time-intensive, is that the subsequent determina-
tion of the total liberated choline becomes significantly more
straightforward than the analysis of individual choline esters.
Classically, colorimetric approaches have been widely used
for choline determination. Following the release of choline-
containing compounds from foods using three cycles of hot
methanol extraction, liberated choline can be reacted to form a
Reinecke salt, which is easily detected by absorbance at
520 nm. While this method is simple and straightforward,
losses have been observed during the preparation of the pre-
cipitate, and the method lacks specificity as the reagent forms
the colored product with any amine. The AOAC Official
Method of Analysis for choline in infant formula and milk
(999.14) is also a colorimetric reaction with choline liberation
by acid and enzyme hydrolyses. The reaction mixture includes
choline oxidase to release hydrogen peroxide, which will
react with peroxidase, 4-aminoantipyrine, and phenol to
form a quinone imine dye with absorbance at 505 nm. Mod-
ifications to this approach have replaced the phenol and
4-aminoantipyrine with dichlorofluorescein, added a basic
hydrolysis step to release PtdCho from lecithin, and added
acid-washed decolorizing carbon (Norit) to the sample to
remove interference from vitamin C, making the approach
more broadly applicable to foods and dietary supplements
other than infant formula and milk. Methods based on choline
oxidase are more specific for choline, but as demonstrated by
the number of necessary modifications, interferences are still
possible in the formation of the detectable colored compound.
To increase specificity for choline and remove interferences
from other sample components that might be simultaneously
extracted, separation-based approaches have been developed.
Some methods utilized base hydrolysis to generate triethyla-
mine, which can be determined by GC with flame ionization
detector (FID) or MS. Like the colorimetric methods described
previously, however, these methods are specific only for qua-
ternary amines and may result in a positive bias from other
compounds in the food matrix. Other methods utilized acid
hydrolysis or a combined acid and enzyme hydrolysis followed
by LC-IDMS or ion chromatography with conductivity or ion-
exchange membrane detection. Approaches that did not utilize
an enzymatic step with the acid hydrolysis yielded results up to
15% lower than those using a combined approach, because
PCho is not completely hydrolyzed under acidic conditions.
However, in one modification that conducted the acid hydro-
lysis in a microwave digestion chamber, an additional enzyme
hydrolysis step was not necessary to completely extract total
choline from food products. Another approach has also been
reported utilizing base hydrolysis, followed by capillary
electrophoresis with indirect absorbance detection in the pres-
ence of 1-methylimidazole as a carrier electrolyte.
Other less common methods for quantitation of choline in
foods and food products have utilized the reaction of choline
oxidase with choline from sample extracts by monitoring the
consumption of molecular oxygen or the generation of hydro-
gen peroxide. More specifically, molybdenum-catalyzed oxida-
tion of iodide by hydrogen peroxide has been detected
potentiometrically for quantitation of total choline in foods.
Choline: Properties and Determination 77
Biosensors have also been utilized for the detection of O2
decrease or H2O2 increase upon introduction of a choline-
containing sample to a reactor containing choline oxidase, in
either a standalone or flow-through design. One potential
benefit of the biosensor approach lies in the ability to deter-
mine choline and choline esters using the same biosensor, by
analyzing the same sample pre- and post-hydrolysis. These
methods have not been widely adopted, however, as LC and
LC-MS equipment are more common in food laboratories, and
the use of biosensors for routine analysis has not yet been
demonstrated as robust.
Determination of Choline for Health Marker Status
For evaluation of choline health status, the most commonly
measured biomarkers are free choline and PtdCho in the
serum and/or plasma. Free choline and PtdCho are often sep-
arated from the serum and/or plasma matrix by liquid–liquid
extraction as described previously for foods; free choline can
also be isolated from serum or plasma using ion-exchange
resins. Early methods for determination of free choline and
PtdCho in plasma and serum involved a radioenzymatic assay
in which free choline is extracted into the aqueous layer by
liquid–liquid extraction (as described previously) and con-
verted to 32PCho using ATP-g-32P in the presence of a choline
kinase catalyst or 32P-labeled acetyl-CoA in the presence of
choline acetyltransferase. Following the separation from the
labeled precursor via electrophoresis or ion-exchange chroma-
tography, the radioactive decay of 32PCho is measured by
scintillation counting. The organic phase can be analyzed sep-
arately for PtdCho by isolation with preparative LC or TLC,
hydrolysis to release choline, and repetition of the free choline
determination procedure by radioenzymatic assay. PtdCho can
also be measured via total phosphorus content by colorimetric
assay. These assays are simple, fast, reproducible, and specific
for choline and PtdCho and have been demonstrated to have
little interference from other choline esters; the only significant
drawback is the sensitivity of the assay to the source of the
enzymes used in the various hydrolysis and reaction steps.
Several other traditional chemical methods have been uti-
lized as well for determination of choline and PtdCho in
biological fluids. Chemiluminescence approaches have been
explored, utilizing the reaction of choline with choline oxidase
to form peroxide and the subsequent oxidation of acridinium-
9-carboxamide or luminol catalyzed by peroxidase. These
methods also described the option for hydrolysis of PtdCho
by phospholipase D to form free choline, allowing both bio-
markers to be determined by the same approach. Another
approach utilized the decay of the fluorescence of choline
oxidase (native or derivatized) as the reaction with free choline
progresses. While these traditional methods have the benefit of
simplicity and low instrumentation demand, developing tech-
nologies demand demonstration of specificity and increased
accuracy and precision for true understanding of clinical
health.
Instrumental approaches have been explored more recently
to give a more accurate profile of choline and choline esters in
plasma and serum. Some of the methods used for food analysis
(described previously) have also been applied to clinical ana-
lyses, separating the various choline-containing compounds
78 Choline: Properties and Determination
using preparative chromatography such as LC or TLC. Isolated
compounds are then hydrolyzed individually to release cho-
line, and the free choline is measured by a technique such as
GC-FID or GC-MS. GC can also be coupled to radiometric
assays described previously, by using GC to separate the radi-
olabeled compounds of interest and monitoring the radioac-
tivity at the column exit. To utilize any GC-based technique,
the choline must first be volatilized. Although one pyrolysis–
GC-MS technique has been described, the most common
approach to volatilization is via chemical derivatization,
which can add lengthy sample preparation time and potential
for the introduction of error or bias. To reduce error or bias that
may result from sample preparation, techniques using stable
isotope-labeled compounds as internal standards have been
described. By spiking 2H-, 13C-, or 15N-labeled variants of each
compound of interest into samples at the start of processing,
inefficiencies in extraction, sample losses, and variability in
molecular ionization can also be mitigated.
Other approaches have utilized LC with electrochemical
detection following postcolumn enzymatic reaction with
choline oxidase to generate and electrochemically detect
hydrogen peroxide. Free choline has also been derivatized
with 1-naphthyl isocyanate prior to LC to utilize fluores-
cence detection or with 3,5-dinitrobenzoyl chloride prior to
LC to utilize absorbance detection. Because choline is a
relatively small, hydrophilic molecule, early approaches to
LC often included the addition of an ion-pairing agent,
which led to challenges in maintaining a robust, reproduc-
ible separation. As described for GC-based techniques, the
utilization of an internal standard is critical in LC determi-
nation of choline as well. The numerous sample prepara-
tion and analysis steps allow the possibility for various
sources of bias and error in the final analytical determina-
tion of choline, and the proper use of an internal standard
can reduce the impact.
Taking advantage of the benefits of an internal standard
approach, most recent methods for choline determination in
serum and plasma have utilized LC with MS or MS/MS detection
and isotope dilution for quantitation. As described previously
for food analysis, emerging technologies such as HILIC allow
simultaneous analysis of choline and PtdCho. Many current
approaches utilizing isotope dilution describe the addition of
an acetonitrile or methanol solution containing the internal
standards to force protein precipitation from serum or plasma.
Following centrifugation, these samples can then be directly
analyzed by HILIC LC-MS/MS. Because similar methods have
been demonstrated in foods for the profiling of numerous
choline-containing compounds, it stands to reason that an
HILIC LC-MS/MS method could be used to screen serum or
plasma samples if a new choline biomarker is discovered.
Conclusions
Choline is a small quaternary ammonium compound that
plays an important role in numerous metabolic processes and
can be present in numerous forms, each having distinct chem-
ical and nutritive properties. To understand the role of choline
in health and function, foods (intake) and biological fluids
(output) must be accurately monitored for choline content. As
a result, understanding and interpretation of the role of cho-
line in human health rely heavily on the availability of appro-
priate analytical methodologies.
See also: Choline: Physiology; Eggs: Composition and Health Effects;
Folic acid and Folates: Physiology and Health Effects; Food
Composition Databases; Human Milk: Composition and Nutritional
Value; Infants: Nutritional Requirements; Phospholipids: Physiology;
Phospholipids: Properties and Occurrence; Pregnancy: Dietary
Guidance for Pregnancy; Sports Nutrition.
Further Reading
Bligh EG and Dyer WJ (1959) A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry and Physiology 37(8): 911–917.
Howe JC, Williams JR, Holden JM, Zeisel SH, and Mar M-H (2004) USDA database for
the choline content of common foods. Beltsville, MD: US Department of Agriculture.
Institute of Medicine (1998) Dietary reference intakes for thiamin, riboflavin, niacin,
vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington,
DC: National Academy Press.
Koc H, Mar M-H, Ranasinghe A, Swenberg JA, and Zeisel SH (2002) Quantitation of
choline and its metabolites in tissues and foods by liquid chromatography/
electrospray ionization-isotope dilution mass spectrometry. Analytical Chemistry
74(18): 4734–4740.
Phillips MM (2012) Analytical approaches to determination of total choline in foods and
dietary supplements. Analytical and Bioanalytical Chemistry 403(8): 2103–2112.
Teng Y-W and Zeisel SH (2011) Choline. In: Obeid R and Hermann W (eds.) Vitamins in
the prevention of human diseases, 1st ed., pp. 599–628. Berlin: Walter de Gruyter
GmbH.
Woollard DC and Indyk HE (2000) Determination of choline in milk and infant formulas by
enzymatic analysis: collaborative study. Journal of AOAC International 83(1): 131–138.
Xiong Y, Zhao Y-Y, Goruk S, Oilund K, Field CJ, Jacobs RL, and Curtis JM (2012)
Validation of an LC-MS/MS method for the quantification of choline-related
compounds and phospholipids in foods and tissues. Journal of Chromatography B
911: 170–179.
Zeisel SH (2006) Choline: critical role during fetal development and dietary
requirements in adults. Annual Review of Nutrition 26: 229–250.
Relevant Websites
http://ars.usda.gov/Services/docs.htm?docid¼6232 – USDA Database for the Choline
Content of Common Foods, Release 2 (2008).
http://lpi.oregonstate.edu/infocenter/othernuts/choline/ – Micronutrient Information
Center, Linus Pauling Institute.
 Chromatography: Combined Chromatography and Mass Spectrometry
Z Zhang, X Hu, and P Li, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, China
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The advanced analytic technologies are revealing a complex pro-
file regarding the food and health research. Most foodstuffs are
produced from living organisms and tissues, thus reflecting the
complexity of the biological systems they are coming from.
Generally, two types of opposite food components, namely, ben-
eficial and hazardous ones, gain the emerging attention. These
components play a key role in food nutritional, functional, or
hazardous properties. Among the toolkits of techniques devel-
oped to investigate food at wide scale including small molecules,
peptide, and proteome, chromatography combined with MS has
gained popularity especially because of its ability to handle com-
plex food matrices. The chromatography–MS methods demon-
strate high resolution ratio, specificity, speed, and reliability of the
analytic response in a high-throughput mode. For these reasons,
chromatography–MS methods have been extensively employed
in food analysis.
In this article, applications of chromatography–MS to food
safety and quality is discussed in detail, including detection,
identification, and quantification. In this way, the identifica-
tion and determination of food could be addressed, especially
for the certification and traceability of foodstuffs. Moreover,
the relationship between structure and function in food
systems could be clarified.
Combined Chromatography to MS Strategies
MS method is based on the production of ions, which are
subsequently separated or filtered according to their mass-to-
charge (m/z) ratio and then detected. The resulting mass
spectrum is a plot of the (relative) abundance of the generated
ions as a function of the m/z. It provides excellent selectivity,
which is of utmost importance in quantitative trace analysis.
The mass spectrometer is a highly sophisticated and comput-
erized instrument, which basically consists of ion source, a
mass analyzer, and a detector. In principle, liquid chromatog-
raphy (LC), gas chromatography (GC), and capillary
electrophoresis (CE) are used as the separation technique,
while the mass spectrometers are employed as a detector. The
different physical and chemical properties between analytes
and their relative affinity for the stationary/mobile phases
promote their separation. The molecules are retained in the
column and then elute (come off) from the column at different
times (retention time) that depend on the analyte affinity for
the mobile phase. This allows the mass spectrometer down-
stream to capture, ionize, accelerate, deflect, and detect the
ionized molecules separately, by breaking each molecule into
ionized fragments and detecting these fragments using their
m/z. However, online chromatography–MS systems offer addi-
tional value, especially in terms of selectivity that allows sensi-
tive and specific analysis in food for various purposes.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00158-6
Mass Spectrometer
A mass spectrometer contains three major components,
namely, an ion source, a mass analyzer, and a detector. The
analyte previously separated from other analytes and
the matrix bulk eluted to the ion interface, where a portion
of the sample is converted into ions. The ions that meet
certain characteristics are trajected through the mass analyzer
and onto the detector. The mass analyzer is used to sort the
ions (according to their m/z values) and to calculate their
abundances.
With the ion source, the target of interest is ionized and
then transported by magnetic or electric fields to the mass
analyzer. For gases and vapors, electron ionization (EI) and
chemical ionization (CI) can be applied. For liquid and solid
biological samples, electrospray ionization (ESI) and matrix-
assisted laser desorption/ionization (MALDI) are the most
commonly used ionization sources. Inductively coupled
plasma (ICP) sources can carry out cation analysis, although
it appears seldom in food analysis. In the combination of
HPLC/GC to MS, the ionization sources are classified accord-
ing to hard and soft ionization of the molecule. Hard ioniza-
tion types, such as EI, provide a high degree of fragmentation
and important information for structure of unknown com-
pounds. However, the EI source is not the most appropriate
for HPLC–MS, because of the short EI shelf life at atmospheric
pressure. On the country, EI source can be used in GC–MS
due to its high vacuum. Soft ionization indicates that ions are
formed using lower residual energy, such as fast atom bom-
bardment (FAB), chemical ionization (CI), atmospheric pres-
sure chemical ionization (APCI), electrospray ionization
(ESI), and MALDI. These sources provide mostly the molecu-
lar ions (or protonated or deprotonated molecules and few
fragments).
Mass analyzers separate the ions according to their m/z ratio
by using an electric and/or magnetic field. There are different
types of mass analyzers. Time-of-flight (TOF) analyzer records
the pass-through time by using an electric field to accelerate the
ions with the same potential, thus allowing the most rapid
reach of lighter ions. Nowadays, TOFs provide accurate m/z.
Quadrupole mass filter can chose ions in a certain range of m/z
by using oscillating electrical fields and can filter m/z as the
quadrupole ion trap. Three-dimensional quadrupole ion trap
can trap and eject ions sequentially. A linear quadrupole ion
trap can trap ions in a two-dimensional quadrupole field,
instead of a three-dimensional quadrupole field as in a 3-D
quadrupole ion trap. In the Orbitrap method, ions are electro-
statically trapped in an orbit around a central, spindle-shaped
electrode.
The final element of the mass spectrometer is the detector.
The detector records the charge induced or the current pro-
duced when an ion passes by or hits a surface. The electron
multiplier, Faraday cups, or ion-to-photon detectors are com-
monly used.
79
80 Chromatography: Combined Chromatography and Mass Spectrometry
GC–MS
GC–MS method combines the features of GC and MS to iden-
tify different substances within single food sample. The use of a
mass spectrometer as detector in GC was developed during the
1950s after being initially assembled by James and Martin in
1952. GC involves an oven and inside of it capillary column
with proper dimensions (length, diameter, and film thickness)
and optimized phase properties that helps to separate the
targets of interested by volatilization using a temperature gra-
dient of the oven. The mobile phase is an inert carrier gas
(as helium or nitrogen), while the stationary phase is a micro-
scopic layer of liquid or polymer on an inert solid support
(silica). In principle, the targets of interest are volatilized and
then interact with the GC stationary phase coated in the col-
umn walls. Each compound elutes at a different time (reten-
tion time) depending on their boiling point and affinity by the
stationary phase.
The typical components of GC instruments are carrier gas,
flow controller, sample injector, column oven, column, and
detector (any type of mass analyzer). In the GC separation
process, a certain volume of gaseous or liquid sample is
injected into the column head by using, usually, a microsyringe
(solid-phase microextraction fibers can also be used). The
carrier gas sweeps the targets through the GC column and,
then, the analyte elute of the column when the oven temper-
ature raises its boiling point. Different adsorption strengths
between the targets and stationary-phase materials allow also
variations in retention time, in favor of the identification and
determination by MS.
Recently, comprehensive two-dimensional (2-D) GC, or
GC
GC  originally developed in 1991 by Professor
Phillips  was employed to separate targets that are difficult
to separate by conventional GC. The two GC columns are
connected sequentially, the 1-D column is a conventional
one and the 2-D column is a short fast one. Between the 2-D
columns, a modulator is employed to collect small fractions of
the effluent from 1-D, focus them as a narrow pulse, and
transfer them to the 2-D. This so-called modulation cycle is
repeated throughout the 2-D GC run. For example, a typical
modulation is the thermal modulation. The liquid nitrogen is
used to immobilize all the components eluting from 1-D. A
hot stream pulse mobilizes a part of the compounds through
the second column, where the 2-D elution starts again.
GC–MS method offers high sensitivity and resolution
power, excellent reproducibility, and extensive and highly
reproducible fragmentation, providing excellent identification
potential through well-established databases, such as the NIST
library. Other advantages are the easy use of the technique and
its low cost. The major disadvantage is that GC–MS is by its
nature limited to the analysis of small volatile molecules and
molecules that can be made volatile. The problem of by-
product formation and degradation needs consideration.
The GC-MS methods allow much finer degree of substance
identification than either of the unit used separately. It is
difficult to make an accurate identification by single GC or
MS methods. The MS process normally requires a pure sample,
while GC using a traditional detector (e.g., flame ionization
detector) cannot differentiate between molecules that coeluted
in the same peak. GC–MS method reduces the possibility of
error, because two different molecules that behave in the same
manner in GC and MS are very rare. Therefore, when an
identifying mass spectrum appears at a characteristic retention
time in a GC–MS analysis, it typically increases certainty that
the analyte of interest is in the sample. Two different molecules
can also have a similar pattern of ionization in a mass spec-
trometer (isobaric interferences). However, accurate mass
instruments reduce or eliminate the possibility of these
interferences.
LC–MS
LC–MS is a powerful technique that has very high sensitivity
and selectivity and so is useful in many applications such as
food safety. This separation principle is similar to GC (different
affinities of the analytes for the stationary/mobile phases).
However, in GC, the analyte separation is between the liquid
stationary phase and the gas mobile phase, while, in HPLC, the
analyte separation is between the solid stationary phase and
the liquid mobile phase. Then, analytes are separated accord-
ing to their polarity independently of whether they are volatile
or not. In HPLC, the separation is forced by a liquid at high
pressure (as the mobile phase) through a column. This HPLC
column is previously packed with a stationary phase generally
composed of irregularly or spherically shaped particles. Usu-
ally, octadecylsilyl (C18) is used as stationary phase with pure or
pH-adjusted water–organic mixtures (as water–acetonitrile or
water–methanol), which is termed reversed-phase liquid chro-
matography (RP-LC). Silica gel is also can be applied as station-
ary phase with neat or mixed organic mixtures, which is called
normal-phase liquid chromatography (NP-LC). RP-LC is most
often used because of its superior separation capabilities.
LC–MS is currently (probably) the most widely used mass
spectrometry technology, due to its ability to separate and
detect a wide range of molecules. The method allows for the
collection of both qualitative and quantitative data and can
achieve pgL1level of detection.
The combination of the separating potential of liquid chro-
matography and the analyzing power of mass spectrometry
makes LC–MS a highly useful tool for analytical chemists.
Due to its high selectivity and sensitivity, it is finding increasing
use in the analysis of a wide range of substances in complex
mixtures. The major challenge in coupling of LC with MS is
posed by the fact that gas-phase ions must be produced in
order to obtain a mass spectrum.
There are many types of LC–MS interface, such as ESI, APCI,
atmospheric pressure photoionization (APPI), and MALDI.
Currently, there are mainly two types of API interfaces. One is
ESI, which is best suited to ionic compounds with high polarity
and the other APCIs. As already mentioned, ESI is a soft ioni-
zation technique, producing little fragmentation. For better
structural information, ESI interface can be coupled with
tandem MS (ESI-MS/MS).
CE–MS Method
CE includes a group of electrokinetic separation methods car-
ried out in submillimeter capillaries and in micro- and nano-
fluidic channels. In these methods, analytes migrate through
electrolyte solutions under the influence of an electric field.
 Chromatography: Combined Chromatography and Mass Spectrometry
Analytes can be separated according to ionic mobility;
additionally, they may be concentrated by means of gradients
in conductivity and pH. The identity of sample components
can be established by direct coupling of the capillary electro-
phoresis with mass spectrometers. Commonly, to connect
both instruments, the capillary outlet is introduced into an
ESI source modified to introduce a sheath liquid that closes
the electrical circuit. CE-MS offers a powerful tool for the
targeted analysis of polar metabolites, such as amino acids,
organics acids, and nucleotides with superior resolution and
sensitivity.
Application in Food Analysis
Food products can be regarded as complex mixtures and
consist of beneficial and hazardous compounds that naturally
occur in food or are introduced during food processing. Bene-
ficial food components include lipid, carbohydrate, polyphe-
nols, carotenoid, amino acids, peptides, and proteins, while
the hazardous ones consist of mycotoxin, marine toxin, plant
toxins, pesticide, and veterinary drugs. As example of natural
beneficial food compounds, lipids such as fats and sterols are
able to store energy and act as structural components of cell
membranes. The positive health benefits associated with
the consumption of omega-3 fatty acids on infant develop-
ment, cancer, cardiovascular diseases, and various mental
illnesses, such as depression, attention-deficit hyperactivity
disorder, and dementia, should also be kept in mind. On the
contrary, toxic substances generally originating from crop pro-
tection treatments against pest, agrochemical treatments, or
packaging materials have been frequently found in foodstuffs.
Pesticide residues can cause acute and chronic health effects in
human and livestock, ranging from simple irritation of the skin
and eyes to the destruction of the nervous system, reproductive
problems, and even cancer. All these compounds, toxic or
beneficial, can be determined using chromatography com-
bined with mass spectrometry. In the next section, the deter-
mination of both, beneficial and hazardous compounds, in
food is discussed.
Analyzing Beneficial Food Components via
Chromatography–MS
Lipid
Lipids are macronutrients that contribute significantly to the
nutritional and sensory value of food. Lipids can be found in
various forms, such as free fatty acids, acylglycerols, phospho-
lipids, sphingomyelins, glycosphingolipids, steroids, and bile
acids. Some lipids have simple structure (as fatty acids), but
many others have complex and variable structures. The struc-
tures can be ascertained and completely resolved by HPLC–MS
or GC–MS methods, helping to establish the structure/func-
tion relationship. HPLC–MS method is one of the most pow-
erful tools for the analysis of lipid components in food. Lipid
content from various foodstuffs has been extensive investi-
gated. To simplify sample preparation, HPLC–MS method
has been an attractive alternative. The main trend of diverse
lipids’ analysis is the employment of HPLC–MS methods.
Triacylglycerols, fatty acids, carotenoids, and phospholipids
81
found in various foodstuffs have been studied in detail via
HPLC–MS method. In addition, the use of tandem mass spec-
trometry (MS/MS) enhances dramatically their structural elu-
cidation, for example, individual triacylglycerols. Besides,
GC–MS is also used to analyze free fatty acids and triacylgly-
cerols, although it required derivatization procedures (e.g.,
transformation of free fatty acids in the methyl esters).
Carbohydrate
Carbohydrates, the most abundant natural products, can be
classified according to their degree of polymerization (DP).
They can be divided initially into three principal groups, namely,
simple sugars (DP 1–2), oligosaccharides (DP 3–9), and
polysaccharides (DP > 9). They are one of the most important
components for nutrition and health, because they have impor-
tant physiological role. They can serve as structural elements in
plants (as cellulose), important sources of dietary fiber, and
major sources of energy (as starch or glucose) in food. For the
reasons mentioned earlier, carbohydrates are specially targeted
via HPLC–MS or GC–MS methods in food. Monosaccharides
and small oligosaccharides have been traditionally analyzed via
GC–MS. However, the use of derivative reaction in GC–MS
method hampered its application to larger oligosaccharides
and molecular conjugates. Due to the high polarity and low
volatility of carbohydrates, ESI ionization is usually preferred
over APCI ionization. Carbohydrates can be hardly observed in
negative ionization, due to its lower sensitivity, while they can be
sensitive in the positive ionization mode. For example, the addi-
tion of Li salts with low concentration to the HPLC eluents
produces [M þ Li]þ ions and can increase the sensitivity of
carbohydrate compounds in food.
Carotenoids
Carotenoids are lipid-soluble pigments responsible for the color
of a wide variety of fruits and vegetables. Some of them are
provitamin A carotenoids, subsequently transformed into vita-
min A, which can prevent serious eye diseases, such as night
blindness; susceptibility to infection; rough, scaly skin; and
retarded tooth and bone development. There are about 700
carotenoids in nature, but only about 50 have provitamin activ-
ities. Of those 50 compounds, the three most important pre-
cursors of vitamin A in humans are a-carotene, b-cryptoxanthin,
and b-carotene.
A range of LC-based techniques have been used to analyze
carotenoids, most of them coupled to a PDA or UV–vis detec-
tor. Although LC separation coupled to UV–vis instruments
has been the most common analytic method for determining
carotenoids qualitatively and quantitatively, the spectra of
many carotenoids are very similar, so many researchers
have complemented the identification of carotenoids using
other detectors, such as MS. With UV and PDA systems, it is
impossible to provide molecular structure information for
identification, especially for unknown carotenoids in complex
sample matrices. The MS instruments are used to overcome
spectral interferences in UV–vis and, therefore, to achieve
high sensitivity in complex mixtures and to obtain molecular
structure information on the basis of the molecular mass and
fragmentation pattern under tandem MS (MS/MS and MS/
MS/MS).
82 Chromatography: Combined Chromatography and Mass Spectrometry
For HPLC–MS, usually ESI, sometimes APCI ionization, is
used. There are many examples of analysis and quantitation of
vitamins in food via HPLC–MS. Most carotenoids are apolar
compounds, so APCI (possibly also APPI) ionization may be
more advantageous than the more commonly used ESI.
HPLC–MS/MS has also been used to distinguish between
structurally related molecules and their epoxidized forms, prod-
ucts of carotenoid oxidation, that are potential oxidative stress
markers and difficult to profile due to their small amounts and
the difficulty in separating them from hydroxyl carotenoids.
Amino Acids, Peptides, and Proteins
There is currently a general trend in food science towards the
consideration of food as an affordable way to prevent diseases. In
this sense, one of the main challenges is to improve our limited
understanding on the interaction of food compounds with genes
and their subsequent effect on proteins and metabolites; this
knowledge should allow a rational design of strategies to manip-
ulate cell functions through diet, which is expected to have an
extraordinary impact on our health in the nondistant future.
Proteomics is the large-scale analysis of a proteome that
includes all the expressed proteins in a particular biological
system at a given time, whereas peptidomics is the analysis of
all peptide content within an organism, tissue, or cell (pepti-
dome) including not only the peptide present in the system but
also the transient products of protein degradation. In general,
the major difficulty in the analysis of protein and peptides
comes from the different physicochemical properties of pro-
teins, the high number of peptidic sequences that can become
available, and the huge dynamic concentration range of both
families of compounds in real samples. Proteomics and pepti-
domics offer multiple applications in food science including
food processing, food quality, food safety, and characterization
of healthy food ingredients.
LC–MS has been extensively used in the identification and
determination of amino acids, peptides, and proteins. In earli-
est studies, for example, conventional RP-HPLC with chiral
derivatization agents was used for the determination of
amino acids. However, these methods were not applicable in
real food samples because of the insufficient sensitivity and the
interference from the numerous coexisting amino acids. For
the determination of peptides and proteins, HPLC–MS plays a
critical role. By using RP-HPLC, ion exchange, hydrophobic
interaction chromatography, etc., peptides and proteins can be
separated via the differences in surface hydrophobicity or
surface charge. These methods provide the practical technolo-
gies to separate complex food matrices, and the affinity
chromatography allows the purification of targeted peptides
and proteins. After HPLC separation, most peptides and pro-
teins in the low concentration range can be identified by MS
techniques.
CE-MS has been mainly applied for proteomics,
peptidomics, and metabolomics studies. Capillary electropho-
resis techniques coupled to MS are ideal analytic techniques for
different ‘omics’ approaches such as metabolomics, proteo-
mics, and peptidomics mainly due to the particular character-
istics of this separation technique. CE provides fast and highly
efficient separations, low reagent and sample consumptions
(CE is particularly well suited for samples that are volume-
limited), and high versatility considering the different CE
modes available. The main drawback of CE is its poor
sensitivity, but it can be improved by combining CE with MS
detection, while the use of preconcentration strategies can give
further sensitivity gain. Besides, the use of MS as detector pro-
vides additional selectivity and structural information of the
detected compounds.
Polyphenols
Flavonoids are polyphenolic compounds, usually found in
food and beverage. The motivation of flavonoids analysis
using HPLC–MS or GC–MS methods arises from their
potential beneficial effects on human health. Flavonoids are
usually present in low amounts in a complex matrix of plant
extracts and thus generally are difficult to isolate in higher
quantities. This, combined with their good sensitivity in elec-
trospray, makes HPLC–MS the method of choice for flavonoid
analysis. To increase selectivity (often a critical aspect) and
high mass resolution and to increase structural information,
tandem mass spectrometry is often used in HPLC–MS analysis
of flavonoids.
In the other side, several CE-ESI-MS methods have been
developed for the analysis of flavonoids, using high-pH run-
ning buffers containing ammonium acetate and MS detection
in the negative-ion mode. Other natural compounds bearing
phenol structures have been analyzed by CE-MS. Thus, a com-
parison between HPLC-ESI-MS and CE-ESI-MS for the analysis
of phenolic compounds from red wines showed that in spite of
CE providing much higher separation efficiency than HPLC, 24
compounds could be identified by HPLC–MS vs. 13 com-
pounds identified by CE-MS in the mentioned red wine
extracts.
Food additives
Food additives are strictly limited; only additives explicitly
authorized may be used in food. Monitoring foodstuffs for
additives is an area of increasing concern and importance.
Due to its excellent figures of merit, HPLC–MS is often a
method of choice. Food additives are groups of substances
commonly classified according to their application and not
to their chemical structure. Some of these are small molecules
(like benzoic acid used for conservation), some are macromol-
ecules (like the ‘infamous’ guar gum, causing concern a few
years ago), some are synthetic products, while others are
natural extracts. For these reasons, it is difficult to generalize
the ‘proper’ analytic method to be used for their characteriza-
tion. Some methods are compound-specific (and do not
estimate total amount of a given class of compounds); some
characterize groups of substances. Often, both identification
and quantification are required. Generic procedures for the
simultaneous extraction of various classes of food additives
and residues in various matrices are in use.
Analyzing Hazardous Food Components via
Chromatography–MS
Mycotoxin
Mycotoxins are toxic secondary metabolites of fungi (usually
molds) that readily colonize crops or foodstuffs during storage.
Most often, these are present in cereals and oil seeds. A
 Chromatography: Combined Chromatography and Mass Spectrometry
database reported 474 mycotoxins and fungal metabolites,
while previously unknown metabolites have recently been
created. These toxins are dangerous even in very small
amounts. Therefore, for some mycotoxins, maximum residue
level of aflatoxins B1, for example, as low as 0.1mgkg1 has
been established by the EU for baby foods and processed
cereal-based foods for infants and young children (please see
EC No 1881/2006). For mycotoxin analysis, two different
strategies can be followed to develop and validate analytic
methods. One is to set up a screening method to detect and
roughly quantify as many metabolites as possible, reducing the
selectivity in extraction and cleanup steps. The other approach
is to develop a high-performance method to accurately quan-
titate a few selected mycotoxins in selected cases. For both
approaches, a number of methods have been developed and
validated by diverse research groups. The combination of
HPLC with MS/MS has proved to be a powerful tool for the
simultaneous determination of different classes of mycotoxins.
Besides mycotoxins, also their metabolites often formed in
plants and animals might be hazardous for man. Such conju-
gated or masked mycotoxins can also be analyzed with
LC–MS/MS methods, very well suited for high sample through-
put, and should be included into multimycotoxin analysis for
the direct detection and characterization of metabolites in
complex biological and food matrices.
Mycotoxin analysis uses a variety of mass spectrometric
methods. Electrospray (in both positive-ion mode and
negative-ion mode), APCI, and APPI ionizations are all fre-
quently used. Triple-quadrupole instruments are often used
for targeted analysis, while high-resolution analysis is used to
look for unexpected derivatives. When quantitation is needed,
using isotope-labeled standards, whenever available, is highly
advantageous for accurate and precise analysis.
Pesticide residues
Pesticide residues in food are a growing concern, both for the
consumers and for legislation. Widespread use of pesticides
necessitates their trace analysis in vegetables and various food
products. There are several hundred active ingredients and
thousands of formulations currently in use. Like in the case
of mycotoxins, a large number of compounds need to be
screened and, if found, accurately quantified. Simultaneous
analysis of pesticides requires development of efficient high-
throughput methods.
A significant fraction of pesticide trace analysis is based on
HPLC–MS. In most cases, tandem mass spectrometry is uti-
lized, as it allows simplification of sample treatment prior to
the analysis and achieves multiresidue analysis in a single
chromatographic run. When quantitation is needed, using
isotope-labeled standards, whenever available, is highly advan-
tageous for accurate and precise analysis.
For chromatography, often, UHPLC (ultrahigh-performance
liquid chromatography) is used. UHPLC allows fast and effi-
cient separation and analysis is usually performed in less than
15 min chromatograms. For analysis, most often, a C18 column
with small particle size (1.7 m) is used. The most often used
mass spectrometer is the triple quadrupole (QqQ) that achieves
tandem mass spectrometry. The QqQ literally has three quadru-
pole mass analyzers in the same instrument. The first analyzer is
set to transmit the precursor (usually the molecular) ion; in the
83
central quadrupole, collision-induced dissociation (CID) takes
place, while the second analyzer transmits the expected product
ion, which is detected. In this instrument, several precursor
ion ! product ion transitions are selected (selected reaction
monitoring (SRM)) to increase sensitivity.
Increasing selectivity is of prime importance for analyzing
trace components in complex materials. For this reason, con-
ventional selected ion monitoring (SIM) in a single quadru-
pole is usually not considered sufficiently selective to identify
pesticide traces in food. Besides tandem mass spectrometry,
analysis using high mass resolution (HRMS) is another direc-
tion to increase selectivity. This can be carried out using
time-of-flight (TOF), Orbitrap, or Fourier transform (FT)
mass spectrometers.
A further option to increase accuracy and reliability of
pesticide analysis is the use of isotope dilution (that means to
use the isotopically labeled analyte as internal standard). This
enhances accuracy of analysis and at the same time may allow a
simplified sample preparation process. However, it increases
the costs of analysis significantly, so for this reason, it is rarely
used for routine pesticide analysis in the agrifood sector.
Pesticide analysis is one of the most important applications
in the agrifood sector. A large variety of different foodstuffs are
routinely analyzed, including baby food, vegetables, vegetable
oil, honey, fruit juice, wine, and milk.
Conclusion
In the past years, there has been an emerging investigation for the
food safety and food quality analysis using chromatography–MS
methods, including HPLC, GC, and CE. These combined
methods allow the rapid, reliable, and accurate analysis,
identification, and quantification in food matrices. State-of-the-
art MS instruments enhanced analytic sensitivities for the identi-
fication of trace components of food and provide a powerful tool
for analyzing various foods to meet the requirements of food
legislation or the concerns about toxicity. Combined MS
(LC–MS, GC–MS, etc.) and MS/MS methods can reduce the
time and labor-consuming sample preparation processes and
improve the selectivity for both qualitative and quantitative ana-
lyses of food samples present in complex matrices. The advances
of simple, robust, and reliable portable chromatography–MS
method can promise an on-site, rapid, accurate identification
and quantification of unknown compounds in food.
See also: Chromatography: Focus on Multidimensional GC;
Chromatography: High-Performance Liquid Chromatography; Food
Poisoning: Tracing Origins and Testing; HACCP and ISO22000: Risk
Assessment in Conjunction with Other Food Safety Tools Such as
FMEA, Ishikawa Diagrams and Pareto.
Further Reading
Agrawal GK, Sarkar A, Righetti PG, et al. (2013) A decade of plant proteomics and mass
spectrometry: translation of technical advancements to food security and safety
issues. Mass Spectrometry Reviews 32: 335–365.
84 Chromatography: Combined Chromatography and Mass Spectrometry

Arena S, Salzano AM, Renzone G, Dambrosio C, and Scaloni A (2014) Non-enzymatic Mohamed R and Guy PA (2011) The pivotal role of mass spectrometry in determining
glycation and glycoxidation protein products in foods and diseases: an the presence of chemical contaminants in food raw materials. Mass Spectrometry
interconnected, complex scenario fully open to innovative proteomic studies. Mass Reviews 30: 1073–1095.
Spectrometry Reviews 33: 49–77. Sandrin TR, Goldstein JE, and Schumaker S (2013) MALDI TOF MS profiling
Botitsi HV, Garbis SD, Economou A, and Tsipi DF (2011) Current mass spectrometry of bacteria at the strain level: a review. Mass Spectrometry Reviews 32: 188–217.
strategies for the analysis of pesticides and their metabolites in food and water Wang J (2009) Analysis of macrolide antibiotics, using liquid chromatography-mass
matrices. Mass Spectrometry Reviews 30: 907–939. spectrometry, in food, biological and environmental matrices. Mass Spectrometry
Garcia-Canas V, Simo C, Leon C, Ibanez E, and Cifuentes A (2011) MS-based analytical Reviews 28: 50–92.
methodologies to characterize genetically modified crops. Mass Spectrometry
Reviews 30: 396–416.
Hernandez F, Portoles T, Pitarch E, and Lopez FJ (2011) Gas chromatography coupled
to high-resolution time-of-flight mass spectrometry to analyze trace-level organic
compounds in the environment, food safety and toxicology. Trac-Trends In Relevant Websites
Analytical Chemistry 30: 388–400.
Herrero M, Simo C, Garcia-Canas V, Ibanez E, and Cifuentes A (2012) Foodomics: MS- http://www.asms.org/ – AAMS.
based strategies in modern food science and nutrition. Mass Spectrometry Reviews http://www.bmss.org.uk/index.html – BMSS.
31(1): 49–69. http://www.bmb.leeds.ac.uk/esms/ – ESMS.
Li P, Zhang Z, Hu X, and Zhang Q (2013) Advanced hyphenated chromatographic-mass http://www.imss.ie/index.htm – IMSS.
spectrometry in mycotoxin determination: current status and prospects. Mass http://www.chem.purdue.edu/cooks/MS%20Links.htm – Mass Spectrometry Links.
Spectrometry Reviews 32: 420–452. http://www.saams.up.ac.za/ – SAAMS.
Malik AK, Blasco C, and Pico Y (2010) Liquid chromatography-mass spectrometry in http://www.ualberta.ca/gjones/mslib.htm – Mass Spectrometry Database, American
food safety. Journal of Chromatography A 1217: 4018–4040. Academy of Forensic sciences.
 Chromatography: Focus on Multidimensional GC
C Cordero, C Cagliero, E Liberto, B Sgorbini, P Rubiolo, and C Bicchi, Università degli Studi di Torino, Torino, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Over the last decades, consumers’ preferences have been
addressed to healthier and flavorsome food with higher nutri-
tional value: the driving force has been food quality. Primary
condition for quality is safety that is closely related to the
compliance with legal standards on human health risks, the
environment, animal welfare, protection of natural resources,
and ethical requirements. On the other hand, the sensory
impact due to flavor, smell, and appearance has assumed an
equally important role. In this perspective, food end products,
semifinished products, and raw food matrices require control
analyses to establish quality requisites and to verify compliance
with legal standards.
Since its introduction in the early 1950s, gas chromatogra-
phy (GC) demonstrated to be an effective, flexible, and sensi-
tive technique for food analysis and has been successfully
adopted as the ‘core’ of the analytic process for
• flavor and aroma characterization (sensomics);
• studies of composition and of origin authentication;
• characterization of the volatile compounds from vegetable
matrices, essential oils, and differently produced extracts;
• fat analysis and characterization (short-chain fatty acids,
fatty acids methyl esters (FAMEs), triglycerides, sterols, etc.);
• residue and contaminant determination (pesticides, veteri-
nary drugs, endocrine-disrupting chemicals (ECDs), food
packaging migration products, and environmental contam-
inants such as polychlorinated biphenyls (PCBs) and poly-
aromatic hydrocarbons (PAHs).
However, one-dimensional GC not always provides adequate
efficiency and selectivity to obtain the required results in terms
of analyte separation (and as a consequence identification and
quantitation), in particular when complex samples have to be
analyzed. On the other hand, although the new generation of
mass spectrometric detectors thanks to improved perfor-
mances (new ionization sources/interfaces and analyzers)
enables reliable identity confirmation and quantitation even
without a separative step, chromatographic separation pro-
vides additional information on analyte physicochemical
properties that in some cases are necessary for unknown iden-
tification (i.e., isomers).
Multidimensional chromatography (MDC) has therefore
emerged as a valuable alternative thanks to the peak capacity
enhancement as well as the possibility to combine orthogonal
discrimination principles (including those provided at the
detection level) to investigate sample composition and to pro-
cess results to obtain higher and further levels of information.
MDC combines two, or more, independent (or nearly inde-
pendent) separation steps, increasing significantly the separa-
tion power of the resulting system. In the early 1980s, the
comprehensive definitions of MDC already distinguished
those system configurations providing a simultaneous zone
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00157-4
displacement from those adopting coupled columns that
provide sequential displacement of analytes.
More recently, because of modern instrumental innova-
tions and MDC developed applications, a more general
definition of MDC as ‘n-dimensional analysis that generates
n-dimensional displacement information’ was proposed.
When gas-phase separations are considered, the most intui-
tive and descriptive definition was given by Marriott, who has
defined multidimensional gas chromatography (MDGC) as “the
process of selecting a (limited) region or zone of eluted com-
pounds issuing from the end of one GC column, and subse-
quently subjecting the zone to a further GC displacement.”
A typical MDGC separation focuses on a fixed number of
fractions eluting from the first dimension (1D), the so-called
heart-cuts (H/C) (the approach is referred to as heart-cut multi-
dimensional gas chromatography (H/C MDGC)) that are sub-
sequently analyzed in the second dimension (2D), with the
aim to improve separation by combining different discrimina-
tion principles exploiting 2-D-system selectivity. Alternatively,
when a fast and continuous heart-cutting (named modulation)
is implemented by applying sampling periods compatible with
the 1D peak width, the resulting MDGC approach is named
comprehensive 2-D GC or GC
GC.
Figure 1 shows a schematic description of mono-
dimensional and different multidimensional GC instrumental
configurations; Figure 1(a) reports a conventional mono-
dimensional GC system, and Figure 1(b) a typical H/C MDGC
including a switching system (or valve V) located at the junction
between the two columns. The system also includes an auxiliary
gas line from an electronic pressure controller (aux EPC) that
provides flow for the switching valve and/or a makeup flow for
the 2D column that is located in a separate oven. The system is
also equipped with a primary detector (Det 1) and with a
monitor detector (Det 2), optional. Figure 1(c) shows a com-
prehensive two-dimensional system configuration that includes
a modulator (M) that interfaces the two columns (1D and 2D)
coupled in series. The detection is placed at the end of the 2D
column.
The next sections present basic operative principles, inst-
rumental configurations, and applications of H/C MDGC
and comprehensive two-dimensional gas chromatography
(GC
GC).
Heart-Cut Multidimensional Gas Chromatography
Basic Operative Principles and Instrumental Configurations
A classical H/C MDGC configuration basically consists of two
conventional capillary columns (e.g., 30 m
0.25 mm
ID
0.25 mm film thickness – df) coated with stationary phases
differing in selectivity, connected in series. Columns can be
located in a single or in two separate ovens, and a suitable
interface is located between the two dimensions enabling to
85
86 Chromatography: Focus on Multidimensional GC

Inj Det

One-dimensional GC system
Capacity = n peaks

n peaks

(a)
Inj Aux EPC Det 2 Det 1

H/T multidimensional GC system

V Capacity = n peaks + m peaks

m peaks

m peaks
1D 2D

(b)

Inj Det

Comprehensive two-dimensional GC system

Capacity = n peaks x m peaks

1D 2D

(c)

Figure 1 Schematic visualization of mono- and multidimensional GC instrumental configurations and their actual peak capacity: (a) shows a typical
one-dimensional system, (b) shows an H/C MDGC, while (c) shows a comprehensive two-dimensional system. Inj, injector; aux EPC, auxiliary gas
electron pressure controller; Det 1, primary detector; Det 2, monitor detector; V, switching interface; M, modulator; 1D, first dimension column; 2D,
second dimension column; n1D, peak capacity; m2D, peak capacity.

transfer chromatographic bands from the 1D to the 2D column
(see Figure 1(a)). The use of a cryotrapping device, between
the two capillaries, position V in Figure 1(a), makes the sys-
tem more efficient, affording a better band focussing and re-
concentration that results in an improved 2D peak capacity and
higher system sensitivity for trace analytes.
The transfer devices developed since the beginnings of the
technique can be classified in three groups: in-line and out-line
valves, the most popular systems, and valveless systems.
Valve-based systems, although straightforward in principle,
are affected by several limitations due to efficiency and inert-
ness. Band dispersion caused by dead volumes and retention
time fluctuations may occur. The modern SilTite™ technology
enabled to overcome valves surface activity and improved
mechanical robustness when installed in a hot oven.
Six-port–two-position W-type valves from Valco, live-cou-
pling/switching pieces, and Deans-switch interfaces utilizing
Valco microvolume T-piece are nowadays the most popular
devices for valve-based systems in MDGC.
However, thanks to the evolution electronic pressure con-
trol systems and their integration into the GC, most of the
MDGC systems adopt nonintrusive, valveless flow-modulated
devices (based on pneumatic flow switching). Some examples
of H/C MDGC systems available on the market are the multi-
Deans-switching technology from Shimadzu, the moving
capillary stream switching (MCSS) from Brechbühler, the
capillary flow technology (CFT) from Agilent, the Swafer™
microchannel system from PerkinElmer, and the SilFlow™
microchannel device (MCD) from SGE. Effluent-switching
devices by Agilent, PerkinElmer, and SGE are based on micro-
chips that connect individual switch components into a single,
small deactivated microchip plate.
The Swafer™ system has laser-fabricated microchannels on
circular metal disks present in the device body and is available
 Chromatography: Focus on Multidimensional GC 87

with different configurations allowing flow switching and
splitting between two or four inputs and two or up to four
outputs. With the SilFlow™ technology, the MCD has options
for three-port or four-port GC splitters. Advantages of micro-
fabricated devices rely on the possibility to overcome and
eliminate chromatographic distortions (peak tailing, peak
broadening, and/or analyte loss) thanks to surface inertness,
low thermal mass, and low dead volumes.
From an operative point of view, they are based on a
microfluidic Deans-switch technology; a description of the
principle of operation is reported in section ‘Thermal and
Pneumatic Modulators.’
H/C MDGC Applications in Food Analysis
One of the most remarkable fields of the application of
multidimensional gas chromatographic techniques is the anal-
ysis of medium-to-high complex mixtures of volatiles and
semivolatiles that include aroma-active compounds, botanical
tracers, and origin authentication markers. Thanks to MDGC
technology, food flavor analysis undoubtedly experienced
advances and improvements, in terms of results’ accuracy and
reliability. Key features in flavor analysis include the possibility
to obtain better separations, after 1D prefractionation of com-
plex samples and improved sensitivity for target analyte detec-
tion due to higher system loadability that is not affected by
column or detector overloading; in addition, when separation
is followed by olphactometric detection to screen for sensory-
active compounds, GC effluent can be diverted to a suitable
sniffing port and contemporarily subjected to further separa-
tion and/or detection.
Another field where MDGC is very popular is chiral recog-
nition of chiral food flavors; the adoption of chiral selectors as
stationary phases for capillary columns in the 2D provided a
further approach for flavor authentication.
Figure 2 shows an interesting application of ES-MDGC-MS
for the characterization of wine aroma and in particular in the
identification of the chiral flavor analytes responsible of the
rose note of a Riesling wine.
A further advancement in flavor authentication is repres-
ented by MDGC coupled with isotopic ratio mass spectrometry
(IRMS). MDGC–IRMS affords to define the origin of diagn-
ostic flavor compounds in consideration of the natural variation
in isotope distribution. Constant-flow MDGC–combustion/
pyrolysis–IRMS was successfully applied to the authenticity
assessment of (E)-a(b)-ionone from raspberry cultivars.

Ionone
α β

Cut 1
4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
(a) min

Ionone
Mass 2 1H–1H
α Mass 3 1H–2H

(b) 45 50 55 60 65 min
Figure 2 Isolation and characterization of aroma-active compounds in Riesling wine. (a) One-dimensional GC-FID/O on polar stationary phase
(Carbowax 20M) enables to identify an aroma-active zone with rose notes. The heart-cut window (inset) shows the same region separated on an apolar
(5% phenyl–95% polydimethylsiloxane) 2D column. (b) Combining MS and olphactometric detection, analytes responsible for the rose aroma are
univocally identified. (c) Separation of rose oxide enantiomers from a standard racemic mixture by ES-MDGC-MS on a 6TBDMS-DiAc-b-CD together
with their odor threshold (OT) values. Lower trace reports the enantiomeric distribution of rose oxides found in a Riesling wine. Reproduced from
Marriott, P. J., Chin, S. T., Maikhunthod, B., Schmarr, H. G. and Bieri, S. (2012). Multidimensional gas chromatography. Trends in Analytical Chemistry
34, 1–21.
88 Chromatography: Focus on Multidimensional GC

Figure 3 One-dimensional chromatogram (a) with FID detection and H/C MDGCpyrolysisisotope ratio mass spectrometry (H/C MDGC–P–IRMS)
separation of the ionones eluting region (b) with MS selected ion monitoring (SIM) detection of a raspberry extract. 2H/1H isotope ratios were
measured by H/C MDGC–P–IRMS run on a twin-oven system from HP model 6890 GC with autosampler A200S (CTC Analytics, Zwingen, Switzerland);
multicolumn switching system MCS2 (Gerstel, Mülheim, Germany) coupled to an isotope ratio mass spectrometer (DeltaplusXL) via a
pyrolysis reactor (ceramic tube (Al2O3), length ¼ 320 mm, 0.5 mm i.d., and reactor temperature ¼ 1450  C); and an open split (Thermo Electron,
Bremen, Germany). The 1D was equipped with an Rtx-1701 column (30 m
0.25 mm ID, 1 mm df) (Restek, Bad Homburg, Germany) and 2D column
with a ZB 5 (30 m
0.25 mm ID, 0.50 mm df) (Zebron, Phenomenex, Aschaffenburg, Germany). The following conditions were employed: splitless
injection (injector temperature, 220  C); 1D temperature program, starting from 100  C, isothermal for 2 min, and increasing by 2–220  C min1,
isothermal for 40 min; transfer line temperature, 220  C; and cut, 46–54 min. The 1D retention times of the ionones were determined by a monitor
detector, FID, temperature ¼ 250  C. The 2D column temperature program started from 60  C, isothermal for 2 min; increased by 6–180  C min1,
isothermal for 10 min; and increased by 1.5–220  C min1, isothermal for 10 min; carrier gas flow (helium) was 0.8 ml min1, constant flow.
Reproduced from Sewenig, S., Bullinger, D., Hener, U. and Mosandl, A. (2005). Comprehensive authentication of (E)-a(b)-ionone from raspberries,
using constant flow MDGC-C/P-IRMS and enantio-MDGC-MS. Journal of Agricultural and Food Chemistry 53, 838–844.

Figure 3 reports the total chromatogram (a) and the resulting
H/C MDGC chromatogram (b) of a raspberry extract measured
by MDGC–IRMS. For accurate and precise isotopic measurement
of (E)-a-ionone and (E)-b-ionone, it is necessary to cut exclu-
sively the 1D band eluting in correspondence to (E)-a(b)-ionone.
The chromatographic system of this example includes a 1D Rtx-
1701 column (30 m
0.25 mm ID
1 mm df) and a 2D
equipped with a ZB 5 column (30 m
0.25 mm ID
0.5 mm df).
H/C MDGC has demonstrated to be a valuable tool to solve
targeted analytic problems: quantitation of informative ana-
lytes and identification and abundance assessment of authen-
ticity markers. However, when comprehensive investigation
has to be run, through untargeted approaches, multidimen-
sionality has to be extended to the whole analytic run.
Two-Dimensional Comprehensive Gas Chromatography
(GC
GC)
Basic Operative Principles
In H/C MDGC, dedicated time-programmable interfaces trans-
fer automatically, and online, fractions consisting of a few
components (peaks) eluting from the 1D column for a limited
time fraction of the whole chromatographic run to the 2D
column; conversely, in GC
GC, each component eluting
from the first column is online and automatically trapped,
refocussed, and reinjected into the second column. This oper-
ation is run by a modulator, thermal or valve-based focussing
interface, within a fixed time (4–8 s), which is also the time
allowed for the analysis in the 2D.

Comprehensive MDGC (i.e., GC
GC) was firstly intro-
duced by Phillips in 1996, and today, it is widely accepted.
This technique has superior separation performance than H/C
MDGC due to the comprehensive application of multidimen-
sional separation to the entire sample in every single analytic
run. Figure 1 shows how H/C MDGC (Figure 1(b)) and
GC
GC (Figure 1(c)) effectively exploit system separation
power and the resulting theoretical peak capacity compared
to one-dimensional separations (Figure 1(a)).
The core of a GC
GC system is the modulator that accu-
mulates (or traps), refocuses, and rapidly releases fractions
eluting from the 1D to the 2D column. These operations are
run within a fixed time frame, named modulation period
(PM), and repeated across the entire chromatographic run.
Modulation has to be sufficiently rapid to preserve the original
1
D separation, while 2D separation has to be fast enough to be
finished before the injection of the subsequent fraction from
1
D effluent.
Appropriate selection of columns’ dimensions, combina-
tion of stationary phases, PM, and timing enables the full
exploitation of system potentials, optimized peak capacity,
and high reproducibility in terms of 2-D peaks’ distribution
over the chromatographic plane and accurate mass transfer
(accuracy in quantitative determinations).
Thermal and Pneumatic Modulators
Modulators can be essentially divided into two main categories
on the basis of the operative principle: thermal and pneumatic
modulators. Thermal modulators use positive and/or negative
temperature difference, with respect to the GC oven, to transfer
fractions from the 1D to the 2D. Common feature of these
systems is the sensitivity enhancement, obtained by the band
refocussing promoted by temperature difference, being their
duty cycle equal to 1, meaning that the mass transfer is com-
plete without leaks.
Conversely, pneumatic interfaces adopt valves connected
either in-line or out-line with the column set, for band transfers.
Until 1998, when a hybrid cryogenic/pneumatic interface
was firstly implemented, all modulators were thermal and
basically inspired to or developed on Phillips models.
Above all, and excluding prototypes and/or innovative ther-
mal interfaces still close in research and development labora-
tories, commercial instrumentation is nowadays equipped
with different design two-stage thermal modulators.
Briefly, fundamental steps include the following:
I step: The narrow band eluting from the 1D (generally a
25 m
0.25 mm ID
0.25 mm df) is focussed under a cold
jet stream (CO2, liquid N2, or compressed air for high-
molecular-weight analytes). Focussing can be done on a
portion of the 1D column, in a deactivated tubing of suit-
able inner diameter (generally a narrow bore capillary
0.10 mm ID), or at the head of the 2D column (generally
a 1 m
0.10 mm ID
0.10 mm df).
II and III steps: Reinjection is achieved by heating and volatil-
izing the condensed fraction; this operation is typically
done by switching off the cold jet and letting modulation
capillary to be heated by oven temperature (longitudinally
modulated cryogenic system – LMCS and dual-jet CO2
Chromatography: Focus on Multidimensional GC 89
developed by Beens) or by activating an hot jet upstream
(loop-type dual jet from Zoex and quad-jet modulators) as
depicted in Figure 5. Typically, remobilization has a pulse
duration from 10 to a few hundred milliseconds depending
on the analyte volatility and on the differential temperature
achieved in the cold–hot jet duty cycle.
Analytes are then subjected to a further cycle of focussing and
reinjection (IV step) to achieve an optimized band width at the
entrance of the 2D column. When orthogonal displacement is
applied between the two dimensions (by adopting different
stationary phases), analytes, not separated in the 1D, can be
separated in the 2D.
Despite the superior effectiveness of cryogenic modulation,
it is also rather expensive due to the cryogenic fluid consump-
tion and instrumental management. The development of
cryogenic-free, low-cost interfaces has therefore been a field
of active research in the last few years. Pneumatic modulation
has experienced an increasing interest, grown in parallel with
the spreading of the technique both in the scientific commu-
nity and in the industry.
In-line connected pneumatic interfaces adopt multiport dia-
phragm valves or solenoid valves, some of them also applied in
H/C MDGC. The first example of a diaphragm valve-based
system was able to sample only very narrow timed fractions
from the effluent of the first dimension. With early pneumatic
modulators, the band focussing was done by rapid sampling of
narrow fractions (50 ms wide, twice a second) from the 1D;
being so sharp, they do not need further focussing to enter the
2
D column. This interface is not mass-conservative and about
90% of the first column effluent is lost. A step forward was done
by the introduction of valve-based systems with flow diversion,
which implement the band focussing ‘in time’ thanks to the
adoption of a sampling loop, connected for the 80% of the
time in series with the 1D column, and an extra flow of carrier
gas toward the 2D column. This system maintains a 2D linear
velocity at least 20 times higher than that of the 1D column; the
injected band therefore becomes compressed and about 80% of
the original mass passes to the second dimension. Alternative
configurations may include two parallel 2Ds and consequently
two detection systems.
Out-line pneumatic interfaces, located outside the GC oven
and not along the sample path, are based on the principles of
flow switching implemented in the Deans-switch technology.
The device, in its first construction, consisted of a planar metal
plate housing a collection chamber connected (via two metal
branches) to a three-way solenoid valve. More recently, to
attain flexibility and differential flow velocity required by
GC
GC, the fixed volume chamber has been replaced by a
flow restrictor (a capillary) with selectable inner diameter and
length (also define as flexible loop). An electronic pressure-
control module supplies the auxiliary gas through a solenoid
valve that can be switched. The collection chamber (or the
restrictor) is filled periodically with 1D column effluent when
the solenoid valve is in the bypass mode. At the end of the
collection period, the internal ‘loop’ is flushed for 100–200 ms
by a very high gas flow (typically 20 ml min1), generated by
switching the valve to the inject mode. By setting the first- and
second-column flow at a ratio of F1:F2 ¼ 1:20, the 1D column
band can be compressed (in time) to form a 20-fold narrower
90 Chromatography: Focus on Multidimensional GC
2
D column pulse (1.0 s pulse can be injected as a 50 ms pulse).
This modulation interface has theoretically no temperature
restriction and can be considered truly comprehensive being
able to transfer the 100% of the column effluent of the first
column into the second column.
Other pneumatic modulators adopting flexible loops and/
or multichannel chip-based capillaries, connected via multi-
port valves to auxiliary gas controllers, have been introduced
but to date are not available as commercial products.
Detectors for GC
GC
Narrow peaks of about 50–300 ms base peak width necessitate
fast acquisition rates (i.e., at least 50–100 Hz). Flame-
ionization detection (FID) with its high maximum frequency
of acquisition (50–300 Hz) has been used for years, while
element-selective detectors, such as electron capture detector
(mECD), nitrogen- and sulfur-chemiluminescence detectors
(NCD and SCD), and nitrogen–phosphorous detector
(NPD), need careful optimization although in specific field
of application they provide useful complementary information
for analyte identification. For example, GC
GC with dual
detection by NPD and ECD, obtained thanks to microfluidic
splitting interfaces, has been successful for multiclass pesticide-
residue analysis in vegetable samples.
Mass spectrometry, on the other hand, can be considered
the elective detector for GC
GC platforms enabling univocal
analyte identification, in combination with retention data,
and accurate quantitation. Time-of-flight mass spectrometry
(TOF-MS) operating in low-resolution mass detection and
fast acquisition is the preferred detector because of its high
data-acquisition rate, selectivity, reliable deconvolution of
overlapping peaks, and ability to scan for broad mass range.
On the other hand, high-resolution TOF-MS, although in prin-
ciple very straightforward in terms of information potentials
related to accurate mass detection, is characterized by slower
acquisition speed. The recent introduction of (HR)TOF-MS
benchtop instrumentation has opened new perspectives for
metabolomics and foodomics applications.
Quadrupole MS (qMS) has experienced in the last years a
renewed interest for a number of applications, including qual-
itative and quantitative. Modern fast quadrupoles (operating
with acquisition rate up to 50 Hz for scan and selected ion
monitoring (SIM) mode) have in fact demonstrated to be
really competitive in terms of quality and reliability of the
results provided; in addition, commercial MS databases, pre-
vailing in qMS spectra, can successfully be adopted for
unknown identification.
Data Visualization and Elaboration
Although GC
GC is a true two-dimensional separation, the
process produces data values that are sequential: chromato-
graphic bands eluting from the 1D are collected, focussed,
and reinjected in the 2D where they undergo further separation
before detection. In the detector, an analog-to-digital (A/D)
converter collects the chromatographic signal at a certain fre-
quency. The digitalized data and related metadata (informa-
tion about the data) are stored in a file with a specific format
for subsequent access. GC
GC systems use an A/D converter
to map the intensity of the chromatographic signal to a digital
number as a function of time. Single-channel detectors (i.e.,
FID, ECD, and SCD) produce a single value at each time
sample of the chromatogram, while multichannel detectors
(such as MSs) produce multiple values (typically over a spectral
range) for each time sample.
Data collected are generally stored under proprietary data
file format but can be converted to text, as ASCII format
comma-separated values (CSV), or to the ASTM format
named analytical data interchange (ANDI) that is a standard
for chromatography and MS data. Although these standards
lack some information specific for GC
GC metadata, as, for
example, the modulation cycle, they are commonly used to
communicate raw data and to enable elaboration within dif-
ferent software platforms.
Visualization is the next step and enables, at first, a quali-
tative inspection of chromatographic separation. It can be
implemented through rasterization by arranging data values
acquired during a single modulation cycle as a column of
pixels (picture elements), so that the ordinate (Y-axis, bottom
to top) is the elapsed time for the 2D separation, and then, by
arranging these pixel columns so that the abscissa (X-axis, left
to right) is the elapsed time for the 1D separation. This ordering
presents the data in the commonly used right-handed Carte-
sian coordinate system, with the 1D retention times as the first
index into the array.
Data processing extracts higher level of information from
raw data enabling peak detection, peak identification, quanti-
tative analysis, and sample comparison, classification, and
recognition based on 2D patterns. A detailed description of
data processing approaches is beyond the scope of this
contribution, but information can be retrieved in suggested
further readings. However, an example of advanced finger-
printing is presented in section ‘GC
GC Applications in
Food Analysis’ for food origin authentication.
GC
GC Applications in Food Analysis
GC plays an important role as an analysis tool for aroma
extracts and essential oils, due to the complexity of the matrix
and the variable abundance of components from trace (ng/g)
to several per cent (g/100 g). Groups of chemically related
components (e.g., monoterpenoids and sesquiterpenoids, sat-
urated and unsaturated hydrocarbons, alcohols, carbonyl
derivatives, and esters) are common. These compound classes
possess similar chromatographic behavior and may have com-
mon isobaric molecular ions and/or MS spectra, which makes
MS interpretation and analyte identification difficult. The capa-
bility to adopt different GC
GC separation mechanisms, as
well as producing rationalized 2D patterns, can overcome these
problems.
GC
GC in food analysis covers a wide range of applica-
tions from the identification and profiling of fats, oils, essential
oils, and extracts to food contaminant determinations. Very
recently, several authors have demonstrated its potentials in
sensomics applications by combining advanced sample prepa-
ration with multiple detection, including olfactometry in GC
(O)
GC-MS platforms.
A typical application of GC
GC in fat and oil characteri-
zation is the detailed analysis of FAMEs. FAMEs differ among
each other primarily in their chain length, their number of
unsaturations, and the cis/trans configuration of their double
 c11 22:1 22:4 22:5
22:5 22:6
c13 22:4 n-3 n-3
n-6 n-6

2.50
c15 n-3
22:0
2D,2.5 m x 0.10 mm SLB-IL111, sec
2.00 21:5
n-3

21:0

20:2 20:3 n-6 20:4 20:5

20:1 n-3
NMI n-6 n-6
n-4 n3
n-3
1.50

20:0

19:1
19:0
18:3
18:1 18:2 n-3 18:4 n-3
1.00

n-6 n-4 n-6 n-1

18:0
17:1
17:0 c15-24:1
16:1 16:2 16:3 16:4 24:0
16:0
0.50

15:0 14:1
14:0
0.00

18.96 30.64 42.31 53.98 65.65 77.32 88.99 100.66 18.96 18.96 18.96 18.96 18.96
1D, 200 m x 0.25 mm SLB-IL111, min
Figure 4 GC
GC separation of FAMEs from a menhaden fish oil. System consisted of an Agilent 7890A GC (Agilent Tech., Wilmington, DE) combined
with a Zoex ZX2 dual-stage cryogenic modulator (Houston, TX). The injection port of the gas chromatograph was connected to a 1034 kPa
electronic pressure control module, and hydrogen was used as the carrier gas. Supelco SLB-IL111 capillary column (200 m
0.25 mm ID
0.2 mm df,
Bellefonte, PA) was used for 1D separation, and the same capillary column but with different dimensions (2.75 m
0.10 mm ID
0.08 mm df) was
used for the 2D separation. The cryogenic modulator first loop consisted of a 2 m
0.10 mm deactivated capillary. The 50 cm
0.15 mm Pd capillary
reactor was placed between the 1D column and the modulator. The timing of the modulation was set to 3.5 s; the temperature of the hot jet was
set at 350  C with a pulse of 350 ms. The column oven temperature was set to 170  C, and the injector port at 300  C. Analysis was acquired in constant
pressure at 690 kPa and the split flow was set to 125.69 ml min1. Reproduced from Delmonte, P., Fardin-Kia, A. R. and Rader, J. I. (2013)
Separation of fatty acid methyl esters by GC-online hydrogenation
GC. Analytical Chemistry 85, 1517–1524.

(a) (b)

(c)

Figure 5 2-D plots of the volatile fraction from an Italian extra virgin olive oil sample and from a defected sample where muddy notes where perceived
by the official panel (b). Image comparison (c) results, visualized in a grayscale ratio mode (GC-Image™ software), reveal compositional
differences by lighter and darker areas. Analysis conditions: HS-SPME sampling, 2 cm StableFlex 50/30 mm DVB–Carboxen–PDMS fiber (Supelco,
Bellefonte, the United States); sample amount, 1.000 g; vial volume, 20 ml; sampling time, 40 min; and temperature, 40  C. GC
GC analyses:
GC
GC-MS system: Agilent 6890 GC–Agilent 5975 MSD ionization mode: EI 70 eV (Agilent, Little Falls, DE, USA); transfer line temp., 280  C; scan
range, m/z 35–250 in fast scanning mode (10 000 amu s1). GC
GC interface: KT 2004 loop modulator (Zoex Corporation, Houston, TX, USA);
modulation time, 4 s. Column set: 1D, CW20M column (30 m
0.25 mm ID, 0.25 mm df); 2D OV1701 column (1 m
0.10 mm ID, 0.10 mm df)
(MEGA – Legnano (Milan), Italy). Analysis conditions: injection mode, split; ratio, 1/20; temp., 250  C; carrier gas, helium; T. program, 40  C (1 min)/
2.5  C/min/260  C (5 min).
92 Chromatography: Focus on Multidimensional GC
bonds. Recently, a straightforward investigation approach was
proposed. This approach uses identical highly polar separation
columns based on ionic liquids (i.e., SLB IL-111 from Supelco)
with an online chemical derivatization made on a Pd tubing in
the presence of hydrogen carrier gas. The hydrogenated deriv-
atives, which are the saturated forms of eluting FAMEs, are
successively separated in the 2D on the basis of their chain
lengths. Operating under isothermal conditions, saturated
FAMEs lie on a straight diagonal line bisecting the separation
plane, while FAMEs with the same carbon skeleton but differ-
ent number, geometric configuration, or position of double
bonds lie on lines parallel to the 1D time axis. Figure 4 reports
the pattern of menhaden oil analyzed under isothermal
conditions.
2
D separation patterns, obtained by comprehensive
methods, have an intrinsic potential as unique and highly
informative sample fingerprints, being potentially useful for
sample characterization, differentiation, discrimination, and
classification. However, this improvement in the informative
content produces large and complex datasets (consisting of
bidimensional retention data, detector responses, and MS
spectra) that require suitable data mining: (1) to interpret the
higher level of information and (2) to extract useful and con-
sistent data on sample compositional characteristics.
2 Pierce KM, Kehimkar B, Marney LC, Hoggard JC, and Synovec RE (2012) Review of
D fingerprint analysis can be adopted in food authentica-
tion as well as in flavor and aroma characterization, to define
the so-called product signature in terms of sensory properties
(sensomics) and botanical/geographic origins and/or to study
modifications induced by thermal treatment, through the vol-
atile fraction of the matrix investigated. Figure 5 reports the
results of a pairwise comparison of the volatile fraction of two
olive oil samples from Italian productions: an edible extra
virgin olive oil and a defected one classified by the official
panel as muddy. Differences, visualized in the comparative
image analysis, correspond to green and red areas of the chro-
matographic plane where analytes differing for their abun-
dance between samples are present. Black and white regions
correspond to analytes present in just one of the two samples.
Brighter areas correspond to 2D peaks with a larger differ-
ence between the reference (muddy sample) and the analyzed
(extra virgin olive oil) patterns and more abundant in the
analyzed image; darker areas conversely correspond to 2D
peaks whose abundance is higher in the reference (muddy
sample) image.
See also: Chromatography: Combined Chromatography and Mass
Spectrometry; Essential Oils: Isolation, Production and Uses; Essential
Oils: Properties, Composition and Health Effects; Fatty Acids:
Determination and Requirements; Fatty Acids: Trans Fatty Acids;
Quality Control in Food Processing.
Further Reading
Marriott PJ, Chin ST, Maikhunthod B, Schmarr HG, and Bieri S (2012)
Multidimensional gas chromatography. Trends in Analytical Chemistry 34: 1–21.
Meinert C and Meierhenrich UJ (2012) A new dimension in separation science:
comprehensive two-dimensional gas chromatography. Angewandte Chemie
International Edition 51(42): 10460–10470.
Mondello L, Lewis AC, and Bartle KD (2002) Multidimensional chromatography.
Chichester: Wiley.
Murray JA (2012) Qualitative and quantitative approaches in comprehensive two-
dimensional gas chromatography. Journal of Chromatography A 1261: 58–68.
chemometric analysis techniques for comprehensive two-dimensional separations
data. Journal of Chromatography A 1255: 3–11.
Reichenbach SE (2009) Data acquisition, visualization, and analysis. In: Ramos L (ed.)
Comprehensive two-dimensional gas chromatography. Comprehensive analytical
chemistry book series, Vol. 55, pp. 77–106. The Netherlands: Elsevier, Chapter 4.
Reichenbach SE, Tian X, Cordero C, and Tao Q (2012) Features for non-targeted cross-
sample analysis with comprehensive two-dimensional chromatography. Journal of
Chromatography A 1226: 140–148.
Seeley JV (2012) Recent advances in flow-controlled multidimensional gas
chromatography. Journal of Chromatography A 1255: 24–37.
Tranchida PQ, Purcaro G, Dugo P, and Mondello L (2011) Modulators for
comprehensive two-dimensional gas chromatography. Trends in Analytical
Chemistry 30: 1437–1461.
Tranchida PQ, Sciarrone D, Dugo P, and Mondello L (2012) Heart-cutting
multidimensional gas chromatography: a review of recent evolution, applications,
and future prospects. Analytica Chimica Acta 716: 66–75.
Tranchida PQ, Donato P, Cacciola F, Beccaria M, Dugo P, and Mondello L (2013)
Potential of comprehensive chromatography in food analysis. TrAC Trends in
Analytical Chemistry 52: 186–205.
 Chromatography: High-Performance Liquid Chromatography
H Gika, Aristotle University Thessaloniki, Thessaloniki, Greece
G Kaklamanos, Institute of Food Safety, Wageningen, The Netherlands
P Manesiotis, Queen’s University Belfast, Belfast, UK
G Theodoridis, Aristotle University Thessaloniki, Thessaloniki, Greece
ã 2016 Elsevier Ltd. All rights reserved.
HPLC: The Separation Workhorse
HPLC is a widely applied analytic technique as evidenced by the
number of instruments installed, practitioners, analyses
performed, and research papers published annually. HPLC is
applied in quality control and is the workhorse of the analytic
departments within the pharmaceutical and food industries and
environmental control. Furthermore, HPLC holds a central place
in most research laboratories: HPLC methods are developed to
study drug delivery, adsorption and metabolism, genetic expres-
sion of proteins, emerging pollutants in the environment, and
nutrients in foodstuffs or in the gastrointestinal tract of animal
models or humans, to name only a few characteristic exam-
ples. Figure 1 depicts the continuous increase in the number of
published articles per year based on a literature search on Sco-
pus, using the terms ‘liquid chromatography’ in combination
with one of the terms ‘clinical,’ ‘pharmaceutical,’ ‘food,’ and
‘review.’ The fact that more than 1000 reviews are published
annually on HPLC and its applications is impressive and indic-
ative of the width and significance of the technique. Indeed, the
corresponding numbers for atomic spectroscopy or electrochem-
istry for the year 2013 are approximately five times less.
With the advent of systems biology and the development of
the ‘omics’ fields, for example genomics, proteomics, and
metabolomics, HPLC maintains its major position as an indis-
pensable research tool: new high-resolution mass spectrometry
(HRMS) instruments such as the time of flight (TOF) and the
Orbitrap MS are ideally combined with different modes of
HPLC to map the protein or small metabolite content of sam-
ples, cells, or organs. As explained later in this article, difficult
analytic tasks require extensive method development in both
LC and MS and cannot be served by a single technique.
In HPLC, sample constituents are separated as they migrate
through a column in different velocities and elute from the
system in different times. The analytes take part in a three-
mode interaction between the stationary phase, the sample,
and the mobile phase. Analytes that are strongly retained on
stationary phase elute late, while analytes that do not interact
strongly migrate faster and elute sooner through the system.
HPLC utilizes a variety of column formats, dimensions, and
chemistries, while mobile phases include a wide range of
organic solvent and buffer mixtures. Detection of eluted ana-
lytes can be achieved by a single detector or multiple detectors
connected in series. Figure 2 depicts a schematic of an HPLC
system with its components.
Modes of HPLC
HPLC is practiced in discrete modes that employ different
separation mechanisms and find application in different
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00159-8
areas. The separation mechanism is governed by the selection
of the stationary phase. The two major separation mechanisms
are the following:
(a) Normal phase (NP), where analytes partition between a
polar stationary phase (e.g., silica) and a low-polarity
mobile phase (e.g., hexane, chloroform, or dichloro-
methane). Dipole–dipole and hydrogen bonding interac-
tions are the major contributors; nonpolar analytes elute
quickly, while polar analytes interact strongly with the
stationary phase eluting later.
(b) Reverse phase (RP), where polarities are reversed, with the
stationary phase being less polar than the mobile phase.
Polar analytes remain mostly in the mobile phase showing
little attraction towards the stationary phase and elute
early. Nonpolar analytes are retained by the stationary
phase by a combination of interactions, including non-
specific hydrophobic interactions.
Reversed-phase liquid chromatography (RPLC) is by far the
widest applied mode largely due to its more robust nature
and its compatibility with aqueous samples such as biological,
environmental, and food samples and extracts. However, RPLC
is restricted to the analysis of medium-polarity and medium-
molecular-weight compounds. Polar or high molar mass com-
pounds are usually problematic. The former include some of
the most important metabolites in the biochemical pathways:
amino acids, nucleotides, organic acids, and amines. These are
not retained in RPLC and elute rapidly with the solvent front.
One solution towards the analysis of polar molecules is
derivatization, which is used to modify analyte molecules
and improve retention and detectability; for example, amino
acids are analyzed as derivatives that absorb in UV or exhibit
fluorescence. However, derivatization as a reaction of organic
molecules imposes a number of drawbacks:
1. Analytes react at different rates.
2. Molecules can behave differently in different environments
(different pH or salinity values may yield inconsistent
results).
3. An excess of reagents is introduced to the system.
4. Derivatives have a limited lifetime, thus introducing a
potential error source.
Rapid advances in MS enabled the development of multi-
analyte methods that can determine hundreds of analytes in
a single run, thus partially overcoming the need for improved
analyte detector response. Nevertheless, nonderivatized polar
molecules will show poor retention and thus, significant
efforts are invested in the development of separation modes
appropriate for the analysis of polar analytes and ionic or
ionizable species. These include aqueous normal-phase
93
94 Chromatography: High-Performance Liquid Chromatography
Clinical
3500
3000
Number of published articles
2500
2000
1500
1000
500
0
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014
Figure 1 Number of published articles per year, returned when using the terms ‘liquid chromatography’ and ‘clinical,’ ‘food,’ ‘pharmaceutical,’ or
‘review.’ Search performed on Scopus on April 27, 2014.
(a)
(c)
(b)
Figure 2 Schematic representation of an HPLC instrument. a: Solvent compartment; b: pump; c: sample injection port or autosampler; d: column
compartment; e: detector(s); f: data recording and processing; g: waste.
(ANP) chromatography, ion pair chromatography (IPC), ion
exchange chromatography (IEC), and hydrophilic interaction
liquid chromatography (HILIC). HILIC can be considered
as a reversed reversed-phase chromatography: analytes are
separated using principally organic mobile phases and a
hydrophilic stationary phase, eluting in order of increasing
hydrophilicity. HILIC phases are categorized as neutral
(where electrostatic interactions are not evident), charged
(where electrostatic forces dominate the separation), and zwit-
terionic. HILIC is increasingly used in the analysis of polar
compounds, providing the elution order reversed to the order
provided by RPLC. However, certain polar groups, such as
phosphorylated molecules, may remain problematic, so other
LC modes are employed: IEC and IPC make use of mobile
phases that contain ionic species typically dissolved in the
mobile phase (in IEC, ionic species may be immobilized in
the stationary phase).
Ionic interactions are stronger and thus harder and slower
to disrupt, and as a result, IEC generally provides wider peaks
compared with RPLC. IEC is extensively used in the analysis of
proteins, taking advantage of ionic interactions between
charged proteins and the stationary phase. IEC can be further
divided into cation or anion exchange chromatography, while
certain stationary phases encompass ampholytes or species
that may appear at a neutral or charged state: tertiary amines
Food Pharmaceutical Reviews
Year
(f)
(d) (e) (g)
will acquire a positive charge at low pH but will be neutral at
high pH. The strength of the ionic interactions between ligands
and analytes is determined by the number and location of the
corresponding charges. To facilitate analyte binding, mobile
phases of low to medium salinity are initially employed (load-
ing phase). To achieve analyte elution, the salt concentration in
the mobile phase is increased, thus eluting analytes in the order
of strength of ionic interactions.
On the other hand, IPC uses ion pair reagents to modify the
stationary phase: these are dissolved in the mobile phase and
interact with the hydrophobic ligand of RP stationary phases.
The polar part of the ion pair reagent is exposed to the mobile
phase, ready to interact with polar metabolites increasing their
retention. IPC when coupled to MS faces severe limitations due
to the high background signal generated by many widely used
ion pair reagents. The latter contaminate MS systems almost
permanently, rendering the MS system practically nonusable
for different analyses. IPC has recently found an important
application area in the analysis of metabolites involved in
central metabolism: intracellular metabolites such as nucleo-
tides, coenzyme A esters, nucleosides, phosphorylated sugars,
and metabolites involved in energy metabolism such as AMP,
ATP, and NADP.
Additional liquid chromatographic techniques relevant to
food and clinical analyses include affinity chromatography
 Chromatography: High-Performance Liquid Chromatography 95

Target
Matrix components
2
Competing agent binding to ligand
Competing agent binding to target
Affinity ligand

3 6

4 5

Figure 3 Schematic representation of the binding mechanism and elution protocols applied in affinity chromatography. 1: Sample loading;
2: targeted analytes bound, other matrix components eluted; 3: elution of targeted analyte by change of conditions (pH and ionic strength); 4: isocratic
elution; 5: addition of competing agent binding on the affinity ligands; 6: addition of competing agent binding on the targeted compound.

(AC) and size exclusion chromatography (SEC). In AC, sepa-
ration is based on analyte affinity to ligands or receptors bound
on the stationary phase. AC shows potential in the isolation of
valuable compounds of biological interest from complex
matrices taking advantage of the natural affinity between
two counterparts, such as an enzyme for its substrate/inhibitor
or an antibody for its antigen. One of the counterparts is
immobilized on a chromatographic column, acting as a
receiver, binding its specific partner over other compounds.
The captured analyte is eluted by changing the conditions in
the mobile phase (pH, ionic strength, and competitive analyte)
isolated in pure form (Figure 3).
SEC utilizes stationary phases of a controlled porosity that
physically block the flow path of the analytes as they migrate
through the column. Analytes of molecular volume smaller
than the particle pores penetrate deeper and elute slower,
compared with relatively large molecules that will be excluded
from the pores and elute faster. Using appropriate standards,
SEC can be used to determine the molecular weight of syn-
thetic and natural macromolecules, while both AC and SEC are
widely used in the separation and isolation of proteins and
polysaccharides from complex mixtures.
A recent development is two-dimensional LC (2-D-LC),
which is employed to address the need for additional separa-
tion power. 2-D-LC is increasingly used in biomarker discovery
and identification, in the oil industry, in the analysis of poly-
mers and biopolymers, and wherever additional separation
power is necessary. 2-D-LC implements two orthogonal sepa-
ration mechanisms that run sequentially. The two modes
should be based on different complementary separation mech-
anisms. For example, in protein/peptide analysis, the ion
exchange or size exclusion column is coupled online with RP:
analytes are separated in the first column and subsequently in
the second column (RPLC). The second column is typically
smaller in dimension so that analysis is rapid to cope with
the continuous flow of analytes eluting from the first column.
2-D-LC requires sophisticated instrument control and data
handling in order to synchronize the pumping of different
solvents through two columns, movement of the valves to
alter the flow path, switch from column to column for loading,
and elution and washing. Results are depicted as heat maps or
contour plots.
Stationary Phases
The vast majority of HPLC columns utilize porous, spherical
octadecyl (C18)-functionalized silica particles of 3–5 mm diam-
eter, while other chemistries include octyl (C8), phenyl-hexyl,
and amine-modified phases. Fluorinated phases provide alter-
native selectivity and separation characteristics, exhibiting
higher retention for polar compounds.
These phases have typical surface areas up to 400 m2 g 1
and pore diameters of 100 Å. Such materials owe their pop-
ularity to their relative ease of manufacturing in large quanti-
ties, simplicity of surface modification, chemical and
mechanical stability, excellent batch-to-batch reproducibility,
and extensive documentation on their utilization as separation
media. However, the growing demand for analysis of increas-
ingly complex samples and higher sample throughput has
driven the development of new stationary phases, with numer-
ous new materials introduced every year. Perhaps, the most
important recent development was the introduction of ultra-
high-pressure LC (UHPLC) systems in the early 2000s. UHPLC
employs sub-2 mm particles and accommodates operating pres-
sures higher than 1000 bar. This development necessitated
major advances in column technology and instrumentation.
Apart from the obvious advantages of higher separation power
that lead to higher specificity (analytes are less likely to coelute
with interferences) and sensitivity (lower background noise),
UHPLC can increase the analytic throughput: an HPLC
method of 10 min per sample may be reduced to 3–4 min in
UHPLC. The major advantages and operating parameters of
UHPLC are presented in Table 1.
96 Chromatography: High-Performance Liquid Chromatography
Table 1 Characteristics and typical operating parameters of UHPLC
Characteristics of
UHPLC Comments
Operating Pressure: 20 000 psi (1400 bar)
parameters Flow rate: 5 ml min 1
Particle size: sub-2 mm particles
Column dimensions: to 2 mm i.d.
Detection rate:  40 points s 1
High throughput 3–10-fold increase in throughput vs. HPLC,
without loss of resolution
Rapid method Ideal for rapid column and mobile phase
development screening and method optimization
High resolution Up to 3-fold increase compared with HPLC
Reduced solvent Typical 5–15-fold reduction vs. HPLC due to
consumption shorter analysis times and smaller i.d.
columns
High sensitivity 3–10-fold increase of mass sensitivity
Higher precision 2–3-fold increase in retention time and peak
area precision
Compatibility Compatible with high temperatures, 2-D-LC and
core–shell columns
Despite its clear advantages, UHPLC is associated with sig-
nificant equipment costs that hinder global transition from
HPLC. To this end, core–shell or superficially porous particles
have recently attracted significant interest. These have a solid,
impenetrable core that supports a thin, porous layer, typically
from 0.2 to 0.6 mm (Figure 4). In totally porous particles,
diffusion paths within the pores are longer, increasing peak
broadening and decreasing column efficiency; superficially
porous particles exhibit superior mass transfer properties,
reducing peak broadening and enhancing column perfor-
mance. In fact, the efficiency for columns of 2.7 mm core–shell
particles rivals that for sub-2 mm totally porous particles, while
the operating back pressure is halved, eliminating the need for
UHPLC instruments. An example of the performance of 5 mm
core–shell vs. fully porous particles is shown in Figure 5.
Monolithic columns were introduced mainly aiming to
overcome limitations of mass transfer described as the C term
in the Van Deemter equation. Compared with particulate
materials, monoliths exhibit very low C values that do not
vary significantly within the flow rate. Monoliths comprise a
polymer or silica network where pores are interconnected gen-
erating an unobstructed flow path that in contrast to particu-
late columns does not have closed pores. Mass transfer is
enhanced enabling higher flow rates. Monoliths have found
application mostly in the analysis of larger molecules (e.g.,
proteins and polynucleotides) and in large-scale biotechnol-
ogy applications, for example, in the form of convective media
conveniently used for the purification of target proteins pro-
duced in bioreactors.
Furthermore, nano-LC and chip-based LC are increasingly
used especially in the analysis of biomolecules such as pro-
teins. Nanoflow enhances analyte sensitivity and at the same
time is well suited to handle reduced sample volumes. Recent
developments bring HPLC in the core of the MS tip (e.g., in the
form of ionKey/MSTM). While developments such as the latter
appear attractive, especially for use by less-trained personnel,
their adaptation requires significant capital investment costs.
Abs
Abs
t t
Figure 4 Schematic depicting the cross sections of fully porous (left) and
core—shell (right) silica particles with the corresponding chromatographic
traces showing sharper peaks on the core—shell material.
Column chromatography will most likely continue to domi-
nate as it provides unparalleled versatility and peak and load-
ing capacity. Chip-based LC and other such formats that have
been presented in recent years are still in the development
stages and, although some promise has been shown, are still
a long way from claiming the HPLC market.
Instrumentation
Analytical instrumentation of (U)HPLC shows continuous
development since the introduction of the method in mid-
1970s. Instrument manufacturers have invested in the devel-
opment of higher quality materials, instruments, and software,
and by the end of the twentieth century, HPLC technology had
overcome most reproducibility and stability issues faced in its
first decades. In the early days, HPLC suffered from issues such
as the use of additives, the blockage of tubes and columns, the
introduction of unnecessary peak broadening, and poor qual-
ity of frits, ferules, connectors, etc. Such issues have now been
addressed with the development of advanced quality or fit-for-
purpose media: Metal-free systems can be used for the analysis
of proteins; sample preparation removes particulate matter
before clogging the system; developments in the production
phase allow high column batch-to-batch reproducibility:
change to a new column of the same material will not result
in difference in method performance. As a result, analysis
throughput has increased dramatically, while method transfer
and interlaboratory repeatability are greatly enhanced.
Detection and Hyphenation
Detection in HPLC typically utilizes optical, electrochemical,
and spectroscopic detectors. These should provide fast data
acquisition to cope with the flow dynamics of LC. Given that
ten or more data points are required for optimum peak quan-
titation, fast data acquisition becomes of primary importance
for accurate quantitative analysis in HPLC and UHPLC in
particular, where peaks may have widths of less than 5 s.
In practical terms, a spectrophotometric detector is an inte-
gral part of the modern (U)HPLC instrument and thus
hyphenation means coupling LC to a mass spectrometer.
UHPLC–MS dominates the field of biopharmaceutical, food,

5 µm HALO Fused-Core
Pressure = 240 bar
Surface area = 100 m2 g−1
N = 16 400
Absorbance
N = 10 000
0 2 4 6
Figure 5 Comparative separations with 5 mm particles: fused-core vs. totally porous. Column dimensions: 4.6 mm  150 mm; temperature: 35  C;
flow rate: 2.0 ml min 1. Reprinted with permission from Elsevier from DeStefano, J. J., Schuster, S. A., Lawhorn, J. M. and Kirkland, J. J. (2012).
Performance characteristics of new superficially porous particles. Journal of Chromatography A 1258, 76–83.
and environmental analyses. MS spectroscopists may consider
HPLC as merely an introduction step to the MS system, while
chromatographers argue that MS is another type of LC detector;
however, the truth is that separation is necessary prior to MS
for a number of reasons, such as the need to separate isomeric/
isobaric substances and the drive to avoid ion suppression
effects. No matter the sophistication and the cost of the MS
analyzer, such incidents would lead to poor or no analyte
detection even for analytes of high concentrations. Implemen-
tation of HPLC, and UHPLC in particular, reduces this threat,
albeit it cannot eliminate it, by adding an orthogonal element
of separation: molecules are first separated according to phys-
icochemical properties and are next introduced to MS.
Applications
This section aims to illustrate selected examples of HPLC appli-
cations in the analysis of biological and food samples. LC–MS
has become the gold standard in a number of application fields
and this has been recognized by regulatory authorities, which
require its application for the majority of analyses of primary
importance such as contaminants in foods, prohibited sub-
stances (e.g., steroids in foods or biological samples), nar-
cotics, pharmaceuticals, and endogenous metabolites.
Adaptation of the technology is enforced in order to ensure
that ion suppression effects are minimized in the analysis of
unknown samples and that analyte identification by a combi-
nation of retention time and MS signal is unambiguous: iden-
tification is verified by retention time, precursor and product
ion mass, and product ion ratios and is corroborated against
reference standards analysis using explicitly set tolerance levels.
Chromatography: High-Performance Liquid Chromatography 97
Peak Identities ( in order)
1. Acetaminophen
2. Aspirin
3. Salicilic acid
N = 20 500 4. Tolmetin
5. Ketoprofen
6. Naproxen
7. Fenoprofen
8. Dichlofenac
9. Ibuprofen
5 µm totally porous
Pressure = 215 bar
Surface area = 300 m2 g−1
N = 11 000
8 10 12 14 16 18
Time, min
Application of HPLC in Food Analysis
Our food supply includes a variety of increasingly processed
materials from various sources. Substances such as fertilizers,
pesticides, veterinary drugs, growth promoters, colorants, anti-
oxidants, natural and artificial sweeteners and flavors, natural
and synthetic vitamins, and carbohydrates are used to improve
productivity and thus increase competitiveness and profit mar-
gins of the food industry. To ensure food safety and nutritional
quality, regulations have been established for the assessment of
food quality, the assessment of nutritional intake, and trace-
ability especially for foods and products with designation of
origin and so forth. Regulation agencies often deal with the
detection of prohibited substances and substances with maxi-
mum permitted levels such as chemical additives, residues, and
contaminants in food products explicitly specifying methodo-
logical considerations to harmonize methods and ensure inter-
changeability of results across different laboratories and states.
Increasingly, food analysis methods are built around HPLC,
which has proved to be an optimal technology for detecting
and/or quantifying the vast majority of food analytes. In recent
years, the demands for high sample throughput, high
efficiency, and high resolving power have significantly boosted
(U)HPLC technology in areas of analytic research. Several
multianalyte methods have been developed offering simplicity
and reduction in cost. The key requirement for developing such
multimethods is the application of generic extraction proce-
dures with a careful examination of matrix effects. Recently,
methods covering more than 250 veterinary drugs, pesticides,
mycotoxins, and other potential chemical contaminants have
been developed and are applied in routine analysis.
Fused-core columns have been applied in the analysis of
bisphenols in canned food and soft drinks, phenolic
98 Chromatography: High-Performance Liquid Chromatography
compounds in beverages, and banned substances in egg,
honey, and milk. HILIC has proved to be complementary to
RPLC in the analysis of foods: meat, milk and its products,
eggs, packaged food, natural products, honey, and cereals.
Fluorinated phases found use in the separation of tocopherols,
alkaloids in natural products, and photoinitiators in packaged
food. Higher temperatures (i.e., >60  C) may expedite
analysis due to the reduction of solvent viscosity and back
pressure. Using elevated temperatures, 12 triazine herbicides
were separated in 2.5 min. Other applications showed
separation improvements (increase in resolution) at very low
temperatures (5  C) for the analysis of photoinitiators in
packaged food.
Sample preparation is still one of the most important steps
of an analytical method. Effective sample preparation is impor-
tant to reduce matrix interferences and achieve reliable analytic
results. A sample preparation method should be fast, simple,
accurate and facilitate the analysis of a large number of sam-
ples. Modern sample preparation techniques such as on-line
solid-phase extraction (SPE), supercritical fluid extraction
(SFE), turbulent flow chromatography, and molecularly
imprinted polymers (MIPs) can permit high-sensitivity HPLC
analysis in the parts per trillion (ppt) range. Utilization of
advanced techniques in the determination of target analyte
groups such as antibiotics and growth promoters in a variety
of food products has improved sensitivity and selectivity,
cleaning up target analytes and removing matrix-interfering
compounds.
Current rapid development of UHPLC has led to its increas-
ing adoption as the approach of first choice for confirmatory
analysis for multiple-target analysis in a variety of food prod-
ucts. Despite technological challenges, such as narrower peaks,
and the impact of sample matrix effects, the overall injection
cycle times have shortened, detection limits have improved,
and chromatographic resolution and the number of com-
pounds in multianalyte methods continue to increase. We
expect the speed of analysis to increase further by using even
higher temperatures and pressures, allowing the separation of a
much larger number of analytes in the field of food analysis.
Application of HPLC in Health-Related Analyses
Contemporary bioanalytical laboratories are equipped with
HPLC or UHPLC systems used in the analysis of small mole-
cules or macromolecules in trace or semipreparative quantities.
A major advantage is that basic instrumentation may be con-
veniently modified for a variety of purposes. Hence, a single LC
pump can deliver the mobile phase or can be combined with
additional pumps in 2-D-LC systems for, for example, proteo-
mics or for purification and isolation of analytes of interest for
characterization purposes.
Two decades ago, pharmaceutical industry laboratories
relied on HPLC and immunoassays for drug discovery and
development: HPLC assays accounted for more than 80% of
pharmacokinetics/pharmacodynamics (PK/PD) needs and
toxicity studies. The landscape is now totally dominated by
LC–MS/MS where RPLC is a key element of method develop-
ment and validation. Regulatory bodies such as the FDA and
EMEA publish explicit guidelines for the validation of LC-
based methods.
The development of UHPLC facilitates high-throughput
screening (HTS), a powerful tool that enables the performance
of routine analysis in, for example, food quality control or the
environmental monitoring sector. The pharmaceutical indus-
try utilizes HTS to promote products to the next development
step at a faster rate: from combinatorial synthesis to drug target
finding and lead finding, structure–activity evaluation, in vitro
and in vivo application, and PK/PD assessment. As a drug
candidate moves through the different sections of a pharma-
ceutical industry, assays are developed, and for all these
purposes, the advantages offered by (U)HPLC remain
unparalleled.
HPLC and LC–MS have achieved significant penetration in
clinical laboratories, substituting other types of assays, such as
ELISA or photometric assays. The major drivers behind this
development are the benefits offered by HPLC and LC–MS
with regard to detection specificity compared with cross-
reactivity often observed in enzymatic assays or immunoassays.
HPLC and LC–MS in particular can offer unambiguous
detection and wide dynamic ranges for quantitation even
when multianalyte methods are applied. HPLC is now used in
parallel to GC–MS, and both methods cover the majority of
potential target analytes in the clinical environment. GC–MS
remains indispensable in the analysis of small volatile mole-
cules, organic acids, and other key analytes in clinical and
forensic toxicology; however, HPLC–MS/MS offers higher sen-
sitivity while reducing the time and labor required for sample
preparation (e.g., for derivatization necessary prior to introduc-
tion in GC). A major limitation however, is the lack of com-
mercial mass spectral libraries, which necessitates the
generation of local databases (lab-built libraries). At present,
HPLC is practiced in virtually all hospitals in the developed
world, while most of the major hospital laboratories are
equipped with a variety of LC–MS and LC–MS/MS systems.
Analytic tasks are distributed to specialized laboratories that
are national reference centers for the analysis of groups of key
analytes (e.g., amino acids, endocrine disruptors, and
antibiotics). HPLC–MS/MS systems are widely used in clinical
chemistry laboratories and pharmacology, toxicology, and
forensic facilities. These laboratories are tasked with the analy-
sis of toxins and drugs of abuse, therapeutic drug monitoring
(TDM; such as the quantification of pharmaceuticals with a
narrow window of therapeutic level concentration, e.g., certain
groups of antibiotics and the majority of immunosuppressants
and antipsychotic drugs), clinical trials of new pharmaceuticals,
and the emerging field of bioequivalence of generic drugs.
Emerging fields such as the assessment of occupational expo-
sure and workplace drug testing represent new application
areas.
Conclusions
HPLC offers an indispensable analytic tool in modern science,
with thousands of new methods and protocols published every
year. Instrument and column manufacturers continue to
improve the specifications, performance, and quality charac-
teristics of their products making the technology more robust,
reliable, and capable to address emerging analytic problems
resulting from modern human activities. As HPLC represents a
 Chromatography: High-Performance Liquid Chromatography
key element in the majority of bioanalytical and biotechnology
fields, the central position of the technology seems secure for
many years to come. A general advice for the practitioners,
especially those coming from other scientific fields and using
HPLC only as a tool for a certain purpose, is to remember that
LC represents a dynamic process; hence, results can be influ-
enced by different physicochemical factors. The sophistication
of current state of the art is based on sound scientific principles
and decades of dedicated work from thousands of scientists
from the manufacturer and the research sectors. This striking
science can be recognized every day in the separations achieved
in thousands of LC systems globally. Separations undreamed
of some years ago can now be achieved conveniently and
offered as kits or standard operating protocols (SOPs). The
continuing investments from the academia and the manufac-
turers will maintain the drive toward lower detection limits,
higher resolution power, and higher quality separations.
See also: Antibiotics and Drugs: Residue Determination;
Chromatography: Combined Chromatography and Mass Spectrometry;
Chromatography: Focus on Multidimensional GC; Mass Spectrometry:
Principles and Instrumentation.
Further Reading
Bernal J, Ares AM, Pól J, and Wiedmer SK (2011) Hydrophilic interaction liquid
chromatography in food analysis. Journal of Chromatography. A 1218: 7438–7452.
Cazes J (2009) Encyclopedia of chromatography, 3rd ed. New York: Taylor & Francis.
Corradini D (2010) Handbook of HPLC, 2nd ed. Boca Raton, FL: CRC Press.
DeStefano JJ, Schuster SA, Lawhorn JM, and Kirkland JJ (2012) Performance
characteristics of new superficially porous particles. Journal of Chromatography. A
1258: 76–83.
Fanali C, Dugo L, Dugo P, and Mondello L (2013) Capillary-liquid chromatography
(CLC) and nano-LC in food analysis. TrAC-Trends in Analytical Chemistry
52: 226–238.
Farré M and Barceló D (2013) Analysis of emerging contaminants in food. TrAC-Trends
in Analytical Chemistry 43: 240–253.
99
Gika HG, Theodoridis GA, Plumb RS, and Wilson ID (2014) Current practice of liquid
chromatography–mass spectrometry in metabolomics and metabonomics. Journal
of Pharmaceutical and Biomedical Analysis 87: 12–25.
Jandera P (2013) Advances in the development of organic polymer monolithic columns
and their applications in food analysis—A review. Journal of Chromatography. A
1313: 37–53.
LeDoux M (2011) Analytical methods applied to the determination of pesticide residues
in foods of animal origin. A review of the past two decades. Journal of
Chromatography. A 1218: 1021–1036.
Malik AK, Blasco C, and Picó Y (2010) Liquid chromatography–mass spectrometry in
food safety. Journal of Chromatography. A 1217: 4018–4040.
Maurer HH (2013) What is the future of (ultra) high performance liquid chromatography
coupled to low and high resolution mass spectrometry for toxicological drug
screening? Journal of Chromatography. A 1292: 19–24.
McMaster M (2005) LC/MS: a practical user’s guide. Hoboken, NJ: John Wiley & Sons.
Meyer VR (2010) Practical high-performance liquid chromatography, 5th ed.
Chichester: John Wiley & Sons.
Neue UD (1997) HPLC columns: theory, technology, and practice. New York: John
Wiley & Sons.
Nunez O, Gallart-Ayala H, Martins CPB, and Lucci P (2012) New trends in fast liquid
chromatography for food and environmental analysis. Journal of Chromatography.
A 1228: 298–323.
Olsen BA and Pack BW (2013) Hydrophilic interaction chromatography: a guide for
practitioners. Hoboken, NJ: John Wiley & Sons.
Shephard GS (2009) Aflatoxin analysis at the beginning of the twenty-first century.
Analytical and Bioanalytical Chemistry 395: 1215–1224.
Snyder LR, Kirkland JJ, and Dolan JW (2010) Introduction to modern liquid
chromatography, 3rd ed. Hoboken, NJ: John Wiley & Sons.
Snyder LR, Kirkland JJ, and Glajch JL (2012) Practical HPLC method development, 2nd
ed. Hoboken, NJ: John Wiley & Sons.
Spagou K, Tsoukali H, Raikos N, et al. (2010) Hydrophilic interaction chromatography
coupled to MS for metabonomic/metabolomic studies. Journal of Separation
Science 33: 716–727.
Zhan J, Yu XJ, Zhong YY, et al. (2012) Generic and rapid determination of veterinary
drug residues and other contaminants in raw milk by ultra performance liquid
chromatography–tandem mass spectrometry. Journal of Chromatography B
906: 48–57.
Relevant Websites
http://www.chromacademy.com/hplc-training.html – LCGC Chromacademy.
http://www.chromatography-online.org/index.php – Chromatography-online.
http://www.chromforum.org/index.php – Chromatography Forum.
http://www.chromsoc.com/ – the Chromatographic Society.
http://www.lcresources.com/resources/getstart/index.htm – LC Resources.
 Chromatography: Supercritical Fluid Chromatography
C Galea, D Mangelings, and YV Heyden, Vrije Universiteit Brussel (VUB), Brussels, Belgium
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Supercritical fluid chromatography (SFC) is a variant of high-
performance liquid chromatography (HPLC) in which a super-
critical fluid is used to replace the liquid mobile phase. HPLC,
in particular reversed-phase liquid chromatography, has long
established itself as a pertinent analytical technique in the food
and health areas. Decades of application and research have led
to this established technique being the mainstay of analysis in
all steps of food analysis and health analysis.
SFC is considered as a valuable alternative over HPLC
because the mobile phase properties differ significantly. A
supercritical fluid has properties intermediate between those
of a liquid and a gas. The temperature and pressure of a
component in its supercritical state should be higher than
the critical values, Tc and Pc, respectively, as seen in Figure 1.
When a component is in its supercritical state, it cannot be
liquefied by increasing the pressure. Supercritical carbon dio-
xide (CO2) is most commonly used in SFC, mostly because it
is cheap, readily available, and environmentally friendly.
Moreover, it is very safe to be used in the food industry since
it is easily removed from samples by simple expansion and
evaporation.
SFC was from its early years recognized as a hybrid of HPLC
and gas chromatography (GC). SFC was acknowledged as a
technique that enables rapid separations, comparable to
HPLC, and allows separation of substances with high boiling
points, which is also possible in GC. The properties of super-
critical CO2 differ from those of liquids used in HPLC, in the
sense that CO2 is less viscous and has a higher diffusivity than
liquids. This leads to rapid mass transfer, therefore resulting in
a higher sample throughput, faster equilibration, and shorter
cycle times. Since solvent consumption is low, waste genera-
tion is also negligible.
Supercritical fluids are used for extraction and for chroma-
tography. Food products analyzed or produced by the utiliza-
tion of supercritical fluids are seen in everyday life. Some of
these products are highlighted in Figure 2.
The main difference between an HPLC and SFC system is
that in SFC, the pumping system needs to be adapted to allow
the pumping of a compressible fluid (Figure 3). The second
difference is that there is a pressure regulation system, which
restricts the column outlet flow and creates a system back
pressure, keeping CO2 in its super-/subcritical state. It is
known that the solvent strength of the supercritical mobile
phase is strongly related to its density (being controlled by
temperature and pressure). It is also well known that the
polarity of the nonpolar carbon dioxide can be increased by
the addition of polar organic solvents, such as methanol.
Hence, the partition between the mobile phase and stationary
phase, of analytes with varying polarities, can be changed and
their retention altered.
100 Encyclopedia of Food and Health
SFC can be coupled with a number of other techniques,
for example, mass spectrometry (MS), ultraviolet (UV)–Vis
spectroscopy, flame ionization detection (FID), LC, and super-
critical fluid extraction (SFE). These numerous advantages are
quite appealing and are possibly the reasons why there has
been an increase in the utilization of this technique in both the
food and health sectors. This is reflected by the number of
publications in the area, which has shown a steady increase
in the last years.
SFC is a versatile technique and can be used to analyze a
large number of compounds. However, it has the unique ability
of analyzing thermally labile and nonvolatile substances. Sub-
stances with such properties are also found in the food and
health industries, for example, thermolabile pesticides, fatty
acids, and numerous medicinal drugs. Moreover, SFC is a pow-
erful technique in the separation and analysis of chiral sub-
stances. A chiral compound is one that consists of stereoisomer
pairs referred to as enantiomers that have nonsuperimposable
mirror images. Chirality may lead to one enantiomer being
responsible for the biological activity, while the other might
be much less potent as in the case of adrenaline. Antagonistic,
toxic, and no effects are also possible with the other enantio-
mer. Twenty-five percent of pesticides used in the food industry
are also chiral. The importance of chirality was highly recog-
nized after the thalidomide (Softenon®) disaster in the early
1960s. Thalidomide was being used by pregnant women to
treat morning sickness. However, it was later shown that one
enantiomer was responsible for the teratogenic effect, affecting
fetuses during their development, resulting in babies with miss-
ing extremities. This article will focus on the applications of SFC
in food, health, and drug analysis. The studies presented are not
exhaustive, but they will give the reader a good understanding
of the applicability of SFC in different domains.
SFC in Food Analysis
Food analysis is a sector of vital importance in analytical chem-
istry, which may sometimes prove to be a challenge due to the
number and wide variety of compounds that must be analyzed.
These compounds range from nutrients and compounds with
bioactivities to pesticides, contaminants, or illicit substances
that are considered harmful. Food analysis with SFC focussed
mostly on lipids and fat-soluble vitamins, probably due to the
high solubility of these analytes in CO2. However, the trend is
moving to use SFC also for the analysis of more polar com-
pounds, for example, amino acids and carbohydrates, and the
analysis of compounds with low volatility or UV absorbance
such as triterpenoid compounds. This section presents an over-
view of different applications of SFC in food analysis. Table 1
shows a summary of SFC applications in food analysis, which
will be discussed shortly.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00160-4
 Chromatography: Supercritical Fluid Chromatography 101

Analysis of Lipids
Supercritical Lipids play a vital role in cellular signaling and energy storage,
Solid Liquid
fluid while phospholipids are the main cell membrane constituent.
Lipids are hydrophobic compounds, but they vary greatly in
their polarity due to the diverse moieties that can form part of
Pressure (bar)

Critical point- Tc, Pc lipids. Some include hydrophobic acyl chains; others, hydro-
(31.1 ⬚C, 73.8 bar) philic phosphoric acid or sugar moieties. Therefore, analyzing
lipids in a mixture is not easy. The study of lipid classes in their
Vapor/gas natural environment is referred to as lipidomics.
An SFC–MS method that allows the simultaneous analysis of
Triple point lipids with diverse structures and polarities has been established
(-56.4 ⬚C, 5.11 bar) by Bamba et al. SFC–MS was used to analyze a complex mixture
of 14 lipids, including phospholipids, glycolipids, neutral lipids,
Temperature (⬚C) and sphingolipids, extracted from the leaf of Catharanthus roseus.
All lipids were successfully separated within 15 min. This system
Figure 1 Phase diagram for carbon dioxide. is suitable for studies on lipidomics because it is useful as a
fingerprinting method for the screening of diverse lipids and
also for the profiling of the specific constituents.

Drinks
• Decaffeinated tea and coffee
• Flavor-enhanced orange juice
• De-alcoholized wine and beer
• Beer brewed with CO2-hop extracts

Food items
• Defatted meat
• Defatted french fries
• Parboiled rice with CO2

Others
• Extraction of antioxidants (e.g., coriander, rosemary, and
vitamin E)
• Removal of pesticides
• Fried oil purification

Figure 2 The application of supercritical fluids to daily food production.

CO2 supply Condenser CO2 pump Mixer

Autosampler

Co-solvent supply Co-solvent pump Column

Detector

Pressure regulation

Waste

Figure 3 Supercritical fluid chromatography process diagram.

102 Chromatography: Supercritical Fluid Chromatography

Table 1 Summary of some SFC applications in food analysis (abbreviations are explained in the text)

Analyte Sample Detector Stationary phase Mobile phase References

Lipid analysis
DHA and EPA Transesterified UV Octadecyl silica Pure CO2 [1]
concentrates tuna oil
EPA-EE Fish oil Not Silica Pure CO2 [2]
mixtures specified
Phospholipids Soybean flakes Not Alumina (preparative) CO2 modified with a 5–30% [3]
specified ethanol/water 9:1(v/v) eluant
Fat-soluble vitamins
b-carotene Standard DAD ODS and C30 CO2 and varying percentages of [4]
mixture methanol
Tocopherols Olive by- MS Capillary column pack with silica particles Pure CO2 [5]
products and 10% polyethylene glycol
Tocopherols Standard UV Silica CO2 and varying percentages of [6]
mixtures ethanol
Carotenes, Palm oil UV Silica CO2 with 4% ethanol [7]
vitamin E,
sterols, and
squalene
Carotenoids and Human serum MS Silica, bonded phenyl, carotenoid, and ODS CO2 and varying gradients of [8]
their and LDL methanol with 0.1% ammonium
epoxidized samples formate as additive
products
Nutraceuticals
Phenol Rosemary UV Specially designed column packed with CO2 and varying percentages of [9]
diterpenes silica particles coated with nonpolar SE- ethanol
54 (5% phenyl silicone, 95% methyl
silicone)
Carnosic acid Rosemary FID Specially designed column packed with Pure CO2 [10]
and carnosol silica particles coated with nonpolar SE-
54
Davanone Natural davana UV 2-Ethylpyridine CO2 and a methanol gradient [11]
oil (1–20%)
Polyphenols Grape seed UV 2 Diol columns coupled in series CO2 and a methanol gradient [12]
extract (17–45%) with different
additives
Glucobrassicin White cabbage UV Silica CO2 and varying gradients of [13]
methanol
Sinalbin Mustard UV Silica CO2 and a methanol gradient [14]
(4–15%) with trifluoroacetic
acid as additive
Monitoring of pesticides
Triazole Standard UV Chiralpak AD CO2 and varying percentages of [15]
pesticides preparations methanol, ethanol, or 2-
propanol and with triethylamine
and trifluoroacetic acid as
additives
Multiple Canned foods, UV Silica CO2 and varying percentages of [16]
pesticide fruit, and methanol
residues vegetables
Other compounds
Underivatized Standard ELSD Diol bonded to silica CO2 and varying gradients of [17]
amino acids mixtures methanol and water,
triethylamine and trifluoroacetic
acid as additives
Caffeine, Standard ELSD Ethylpyridine bonded to silica CO2 and a methanol gradient [18]
fructose, mixture (5–50%)
glucose,

(Continued)
 Chromatography: Supercritical Fluid Chromatography 103

Table 1 (Continued)

Analyte Sample Detector Stationary phase Mobile phase References

sucrose, and
NHDC
Triterpenoid Apple pomace ELSD Ethylpyridine bonded to silica and CO2 and varying percentages of [19]
compounds extracts phenyloxy propyl methanol

[1] Alkio, M., Gonzalez, C., Jäntti, M. and Aaltonen, O. (2000). Purification of polyunsaturated fatty acid esters from tuna oil with supercritical fluid chromatography. Journal of the
American Oil Chemists’ Society 77, 315–321. [2] Pettinello, G., Bertucco, A., Pallado, P. and Stassi, A. (2000). Production of EPA enriched mixtures by supercritical fluid
chromatography: from the laboratory scale to the pilot plant. The Journal of Supercritical Fluids 19, 51–60. [3] King, J. W. and Srinivas, K. (2009). Multiple unit processing using
sub- and supercritical fluids. The Journal of Supercritical Fluids 47, 598–610. [4] Lesellier, E., Gurdale, K. and Tchapla, A. (1999). Separation of cis/trans isomers of beta-carotene by
supercritical fluid chromatography. Journal of Chromatography A 844, 307–320. [5] Ibanez, E., Palacios, J., Senorans, F.J., Santa-Maria, G., Tabera, J., and Reglero, G. (2000).
Isolation and separation of tocopherols from olive by-products with supercritical fluids. Journal of the American Oil Chemists’ Society 77, 187–190. [6] Jiang, C., Ren, Q. and Wu, P.
(2003). Study on retention factor and resolution of tocopherols by supercritical fluid chromatography. Journal of Chromatography A 1005, 155–164. [7] Choo, Y.M., Ng, M.H., Ma, N.M.,
Chuah, C.H., and Hashim, M.A. (2005). Application of supercritical fluid chromatography in the quantitative analysis of minor components (carotenes, vitamin E, sterols, and squalene) from
palm oil. Lipids 40, 429–432. [8] Matsubara, A., Uchikata, T., Shinohara, M., Nishiumi, S., Yoshida, M., Fukusaki, E., and Bamba, T. (2012). Highly sensitive and rapid profiling method for
carotenoids and their epoxidized products using supercritical fluid chromatography coupled with electrospray ionization-triple quadrupole mass spectrometry. Journal of Bioscience and
Bioengineering 113, 782–787. [9] Ramirez, P., Santoyo, S., Garcia-Risco, M.R., Senorans, F.J., Ibanez, E., and Reglero, G. (2007). Use of specially designed columns for antioxidants and
antimicrobials enrichment by preparative supercritical fluid chromatography. Journal of Chromatography A 1143, 234–242. [10] Ramı́rez, P., Señoráns, F. J., Ibañez, E. and Reglero, G.
(2004). Separation of rosemary antioxidant compounds by supercritical fluid chromatography on coated packed capillary columns. Journal of Chromatography A 1057, 241–245. [11]
Coleman, W. M., Dube, M. F., Ashraf-Khorassani, M. and Taylor, L. T. (2007). Isomeric enhancement of davanone from natural davana oil aided by supercritical carbon dioxide. Journal of
Agricultural and Food Chemistry 55, 3037–3043. [12] Kamangerpour, A. and Ashraf-Khorassani, M. (2002). Supercritical fluid chromatography of polyphenolic compounds in grape seed
extract. Chromatographia 55, 417–421. [13] Buskov, S., Olsen, C. E., Sørensen, H. and Sørensen, S. (2000). Supercritical fluid chromatography as basis for identification and quantitative
determination of indol-3-ylmethyl oligomers and ascorbigens. Journal of Biochemical and Biophysical Methods 43, 175–195. [14] Buskov, S., Hasselstrøm, J. and Olsen, C (2000).
Supercritical fluid chromatography as a method of analysis for the determination of 4-hydroxybenzylglucosinolate degradation products. Journal of Biochemical and Biophysical Methods
43, 157–174. [15] Toribio, L., del Nozal, M.J., Bernal, J.L., Jimenez, J.J., and Alonso, C. (2004). Chiral separation of some triazole pesticides by supercritical fluid chromatography. Journal
of Chromatography A 1046, 249–253. [16] El-Saeid, M. H. (2003). Pesticide residues in canned foods, fruits, and vegetables: the application of supercritical fluid extraction and
chromatographic techniques in the analysis. The Scientific World Journal 3, 1314–1326. [17] Camel, V., Thiebaut, D., Caude, M. and Dreux, M. (1992). Packed column subcritical fluid
chromatography of underivatized amino acids. Journal of Chromatography A 605, 95–101. [18] Lefler, B. J. L. and Chen, R. (2008). A feasibility study of using supercritical fluid
chromatography (SFC)-UV-ELSD for food and beverage analysis. LC-GC North America 6, 42–47. [19] Lesellier, E., Destandau, A., Grigoras, C., Fougere, L., and Elfakir, C. (2012). Fast
separation of triterpenoids by supercritical fluid chromatography/evaporative light scattering detector. Journal of Chromatography A 1268, 157–165.

The following three studies concerning lipids were devel-
oped at analytical scale, optimized, and then upscaled to the
preparative level. The first study ([1] – refer to Table 1) focuses
on the isolation of eicosapentaenoic acid (EPA) and docosa-
hexaenoic acid (DHA) ethyl ester concentrates from transester-
ified tuna oil using SFC. The interest in these compounds is
coming from the fact that EPA and DHA polyunsaturated fatty
acids (PUFAs) are found in fish oils in ranges between 6% and
26% weight of the total fatty acids. These fatty acids have
shown clinical benefits in cardiology, neurology, and mental
health, among others. They continue to be studied due to their
market in food additives and supplements and also in phar-
maceutical products.
Pettinello et al. ([2] – refer to Table 1) investigated the
attainment of enriched fractions of eicosapentaenoic acid
ethyl ester (EPA-EE). Different operating conditions were
investigated at bench scale. The final conditions (Table 1),
which gave a purity of EPA-EE of 90% and a recovery of 49%,
were used as a starting point for pilot plant experiments. This
study continues confirming the versatility of SFC, in that it can
be used for a number of purposes, starting from separation and
analysis to large-scale production.
A subgroup of lipids is the phospholipids. These are com-
ponents of living cell membranes and play an important part
in enzyme activation – thus, phospholipids are important in
nutrition. SFC was used to fractionate phospholipids in a
multiunit process after being extracted from soybean flakes
([3] – refer to Table 1). This process produced purities above
75% for the individual phospholipids, phosphatidylcholine,
and phosphatidylethanolamine.
Analysis of Fat-Soluble Vitamins, Including Carotenoids
Fat-soluble vitamins (A, D, E, and K) play a major role in
anabolism and catabolism. Deficiency of these vitamins can
lead to serious diseases, such as night blindness (vitamin A),
rickets (vitamin D), rupturing of blood cells and cancer (vita-
min E), and blood coagulation disorders (vitamin K). Methods
for the extraction and enrichment of these vitamins from nat-
ural sources like plants and seeds are constantly sought after, to
be able to produce food supplements. SFC has been widely
used for this group of compounds as can be seen from the
following examples.
Tocopherols (a, b, and g) are components of vitamin E and
are particularly interesting for the food industry because of their
antioxidant and nutraceutical activities. SFE and SFC were used
to isolate tocopherols from olive by-products ([5] – refer to
Table 1). The residue obtained from olive pomace was subjected
to SFE with subsequent fractionation by two successive depres-
surizations. Tocopherols were separated and quantified using
SFC subsequently. This study thus resulted in the isolation
of tocopherols in an environmentally friendly way.
In a study by Jiang et al. ([6] – refer to Table 1), the effects of
temperature, pressure, and modifier concentration in the
mobile phase on the retention factor and resolution of tocoph-
erols were studied. It was concluded that the separation of
104 Chromatography: Supercritical Fluid Chromatography
a-tocopherol from g- and d-tocopherols was best at 18 MPa
and 80  C and with 5% ethanol in the mobile phase. This
study also showed that the resolution of tocopherols increases
when the temperature increases and the pressure decreases.
Another study ([7] – refer to Table 1) investigated the appli-
cation of SFC to quantitatively analyze minor components
found in palm oil. Carotenes, five isomers of vitamin E, sterols,
and squalene were separated in < 20 min. Results obtained
from this study were comparable with those from an estab-
lished analytical technique, such as GC. However, the SFC
method showed several advantages. The conventional analysis
of palm oil components requires the use of several techniques,
such as GC, HPLC, and UV–Vis spectroscopy since no tech-
nique is able to identify all components. Moreover, the differ-
ent techniques require different sample preparations. Using
SFC, labor and time were saved for the sample preparation
and separation of all components ( 25 min) from the palm
oil. This study also showed that there was a significant differ-
ence in the concentrations of vitamin E isomers analyzed by
SFC and HPLC. Prolonged exposure of vitamin E isomers to
organic solvents in HPLC resulted in destruction of the com-
pounds, leading to lower levels found than in the SFC analysis.
Carotenoids are naturally occurring fat-soluble pigments,
of which some act as provitamin A. Their potent antioxidant
properties have led to increased investigations as potential anti-
cancer, antiaging, and cardiovascular drugs. Carotenoids are
known to be sensitive to temperature; therefore, low tempera-
tures have to be employed in their analysis. This means that
analysis takes place in subcritical conditions. Lesellier’s research
group ([4] – refer to Table 1) worked on the method develop-
ment and improvement of the separation of b-carotene cis/trans
isomers. The factors that affected retention differences were the
dielectric constant of the modifier, solubility parameters, type
of mobile phase, and stationary phase conformation.
Oxidation of carotenoids leads to the formation of epoxy-
carotenoids, which may be better markers for low-density
lipoprotein (LDL) oxidation, an important process in athero-
sclerosis. A decrease in the carotenoid levels is seen before the
increase in lipid peroxide levels during the oxidation of LDL.
However, the low levels of carotenoids in the blood cannot be
directly correlated to oxidative stress levels, because of indi-
vidual differences in dietary intake. A method employing
SFC–MS was used to identify epoxycarotenoids proving the
sensitivity of the technique and therefore its applicability to
biomarker screening ([8] – refer to Table 1).
Nutraceuticals
A number of natural products, including plants, contain con-
stituents that show biological activities. A nutraceutical is
defined as a food that possesses medicinal or nutritional prop-
erties, including the prevention and/or cure of an illness. Rose-
mary, davana, and grape seed extract are examples of such
harvests, from which beneficial products can be separated
and isolated by SFC as discussed in the succeeding text. Pro-
cesses can then be scaled up to ease their productivity.
Rosemary is well known for its antioxidant properties. The
most active compounds are the phenol diterpenes, primarily
carnosic acid and also carnosol, rosmanol, and epi- and iso-
rosmanol. Ramirez et al. ([10], [11] – refer to Table 1) worked
on the separation and isolation of compounds with anti-
oxidant and antimicrobial properties. Initially, the group
focussed on the optimization of the separation of carnosic
acid from other antioxidants in rosemary extracts obtained by
SFE. Then, semipreparative SFC was used to obtain fractions
with enhanced functional activities. The biologically active
fractions were then shown to have improved antioxidant and
antimicrobial properties. The optimized method resulted in
two very active fractions. The CO2-based mobile phase with a
low percentage of modifiers has advantages for upscaling, since
the lower the percentage, the lower the costs and residues
incurred and the easier the evaporation of the mobile phase.
Davana is an aromatic herb from India. The oil of davana
was initially only recognized for its importance in the perfume
industry. However, davanone, a sesquiterpene, is a principal
component of this oil. Davanone exhibits antifungal and anti-
spasmodic properties. This compound was separated from the
bulk matrix or natural davana oil by SFC ([11] – refer to
Table 1). The final sample was nearly 100% optically pure.
Grape seed extract is a popular nutritional supplement with
polyphenols that have antioxidant properties. Phenolics vary
in their distribution and content depending on the raw mate-
rial. Kamangerpour et al. ([12] – refer to Table 1) worked on
the development of a fast SFC method for the isolation and
identification of these polyphenols. The method was then used
to separate phenolics in a grape seed extract.
Two other examples of nutraceuticals extracted from plants
are glucobrassicin and sinalbin, both glucosinolates, with anti-
cancer properties, coming from the plants of the Brassicaceae
and the seeds of the Sinapis alba L. families, respectively ([13],
[14] – refer to Table 1). White cabbage was used as a source of
glucosinolates, while mustard was used as a source of sinalbin.
The developed SFC methods can be used to separate and
isolate the individual compounds of interest.
Monitoring of Pesticides
Pesticide or chemical residues in food, added during planting
or processing phases, put the health of consumers at risk of
adverse effects. It is therefore vital to monitor, identify, and
quantify pesticide levels in food samples. A number of studies
have used SFC to separate and analyze pesticides, for instance,
contaminants in actual food samples. Toribio et al. ([15] –
refer to Table 1) worked on the enantiomeric separation of
six triazole fungicides, namely, cyproconazole, propiconazole,
diniconazole, hexaconazole, tebuconazole, and tetraconazole.
A polysaccharide-based chiral stationary phase was used and
the effect of different modifiers studied. It was found that the
organic modifier that provided the highest resolutions
depended on the analyzed pesticide.
El-Saeid ([16] – refer to Table 1) monitored pesticide residues
in canned foods, fruits, and vegetables, obtained from a local
market in Houston, Texas. SFE was used to extract the pesticides
from the samples, which were then analyzed by SFC. A total
of 39% of foods tested were found to contain four pyrethroids,
four herbicides, four fungicides, and six carbamates in different
food samples. All the tested foods had different levels of pesticide
residues ranging from 0.03  0.005 to 0.8  0.12 ppm. This
study helped to promote consumer safety by assaying pesticide
residues in food items.

Analysis of Other Compounds
Due to the nonpolarity of CO2, SFC is mainly used in the
analysis of hydrophobic or weakly polar compounds. However,
by modifying the mobile phase polarity, a wider range of com-
pounds can be analyzed. Figure 4 shows a chromatogram of a
separation of amino acids, which are very polar compounds:
proline (Pro), threonine (Thr), hydroxyproline (Hyp), serine
(Ser), glutamic acid (Glu), and aspartic acid (Asp). Methanol,
with water and triethylamine as additives, was used as a modi-
fier to separate mixtures of underivatized amino acids. When a
modifier gradient was used, even a higher number of amino
acids were resolved in < 10 min ([17] – refer to Table 1).
Carbohydrates were also separated with SFC ([18] – refer to
Table 1). Mixtures of caffeine, fructose, glucose, sucrose, and
neohesperidin dihydrochalcone (NHDC), which is an artificial
sweetener, are commonly found in soft drinks. The mixture
was separated in < 5 min when SFC was used. Another sweet-
ener, sucralose, was also identified and quantified in two sports
drinks using SFC. Sucralose is roughly 600 times sweeter than
sucrose with only 12% of the calorie count. The beverage
marketed as ‘low calorie’ was indeed found to have a small
amount of the sweetener.
Triterpenoids are recognized to possess a variety of bio-
logical activities, such as antibacterial, antioxidant, antitumor,
antihyperglycemic, anti-HIV, antisclerotic, and cardiovascular
Figure 4 SFC chromatogram of a standard mixture of polar amino
acids. Diol stationary phase; flow rate, 5 ml min 1; inlet pressure,
230 bar; temperature, 0  C; CO2 modifier (80:20, v/v); modifier,
methanol/additives (87.95:12.05, v/v) with water–triethylamine–pyridine
(7:0.05:5, v/v) ([17] – refer to Table 1). Reprinted from Packed column
subcritical fluid chromatography of underivatized amino acids, Camel, V.,
Thiebaut, D., Caude, M. and Dreux, M. (1992). Packed column subcritical
fluid chromatography of underivatized amino acids. Journal of
Chromatography A 605, 95–101. Copyright (1992), with permission from
Elsevier.
Chromatography: Supercritical Fluid Chromatography 105
activities ([19] – refer to Table 1). These compounds can be
found in natural plants and in resinous natural materials.
However, these compounds have a low UV absorbance; thus,
a UV detector could not be used in this case. An alternative
detector, evaporative light scattering detector (ELSD), was
used, which gave acceptable peak intensities.
SFC in Health Analysis
The determination and quantification of drug levels in plasma
play an important role in the monitoring and development of
modern pharmaceutical products. Data generated from these
measurements help in the understanding of toxicological effects
and offer insight to a drug’s mode of action (pharmaco-
dynamics) and are used to establish pharmacokinetic parame-
ters related to the absorption, distribution, metabolism, and
elimination of the drug. Such studies are needed for all drugs
in order to obtain regulatory approval. The following are just
three examples of the use of SFC for health analysis, which help
contribute to the safety and efficacy of medicinal products in
patients.
Malaria is still prevalent in many areas of the world, and it
continues to be studied to find treatments. Artemisinin, also
known as qinghaosu (QHS), mefloquine, and sulfadoxine are
all compounds recognized as having antimalarial activity.
Developing GC and HPLC methods is challenging because
on the one hand, these compounds are thermally labile,
while on the other hand, they do not contain a UV, visible of
fluorescent chromophore to facilitate their detection. Acidic
hydrolysis or basic hydrolysis of QHS was done to produce a
UV chromophore, but acidic hydrolysis lacked specificity,
while the basic hydrolysis method required a lot of experimen-
tal work. An SFC–ECD (electron capture detection) method
proved to be sensitive enough to measure QHS levels for
therapeutic drug monitoring by Mount et al. This method
had a limit of detection of 20 ng ml 1. However, this limit
can be lowered further by decreasing the baseline noise of the
detector, possibly by using carbon dioxide of a better quality.
This SFC–ECD method was also used to quantify mefloquine
in blood samples. More work was required for sample prepa-
ration when compared with HPLC–UV and GC–ECD
methods, but the method proved to be more sensitive and
selective than HPLC–UV and more robust than GC–ECD. In
another study by Bhoir et al. concerning the method develop-
ment for the measurement of sulfadoxine levels in human
plasma, SFC was found to be sensitive, rapid, and re-
producible. The SFC method was found to be superior to an
HPLC–UV method in terms of speed and cost.
Ketotifen, an analgesic, is another medicine where pharma-
cokinetic studies were done by SFC. A method for the determi-
nation of (R)- and (S)-ketotifen in human plasma was
developed. Oral ketotifen shows a bioavailability above 92%;
however, the maximum plasma concentrations reach low
microgram per milliliter levels after a 25 mg dose. SFC was
used to plot a plasma drug concentration versus time curve
for both enantiomers by Hoke et al. A chiral stationary phase
consisting of (R)-1-naphthylglycine and 3,5-dinitrobenzoic
acid was used. The method was shown to be valuable to
106 Chromatography: Supercritical Fluid Chromatography
measure ketotifen levels in human plasma, between 0.05 and
2500 ng ml 1, after both topical and oral administration.
A final example concerns the use of the antineoplastic anti-
metabolite cytarabine, which is used in the treatment of leuke-
mia. To understand better the pharmacodynamics of cytarabine,
its plasma concentrations were monitored using an SFC–MS/MS
method and an HPLC–MS/MS method by Hsieh et al. The
results obtained from both methods displayed equivalent
accuracy, and they can thus be used in an alternative manner.
SFC for Preparative Use
Preparative chromatography has had a significant impact on
both food and pharmaceutical industries. The advantage of
this technique arises from the ease of scalability, robustness of
the equipment, and the possibility to separate diverse mixtures.
Moreover, this technique can be used routinely for purification,
from a few grams to tons per day. When SFC is used for prepar-
ative purposes, once again, the technique exhibits advantages
over HPLC. In preparative SFC, the mobile phase is recycled
online, which is not the case for preparative HPLC. Other
advantages of prep-SFC stem from the inherent properties of
CO2 like the higher diffusivity, low viscosity, and retention
control by the variation of pressure and temperature. Productiv-
ity in preparative chromatography is increased by the use of
overlapping or stacked injections. This is a technique where a
second injection is made before all the substances from the first
injection have eluted from the stationary phase. Restrictions of
prep-SFC include samples that are not soluble in methanol or
CO2, high-polarity compounds, and, just like in HPLC, cases
where impurities coelute with a particular product.
Prep-SFC is used for the separation of diverse compounds in
given mixtures, for example, the production of functional food
ingredients. It is commonly utilized for the fractionation and
purification of extracts acquired with SFE. Triglycerides, diglycer-
ides, free fatty acids, carotenes, tocopherols, and tocotrienols
were successfully separated from crude palm oil. Purified PUFA
ethyl esters were obtained from tuna oil, while free sterols and
ferulate phytosterol esters were attained from corn bran. The oil
that is obtained as a by-product of corn bran dry milling contains
cholesterol-lowering phytosterols – which are thus interesting
for the nutraceutical market. Prep-SFC was also applied by Li
et al. to the separation of polymethoxyflavones (PMFs), mainly
nobiletin, tangeretin, 3,5,6,7,3’,4’3-heptamethoxyflavone, and
5,6,7,4’-tetramethoxyflavone, from orange peel extracts. PMFs
show anti-inflammatory, anticarcinogenic, and antiatherogenic
properties. The botanical species, Thymus vulgaris L. is well
known for its food flavoring properties. However, thymol,
which is a monocyclic terpenoid found in thyme, possesses
antiseptic, analgesic, and anti-inflammatory properties. This ter-
penoid was extracted and isolated by prep-SFC, with a recovery
of up to 97% of the total thymol content by Garcia-Risco et al.
Moving to drug analysis, prep-SFC has already been imple-
mented as a routine tool in a number of pharmaceutical labo-
ratories. White has described the integration of prep-SFC for
large-scale chiral purification. The technique proved to reduce
the overall sample throughput time and running costs while
delivering high-purity fractions and acceptable recoveries. In
another study by Toribio et al., the semipreparative chiral
separation of lansoprazole, pantoprazole, and rabeprazole
was presented. The recoveries and production rates obtained
for the first eluted enantiomer were lower than those obtained
for the second, especially if high-purity levels were aimed at.
Several fractions of the first enantiomer were contaminated
with the second, decreasing the recovery and production
rates. This is possibly due to overloading conditions, where
the peaks were distorted in the front.
Other Use of Supercritical Fluids in Food and Health:
Extraction
SFE is the most common application for supercritical fluids.
The process takes advantage of the properties of supercritical
fluids, just like SFC. The major parameters on which the extrac-
tion depends are temperature, pressure, and flow rate. Super-
critical CO2 has been used to extract a wide range of analytes,
ranging from essential oils to phytochemicals. These extracts
can then be used for analytical purposes, for supplementation,
and for flavor and fragrance purposes.
In addition to the benefits of using CO2 in SFC, namely, the
higher speed and lack of chemical residue, still, a few more
advantages can be seen in SFE. Supercritical CO2 shows no
surface tension. Therefore, it penetrates the pores of heteroge-
neous matrices rapidly, helping to enhance the extraction effi-
ciency. Moreover, the CO2 used in an extraction can be
recycled and reused. Products obtained from the extraction
can then be further separated into different components by
SFC. From the analytical point of view, SFE is compatible with
SFC since the two techniques can share the mobile phase and
some devices, like the CO2 pump and condenser.
Some constituents extracted by SFE include lycopene from
tomato seeds and skins, ginger oleoresin from ginger, oil from
coriander seeds, and caffeine, theobromine, and cocoa butter
from Brazilian cocoa beans. In the pharmaceutical sector,
hyperforin and adhyperforin have been extracted from Hyper-
icum perforatum L., commonly known as St. John’s wort. Prod-
ucts from St. John’s wort are known to have anti-inflammatory,
antidepressive, antiviral, and antimicrobial effects.
SFE can also be used to determine the presence of drug abuse
in human hair. Amphetamines, such as methylenedioxyethy-
lamphetamine, methylenedioxymethamphetamine, and methy-
lenedioxyethylamphetamine, can be investigated with a fast and
reproducible method. The SFC method gives results that are
similar to those obtained from established techniques used to
extract amphetamines from hair, such as solid phase extraction
and liquid–liquid extraction.
Conclusions
This article briefly explained the benefits of using supercritical
CO2 in the food and health sectors, with special reference to
the SFC technique. SFC is considered as an environmentally
friendly technique since the use of supercritical CO2 reduces
the consumption of organic solvents, CO2 is obtained as a by-
product of fermentation processes, and minimal waste is gen-
erated. The high diffusivity and low viscosity of CO2 enable
experiments to be conducted at higher flow rates, therefore
achieving a higher analysis throughput without compromising
on efficiency. These properties are leading to an expansion of

SFC applications in the food and health industries. The use of
SFC continues to expand, and we anticipate it will continue
growing in the future.
SFC is maturing and is establishing its position in analytical
and preparative analyses. The technique provides highly accu-
rate, precise, and sensitive measurements, especially when
coupled with an MS detector. SFC has been conducted on a
broad range of food, plant, and biological samples for the
analysis of minor components, additives, contaminants, and
medicinal products. Moreover, the technique can also be used
to characterize compounds, for example, in lipidomics.
The separation mechanism of SFC can be compared with
that of normal-phase liquid chromatography (NPLC). Just like
in NPLC, in SFC, the polarity of the mobile phase here, super-
critical CO2, can be modified by the addition of polar modi-
fiers. In this way and also by adjusting the density of the CO2,
one can vary the retention of the analytes on the stationary
phase. SFC can be seen as a complementary technique to HPLC
since many separations that are impractical or impossible to
accomplish by HPLC can more easily be achieved by SFC.
Online coupling of SFE with SFC facilitates high through-
put analysis as long as the compounds under study are soluble
in CO2. This system is suitable for the analysis of compounds
that are easily destroyed during organic solvent extraction, for
example, by the high temperature, in a Soxhlet extractor.
The versatility and wide applicability of SFC in the food and
health sectors are very promising. More research is needed to
discover the full application potential of this technique. In the
coming years, more applications of SFC are expected, not only
for research purposes but also more in routine agricultural,
food, and clinical applications.
See also: Fatty Acids: Determination and Requirements; Herbs:
Composition and Dietary Importance; Pesticides and Herbicides:
Residue Determination; Pesticides and Herbicides: Types, Uses, and
Determination of Herbicides; Supercritical Fluid Extraction;
Tocopherols: Properties and Determination.
Further Reading
Bamba T, Shimonishi N, Matsubara A, Hirata K, Nakazawa Y, Kobayashi A, and
Fukusaki E (2008) High throughput and exhaustive analysis of diverse lipids by
using supercritical fluid chromatography–mass spectrometry for metabolomics.
Journal of Bioscience and Bioengineering 105: 460–469.
Chromatography: Supercritical Fluid Chromatography 107
Bernal JL, Martı́n MT, and Toribio L (2013) Supercritical fluid chromatography in food
analysis. Journal of Chromatography A 1313: 24–36.
Bhoir SI, Bhoir IC, Bhagwat AM, and Sundaresan M (2001) Determination of
sulfadoxine in human blood plasma using packed-column supercritical fluid
chromatography. Journal of Chromatography B: Biomedical Sciences and
Applications 757: 39–47.
Brunner G (2005) Supercritical fluids: technology and application to food processing.
Journal of Food Engineering 67: 21–33.
Garcı́a-Risco MR, Vicente G, Reglero G, and Fornari T (2011) Fractionation of thyme
(Thymus vulgaris L.) by supercritical fluid extraction and chromatography. The
Journal of Supercritical Fluids 55: 949–954.
Hoke SH, Pinkston JK, Bailey RE, Tanguay SL, and Eichhold TH (2000) Comparison of
packed-column supercritical fluid chromatography–tandem mass spectrometry with
liquid chromatography–tandem mass spectrometry for bioanalytical determination
of (R)- and (S)-ketoprofen in human plasma following automated 96-well solid-
phase extraction. Analytical Chemistry 72: 4235–4241.
Hsieh Y, Li F, and Duncan CJG (2007) Supercritical fluid chromatography and
high-performance liquid chromatography/tandem mass spectrometric methods for
the determination of cytarabine in mouse plasma. Analytical Chemistry
79: 3856–3861.
Li S, Lambros T, Wang Z, Goodnow R, and Ho CT (2007) Efficient and scalable method
in isolation of polymethoxyflavones from orange peel extract by supercritical fluid
chromatography. Journal of Chromatography B: Analytical Technologies in the
Biomedical and Life Sciences 846: 291–297.
Majewski W, Valery E, and Ludemann-Hombourger O (2005) Principle and applications
of supercritical fluid chromatography. Journal of Liquid Chromatography & Related
Technologies 28: 1233–1252.
Mount DL, Todd GD, and Navaratnam V (1995) Packed-column supercritical fluid
chromatography of artemisinin (qinghaosu) with electron-capture detection.
Journal of Chromatography B: Biomedical Sciences and Applications
666: 183–187.
Rozzi NL and Singh RK (2002) Supercritical fluids and the food industry.
Comprehensive Reviews in Food Science and Food Safety 1: 33–44.
Señoráns FJ and Ibañez E (2002) Analysis of fatty acids in foods by supercritical fluid
chromatography. Analytica Chimica Acta 465: 131–144.
Toribio L, del Nozal MJ, Bernal YL, Alonso C, and Jimenez JJ (2008) Semipreparative
chiral supercritical fluid chromatography in the fractionation of lansoprazole and
two related antiulcer drugs enantiomers. Journal of Separation Science
31: 1307–1313.
Turner C, King JW, and Mathiasson L (2001) Supercritical fluid extraction and
chromatography for fat-soluble vitamin analysis. Journal of Chromatography A
936: 215–237.
White C (2005) Integration of supercritical fluid chromatography into drug discovery as
a routine support tool: I. Fast chiral screening and purification. Journal of
Chromatography A 1074: 163–173.
Relevant Websites
http://www.chromatographyonline.com/lcgc – Supercritical Fluid Chromatography
(SFC): A Review of Technical Developments.
http://cnx.org – Basic Principles of Supercritical Fluid Chromatography and
Supercritical Fluid Extraction.
 Chromium: Physiology
JB Vincent, The University of Alabama, Tuscaloosa, AL, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Recently, a paradigm shift has occurred in the understanding of
the physiological effects of trivalent chromium, Cr3þ. While
initially proposed to be an essential trace element for mam-
mals in the late 1950s, the initial studies have been found in
hindsight to be methodologically flawed. As a consequence of
this and recent data, no unambiguous data for chromium
being an essential element exist. The data demonstrating
beneficial effects are best interpreted in terms of a pharmaco-
logical role for chromium at supranutritional doses. While
guidelines from governmental agencies and textbooks have
not yet caught up with the recent literature, chromium should
be removed from the list of essential trace elements.
Absorption, Transportation, Storage, and Excretion
Chromium Absorption
While aspects of the mechanisms of absorption and transport of
Cr3þ ions are known, overall, the process is poorly elucidated.
Most notably, little is known of the fate of Cr3þ taken orally
before the Cr reaches the bloodstream. Essentially, no data exist
on the forms of Cr3þ in food as a result of its very low concen-
tration. The fate of dietary chromium at a molecular level in the
digestive tract is also essentially unknown, although >98% of
Cr3þ passes through without being absorbed. This lack of
knowledge is in stark contrast to what has been established for
ferric iron (Fe3þ), with a similar charge to size ratio as Cr3þ.
Dietary iron is probably primarily in the ferric form. Absorption
of iron takes place in the proximal portion of the duodenum.
Unlike Cr3þ, whose reduction potential is such that it should not
readily be reduced under the conditions in the gastrointestinal
tract, ferric ions can be reduced chemically or enzymatically by a
brush border ferrireductase of the enterocytes. Subsequently,
iron enters the enterocytes as ferrous iron via the transmem-
brane protein DMT1 (divalent metal transporter 1), which is
probably responsible for the entry of a variety of divalent metal
ions. The transported iron is then stored or is released from the
enterocyte by the basolateral transmembrane protein ferropor-
tin. Iron probably passes through the enterocyte in the ferrous
state (Fe2þ) and is returned to the ferric state (Fe3þ) by ferrox-
idases for transport in the bloodstream by the protein transferrin
(vide infra). The failure of Cr3þ ions to be reduced requires a
unique absorption system to be present for chromium, com-
pared with other proposed essential metals ions, if chromium is
actively absorbed and essential.
However, the preponderance of evidence suggests that
chromic ions are passively absorbed, that is, absorbed via simple
diffusion. In rats, chromium has been established to be
absorbed via passive diffusion. Only a small percentage (<1%)
of dietary Cr is absorbed, while the remainder is excreted in the
feces. Inorganic chromium salts are generally used to mimic
dietary chromium and are absorbed to a similar extent, that is,
108 Encyclopedia of Food and Health
<1%, over a wide range of doses. (The most commonly used salt
is chromium(III) chloride hexahydrate, which is actually the
chloride salt of the trans isomer of the [CrCl2(H2O)4]þ cation.)
Chromium supplementation of the diet results in an increase in
urinary chromium loss, and most of the absorbed chromium is
rapidly excreted. Recently, rats that were gavage-dosed with
CrCl3 were found to exhibit  0.2% absorption of Cr over a
2000-fold range of doses, consistent with the absorption of
simple inorganic chromium(III) salts occurring via diffusion
and not active transport. The concentration of Cr in the urine,
blood, and tissues is proportional to intake. In fact, Cr content
of the liver and kidney for multiple complexes of Cr(III) has
been shown to increase linearly with intake. Urinary Cr loss
appears to be controlled by intake. When followed by isotopi-
cally labeled chromium (50Cr or radioactive 51Cr) in rats or
humans with increased urinary Cr loss from diabetes or strenu-
ous exercise, the increased urinary Cr loss has been found to be
offset by an increase in Cr absorption. In fact, the increased
uptake of water in these conditions may simply result in the
increased diffusion of Cr and the subsequent increase in urinary
chromium loss.
Additional data come from studies of the absorption of
Cr3þ from a rat intestinal perfusate with added Cr as CrCl3. A
double-perfusion technique has been utilized in which the
intestinal vasculature, from the superior mesenteric artery to
the portal vein, and the intestinal lumen, from the duodenum
to the ileum, were perfused simultaneously. Over a greater than
100-fold range of Cr3þ concentrations, Cr absorption was
found to be a nonsaturable process, consistent with animal
studies. These results led to the conclusion that the Cr3þ was
absorbed by the nonmediated process of passive diffusion in
the small intestine of rats fed with a Cr-adequate diet.
However, the fate of dietary chromium(III) organic
complexes could be different to that of the inorganic chro-
mium salts if the complex (or some product thereof) absorbed
is different from the form absorbed using inorganic chromium
(III) salts. For example, the presence of added amino acids,
phyate (high levels), and oxalate in the diet reportedly alters Cr
uptake, as does ascorbic acid. Yet, none of these studies
disproves that absorption is passive, only that the extent of
chromium available for diffusion may be altered by the
addition of these potential chelators to the diet and that the
potential chelators may alter steps subsequent to absorption.
The possibility that complexes of chromium with organic
ligands may have altered the absorption properties is part of
the impetus behind the various Cr(III) complexes used or
proposed as nutritional supplements, although generally, this
has not proven to be the case. In fact, studies comparing the
blood, urine, and tissue concentration in rats of 51Cr from
labeled CrCl3 and the most popular chromium supplements,
chromium picolinate and chromium nicotinate, indicate that
all are absorbed to a similar extent within experimental error.
Another interesting conclusion that can be drawn from the
intestinal perfusate studies is that chromium appears to be
http://dx.doi.org/10.1016/B978-0-12-384947-2.00162-8

actively transported out of the intestinal cells, as nearly all of the
chromium entering the cells is cleared from the cells (leaving
<10% behind to be stored). Yet, no transporter is known for
chromium. Cr3þ bound to some chelating ligand has been
proposed to be transported by monocarboxylate transporters
(MCT) as occurs for chelated Al3þ (a nonessential metal ion).
Chromium complexes with free carboxylates are known; for
example, the complex formed between Cr3þ and EDTA (ethyl-
enediaminetetraacetate) is the violet [Cr(Hedta)(H2O)], where
one of the carboxylate groups is protonated. More importantly,
the common complex of Cr3þ and citrate possesses a free
carboxylate in the solid state and in solution. This idea is also
supported by the perfusate studies. When amino acids were left
out of the solutions perfused through the intestinal lumen, less
chromium was transported into the vascular perfusate, while
additional chromium was retained by the intestinal mucosa.
Thus, potential ligands for the chromium may need to be trans-
ported into the intestinal cells for chromium to be efficiently
transported out of the cells. However, the recent studies showed
that monocarboxylate transporters are not involved in Cr3þ
transport, at least from endosomes in hepatocytes.
Chromium Transport from the Blood to the Tissues
The fate of chromium in the bloodstream is better understood.
In vivo administration of Cr3þ to mammals by injection results
in the chromic ions being bound by the iron-transport protein
transferrin, while administration of 51CrCl3 by stomach tube to
rats results in 80% of the chromium immunoprecipitating with
transferrin. In vitro studies of the addition of chromium sources
to the blood or blood plasma also result in the loading of
transferrin with Cr(III), although under these conditions, albu-
min and some degradation products also bind chromium.
When chromium is added to the blood in vitro, chromium
binds to both transferrin and albumin; in vivo, only transferrin
binds appreciable quantities of chromium. Thus, studies in
which Cr sources are added to the whole blood, blood plasma,
or serum should be avoided.
Transferrin is an 80 kDa blood serum protein that tightly
binds two equivalents of ferric iron at neutral and slightly basic
pH values. The protein exhibits amazing selectively for Fe3þ in
a biological environment because the metal sites are adapted to
bind ions with large charge to size ratios and is primarily
responsible for the transport of iron through the bloodstream.
In humans, transferrin is maintained only on average  30%
loaded with iron; consequently, the protein has been proposed
to potentially carry other metal ions, although data to support
this under physiological conditions are limited. The similar
charge and ionic radii of chromic ions to ferric ions suggest
that chromic ions should bind relatively tightly to the protein.
In vitro studies of the addition of chromic ions to isolated trans-
ferrin reveal that Cr3þ readily binds to the two-metal-binding
sites, resulting in intense changes in the protein’s ultraviolet
spectrum. Two equivalents of (bi)carbonate are concomitantly
bound. Ultraviolet and Raman spectra suggest that each chromic
ion binds to two tyrosine residues from the protein, strongly
indicating that the chromic ions bind in the two ferric ion bin-
ding sites. Visible spectra suggest the d3 chromic centers are in
pseudooctahedral environments. Variable temperature magnetic
susceptibility studies confirm that the chromium centers are
Chromium: Physiology 109
trivalent (i.e., S ¼ 3/2). Estimates of the binding constant for
chromium and the maintenance of transferrin on average only
30% loaded with ferric ions suggest that the protein appears to
be primed to be able to transport chromium through the
bloodstream.
Chromium-loaded transferrin has been demonstrated to
transport chromium in vivo. Injection of 51Cr–transferrin into
rats results in incorporation of 51Cr into tissues. Injection of
labeled transferrin and insulin results in a severalfold increase
in urinary chromium; iron transfer by transferrin had previ-
ously been shown to be insulin-sensitive. Thus, transferrin, in
an insulin-dependent fashion, can transfer Cr to tissues after
which Cr is ultimately excreted in the urine. Cr2–tranferrin
serves as an inhibitor for the binding of Fe2–transferrin to the
surface of reticulocytes, presumably at the site of binding trans-
ferrin to transferrin receptor. Cr-loaded human transferrin is a
better inhibitor than apotransferrin but not as good as mono-
or diferric transferrin.
Chromium Transport from the Tissues to the Bloodstream
and Urine
Cr3þ appears to be moved from the tissues to the bloodstream
and subsequently urine via a peptide named low-molecular-
weight chromium-binding substance (LMWCr or chromodu-
lin). The peptide is 10 or 11 amino acids long; is composed of
glycine, aspartate, glutamate, and cysteine; and tightly binds
four chromic ions. The peptide has been isolated from mam-
mals, chickens, and alligators where all appear to contain the
contiguous peptide of sequence Glu-Glu-Glu-Glu-Gly-Asp-
Asp. The peptide is rapidly cleared from the body after binding
Cr3þ. Spectroscopic studies indicate that the chromic ions are
bound to the peptide primary via the carboxylate side chain of
the aspartate and glutamate residues. The chromic ions appear
to be arranged in a multinuclear assembly.
The movement of Cr(III)–LMWCr is stimulated by increases
in blood insulin concentration; thus, increased movement of Cr
from the bloodstream to the tissues by transferrin results in a
subsequent elimination of chromium from the body bound to
LMWCr. The peptide appears to be maintained in the tissue in
the apo form; levels of the apo form potentially are under
homeostatic control. At a minimum, LMWCr functions to
rapidly clear Cr from tissues.
The Cr(III)-containing form of LMWCr has been proposed to
be the biologically active form of chromium. In vitro, it has been
found to activate the kinase activity of insulin receptor; this
activation was proportional to the Cr content of LMWCr. In a
proposed mechanistic scheme, at high intakes of Cr, LMWCr
would accumulate to pharmacological levels, bind to the insulin
receptor (helping to maintain the receptor in its active confir-
mation), and result in increased insulin sensitivity. The validity
of this proposal requires testing in vivo.
Metabolic Functions and Deficiency Symptoms
‘Low-Chromium’ Diets
Traditionally, evidence for chromium being an essential
element for mammals has come primarily from four sources:
(i) studies attempting to provide rats with chromium-deficient
diets, (ii) studies examining the absorption of chromium as a
110 Chromium: Physiology
function of intake, (iii) studies of patients on total parenteral
nutrition (TPN), and (iv) studies looking for an association
between insulin action and chromium movement in the body.
The field of Cr nutrition had its beginnings in 1955 when
rats fed with a Torula yeast-based diet apparently developed
impaired glucose tolerance in response to an intravenous glu-
cose load from which a new dietary requirement, coined glu-
cose tolerance factor (GTF), absent from the Torula yeast-based
diet and responsible for the glucose intolerance was proposed.
The active ingredient of GTF was Cr3þ. Inorganic compounds
containing over 40 different elements (200–500 mg kg1 body
mass) could not restore glucose tolerance, while several inor-
ganic Cr(III) complexes (200 mg Cr per kilogram body mass)
restored glucose tolerance. Unfortunately, these Cr studies
were methodologically flawed. For example, the Cr content of
the diet was not reported. The rats were maintained in wire
mesh cages, possibly with stainless-steel components, allowing
the rats to obtain Cr by chewing on these components.
Consequently, the actual Cr intake of the rats in these studies
is impossible to gauge, putting into question the suggestion
that the rats were Cr-deficient. The use of the large amounts of
the metal ions is also of concern; as will be discussed (vide
infra), supranutritional doses of Cr pharmacological effects in
rats with insulin resistance. These doses are probably about
103 times the typical daily content of a rat’s diet.
Details of the ‘isolation’ of Brewer’s yeast GTF were reported
in 1977. However, the isolation procedure included harsh
procedures such as an 18 h reflux in 5 M HCl, which would
hydrolyze any proteins, complex carbohydrates, or nucleic
acids. Nicotinic acid was apparently sublimed from the mate-
rial (although no data or experimental details were presented
for the mass spectral, sublimation, or extraction studies).
Amino acid analyses indicated the presence of glycine, gluta-
mic acid, cysteine, and other amino acids, although the relative
amounts were not reported. The results were interpreted to
indicate that GTF was a complex of Cr, nicotinate, glycine,
cysteine, and glutamate. In paper chromatography experiments,
the material on which the studies in the preceding text were
performed gave several bands, only one of which was active in
bioassays. Based on this work, GTF has been proposed to be a Cr
(III)–glutathione–nicotinate complex as glutathione is a tripep-
tide of glutamate, glycine, and cysteine. A three-dimensional
structure has also been proposed for Brewer’s yeast GTF that
has two trans N-bound nicotinic acid ligands and amino acids
occupying the remaining four sites of an octahedral around the
chromic center. However, Cr3þ has been demonstrated repeat-
edly to be separable from agents in yeast responsible for in vitro
stimulation of glucose metabolism in adipocytes. Thus, the com-
ponent of yeast that is active in the bioassays apparently does not
contain Cr. The characterization of a Cr fraction from yeast was
performed on a mixture that contained multiple Cr-containing
species and other components, and the Cr-containing compo-
nents of the mixture probably do not reflect what bound Cr
initially in the intact yeast. Yet, despite the numerous publica-
tions refuting the GTF studies, these studies unfortunately con-
tinue to be cited as evidence of the essential role of Cr.
Since the initial GTF studies and prior to 2011, the most
notable efforts of work with rats to generate a chromium-
deficient diet were studies utilizing high-sugar or high-fat diets.
For the high-sugar diet studies, rats in plastic cages (with no
access to metal components) were given a purified diet consist-
ing of 55% sucrose and providing 33  14 mg Cr per kilogram
diet. A supplemented pool of rats was given with water contain-
ing 5 ppm CrCl3. At 12 weeks, Cr-deficient rats had lower fasting
plasma insulin concentrations and similar fasting plasma
glucose levels compared with supplemented rats; yet, both
concentrations were similar after 24 weeks. In intravenous
glucose tolerance tests after 24 weeks on the diet, plasma insulin
levels tended to be higher in Cr-deficient rats; rates of excess
glucose clearance were statistically equivalent. Thus, a high-
sucrose diet can lead to hyperinsulinemia that appears to be
partially corrected by chromium. This research group also
obtained similar results using a high-fat diet that contained
33 mg Cr per kilogram diet. After 16 weeks on the diet alone,
rats had higher fasting plasma insulin levels, compared with rats
also receiving drinking water containing 5 ppm Cr. Thus, the
high-fat diet also appears to induce increased fasting insulin
levels, which can be corrected with chromium administration.
Some calculations are in order to put this work into perspective.
Humans lack signs of Cr deficiency with a daily intake of 30 mg
Cr; assuming an average body mass of 60 kg, 30 mg day1
corresponds to 0.5 mg Cr per kilogram body mass per day.
Thus, for a rat, this is equivalent to 5 mg Cr per kilogram body
mass per day, ten times what a human intakes. Thus, the ‘low-Cr’
high-sugar and high-fat diets cannot be said to be deficient at all
unless rats require more than ten times the chromium that
humans do on a per kilogram body mass basis. Consequently,
the effects of the diet cannot be attributed to Cr deficiency; the
high doses of Cr in the Cr-supplemented rats can only be con-
sidered as having a pharmacological role on those rats whose
physical condition were impaired by the high-sucrose or high-fat
diets and/or the other mineral stresses. In 2011, the effects of a
purified diet (AIN-93G without Cr in the mineral supplement,
16 mg Cr per kilogram diet) on male Zucker rats in metal-free
cages for 16 weeks were examined. Zucker rats also received the
normal AIN-93G diet (i.e., with 1000 mg Cr added per kilogram
diet) and the normal diet supplemented with an additional 200
or 1000 mg Cr per kilogram diet. No ill health effects were noted
for the rats on the low-chromium diet. Plasma insulin concen-
trations in glucose tolerance tests decreased as a function of
chromium dose indicating a pharmacological effect for the die-
tary chromium.
Thus, with no added stresses, a purified diet with as little
chromium content as possible does not result in symptoms
that can be attributed to chromium deficiency, although effects
on insulin sensitivity can be observed at supranutritional, that
is, pharmacological, doses of chromium. Hence, establishing
whether chromium is an essential nutrient is apparently not
feasible from traditional nutrition studies, because developing
a chromium-deficient but otherwise sufficient diet appears not
to be possible. This could be simply because chromium is not
an essential element or uniquely because such low amounts of
chromium are required that generating a deficiency is not
possible. Similarly, no genetic disorder that alters chromium
transport or distribution or other function has been identified;
the study of such disorders for other metals has pointed to the
essential nature of those elements and the biomolecules that
utilize the metal. How then can the potential essential nature
of chromium (or other elements such as silicon, vanadium,
arsenic, and boron that have been proposed to be essential but

in such miniscule amounts that generating a dietary deficiency
is impossible) be established? The most obvious method is
establishing that a biomolecule containing the element
in vivo performs an essential biological function at physiolog-
ical levels of the element. For chromium, this has proved less
than straightforward, as several mechanisms for chromium
action at a molecular level have been proposed, while none
have been adequately established in vivo. In fact, major ques-
tions shroud each proposal, and open scientific discussions of
these issues and the other issues described in the preceding text
have proved to be quite difficult given the financial implica-
tions of the outcomes.
Total Parenteral Nutrition
Evidence for an essential role of chromium in humans has also
been inferred from studies of patients on TPN. Patients on TPN
have developed impaired glucose utilization or glucose intol-
erance and neuropathy or encephalopathy. These symptoms
were reversed by chromium infusion while unaltered by other
treatments. While limited to about ten individual cases, these
studies have been interpreted as providing evidence of clinical
symptoms associated with chromium deficiency that can be
reversed by supplementation. Curiously, the development of
the symptoms that were reversible by chromium supplemen-
tation does not correlate with serum chromium levels, indicat-
ing either that serum chromium levels are not an indicator of
chromium deficiency or that another factor is in operation.
Additionally, these incidences of diagnosed potential chro-
mium deficiency have been questioned as they lack consistent
relationships between the chromium in the TPN, time on TPN
before symptoms appear, serum chromium levels, and symp-
toms. Yet, subjects, albeit a limited number, with certain
conditions not responsive to other treatments appear to have
improved glucose control and reduction in insulin needs in
response to intravenous chromium.
The level of Cr administered must be examined carefully. In
the cases where reversal of symptoms was reported, the TPN
solutions provided 2–10 mg Cr per day. All the Cr in the TPN is
introduced into the bloodstream, while only 0.5% of Cr in
the regular diet is absorbed into the bloodstream. Thus, 30 mg
of Cr in a typical daily diet results in only  0.15 mg of Cr
entering the bloodstream. The TPN solutions utilized in these
studies are consequently presenting 13–67 times the required
amount of chromium; thus, based on these data, the TPN
solutions cannot be considered Cr-deficient. For the treat-
ments, the subjects received 40–250 mg Cr per day added to
the TPN solution to alleviate their conditions, clearly pharma-
cological doses as the largest dose provided 1.7  103 times
more chromium than a standard diet when one considers
that this administration corresponds to the equivalent of
100% absorption so that the values should be multiplied by
100 for comparison against oral studies. Consequently, the
results with the insulin-resistant TPN patients can only be
considered as providing evidence for a pharmacological role
of chromium. The data are not relevant for examining whether
chromium is an essential element.
Not surprisingly, as TPN provides ten or more micrograms
of chromium per day, TPN patients accumulate chromium in
Chromium: Physiology 111
their tissues. Calls are appearing for the reexamination of the
chromium levels in TPN solutions in terms of a need to reduce
recommended levels.
Absorption
The absorption of Cr as a function of intake by humans is still
widely cited as evidence for the essentiality of chromium,
although this is based on a single study. An inverse relationship
between dietary chromium intake and degree of absorption
was observed, in contrast to rodent studies described in the
preceding text. Cr intake was determined from the amount of
various food consumed where the Cr content of each food had
been previously determined; Cr absorption was estimated from
the Cr content of the urine as use of radiolabeled Cr to track the
fate of all the administered Cr is not possible in humans. The
data suggested that absorption of Cr varies approximately from
0.5% to 2.0% for Cr intakes of  15–50 mg day1. This difficult
to perform study is far from definitive and desperately requires
repeating. For example, a distinct difference is found when the
data are separated into male and female subjects. For males, no
statistical variation occurs for apparent chromium absorption
as a function of intake, while an inverse trend was observed for
the female subjects. A thorough statistical treatment including
a propagation of error analysis is needed. Additionally, these
data are in striking contrast to a similar human study.
Chromium absorption was determined to be 0.4% for free-
living individuals; when Cr intake was increased by over four-
fold, urinary chromium excretion increased over fourfold
while maintaining 0.4% absorption of chromium for both
males and females. The difference between the two studies lies
in the range of Cr intakes of 15–50 mg day1 for the former
and 60–260 mg day1 for the latter, suggesting that an inverse
relationship between Cr intake and absorption, if it exists,
exists only at the lowest portion of the range of intakes. These
results have led to the proposal that Cr homoeostasis is main-
tained at the level of excretion, not absorption, and that the
mechanism of Cr uptake by rats may be different from that in
humans. However, the assumption that chromium is in a state
of homoeostasis is unproven. In fact, the extent of absorption
appears to determine the amount of urinary excretion. As
noted in the preceding text, absorption appears to be directly
determined by intake – the amount of chromium intake deter-
mines the amount of chromium excretion. No evidence for
chromium homoeostasis can be derived from these studies
(with the exception of the data for female humans).
Thus, in summary, Cr appears to be absorbed by diffusion,
not actively transported. The process of chromium absorption
could possibly be different in humans (i.e., at least females)
from that in rats, but this would seem unlikely and would
require more research for this to be firmly established. If chro-
mium intake by humans is active, this transport system would
only play a significant role at low chromium intake and would
be overwhelmed by diffusion at higher intakes. Clearly, chro-
mium absorption in rodents is not inversely proportional to
intake, distinct from that of other reported essential elements.
Based on absorption studies, at least in rodents, chromium
does not appear to be an essential element; and this probably
extends to other classes of mammals.
112 Chromium: Physiology
Chromium Movement
The rates of absorption and transport of Cr are altered in
humans with type 2 diabetes and in rodent diabetes models.
Increases in blood insulin concentration (such as those follow-
ing a glucose challenge or similar dietary stress) result in
decreases in plasma glucose concentration followed by increases
in urinary Cr loss with the amount of increased urinary Cr loss
roughly equivalent to the decrease in the amount of blood
chromium. Serum chromium levels of diabetic patients are
lower than those of healthy subjects, while urinary Cr loss is
increased in diabetic subjects. As noted in the preceding text, the
increases in chromium loss are accompanied by (if not the result
of) increases in chromium absorption. While the association
between insulin insensitivity/diabetes and rates of chromium
transport is suggestive, it is not proof of an essential role for
chromium and is explained in part by the transport of chro-
mium by the iron-transport protein transferrin. A role for trans-
ferrin in the detoxification of chromium resulting from increases
in chromium absorption associated with insulin insensitivity/
diabetes could also explain its phenomenon.
Pharmacological Effects
Supplementation with Cr has been proposed to result in
beneficial responses in mammals with demonstrated glucose
intolerance or insulin insensitivity, including type 2 diabetes,
cardiovascular disease, and related conditions. However,
studies in humans tend to be negative or at best ambiguous.
The clinical studies, while focused primarily on type 2 diabetic
subjects, tend to have small subject pools and not be well
designed. One study has dominated attention; 185 adult-
onset diabetic Chinese patients participated in the study, in
which decreases in the concentration of fasting serum glucose,
insulin, hemoglobin A1C, and total cholesterol and decreased
glucose and insulin levels in response to glucose challenges
were observed as a result of Cr supplementation in a dose-
responsive manner. Unfortunately, attempts to reproduce
these results with other populations, including Western popu-
lations, have been unsuccessful. Two meta-analyses commis-
sioned by agencies of the US government have generated
inconclusive results on whether Cr affects symptoms of type
2 diabetes. One in 2002 with funding from the Office of
Dietary Supplements of the National Institutes of Health
(NIH) identified only four quality studies for analysis and
found that the results were inconclusive as the combined
results except for those of the Chinese study found no effects.
Another in 2007 with funding from the Department of Health
and Human Services identified 18 studies. The authors
concluded that Cr supplementation might have a modest effect
on glucose metabolism in type 2 diabetics but that large
heterogeneity combined with the overall poor quality of the
studies limited the strength of any conclusions. Unfortunately,
the positive effects of the greatest magnitude used in the anal-
ysis came from the 12 studies ranked lowest in quality, includ-
ing the Chinese study. A trend was observed that commercial
industry-sponsored studies were more likely to observe bene-
ficial effects. Consequently, the position of the American
Diabetes Association (ADA) is that insufficient evidence exists
to support the use of chromium to improve glycemic control in
people with diabetes.
In contrast, studies with rodent models of diabetes and
peripheral tissue insulin resistance generally observe beneficial
effects on insulin sensitivity and often also observe beneficial
effects of serum cholesterol and triglyceride levels. These rat
studies generally provided rats between 80 and 1000 mg (or
even more) Cr per kilogram body mass daily for periods of
weeks or months. Based only on mass, this would correspond
to 5.2–65 mg Cr daily for an average 65 kg human. Even when
corrected for the increased metabolic rate of rats compared to
humans, this range corresponds to 1–13 mg Cr daily. Thus,
human clinical trials may have only started to approach the
dose necessary to see a beneficial effect in humans. The amount
of Cr used in clinical trials needs to be increased before deter-
mining whether or not chromium supplementation has an
effect on individuals with type 2 diabetes, insulin resistance,
or related conditions. However, one also cannot rule out that
beneficial effects might be unique to rodents and perhaps
certain other animals.
Requirements
The basis for the use of chromium as a nutritional supplement
stems from chromium being on the list of essential vitamins
and minerals under examination by the NRC (National
Research Council of the National Academies of Science, the
United States) since 1980 when an estimated safe and
adequate daily dietary intake (ESADDI) for 50–200 mg was
suggested. In 2001, the National Academies of Science estab-
lished an adequate intake (AI) of chromium of 35 mg day1 for
men and 25 mg day1 for women. AI is defined as the recom-
mended average daily intake level based on observed or exper-
imentally determined approximations or estimates of nutrient
intake by a group (or groups) of apparently healthy people that
are assumed to be adequate. The AI is more conservative than a
recommended daily allowance and is expected to cover the
needs of more than 97–98% of individuals. Thus, essentially,
all Americans are believed to be chromium-sufficient, and little
if any need exists for chromium supplementation. The bases
for this determination are rather limited. The AI is primarily
based on the Cr content of nutritionist-designed diets (on
average roughly 35 mg Cr for men and 25 mg Cr per day for
women), which turns out to be almost identical to the chro-
mium content of self-selected American diets. Diets in other
developed nations appear to be similar in Cr content, if not
slightly higher. The National Research Council is currently
reviewing dietary requirements of trace elements and vitamins.
What decision is made in terms of the status of chromium as an
essential element will be interesting.
Dietary Sources and Content in Food
Chromium is ubiquitous in foods but at very low concentra-
tions. However, during processing, particularly in stainless-
steel (10–40% chromium) equipment, the concentration of
chromium appears to increase; in fact, most of the Cr in some
foods may come from processing. Foods particularly rich in Cr
(i.e., >100 ppb) include broccoli and black pepper and certain
beers; however, values for vegetables must be considered

carefully because of the variable amount of chromium that
comes from soil contamination. The low concentrations of
chromium in food, the ease of contamination, and the low
suggested dietary requirement for chromium (vide supra)
make preparation of a low-chromium (or chromium-deficient)
diet difficult if even possible (as this assumes that chromium is
actually an essential trace element). Given the amount of Cr in
the diet that comes from modern processing, the Cr intake of
early humans must have been extremely limited.
Reference Doses
No tolerable upper limit (UL) has been set for Cr3þ. No signif-
icant health concerns have been found for chromium supple-
ments other than chromium picolinate at current doses.
However, a study commissioned by the National Toxicology
Program (National Institutes of Health) has found that pro-
viding chromium picolinate up to 5% of the diet of male and
female rats and mice for 2 years had no conclusive deleterious
health effects. This is probably the result of chromium picoli-
nate dissociating in the gastrointestinal tract when provided
orally. In contrast, cell culture study and in vivo studies suggest
that if intact chromium picolinate reaches cells intact that it is
toxic and clastogenic, mutagenic, and possibly carcinogenic,
hence, the supplement should not be used intravenously, for
example, in TPN.
See also: Chromium: Properties and Determination; Cooking:
Domestic Techniques; Dietary References: US; Glucose: Glucose
Intolerance; Iron: Physiology of Iron; Parenteral Nutrition; Trace
Minerals and Trace Elements.
Chromium: Physiology 113
Further Reading
American Diabetes Association (2014) Clinical practice recommendations. Diabetes
Care 37(Suppl. 1): S1–S155.
Anderson RA, Bryden NA, and Polansky MM (1992) Dietary chromium intake: freely
chosen diets, institutional diets, and individual foods. Biological Trace Element
Research 32: 117–121.
Balk EM, Tatsioini A, Lichtenstein AH, Lau J, and Pittas AG (2007) Effect of chromium
supplementation on glucose metabolism and lipids: a systemic review of
randomized controlled trials. Diabetes Care 30: 2154–2163.
Chen Y, Watson HM, Gao J, Halder Sinha S, Cassady CJ, and Vincent JB (2011)
characterizing the organic component of low-molecular-weight chromium-binding
substance and its binding of chromium. Journal of Nutrition 141: 1225–1232.
Di Bona KR, Love S, Rhodes NR, McAdory D, Halder Sinha S, Kern N, Kent J,
Strickland J, Wilson A, Beaird J, Ramage J, Rasco J, and Vincent JB (2011)
Chromium is not an essential trace element for mammals: effects of a “low-
chromium” diet. Journal of Biological Inorganic Chemistry 16: 381–390.
Hopkins LL Jr and Schwarz K (1964) Chromium(III) binding to serum proteins,
specifically siderophilin Biochimica et Biophysica Acta 90: 484–491.
National Research Council (2002) Dietary reference intakes for vitamin A, arsenic,
boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon,
vanadium, and zinc. In: A report of the panel on micronutrients, subcommittee on
upper reference levels of nutrients and of interpretations and uses of dietary
reference intakes, and the Standing Committee on the Scientific Evaluation of
Dietary Reference Intakes, Washington, DC: National Academy of Sciences.
Vincent JB (ed.) (2007) The nutritional biochemistry of chromium(III). Amsterdam:
Elsevier.
Vincent JB (2010) Chromium: celebrating 50 years as an essential element? Dalton
Transactions 39: 3787–3794.
Vincent JB and Love S (2012) The binding and transport of alternative metals by
transferrin. Biochimica et Biophysica Acta 1820: 361–378.
Vincent JB (2013) The bioinorganic chemistry of chromium. Chichester: John Wiley &
Sons.
Relevant Websites
http://www.diabetes.org – American Diabetes Association.
http://www.iom.edu/ – Institute of Medicine of the National Academies.
http://ods.od.nih.gov/ – National Institutes of Health, Office of Dietary Supplements.
 Chromium: Properties and Determination
JB Vincent, The University of Alabama, Tuscaloosa, AL, USA
ã 2016 Elsevier Ltd. All rights reserved.
Physical and Chemical Properties
Chromium(6þ)
Only two oxidation states of chromium, Cr3þ and Cr6þ, are
generally considered biologically and environmentally relevant
and stable; in other words, they are stable in the presence of air
and water. Chromium(III) complexes are both kinetically and
thermodynamically stable; however, chromium(VI) complexes
are kinetically stable but thermodynamically unstable. In the
presence of appropriate reducing agents, Cr6þ can readily be
reduced via Cr4þ and/or Cr5þ intermediates ultimately to Cr3þ.
The biochemistries of both Cr3þ and Cr6þ have controversial
histories. The public is generally more familiar with the chem-
istry of Cr6þ (or chromate) because of its toxicity.
Chromium(6 þ), d0, is most commonly encountered as the
intensely colored chromate, [CrO4]2, or dichromate,
[Cr2O7]2, anions. These two species are interconvertible in
water. Chromate has a distinctive yellow color and exists at
basic pH values. Insoluble PbCrO4 was previously used as the
pigment in paint for yellow highway lines. Below pH 6, chro-
mate is in equilibrium with the yellow-orange dichromate
anion. Acidic dichromate solutions are potent oxidants. The
coordination environment of the chromium centered in both
the chromate and dichromate anions is tetrahedral. The
intense color of both anions stems from ligand to metal charge
transfer bands. Mixed ligand complexes of Cr6þ with oxides
and halides or oxides and amines are well known, as are Cr(VI)
peroxo complexes. The diamagnetic Cr6þ center does not give
rise to ESR (electron spin resonance) spectra, while NMR
(nuclear magnetic resonance) studies of Cr(VI) complexes
with oxo, peroxo, and halo ligands are of limited utility.
While Cr(VI) complexes are known to be potentially potent
carcinogens and mutagens when inhaled, a serious debate has
arisen with regard to the effects of the oral intake of these
complexes, as illustrated in recent years by the popular movie
Erin Brockovich. Chromium(VI) complexes could give rise to
these effects through a number of mechanisms, including oxi-
dation of biomolecules by the complexes or by the subse-
quently generated Cr4þ and Cr5þ intermediates, reactions of
reactive oxygen species generated as by-products of these oxi-
dations, reactions of organic radicals generated in these pro-
cesses, and the binding of the ultimately generated Cr3þ to
biomolecules. The relative importance of these mechanisms
is far from being deciphered. The nature and significance of
the coordination of Cr3þ ions to DNA as a result of Cr6þ
reduction is a current topic of much debate. Chromate can
readily enter cell via sulfate (SO2
4 ) and phosphate (HPO4 )
2
transporters given its similar shape and charge.
Chromium(3þ)
Coordination complexes of Cr3þ are nearly always octahedral.
Consequently, the chromic center has a d3 electron
114 Encyclopedia of Food and Health
configuration with three unpaired electrons (S ¼ 3/2), one in
each of the three t2g orbitals. This configuration is responsible
for the kinetic inertness of Cr(III) complexes, where ligand
exchange half-times are generally in the range of hours. The
hexa-aquo ion of chromium, [Cr(H2O)6]3þ, is purple in aque-
ous solution. Solutions of the ion are acidic; at neutral and
basic pH, the ion readily oligomerizes to give hydroxo-bridged
species starting with the [(H2O)5Cr(m-OH)2Cr(H2O)5]4þ ion.
The Cr3þ ion has a large charge to size ratio and is considered
as a hard Lewis acid, preferring oxygen and nitrogen coordina-
tion. With common biomolecules, coordination to anionic
oxygen-based ligands such as phosphates and carboxylates
would be expected; however, the structure of Cr(III) biomole-
cules generally is poorly characterized to date.
Common commercially available forms of Cr3þ include
chromium chloride (CrCl3  6H2O), chromium nitrate (Cr
(NO3)3  9H2O), and chrome alum (K[Cr(SO4)2]  12H2O),
which are utilized because of their solubility. The commonly
used commercial form of CrCl3  6H2O is actually trans-[Cr
(H2O)4Cl2]Cl2 H2O. Dissolution of this green solid initially
yields green solutions of the [Cr(H2O)4Cl2]þ cation. Chromium
chloride, chromium picolinate, and chromium nicotinate are
the most popular forms of chromium used in nutritional sup-
plements. Chromium picolinate is the meridional isomer of the
tris-chelate complex of Cr3þ and the picolinate anion, [Cr(pico-
linate)3]. Chromium nicotinate is a poorly characterized insolu-
ble polymer of Cr3þ, nicotinate, and hydroxide; its composition
varies depending on the method of synthesis. The hydrochloride
of a complex of Cr3þ and methionine ([Cr(L-methioni-
ne)3]HCl) and ‘chromium propionate’ have been used in nutri-
tional supplements for cattle (‘chromium propionate’ only) and
swine in the United States. Trivalent chromium is not approved
as a feed additive in the European Union. Although originally
characterized as anhydrous chromium propionate, Cr(propio-
nate)3, chromium propionate is actually the trinuclear cation [Cr
(III)3O(propionate)6(H2O)3]þ. The structural, spectroscopic,
and magnetic properties of this cation and its analogs with
other carboxylate ligands have been extensively studied. Limited
characterization has been reported on chromium methionine.
The magnetic and spectroscopic properties of chromium
(III) complexes do not readily lend themselves to providing
much information on the coordination environment of chro-
mic centers in biomolecules. For mononuclear complexes, a
magnetic moment close to the spin-only value for an S ¼ 3/2
center (3.88 BM) is generally observed. While 1H and 13C NMR
spectra can be obtained on Cr(III) complexes, the spin 3/2
center results in greatly broadened and shifted resonances in
NMR spectra. The structure of the complex generally needs to
be known in order to interpret the NMR spectra, rather than
the reverse. In contrast, Cr(III) complexes can give rise to
sharp features in ESR spectra; however, the ESR spectra of bio-
molecules have often proved to be quite broad, providing
limited information. ESR spectroscopy is probably a
http://dx.doi.org/10.1016/B978-0-12-384947-2.00161-6

significantly underutilized technique in characterizing chro-
mium in biological systems. Cr3þ as an impurity in the Al2O3
matrix of emeralds and rubies gives rise to the green and red
color of these gems; yet, the electronic spectra of chromium-
containing biomolecules are usually very simple. Three spin-
allowed d ! d transitions are expected; two normally occur in
the visible region, while the third is expected in the ultraviolet
region (where it can be hidden by ligand-based features). No
charge transfer transitions generally occur while the visible
absorption bands have extinction coefficients of typically less
than 100 M1 cm1. Thus, only relatively concentrated solu-
tions of Cr3þ have observable color. Cr(III) complexes are
generally stable against oxidation or reduction, although the
redox potential can be significantly altered by certain ligands.
Although chromium as the Cr3þ ion was proposed to be an
essential element about 50 years ago, its status is currently in
question, as recent experiments appear to demonstrate that the
element can no longer be considered essential. Supplemental
nutritional doses of Cr3þ have been proposed to result in body
mass loss and lean muscle mass development, leading to an
appreciable nutraceutical industry being built around chro-
mium. However, these claims have been thoroughly refuted.
Chromium has also been suggested to be a conditionally
essential element whose supplementation could lead to
improvements in carbohydrate and lipid metabolism under
certain stress situations, including type 2 diabetes and the
effects of shipment of farm animals; this is currently an area
of intense and hotly debated research with recent findings
suggesting that beneficial effects from Cr3þ supplementation
are pharmacologically, not nutritionally, relevant. However,
while beneficial effects from chromium supplementation
have reproducibly been demonstrated in rodent models of
diabetes and insulin resistance, well-performed meta-analysis
indicates that no clinical effects in humans result from dietary
supplementation up to 1 mg chromium per day. Rodent
studies use proportionally greater doses based on body mass
than the human studies suggesting that clinical trials using
larger doses of Cr3þ are required to establish if the metal ion
has beneficial effects in humans. At the same time, supplemen-
tation of the diet with at least certain Cr(III) complexes has
been proposed to have potentially deleterious effects, although
recent data suggest that oral chromium supplementation
is safe.
Determination in Food and Drink
Chromium levels in tissues, body fluids, food components,
and other biological samples reported prior to c.1978 are
Table 1 Comparison of chromium analysis techniques
GFAAS ICP-AES
Analysis time 2–3 min for only Cr 2–5 min all elements
Detection limit 0.1–0.01 ppb 10–1 ppb
Isotope specific No No
Sample volume Small Moderate
Instrument cost Moderate High
Operating cost Moderate High
Chromium: Properties and Determination 115
problematic and should be ignored. At this time, improve-
ments in analytic techniques revealed several problems,
including appreciable contamination of biological samples
(as these samples were often homogenized in a stainless steel
blender); in fact, measured Cr levels reflected the levels of
contamination not the actual tissue or fluid Cr concentrations,
which were extremely small. Another major problem in atomic
absorption experiments prior to 1978 was that workers were
attempting to measure a tiny signal against a large background;
a linear correspondence was actually found to exist between
background absorbance and the reported apparent Cr content
of samples. Currently, analyses of human blood and urine
samples with Cr concentrations above 1 ppb should be con-
sidered suspect, unless the subjects are taking chromium sup-
plements; even values above 0.5 ppb may be considered
suspect. Consequently, studies prior to 1978 utilizing patients
who were believed to be Cr-deficient based on Cr tissue or fluid
concentrations and that reported Cr levels in tissues, foods, or
fluids of one order to several orders of magnitude too high
must be ignored. Thus, with the exception of some 51Cr-label-
ed tracer studies, the fields of chromium nutrition and bio-
chemistry really began in the late 1970s when chromium
uptake and loss could be followed to a reasonable degree of
accuracy. At present, chromium levels in tissues and biological
fluids are usually determined by graphite furnace atomic
absorption spectrometry (GFAAS), although neutron activa-
tion analysis (NAA) and inductively coupled plasma-mass
spectrometry (ICP-MS) can also be used, with the latter grow-
ing in popularity. Inductively coupled plasma-atomic emission
spectrometry (ICP-AES) has also seen some use although the
detection limit is not suitable for urine and plasma samples so
that the technique will not be discussed further. Neutron acti-
vation and ICP-MS have also been utilized with stable isotopes
of Cr for determining Cr levels in tracer studies, in addition to
the continuing use of radioactive 51Cr for such studies. As most
researchers have limited access to NAA, the technique will not
be discussed further. These techniques are compared in
Table 1.
Chromium is ubiquitous in foods but at very low concen-
trations. However, during processing, particularly in stainless
steel (10–40% chromium) equipment, the concentration of
chromium appears to increase; in fact, most of the Cr in some
foods may come from processing. Foods particularly rich in Cr
(i.e., >100 ppb) include broccoli and black pepper and certain
beers; however, values for vegetables must be considered care-
fully because of the variable amount of chromium that comes
from soil contamination. The low concentrations of chromium
in food, the ease of contamination, and the low suggested
dietary requirement for chromium (30 mg day1) make
ICP-MS NAA
2–5 min all elements days multiple elements
1–0.01 ppb 0.1 ppb
Yes Cr-50
Small Varying
Very high Extremely high
High High
116 Chromium: Properties and Determination
preparation of a low-chromium (or chromium-deficient) diet
difficult if even possible (as this assumes chromium is actually
an essential trace element). Given the amount of Cr in the diet
that comes from modern processing, the Cr intake of early
humans must have been extremely limited.
Tables of chromium content for various foods are probably
of limited utility because of the variability of chromium con-
tent of samples based on history (processing, growth condi-
tions, etc.). For example, a 50-fold difference in chromium
content has been found for different types of oatmeal. Foods
used in nutrition studies need to be analyzed for each study,
rather than depending on tabulated values in the literature. The
chromium content of commercial rodent chows is surprisingly
high and extremely variable, even between lots of the same
brand. The amount of chromium in commercial rodent chows
is normally about 500 mg kg1. The purified AIN-93G diet has
1 mg kg1 of added Cr3þ as potassium chromium sulfate
dodecahydrate.
Precautions to Be Taken in Chromium Determination
Chromium concentrations in urine and blood are in the sub
part per billion range unless subjects receive chromium sup-
plementation. Hence, the concentration of Cr in these fluids is
within about an order of magnitude of the detection limit of
current techniques. Thus, contamination is a major concern.
Similarly, the chromium concentration of tissues and foods
generally range from 1 to 1000 ppb, meaning that contamina-
tion can be a major problem as well. For foods, knowing the
history of the sample is important as processing can provide
more chromium than the food initially contained. Differences
in commercial processing can result in large differences in
chromium content. Similarly, differences in chromium content
of vegetables and other plants in terms of growth conditions
are poorly known.
Because of the chromium content of stainless steel, contact
with stainless steel should be avoided during sample collection
and handling. For example, the use of a stainless steel blender
to homogenize samples transfers appreciable amounts of chro-
mium. Needles for venipuncture, thus, can be a challenge.
Most of the chromium removed from a stainless steel needle
during blood collection appears to be removed by the first
20 ml of blood; consequently, collecting samples after allow-
ing several milliliters of blood to pass through the needle has
been proposed. However, contamination, while reduced, still
appears to be substantial. Siliconized stainless steel needles
seem to be suitable. Plastic cannula or catheters are suitable.
For plasma, anticoagulants, for example, ethylenediamine-
tetraacetic acid, used should also be low in chromium.
Plasticware (such as those made of polyethylene or poly-
propylene) should be used wherever possible in transferring
and storing samples, and all glassware or quartz tubes should
be acid-washed. Rubber stoppers are a source of chromium
and should be avoided. All water and chemical reagents need
to be of ultra high purity. Investigators should wear non-
powdered nitrile or polyvinylchloride gloves. All methods
should be verified with the use of a certified chromium
standard.
Sample Digestion
Discussion will be limited to samples for GFAAS, for which
more data exist. Similar procedures are required for ICP-MS.
Urine
Sample digestion can be avoided for urine samples, although
the use of the method of standard addition may be required to
remove matrix effects and is recommended. Wet ashing can
also be utilized; for example, digestion with HNO3 and 30%
H2O2 at subboiling temperatures for 15 h has been utilized.
The solid can be dissolved in water for analysis.
Blood
For dry ashing, magnesium nitrate has been added as a matrix
modifier to samples in quartz tubes followed by lyophilization
using a stainless steel-free lyophilization apparatus. The solid is
then ashed in a muffle furnace at 480  C and then dissolved in
HCl for analysis. Following this technique because of the uni-
formity of samples, the use of the method of standard addition
has been reported to not be necessary.
Alternatively, blood plasma has also been enzymatically
digested and used directly with the addition of magnesium
nitrate as a matrix modifier; the method of standard addition
was also utilized.
Tissues and Foods
For such samples, digestion of samples is essential. (Numerous
variations of the procedures described have been used.) For dry
ashing, magnesium nitrate is added before placing in a muffle
furnace; the ash is dissolved in HCl for analysis. For wet ashing,
samples can be treated with a mixture of nitric and sulfuric
acids and heated to dryness at subboiling temperatures fol-
lowed by dissolution in water for analysis. A combination of
wet and dry ashing has also been used. Samples in borosilicate
glass tubes are heated in a muffle furnace at 375  C for 48 h;
subsequently, nitric acid is added to the cooled samples that
are then heated in a heating block at 90  C. Additionally,
hydrogen peroxide (50%) is added at time intervals until car-
bon particles are dissolved. Alternatively, the use of microwave
digestion systems has become popular. Nitric acid is added to
samples before heating in the microwave digestion system.
Determination Methods
Graphite Furnace Atomic Absorption Spectrometry
In GFAAS, a furnace dissociates a sample into its component
atoms. Light from a hollow cathode tube (which is element-
specific) is passed through the generated cloud of atoms where
the atoms of the element of interest absorb the light. The
absorption of light is proportional to the concentration of the
element in the sample. The furnace, an electrically heated
graphite tube with an opening for sample introduction,
replaces the flame in traditional atomic absorption spectrom-
etry. The furnace, in contrast to the flame, generates a higher

Table 2 Furnace program for chromium determination by GFAAS
Temperature Ramp time Hold time
Mode Step ( C) (s) (s)
Dry 1 100 5 20
Dry 2 140 15 15
Char 3 1600 10 20
Atomization 4 2500 0 4
Clean 5 2600 1 3
density of atoms with a longer residence time in the cloud,
increasing the sensitivity of the method. The use of modern
instruments with their background corrections removes some
of the pre-1978 difficulties; however, contamination given the
low concentration of chromium in biological samples will
always be a potential problem.
Generally, the protocol of Veillon and coworkers or varia-
tions thereof are utilized. After being injected into the furnace,
samples are subjected to a program of drying (two steps at 100
and 120–140  C), charring (1150–1350  C), atomization
(2500–2700  C), and cleaning (2600–2700  C). A typical pro-
gram is shown in Table 2.
Inductively Coupled Plasma-Mass Spectrometry
ICP-MS generates the constituent atoms and ions using an ICP.
In contrast to GFAAS where the absorption of light is detected,
the generated ions are detected directly. The ions are separated
based on their charge to mass ratio in the mass spectrometer,
generally using a quadrupole analyzer or magnetic sector ana-
lyzer. The detection limit of ICP-MS is normally about an order
lower than GFAAS based on the number of ions generated in
the former compared to the number of atoms excited in the
latter. Contamination becomes a greater concern as the level of
detection is pushed to lower limits. ICP-MS has the advantage
of being isotope-specific, whereas GFAAS is only element-spe-
cific. ICP-MS instruments are considerably more expensive
than GFAAS, which is why GFAAS is currently more commonly
used for Cr analysis. ICP-MS instrument currently also requires
more care in operation, although analysis time is shorter than
for GFAAS.
Chromium(III) and Chromium(VI) Speciation
Given the different chemistries and toxicities of chromium(III)
and chromium(VI) complexes, determining of the concentra-
tion of one or the other rather than the total chromium con-
centration is often desired. However, none of the techniques
described earlier can separate complexes of the two oxidation
states but rather only measure total chromium content. Tech-
niques, such as colorimetric assays that can specifically test for
chromium(IV) species, lack the sensitivity to measure biologi-
cal concentrations. Consequently, some type of separation
technique is required before detection of chromium using
one of the determination methods described earlier. This sep-
aration must be accomplished without the oxidation states of
the chromium changing. The separation also can lead to
Chromium: Properties and Determination 117
dilution requiring preconcentration before chromium analysis.
The concentration of each ion can be measured, or the con-
centration of one ion in combination with the total chromium
concentration can be measured allowing for the calculation of
the concentration of the other ion.
Developing methods to separate these ions at low concen-
trations without altering the oxidation states is an area of active
research. Most research has been focused on water samples that
generally lack the complexity of biological samples. Yet, studies
have shown that Cr3þ and Cr6þ concentrations can be deter-
mined in urine and plasma samples, although nothing has
been adapted for routine use to date. For example, cloud
point extraction has been combined with GFAAS to determine
the concentrations of Cr3þ and Cr6þ in human blood serum
samples, while ion-pair reversed-phase high-performance liq-
uid chromatography (HPLC) has been coupled with ICP-MS to
measure the concentrations of Cr3þ and Cr6þ in human urine
(from chromate workers).
Conclusion
Cr3þ and Cr6þ ions have distinctly different chemistry. Most
notable, Cr(VI) complexes are kinetically stable and thermo-
dynamically unstable, while Cr(III) complexes are both kinet-
ically and thermodynamically stable. In a biological
environment, only Cr3þ is stable as Cr6þ is reduced via Cr5þ
and/or Cr4þ intermediates to Cr3þ. This chemistry results in
Cr6þ being toxic, mutagenic, and carcinogenic, while Cr(III)
complexes are generally nontoxic. Chromium concentrations
in tissues, body fluids, foods and beverages, and other biolog-
ical samples are very low. Chromium concentrations in these
samples reported prior to c.1978 are problematic (except when
radioactive Cr-51 was used as a tracer), and these values must
be disregarded as they are often too low by as much as three
orders of magnitude. Currently, chromium concentrations in
biological samples are generally measured using graphite fur-
nace atomic absorption spectroscopy or ICP-MS, with the for-
mer being more popular because of instrument expense and
ease of maintenance. Sample preparation must be performed
carefully to prevent contamination, particularly from stainless
steel. Currently, research in the separation and determination
of Cr3þ and Cr6þ simultaneously is an active area.
See also: Chromium: Physiology; Cooking: Domestic Techniques;
Trace Minerals and Trace Elements.
Further Reading
Gomez V and Callao MP (2006) Chromium determination and speciation since 2000.
Trends in Analytical Chemistry 25: 1006–1014.
Katz SA and Salem H (1994) The biological and environmental chemistry of chromium.
New York: VCH.
Miller-Ihli NJ (1996) Graphite furnace atomic absorption spectroscopy for the
determination of the chromium content of selected U.S. foods. Journal of Food
Composition and Analyses 9: 290–300.
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 Cider (Cyder; Hard Cider): The Product and Its Manufacture
E Coton and M Coton, Université de Brest, Plouzané, France
H Guichard, Institut Francais des Productions Cidricoles, Le Rheu, France
ã 2016 Elsevier Ltd. All rights reserved.
Definition and Origin
Cider is generally defined as an alcoholic beverage obtained by
apple juice (apple must) fermentation. Noteworthy, in North
America, the term ‘cider’ is rather associated with a cloudy unfer-
mented and unpasteurized apple juice, whereas the fermented
product is called ‘hard cider.’ In Europe, the fermented product is
mainly named ‘cider’ in the UK, ‘cidre’ in France, ‘sidra’ in Spain,
and ‘apfelwein’ in Germany. Another common fruit fermenta-
tion is obtained from pears and leads to a product named ‘perry’
in English or ‘poiré’ in French. It is worth mentioning that cider
can either be a final product ready for consumption or an inter-
mediate product used for apple brandy (e.g., ‘Calvados’ in Nor-
mandy and ‘Lambig’ in Brittany) production by distillation or
cider vinegar via an acetic fermentation. Moreover, Calvados
blended with apple must leads to an aperitif-type beverage (i.e.,
predinner drink) named ‘Pommeau’ in France.
Like the other major fermented beverages (i.e., wine and
beer), cider is one of the oldest alcoholic beverages in the
world; Hebrews called it ‘sichar,’ whereas Romans and Greeks
called it ‘sicera’ and ‘sikera,’ respectively. From an etymological
point of view, the name cider would come from ‘sicera’ (mean-
ing any fermented beverage that is not wine; Cambridge Psal-
ter); this can especially be observed in Normandy, as ‘cidre’ was
originally spelled ‘sidre.’ Although the name cider was not used
at the time, during antiquity, a certain number of writings by
Pliny the Elder or Palladius refer to alcoholic beverages
obtained from apples or pears. During the ninth century, the
term ‘sicetores,’ referring to brewers producing ale but also
‘pomacium’ from apples, was used by Charlemagne. In France,
the first use of the word ‘cidre’ was found in the Conception de
Nostre-Dame (Wace, twelfth century). From this time, and
thanks to the invention of the press (thirteenth century),
cider production extended to various apple-producing
European regions. From the fourteenth to twentieth centuries,
technological practices and processes were optimized and led
to higher volumes and better-quality products. Nowadays,
cider production, although far more limited than wine and
beer, can be found on every continent in apple-growing
regions worldwide. In Europe, the UK (mainly West Country,
West Midlands but also Wales), France (mainly Normandy and
Brittany), Spain (mainly Asturias and Basque country), and
Germany are the main cider-producing countries, although
many others have local productions (e.g., Ireland, Austria,
Poland, Sweden, Norway). In North America, hard cider pro-
duction is done in the United States, Canada, and even Mexico.
Interestingly, in Quebec, a new cider type named ‘ice cider’
(equivalent to icewine in enology and thus using apples with
high sugar contents due to natural frost) has recently appeared.
In South America, cider is produced in Argentina and Chile.
Cider production is also found in Asia (China and Japan),
Africa (South Africa), Australia (Tasmania), and New-Zealand.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00163-X
Although the generic term ‘cider’ is commonly used, the
term ‘ciders’ is more appropriate to reflect the large variety of
fermented apple products. According to production region,
cider can be produced from only apples, with the addition of
pear or from apple concentrate. Additives such as sugar, acid,
or coloring agents can also be used. Sugar content may vary
leading to dry, semidry, or sweet ciders. Carbon dioxide (CO2)
may be absent or present and obtained in the final product by
carbonation or a secondary fermentation. In this context,
beyond the classical definition of the term cider, it is important
to note that several country regulations exist to define cider and
cidermaking. Examples of regulations include the French
“Décret n
87–600 du 29 juillet 1987 modifiant le décret n

53–978 du 30 septembre 1953 relatif à l’orientation de la
production cidricole et à la commercialisation des cidres et
des poirés,” the UK “Customs & Excise Notice 162: cider
production” and the Spanish “Orden de 1 de agosto de 1979
por la que se reglamentan las sidras y otras bebidas derivadas
de la manzana.” In these regulations, authorized products
entering the cidermaking process as well as specific analytical
parameters (e.g., ethanol, volatile acidity, or SO2 levels) are
defined.
Moreover, to ensure cider product quality, several certifica-
tions exist at the national or international (European) level.
They are delivered based on certain product specifications and
requirements to ensure product authenticity and quality for
consumers. Examples of these certifications, which are gener-
ally easily recognizable by specific logos on the label, include
• Protected Designation of Origin (PDO) and covers, as
defined by the European Commission, “agricultural prod-
ucts and foodstuffs which are produced, processed and
prepared in a given geographical area using recognized
know-how” (e.g., Cidre Pays d’Auge, Cidre de Cornouaille
and Sidra de Asturias).
• Protected Geographical Indication (PGI) and covers, as
defined by the European Commission, “agricultural prod-
ucts and foodstuffs closely linked to the geographical area.
At least one of the stages of production, processing or
preparation takes place in the area” (e.g., Cidre de Bretagne,
Cidre de Normandie, Gloucestershire cider, Herefordshire
cider, and Worcestershire cider).
• Organic farming corresponds to products that must comply
with strict EU requirements covering not only production
and processing, but also the control and labeling of organic
foods. This certification has gained interest due to strong
societal demand but also higher commercial value for the
product.
• ‘Label Rouge’ (red label) is specific to France and attests that
the product, examined by a national commission, presents
superior overall quality compared to a similar product (e.g.,
Cidre de Normandie, Cidre Royal Guillevic).
119
120 Cider (Cyder; Hard Cider): The Product and Its Manufacture

Production organoleptic qualities that make them uninteresting for direct

Cider production practices in France (Figure 1) and the UK,
although both leading to products called ciders, exhibit major
differences that in some instances can even be considered
opposite. In the following section, these practices will be
used as examples.
Raw Material
Although cider can be produced from any apple cultivar, in
Europe, a distinction is traditionally made between dessert,
cooking, and cider apples. Cider apples have distinct
consumption linked to their acidity or natural polyphenol
content. On the contrary, these traits provide high product
qualities in terms of body equilibrium (sweetness, acidity,
astringency, and bitterness) that are not obtained using dessert
apples. Different cider apple cultivars exist, and more than
1000 have been described, although only a few are used for
production. They are classified into six categories based on
juice-specific savors:
• Sweet: These apple varieties are the blandest of the six
categories. They have low acid (<60 mequiv. l1) and poly-
phenol (<2 g l1) contents and up to 15% sugar (cultivars:
Douce Coëtligné, Doux Normandie, Muscadet Petit de l’Orne,
Rouge Duret, Sweet Copin, Sweet Alfortd, Nortwood . . .).

Manual or
• Sour: These apples are mildly sour (60–90 mequiv. l1)
with low polyphenols (<2 g l1) (cultivars: Guillevic, Jude-
mechanical harvest
line, Rouget de Dol, Locard blanc . . .).
• Sharp: These apples provide sourness and freshness to cider
Storage to maturity due to their high acidity (>90 and up to 240 mequiv. l1)
and low polyphenol (<2 g l1) contents (cultivars: Petit
Jaune, Judor, Avrolles, Locard vert, Crimson King, Brown’s
Variety blending Apple . . .).
• Bittersweet: This apple type corresponds to intermediate
cultivars with low acidity (<60 mequiv. l1) and high poly-
Washing & sorting phenol (>2 g l1 and <3 g l1) contents providing bitter-
ness and astringency to cider (cultivars: Douce Moën, Bedan,
Binet Rouge, Bisquet, Ashton Bitter, Dabinett, Yarlington Mill,
Milling Tremlett’s Bitter . . .).
• Bittersharp: These apples have high acid (>60 mequiv. l1
pomace
Water leaching and up to 240 mequiv. l1) and total polyphenols (>2 g l1
Cuvage
and up to 6 g l1) (cultivars: Cazo jaune, Kingston Black,
Foxwhelp . . .).
Pressing • Bitter: These apples have low acid (<60 mequiv. l1) and
high total polyphenols (3–9 g l1) (cultivars: Marie
Ménard, Kermerrien, Petit Amer, Fréquin rouge, Bramtot, Ellis
Apple juice Bitter . . .).
Pressing
Traditionally, ciders are not obtained from a single cultivar.
Pre-fermentation Indeed, blending (‘pommage’) of different apple types allows
juice treatments for the production of balanced ciders with various organoleptic
qualities. Also, three maturity stages can be distinguished: early
season (beginning to end of September), midseason (begin-
Fermentation
ning of October to mid-November), and late season (mid-
November to end of December). After harvest, which is rather
Stabilization by mechanically performed nowadays, apples are stored for a
fining or filtration maximum of 5 days before processing to reach full maturity
and to ensure that all starch has been converted into sugar. This
Blending short time period allows good fruit preservation and texture
compatible with optimal pressing.

Bottling
Milling and Pressing
Prise de mousse Before any processing operation, fruit sorting and washing are
or/and carbonation
required to eliminate rotten fruits and potential foreign mate-
rials (e.g., wood pieces, grass, soil, and stones) that could
Commercialization impact overall product quality. Rotten fruits are associated
with mold contamination and originate either directly
Figure 1 French cidermaking diagram. Steps in lighter gray and from the apple tree itself (Monilia spp.) or indirectly after
discontinued lines are optional. impacts due to falling and being in contact with the ground
 Cider (Cyder; Hard Cider): The Product and Its Manufacture
(Fusarium spp.). All damaged fruit, especially in the case of
mechanical harvesting, can also be spoiled by secondary
molds belonging to Botrytis and Penicillium genera. Beyond
the rotten aspect of these fruits and potential associated off-
flavors, it is worth noting that some molds are also able to
produce toxins (mycotoxins) that may have an impact on
human health. Milling (rapâge in French) is then performed
to obtain fruit pulp for more efficient juice (or must) extrac-
tion. In general, a high-speed mill corresponds to a wheel
bearing graters or coarse knives rotating against a fixed surface;
they are calibrated to tear the fruit tissues without smashing the
seeds. Fine milling is used for firm apples, whereas rougher
milling is used for very ripe apples. In France, an extra step
called ‘cuvage’ (pectin leaching for 2–5 h for better pressing
yields) can be performed.
In both the UK and France, the next step involves pressing
the pulp by exerting high pressure. Nowadays, technological
evolutions have led to the use of semicontinuous, continuous,
or even automated presses, although discontinued and more
traditional presses can still be encountered in farm and arti-
sanal cider productions. The main types encountered are
• Horizontal presses: They correspond to compressible cham-
bers, equipped with several juice ducts, in which the pulp is
introduced. Then pressure is applied through the action of a
piston or a membrane. Thanks to several cycles of pressure
and homogenization, high extraction yields (up to
800 l T1) can be reached.
• Continuous belt presses: In this case, the pulp is placed
between two bands that will go through a series of rollers
that apply pressure onto the belt, resulting in a pressing
action. This type of press gives yields ranging from 600 to
750 l T1.
Pressing leads to two products, on the one hand, a liquid phase
corresponding to the juice, also called ‘apple must’ (moût pur
jus in French), and on the other hand, a solid phase called
pomace (marc in French). After coarse screening, the juice is
transported through piping systems to stainless steel, HDPE
(high-density polyethylene) or fiberglass tanks. Less com-
monly, wood tanks can still be encountered in more tradi-
tional cider productions, even though this material is more
difficult to clean and sterilize, thus leading to potential product
spoilage.
The obtained pomace (200–400 kg per ton) still contains
compounds of interest. A second extraction can therefore be
performed by water leaching (countercurrent water percolation
of the pomace) or by pressing the watered pomace. These
actions lead to diluted juice (moût de diffusion or petit jus in
French) that will be added to the first juice obtained. The
extraction step may be facilitated by using enzymatic cocktails
(pectinases and hemicellulases, or pectin methylesterase [PME]
in the presence of calcium) – by themselves or in combination
with the mechanical material.
Prefermentation Juice Treatments
In France, juices are subjected to additional specific steps called
défécation and décantation (keeving and settling in English) to
obtain slow and long fermentations. Keeving corresponds to
121
the traditional juice treatment, and about half of French cider
juices are still treated by this method.
• Keeving relies on the ability of pectins to form a gel via PME
enzyme and calcium action. All compounds necessary for
keeving (pectin, PME, and calcium) are naturally present in
juice. During PME demethylation, an increase of acidic
groups on pectin chains is observed. In the presence of
calcium, bonds will form between pectic chains, thus lead-
ing to gel formation. After 2–6 days, CO2 is generated by
the onset of the fermentation process, and the bubbling
action will force the gel to rise to the top of the tank,
carrying along suspension deposits (rich in nitrogen) that
form a complex structure called chapeau brun. At the same
time, complexed materials will also sediment to the bottom
of the tank. The clarified juice is situated between the gel
and sediment layers. The natural process is slow and not
always reliable. Nowadays, in order to increase the reliabil-
ity of this static system, fungal PME preparations and CaCl2
are added. Recently, a continuous dynamic system, termed
flottation, has also been used by some cider producers. In
this case, the juice is treated for up to 48 h, before being
introduced in a specific device with nitrogen bubbling in
the presence of CaCl2.
• Settling is obtained by pectin hydrolysis, using a combina-
tion of enzymes (PME, endopolygalacturonase and pectin-
lyase). The clarified juice is obtained by decanting; some-
times this is followed by fining by gelatin addition. This
method is widely used by industrial cider producers.
The clarified juice obtained by either method is then trans-
ferred to another tank to begin the next production step.
Prefermentation clarification generates juices without any sus-
pension deposits and low bacterial counts, thus reducing
potential microbial spoilage. Moreover, amino nitrogen con-
tent and yeast counts are also reduced, thus leading to a slow
fermentation. Polyphenols have also been shown to be
affected by these treatments, mainly through a reduction in
both flavanol content and average degree of polymerization.
In the UK, keeving is not performed but juice additions
are. The goal is to obtain a blend of fermentable sugar
sources, including juice but also apple juice concentrates
and/or syrups that give a final product of 10–12% alcohol
after fermentation. Whereas in the case of French cider pro-
duction, everything is done to allow for slow fermentation by
reducing nutrients, reducing yeast counts, and using cold
temperatures. In the UK, the goal is to ferment juices to
dryness in as a short time as wine. To achieve this, several
nutrients are added to the blend. Although there is obviously
no sugar shortage in apple juice, ammonium nitrogen and
vitamins (enzymatic cofactors), which are essential for yeast
development and thus for fermentation, can be limiting,
thereby leading to sluggish or stuck fermentations. This is
especially the case for apple juice concentrates and syrups.
In this context, ammonium phosphate (250 ppm) will be
used to standardize the blend in amino nitrogen content
(about 100 mM). Thiamin, biotin, panthoneate, or pyridox-
ine vitamins can also be added. As performed in France,
British cider makers also depectinize the juice using pectino-
lytic enzyme preparations. This indeed facilitates filtration
operations and prevents haze formation.
122 Cider (Cyder; Hard Cider): The Product and Its Manufacture
Finally, sulfur dioxide (SO2) is an important technological
additive that is added during both French and British fermen-
tation processes. It presents two main properties of interest for
cider making and is widely used in oenology. First, it exhibits
antiseptic activity against bacteria and non-Saccharomyces yeast.
The multiple SO2 targets leading to microbial cell death
explains why no sulfite resistance has been observed to date.
Second, sulfite has antioxidant (leading to sulfate formation in
the presence of oxygen) and antioxidative (oxidase inhibition)
activities. SO2 performance is directly linked to pH as only the
undissociated form (termed ‘molecular SO2’) is active. There-
fore, it is recommended to adjust the pH of the juice to pH 3.75
by adding malic acid, the main organic acid found in apples.
Moreover, SO2 interacts with various chemical compounds
(especially aldehydes), microorganisms, and solids; therefore
only the so-called free SO2 will be active and participate to the
overall juice or blend quality. However, SO2 can negatively
affect cider aroma, in particular due to its action on oxidative
flora, and more important is considered to be an allergen (legal
limits for SO2 contents in ciders have been set in Europe).
Although no efficient alternative has been found to date, ascor-
bic acid (antioxidant properties) addition can help reduce SO2
concentrations.
Fermentation
Fermentation refers to microbial activities that will transform
the juice into cider. Like any other technological process, the
key factor to obtain the desired end product is control. This is
especially true and critical when living organisms are involved.
In the context of cidermaking, several technological levers can
be used. These levers correspond to biotic factors (microbial
inoculation or indigenous flora) and abiotic factors, either
intrinsic (pH, amino nitrogen content, sugar content) or
extrinsic (temperature, oxygen content). As discussed earlier
for amino nitrogen content, the contrast between the French
and British processes is also obvious for the fermentation step.
French production relies exclusively on indigenous flora.
The typical fermentation temperature is between 5 and 15
C,
due to the fact that cider tanks were traditionally situated
outside the buildings and thus directly influenced by seasonal
temperatures. This also prevents or inhibits lactic acid bacteria
(LAB) and spoilage microorganism growth. The combination
of low temperature, low amino nitrogen content, and lower
yeast counts (by centrifugation or filtration at strategical time
points) yields slow fermentation times, in accordance with the
desired organoleptic qualities. In the case of PDO and Label
Rouge ciders, a 6-week minimum fermentation time before
bottling is required. In the cider industry, temperature is regu-
lated between 8 and 10
C by refrigeration systems to control
the fermentation, whereas in traditional ciderhouses, racking,
centrifugation or filtration can still be used for control.
French cider production consists of several phases:
• Oxidative phase: This phase is named oxidative due to the
presence of oxygen in juice and lasts from 5 to 15 days.
From a microbiological point of view, high species biodi-
versity can be observed (Tables 1 and 2) with oxidative or
slow fermentative yeasts especially belonging to the Metsch-
nikowia, Hanseniaspora, and Candida genera (Figures 2(a)
and 2(c)). During this stage, little alcohol is produced
(<1%), but numerous volatile compounds are produced,
especially esters that largely contribute to the aromatic
qualities of the final product.
• Alcoholgenic phase (alcoholic fermentation): During the
transition between the oxidative phase and this phase,
microbial diversity largely decreases and the Saccharomyces
uvarum species becomes dominant (Figures 2(a) and 2(c)
and Table 1). Under anaerobic conditions, this species,
more adapted to cold temperature than Saccharomyces cere-
visiae (the main species carrying out the alcoholic fermen-
tation in many fermented beverages), mainly metabolizes
sugars into ethanol and CO2 as well as contribute to the
organoleptic qualities of the cider by producing fusel alco-
hols, acids, and more important, ethyl esters.
• Maturation phase: This stage actually corresponds to a
slower fermentation period, usually associated with a
decrease in S. uvarum counts and sometimes with the
appearance of other yeast species such as Lachanceae cidri,
S. cerevisiae, Brettanomyces anomala, or B. bruxellensis. In
general, LAB counts can also rise at this time (Figure 2
(b)), due to increased fermentation temperatures (climate
changes) and their metabolism (especially for the Oenococ-
cus oeni species). They induce malolactic fermentation (this
transformation sometimes happens concurrent with the
alcoholic fermentation). LAB species encountered mainly
belong to Oenococcus, Lactobacillus, Leuconostoc, and Pedio-
coccus genera (Table 2). Malolactic fermentation results in
the decarboxylation of the main apple juice acid (malic
acid) to lactate. The loss of a carboxylic (–COOH) function
leads not only to the production of CO2 but also to a
reduction in acidity. Although this metabolic step can pos-
itively contribute to the overall organoleptic quality of
the product, it is not always desired as it may lead to ciders
with high pH values (>3.75), compatible with microbial
spoilage. Malolactic transformation can be controlled by
racking, centrifugation, and filtration operations. The mat-
uration stage can last until bottling.
Except for traditional ciders, British fermentation can be consid-
ered as the complete opposite. Indeed, in the UK cider industry,
rapid and complete fermentations are performed. To do so, not
only are juice compositions adjusted as described earlier, but
they are also inoculated with commercial active dried yeasts
(S. uvarum, S. bayanus, or a mix of both). The commercial yeasts
usually originate from the wine industry. Notable technological
traits of these yeast strains include aroma production, killer
activity (favors strain development in the presence of other
yeasts), and flocculation (for easy clarification). Inoculation
trials, concerning both alcoholic fermentation (even using
mixed cultures for ester production), but also malolactic trans-
formation, have been performed in various countries; however,
to this date, this method has not yet been widespread.
Usually in the UK cider industry, an aerobic phase is per-
formed to favor yeast sterol production, essential to cope with
the final ethanol content. After this step, the inoculated juice is
fermented to dryness under anaerobic conditions for one week
at temperatures ranging between 15 and 20
C.
In all cases, alcoholic fermentation is monitored by follow-
ing density or specific gravity, until the desired residual
 Cider (Cyder; Hard Cider): The Product and Its Manufacture 123

Table 1 Yeast biodiversity in cider during the various production steps

Species Cider type Dominance Fermentation stages when species can be found
a
Arthroascus schoenii (formerly Endomyces French cider þ Cider must, oxidative phase
schoenii)
Cryptococcus sp.a French cider þ Water for apple transport, cider must, oxidative
phase
Candida matritensis French cider þ Cider must, oxidative phase
Candida oleophila French cider, UK cider þ/ Cider must, oxidative phase
Candida parapsilosis Spanish cider þ Cider must, oxidative phase
Candida pomicola French cider, UK cider þ Cider must, oxidative phase
Candida sake French cider þ/ Cider must, oxidative phase
Candida stellata French cider þ Cider must, oxidative phase
Candida tropicalis French cider þ/ Cider must, oxidative phase
Dekkera anomala French cider þþ Maturation phase, bottled cider
Dekkera bruxellensis French cider þ Maturation phase, bottled cider
Hanseniaspora osmophila French cider, UK cider, þ Cider must, oxidative phase
Spanish cider
Hanseniaspora sp. French cider þ Alcoholic fermentation phase, maturation phase
Hanseniaspora uvarum French cider, UK cider, þþþ Cider must, oxidative phase
Spanish cider
Hanseniaspora valbyensis French cider, UK cider, þþ Cider must, oxidative phase or late stages
Spanish cider
Issatchenkia occidentalis French cider þ Water for apple transport, cider must, oxidative
phase
Issatchenkia terricola French cider þ Cider must, oxidative phase
Kloeckera sp. French cider, Spanish cider þ Cider must, oxidative phase
Kluyveromyces marxianus French cider þ Cider must, oxidative phase
Lachanceae cidri (formerly French cider þþ Alcoholic fermentation phase, maturation phase,
Zygosaccharomyces cidri) bottled cider
Metschnikowia pulcherrima French cider, UK cider, þþ Cider must, oxidative phase
Spanish cider
Meyerozyma guilliermondii (formerly Pichia French cider, UK cider, þþ Cider must, oxidative phase
guillermondii) Spanish cider
Pichia delftensis French cider þ Cider must, oxidative phase
Pichia fluxum French cider, Spanish cider þ Cider must, oxidative phase
Pichia membranifaciens French cider þ Cider must, oxidative phase
Pichia misumaiensis French cider, UK cider þ Cider must, oxidative phase
Pichia nakasei French cider þ Cider must, oxidative phase
Rhodotorula ingeniosa French cider þ Water for apple transport, cider must, oxidative
phase
Saccharomyces uvarum French cider, UK cider, þþþ Alcoholic fermentation phase, maturation phase,
Spanish cider bottled cider
Saccharomyces cerevisiae French cider, Spanish cider, þþ Alcoholic fermentation phase, maturation phase,
UK cider bottled cider
Saccharomycetes sp. French cider þ Water for apple transport, cider must, oxidative
phase
Torulaspora delbrueckii French cider þ Cider must, oxidative phase

Dominance based on studies available in the literature and unpublished data (French ciders). þ, punctually identified; þþ, regularly present; þþþ, dominant species.
a
Filamentous fungi.

sugar concentrations are reached (in the UK: dry, in France: <
28 g l1 for ‘Brut,’ between 28 and 42 g l1 for ‘Demi-Sec,’
and >35 g l1 for ‘Doux’ ciders). Malolactic fermentation
is monitored by the follow-up of malate and lactate
using classical biochemical methods (ex. paper thin layer
chromatography).
Postfermentation Treatments
After fermentation, different operations are performed before
bottling. In most cases, cider is racked from the lees to clarify
and stabilize the product. If not, a limpid and stabilized
product is obtained by fining, centrifugation or filtration oper-
ations that can be combined or applied individually. Fining
consists of adding proteins that interact with polyphenols and
form complexes to entrap matter in suspension while settling
out. Typical fining agents correspond to gelatin (animal pro-
tein) or chitosan (prepared from crab-shell chitin); however,
bentonite, a negatively charged absorbent clay, can also be
added to accentuate sedimentation. Filtration is performed
using either kieselguhr, an unconsolidated form of diatomite,
or plate filtration or microfiltration. For traditional ciders,
fining and filtration are not performed, thus leading to yeast
haze and deposit in the bottle.
124 Cider (Cyder; Hard Cider): The Product and Its Manufacture

Table 2 LAB biodiversity in cider during the various production steps

Species Dominance Fermentation stages when species can be found

Lactobacillus brevis þ MLF fermentation phase, maturation phase

Lactobacillus buchneri þþ All stages
Lactobacillus casei/paracasei þ Early and late phases
Lactobacillus collinoides þþþ All stages
Lactobacillus diolivorans þ Occasionally identified
Lactobacillus mali þ All stages
Lactobacillus pentosus þ All stages
Lactobacillus sp. þ Maturation phase
Lactobacillus fermentum þ All stages
Lactobacillus plantarum þ All stages
Leuconostoc mesenteroides þþþ Early, oxidative phase, less frequent in late phases
Oenococcus oeni þþþ Dominant during alcoholic, MLF, and maturation phase, can be present in all stages
Pediococcus ethanodurans þ Occasionally identified
Pediococcus parvulus þ All stages
Pediococcus pentosaceus þ All stages

Dominance based on studies available in the literature and unpublished data (French ciders). þ, punctually identified; þþ, regularly present; þþþ, dominant species.

1.00E+08
1 2 3

1.00E+07

1.00E+06
M. pulcherrima

1.00E+05 S. uvarum
CFU/ml

H. uvarum
1.00E+04
P. membranifaciens M W A D0 D3 D7 D15 D30 D60 D120 D180
1.00E+03 C. pomicola
H. uvarum
Hanseniaspora sp. H. uvarum
1.00E+02
Hanseniaspora sp.
1.00E+01

C. pomicola
1.00E+00
1 4 7 29 60 123 180
Day M. pulcherrima
(a)
P. membranifaciens

1 2 3
1.00E+08

1.00E+07 S. uvarum
1.00E+06 M. pulcherrima
Lactobacillus
1.00E+05 mali
M. pulcherrima
CFU/ml

1.00E+04 Leuconostoc
mesenteroides
1.00E+03 (c)
Oenococcus
1.00E+02 oeni

1.00E+01

1.00E+00
1 3 7 15 24 64 119 180
Date
(b)

Figure 2 Examples of microbial dynamics during cidermaking observed using culture-dependent (PCR-RFLP for yeast (a) and ARDRA for LAB (b)) and
culture–independent (using TGGE for yeast (c)) methods. All methods showed a decrease in biodiversity leading to the dominance of S. uvarum
for yeast (responsible for alcoholic fermentation) and O. oeni for LAB (responsible for malolactic fermentation). 1, 2, 3 correspond to fermentation
stages; W, water used to transport apples; and A, apples in silos.

Ciders are then blended based on the experience of the and Spain, British and French ciders are expected to be effer-
cidermaker. Noteworthy, although limited additions are vescent. In industrial or artisanal ciders, carbonation is per-
allowed in most countries (see detailed legislations), in the formed to saturate the liquid with ca. 5–6 g l1 CO2. Natural
UK, water is added to reach the desired alcoholic level. effervescence is obtained through a supplementary step, called
Other additives permitted include sweetening agents such as prise de mousse in French, and is usually performed during the
sugar, but also acids, coloring agents, and preservatives to maturation phase. To do so, indigenous yeast fermentation, or
obtain expected organoleptic qualities. Unlike in Germany inoculation of active dry yeasts in bottles or in tanks is
 Cider (Cyder; Hard Cider): The Product and Its Manufacture
performed. The yeasts naturally ferment residual sugars to form
CO2. Temperature, yeast counts, and residual amino nitrogen
will be the main factors affecting effervescence in the final
product.
Finally, the product is bottled in glass or PET bottles or kegs
using an isobarometric filling machine to preserve product
effervescence. Pasteurization is possible according to product
type and nature of the container (tunnel pasteurization after
filling for glass bottles, flash pasteurization and chilling before
bottling for PET bottles). Although the temperature and dura-
tion applied for microbiological stabilization are not necessar-
ily high, due to pH and alcohol content, this step may still
impact the organoleptic characteristics of the final products
(typical cooked apples or caramel flavors). The product is
then commercialized.
Cider Spoilage
Different types of spoilage can occur during cidermaking and can
arise from either the presence of undesirable microorganisms or
specific physicochemical parameters. A survey performed in
2004 among French cidermakers showed that microbial spoilage
was of greater importance. In terms of frequency and economic
impact, the main microbial spoilages are
• Phenolic off-flavor: This organoleptic defect is due to volatile
phenols usually associated with ‘animal,’ ‘leather,’ ‘phenolic,’
or ‘spicy’ aromatic notes. This spoilage has been extensively
studied in wine and to a lesser extent in cider. It has been
shown that Brettanomyces spp. yeasts are responsible for the
transformation of hydroxycinnamic acids into volatile phe-
nols via vinyl phenol intermediates (these reactions are
mediated by hydroxycinnamate decarboxylase, and vinyl-
phenol reductase activities). However, in wine as in cider,
hydroxycinnamic acid mainly exists in an esterified form
(with tartaric acid in wine and quinic acid in cider) that
Brettanomyces cannot metabolize. In wine, it was shown
that commercial enzymatic preparations, containing a cin-
namoyl esterase, were mainly responsible for hydroxycin-
namic acid production. In cider, it was recently shown that
Lactobacillus collinoides could metabolize the main hydroxy-
cinnamic ester, chlorogenic acid (¼ caffeoylquinic acid), and
produce the caffeic acid precursor. This precursur can be
further metabolized into ethyl catechol by either L. collinoides
or B. anomala species.
• Cider-sickness: This spoilage, known as ‘framboisé’ in
France, is less frequent but can have a severe economic
impact. It mainly concerns sweet ciders. It occurs in
tanks or in bottles and is characterized by an excessive
production of acetaldehyde (often above regulatory limits,
see following). This leads to unpleasant flavors (rotten
banana, vegetal aromas), haze formation, high pressure in
bottles, and excessive foaming. This spoilage is associated
with the development of the Gram-negative bacterium,
Zymomonas mobilis (the subspecies pomaceae is associated
with British cider-sickness, whereas the subspecies francensis
is associated with French ‘framboisé’ ciders). It vigorously
ferments sugars into multiple end products including exces-
sive amounts of acetaldehyde and CO2. Favorable factors
are residual sugars, absence of SO2 addition, pH >3.75,
125
and high amino nitrogen. Acetaldehyde levels are regulated
in France (<100–120 mg l1).
• Acrolein spoilage: This spoilage is mainly associated with
ciders fermented to dryness and used for apple brandy
production. Spoilage is associated with LAB able to degrade
glycerol (including L. collinoides, although this trait may be
strain-dependent). Glycerol is produced (ca. 5 g l1) by
yeast during fermentation and provides roundness to the
product. Glycerol is degraded via a glycerol dehydratase
into 3-hydroxypropionaldehyde (3-HPA). This molecule is
a precursor for acrolein and can be nonenzymatically
formed via spontaneous dehydration in acidic conditions.
It leads to pepper flavors and a strong bitter taste.
• Lactic acid spoilage: This is due to the natural metabolism of
LAB, associated with their early development before alco-
holic fermentation is complete (in the presence of ferment-
able sugars). It can be easily detected as malolactic
transformation only leads to L-lactate while lactic acid spoil-
age is associated with both D- and L-lactate isomers. Acetic
acid is also formed by heterofementative LAB. The eco-
nomic impact of lactic acid spoilage is rather low.
The following microbial spoilages are less frequent:
• Ropiness: This spoilage is characterized by abnormal viscos-
ity associated with the presence of exopolysaccharides
(sugar polymers). The type of exopolysaccharides depends
on the producing microorganism. Responsible microorgan-
isms in cider have been associated to LAB (e.g., Pediococcus
damnosus, Lactobacillus sicerae), or sporulating bacteria
(Bacillus licheniformis).
• Acetic acid spoilage: This alteration is easily recognizable due
to its characteristic acidic taste and vinegar flavor. It is
associated with the development of acetic acid bacteria
(e.g., Acetobacter, Gluconobacter spp.) that metabolize etha-
nol in the presence of oxygen. Acetic acid can then react
with ethanol to form ethyl acetate, another off-flavor mol-
ecule. Therefore, maintaining the product in anaerobic
conditions will prevent this spoilage.
Concerning, physicochemical spoilage (‘casse’ in French), they
are observed when cider is in contact with air and thus, as
stated earlier, can be controlled by using anaerobic conditions.
In the presence of oxygen, polyphenol oxidation occurs
conferring product instability and color change. This reaction
is observed in the presence of metals (iron or copper)
that contribute to polyphenol complexation. Oxidative
spoilage is associated with the polyphenol oxidase enzyme,
linked to overripened or moldy fruits. Finally, protein haze,
characterized by deposits in bottles, is associated with
protein–polyphenol combinations. Noteworthy, protein con-
centration being low in cider, it is rarely observed.
Cider Composition, Organoleptic Qualities and
Health Impact
Composition
Cider composition (Table 3) first depends on apple juice com-
position and thus not only on the apple varieties but also culture
conditions, fruit maturity, and physical (cracks and bruises) or
126 Cider (Cyder; Hard Cider): The Product and Its Manufacture

Table 3 Physicochemical parameters determined for 150 ciders representative of French cider production

Total
Mass Alcohol L-Malate D-Lactate L-Lactate Acidity Volatile Sorbitol Fructose Glucose Sucrose sugar
1 1 1
density (%vol) pH (mg l ) (mg l ) (mg l ) (mequiv. l1) acidity (mg l1) (mg l1) (mg l1) (mg l1) (mg l1)

All cidersFrom 1000.9 1.4 3.28 0.00 0.04 0.04 1.11 0.03 3.1 8.2 0.1 0.0 8.0
To 1040.2 7.2 4.22 6.79 1.53 4.35 7.09 1.11 14.1 62.7 20.8 11.8 82.3
Mean 1017.4 4.1 3.74 1.29 0.24 1.61 2.63 0.42 6.3 34.5 7.1 0.4 39.6
Median 1016.6 4.2 3.76 0.45 0.16 1.79 2.29 0.43 6.0 34.2 5.8 0.0 37.2
Dry From 1000.9 2.9 3.43 0.00 0.06 0.04 1.26 0.03 3.7 8.2 0.1 0.0 8.0
To 1023.4 7.2 4.03 5.05 1.53 3.14 4.37 1.11 14.1 61.4 20.8 2.4 78.1
Mean 1011.3 4.9 3.75 1.25 0.27 1.48 2.27 0.54 6.5 25.7 4.0 0.1 28.3
Median 1010.6 4.9 3.75 0.67 0.18 1.71 2.01 0.53 5.8 25.5 3.5 0.0 27.0
Semisweet From 1010.4 3.0 3.28 0.00 0.05 0.12 1.11 0.04 3.1 22.7 2.3 0.0 24.9
To 1028.2 5.5 4.22 6.79 0.69 4.35 5.77 0.89 10.5 52.0 15.2 1.1 59.1
Mean 1018.4 4.2 3.77 1.14 0.19 1.94 2.25 0.44 6.4 37.1 7.2 0.1 42.1
Median 1018.3 4.2 3.80 0.38 0.14 2.15 2.09 0.50 6.5 36.9 7.1 0.0 42.1
Sweet From 1014.0 1.4 3.35 0.01 0.04 0.13 1.20 0.04 3.7 30.4 3.7 0.0 11.6
To 1040.2 5.2 4.06 4.19 0.57 3.45 7.09 0.67 7.3 62.7 18.6 11.8 82.3
Mean 1025.2 3.1 3.70 1.93 0.18 1.32 3.47 0.24 5.3 45.3 11.7 2.3 53.8
Median 1025.4 2.8 3.70 2.60 0.14 0.85 3.18 0.15 5.3 46.3 12.4 1.0 54.3

(Source: IFPC)

biological (molds, worm holes) damage. Then, the process and
microbial activity during fermentation impact the product.
Apple juice is mainly constituted of water (80–90% w/v) but
also contains various soluble compounds:
• Sugars: They are the principal component of the soluble
matter, representing from 75% to 90% (80–170 g l1). They
are encountered in various forms: mono-, oligo-, and poly-
saccharides (amylose, amylopectin, pectins). However, the
main sugar is fructose. It is the only one present in the final
fermentation phase, as other fermentable sugars (glucose and
the disaccharide: sucrose) are rapidly metabolized into fruc-
tose during the first two-thirds of the fermentation. Notewor-
thy, glucose can still be found in semisweet or sweet ciders. At
the end of fermentation, all or part of these sugars are trans-
formed into ethanol (generally 2–8% but up to 12% ABV –
alcohol by volume). In ciders, residual sugar concentrations
are generally situated between 8 and 67 g l1. Moreover,
sorbitol, a nonfermentable sugar alcohol, ranges from 3 to
14 g l in cider.
• Acids: The main acid, as noted earlier, is malic acid. It
can occur at concentrations between 1 and 10 g l1, accord-
ing to apple variety (ca. 85% of total acidity). Citric, acetic,
quinic, and citromalic acids can also be detected. Galac-
turonic acid can be detected when enzymatic preparations
have been used due to pectin degradation. In ciders, lactic
acid is detected if malolactic transformation has occurred.
• Polyphenols: Cider phenolic compounds are (1) flavan-3-ols
(composed of the monomers (þ)-catechin et ()-epicatechin
and their corresponding polymer: procyanidin; 2–5 g kg1),
(2) hydroxycinnamic acids (caffeic, p-coumaric and ferulic
acids), and their esters (chlorogenic acid or 5- caffeoylquinic
acid being the most represented; 0.3–2.5 g kg1), (3) dihy-
drochalcones (phloridzin, phloretin et phloretin-20 -O-
xyloglucoside; 0.02–0.10 g kg1) as well as (4) flavonols
(e.g., quercitrin, hyperin, or avicularin) and anthocyanins in
smaller quantities.
• Nitrogenous compounds: They are only present in small
quantities, although their content may vary considerably
according to the considered cultivar (40–150 mg l1).
Yeast-assimilable nitrogen substances are of interest as
they will condition fermentation speed and achievement
(as noted earlier, keeving can be used to control amino
nitrogen content in apple juice). They mainly correspond
to amino acids, in particular asparagine but also aspartic
acid (ca. 50% and 5% of total nitrogen, respectively).
• Vitamins: Vitamin C is the main vitamin found in apple
juice, but is generally lost during the process. Vitamin C
may be added in some productions.
• Minerals: Iron, potassium, as well as calcium and sodium,
are present in both apple juice and cider.
• Volatile compounds: These molecules are either varietal (orig-
inating from the fruit) or fermentative (originating from
microbial metabolism). They belong to various chemical
families: alcohols, esters, ketones, aldehydes, phenols, sul-
fur compounds, and organic acids.
Organoleptic Qualities
Concerning cider taste, the three main components that interact
to give the overall perception in the mouth are sugars, acids, and
polyphenols. Sugars contribute to sweetness and roundness
perception. In this context, fructose, and to a lesser extent sor-
bitol, largely contributes to the sugary flavor of the final product.
Acids contribute to cider flavor and directly influence sweetness
perception. A positive acidity perception is associated with prod-
uct freshness. Finally, polyphenols contribute to several product
traits: bitterness, astringency, and color. Concerning aroma,
many volatile molecules contribute, according to their sensory
perception threshold, to cider aroma. Although some cider
aromas are already present in apple juice (apple cultivar aromas
such as hexanol, ethyl 2-hydroxycaproate, ethyl lactate, or dia-
cetyl), a large majority are produced during fermentation. The
molecules correspond to alcohols like propanol, isobutanol,
isopentanol, benzylic alcohols, and 2-phenylethanol (known
for its rose aroma). Yeasts also produce esters during the alco-
holic fermentation (e.g., ethyl 3-methylbutyrate, 2-phenylethyl
 Cider (Cyder; Hard Cider): The Product and Its Manufacture 127

acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate
among many others). Terpene derivatives, sulfur-containing
compounds, and lactones can also be detected.
All these compounds contribute to the aromatic complexity
of the final product and rely on a subtle balance between them
(Table 4). As stated earlier, this balance can be affected by
several metabolites (e.g., volatile phenol, acetaldehyde, lactic
acid, acetic acid) originating from spoilage microorganisms.
Moreover, this can also be affected by aroma compounds
present at much higher levels than their sensory perception
threshold. Noteworthy, the amount of effervescence, bubble
size, and persistence will also affect overall product perception.
Health Impact
Like any other food product, a ‘benefits and risks’ approach
should be considered. The first aspect to consider is the nutri-
tional value of the product. As stated earlier, there is not one
cider but many ciders; thus a range of nutritional values can be
observed according to the considered cider. Examples of values
for French dry and sweet ciders are as follows: water 92.70 and
94.1, carbohydrates 2.35 and 3.21, fibers 0.5 in both types,
alcohol 3.2 and 1.9 g/100 g, respectively. No fat, starch, or
proteins are found.
Potential Benefits
• Microbial safety: Due to its intrinsic characteristics (low pH,
presence of alcohol and polyphenols – exhibiting bacterio-
static effects – and anaerobic conditions) and, in some
cases, the cidermaking process (pasteurization), cider does
not contain pathogenic bacteria or permit their growth.
Reported cases of Shiga toxin-producing E. coli (serotype
O157:H7) have been reported in the literature, but are
actually associated with the nonfermented and unpasteur-
ized North American nonalcoholic product called cider (by
opposition to hard cider).
• Antioxidant effect: Cider (like apples and apple juice) is rich
in polyphenols that exhibit antioxidant properties. These
dietary molecules have been recognized as having health
benefits by inactivating free radicals (O2 and other reactive
oxygen species) naturally produced in the body. In this
context, like other polyphenol-rich food and beverages,
cider and related apple products may contribute to

Table 4 Organoleptic descriptors associated with cider

Family Group Descriptor Family Group Descriptor

Tastes Acidic Sensation Sparkling

Bitter Astringent
Sweet
Aromas Fresh fruit Apple Aromas Floral White flowers
Pear Honeysuckle
Quince Violet
Apricot Acacia
Peach Honey
Grapefruit Rose
Lemon Spices Clove
Pineapple Cinnamon
Passion fruit Pepper
Mango Anis
Banana Resin
Raspberry Cedar
Strawberry Pine
Blackcurrant Grilled Roasting
Cherry Toasted bread
Complex fruit Crystallized fruits Sweet bread
Cooked fruit Licorice
Applesauce Smokey
Almond Woody Undergrowth
Hazelnut Fresh wood
Chestnut Humus
Fig ‘Brown’ Butterscotch
Prune Caramel
Vegetal Grass Chocolate
Mushroom Vanilla
Hay Mineral Flintstone
Mint Silex
Moss Milky Milk
Tobacco Butter
Yeast Animal Leather
Tea Musk
128 Cider (Cyder; Hard Cider): The Product and Its Manufacture
lowering risks of some chronic medical problems (particu-
larly cardiovascular diseases and cancers).
• Minerals: Apple cider is a good source of potassium and
iron. It is also low in sodium, making it compatible with a
sodium-restricted diet.
• Low calorie content: Ciders possess low calorie contents,
compared to other alcoholic beverages (especially wine).
For example, in France, whereas 79 kcal/100 g were deter-
mined for average wine, the following calorie contents have
been determined for average cider, dry cider, and sweet
cider: 30.2, 32.8, and 27.1 kcal/100 g, respectively. Dry
ciders have more calories than sweet ciders due to the fact
that, for a same quantity, ethanol represents more calories
than sugar (7 vs. 4 kcal g1 for ethanol and sugar,
respectively).
• Gluten-free: Cider is a gluten-free fermented beverage and
thus can be an alternative to beer for gluten-intolerant
people. This is especially used as a marketing argument in
North America, where a higher percentage of the popula-
tion is concerned with celiac disease.
Potential Negative Aspects
• Alcohol: Although ciders possesses low alcohol contents
(generally ranging from 2% to 8%) and observational stud-
ies suggest that moderate alcohol consumption is linked
with lower risks of coronary heart disease, cider should be
consumed in moderation like any other alcoholic beverage.
This is especially true for white ciders (7.5 ABV), an almost
colorless product made either by processing dessert apples
and pomace or by using apple concentrates with glucose or
corn syrup addition for alcohol production. This product,
although often containing little apple juice, falls under the
UK regulation cider definition (Customs Notice 162).
• Sugar: According to the amount of residual sugars or sugar
added, cider may contain little to high amounts of simple
sugars (up to more than 8 g per 100 ml). Therefore, this
should be taken into consideration in a balanced diet.
• SO2 : As stated earlier, sulfites are added during the produc-
tion process to control microbial growth. However, SO2 can
also be considered as an allergen to some individuals. In
Europe, labeling is compulsory if concentrations of more
than 10 mg kg1 or 10 mg l1 (expressed as SO2) of sulfur
dioxide and sulfites are present (Directive 2003/89/EC).
• Patulin: This mycotoxin is a secondary metabolite produced by
a number of fungal species (Aspergillus, Byssochlamys, and Pen-
icillium) with Penicillium expansum being the most frequent.
Although patulin can be found in apples and apple juice,
studies suggest that it is metabolized by Saccharomyces spp.
during alcoholic fermentation to form (E)- and (Z)-ascladiol,
apparently less toxic compounds. Although ciders are less at
risk, similar to fruit juices, concentrated fruit juices and other
fermented drinks from apples or containing apple juice, patu-
lin content is regulated to maximum levels of 50 mg kg1 at the
EU level (Commission regulation (EC) N
1881/2006).
• Biogenic amines: These molecules result from the metabo-
lism of living organisms (LAB in cider), mainly through the
decarboxylation of the corresponding precursor amino
acid. Histamine, tyramine, as well as putrescine and cadav-
erine, can be encountered. They are undesirable due to their
physiological activity, especially in sensitive consumers
(with natural or drug-related mono-amine oxidase deficien-
cies). No legislation exists limiting the presence of these
metabolites in cider.
See also: Alcohol: Metabolism and Health Effects; Alcohol: Properties
and Determination; Apples; Beverage: Health Effects; Beverage: Patterns
of Consumption; Lactic Acid Bacteria; Yeasts.
Further Reading
Dürr P (1986) The flavour of cider. In: Morton ID and Macleod AJ (eds.) Food flavours:
part B: the flavour of beverages, pp. 85–97. Amsterdam, Netherlands: Elsevier.
Jarvis B (2014) Cider (Cyder; hard cider). In: Batt CA and Tortorello ML (eds.)
Encyclopedia of food microbiologyVol. 1, pp. 437–443. Elsevier.
Jolicoeur C (2013) The new cider maker’s handbook: a comprehensive guide for craft
producers. White River Junction, USA: Chelsea Green Publishing Co.
Lea AGH and Drilleau J-F (2003) Cidermaking. In: Lea AGH and Piggot JR (eds.)
Fermented beverage production, 2nd ed., pp. 59–88. New York, NY: Kluwer
Academic/Plenum Publishers.
 Cirrhosis
S Honigbaum, J Lucas, and KB Schwarz, Johns Hopkins University School of Medicine, Baltimore, MD, USA
ã 2016 Elsevier Ltd. All rights reserved.
Malnutrition is a prevalent clinical complication of end-stage
liver disease (ESLD) among pediatric and adult populations.
The spectrum of malnutrition seems to worsen in relation to
the progression of liver dysfunction, further increasing the risk
of mortality especially in the case of the patient awaiting trans-
plantation. Optimizing the nutritional status of patients with
ESLD is therefore a key component of care.
Underlying Pathways Resulting in Malnutrition
Malnutrition is generally recognized in the setting of nutri-
tional losses, increased nutritional requirements, inadequate
ingestion, and/or metabolic disturbances. In patients with
ESLD, it is possible that each of these aspects may contribute
to malnutrition.
Nutritional Losses and Metabolic Disturbances
Lipids
With regard to malabsorption, hepatic dysfunction with cho-
lestasis results in decreased circulation of bile salts causing
nutritional losses of dietary lipids via steatorrhea. Deficiency
of the essential fatty acids (EFAs) can result. Since lipids pro-
vide a substantial amount of kilocalories (9 Cal g 1 of fat),
steatorrhea also leads to losses in overall energy intake and
poor growth. In an effort to promote increased total fat intake
and calorie absorption, medium-chain triglyceride (MCT)-
based products are often utilized because of their ability to be
absorbed without micelle formation. However, this practice
should be weighed carefully for risk–benefit, as this can con-
tribute to a greater degree of displacement of long-chain tri-
glycerides and EFAs (a very high ratio of MCT to total lipid
>80% is particularly risky). It is also of note that elongation
and desaturation of EFAs are decreased in cirrhosis. The liver of
a cirrhotic patient is more likely to depend on fuel from fat
stores versus carbohydrates.
Fat-Soluble Vitamins
Steatorrhea often results in deficiencies of fat-soluble vitamins
(vitamins A, D, E, and K) in ESLD.
Vitamin A
Vitamin A (retinol) deficiency can lead to poor immune func-
tion, growth failure, anorexia, epithelial keratinization, and
night blindness. Nearly all of retinol absorption is facilitated
by emulsification by bile acids and micelle formation. Vitamin
A is available to the tissues primarily via transport by the
retinol-binding protein (RBP). When the synthetic function
of the liver is affected, the production of RBPs is decreased.
However, assessment of true vitamin A status is difficult, since
serum retinol levels are not in accordance with actual hepatic
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00164-1
stores. Therefore, the use of the retinol/RBP molar ratio can be
used to help detect vitamin A deficiency (deficiency defined as
a molar ratio <0.8).
Vitamin D
Vitamin D deficiency can lead to rickets and osteomalacia.
Micelle emulsification is necessary for vitamin D absorption.
It has been estimated that 25–33% of children and adult
patients with chronic liver disease will have vitamin D
deficiency.
Vitamin E
Vitamin E deficiency can result in peripheral neuropathy,
myopathy, and hemolytic anemia. Lipoproteins are required
for vitamin E transport. Therefore, in disease states such as
cholestasis where lipoprotein production may be altered, it is
recommended that vitamin E status is assessed by the ratio of
serum vitamin E to total serum lipids (deficiency defined as the
ratio <0.6). It has been noted that vitamin E may be more
malabsorbed than vitamin D.
Vitamin K
Deficiency of vitamin K can cause bleeding, bruising, and
hemorrhage by way of impairing the synthesis of clotting
factors, which can be compounded by hepatic failure also.
Prothrombin time (PT) is generally used as an indicator of
vitamin K status; however, more sensitive measures of vitamin
K status (protein induced by vitamin K absence-II (PIVKA-II))
are increasingly available.
Carbohydrates
Cirrhotic patients are also more vulnerable to hypoglycemia, in
the setting of decreased hepatic glycogen stores and gluconeo-
genesis. In this case, ESLD patients, especially infants and
young children, are at high risk for catabolism in the fasting
state. Insulin resistance also complicates glucose utilization in
patients with ESLD.
Protein
The production of several key proteins (including albumins,
retinol-binding proteins, protease inhibitors, coagulation fac-
tors, iron-binding proteins, and insulin-like growth factors) is
decreased when the synthetic function of the liver is impacted
by disease. Protein degradation and amino acid oxidation and
proteolysis are all increased. Patients with chronic liver disease
have altered concentrations of amino acids (AAs), namely,
increased concentrations of aromatic AAs and decreased con-
centrations of branched-chain AAs. Moreover, hyperammone-
mia may result from decreased function of the enzymes
involved in the urea cycle. There is also protein malabsorption
due to enteropathy in portal hypertension.
129
130 Cirrhosis
Water-Soluble Vitamins
Pediatric patients may be susceptible to water-soluble vitamin
deficiency due to malabsorption and inadequate ingestion.
Adult patients, particularly those with alcoholic liver disease,
have been found to be deficient in thiamine, folate, vitamin
B12, and niacin. Folate and vitamin B12 deficiencies can result
in macrocytic anemia, while deficiency of vitamins B6 and B12
and thiamine can cause neuropathy. Thiamine deficiency has
been highly associated with alcoholic liver disease, which can
ultimately lead to Wernicke’s encephalopathy.
Minerals
Iron losses may result from gastrointestinal bleeding, and iron
stores may be affected in repeated episodes of bleeding. Zinc
and magnesium losses may affect those patients who are pre-
scribed diuretics. Zinc deficiency may also result due to poor
intake and decreased absorption. Zinc deficiency may result in
altered taste perception and anorexia, which may further con-
tribute to poor intake. Insufficient calcium levels can be found
due to steatorrhea and ‘hungry bone’ phenomenon in vitamin
D deficiency. On the other end of the spectrum, copper and
manganese may accumulate in the liver to the point of hepa-
totoxicity, and those patients who may be susceptible should
be monitored.
Inadequate Ingestion
Inadequate intake can be a significant causative factor of mal-
nutrition in ESLD. Taste changes, lack of appetite, early satiety,
and nausea and vomiting are all possible variables leading to
poor intake. Poor appetite and early satiety may be related to
increased leptin, tumor necrosis factor, and tryptophan. Poor
oral intake may be found in patients who are receiving a salt-
restricted diet due to the perceived unpleasant taste or unpal-
atable nature of the diet. Delayed gastric emptying in the
setting of hepatomegaly and ascites can contribute to nausea
and discomfort with eating.
Increased Nutritional Requirements
Energy
Hypermetabolism and increased energy requirements are char-
acteristic of adult patients with cirrhosis. Historically, predic-
tive equations have been used to estimate caloric requirements,
despite their inaccuracies. A few studies have validated the use
of indirect calorimetry to determine the resting energy expen-
diture (REE) in cirrhotic adult patients. Indirect calorimetry is
the gold standard but is not available in all centers. Among the
few studies in pediatric patients who underwent measurement
of REE, the results generally showed hypermetabolism
(upward of 125–140% of estimated needs). When indirect
calorimetry is not available, periodic serial assessment of
anthropometrics (especially arm circumference and triceps
skinfold) can serve as a guide for nutritional provision.
Protein
Patients with ESLD have increased protein needs. For pediatric
patients, a minimum of protein intakes of 2 g kg 1 day 1 has
been suggested. However, some studies have shown that
intakes of 2 g kg 1 day 1 do not support appropriate growth.
Ranges of 2–4 g kg 1 day 1 are therefore suggested. Age-based
guidelines for children with severe liver disease have not been
established. However, it might be reasonable to say that such
children might require at least twice the US Dietary Reference
Intakes for age. Expressed as g protein/kg/day, these values for
healthy children are as follows: 0.5–1 years 1.0 g, 1–3 years
0.87 g, and 4–8 years 0.76 g; males 9–13 years 0.76 g and
14–18 years 0.73 g; and females 9–13 years 0.76 g and 14–18
years 0.71 g. Protein restriction is not recommended in adult
or pediatric patients unless they evidence hepatic encephalop-
athy. Pediatric patients with cholestasis may require more
branched-chain amino acids (BCAAs). However, children
receiving ready-made commercial formulas, particularly those
that contain hydrolyzed protein, generally meet their BCAA
requirements. In adults, the ASPEN guidelines recommend
the use of standard formulas in critically ill adults with liver
disease, with provision of BCAA formulas only in refractory
encephalopathy. A 2003 Cochrane review evaluated 11 ran-
domized trials and did not find a benefit to the use of BCAAs in
patients with encephalopathy.
Nutritional Assessment in End-Stage Liver Disease
Nutritional assessment in liver disease is challenging yet is the
integral basis of nutritional intervention and management.
Nutrition assessment should be performed at the initial
meeting and routinely for monitoring. Nutrition assessment
can be composed of four constituents – anthropometric, bio-
chemical, clinical, and dietary components.
Anthropometry
Anthropometry is a very challenging component of the nutri-
tion assessment in liver disease. Many of the traditional mea-
sures of nutritional status, primarily weight, can be affected by
fluid status, hepatomegaly, and ascites and therefore may not
be a valid indicator of nutritional status. Monitoring of
abdominal circumference may be helpful. Length or height
may be a better indicator of nutritional status, especially
when measured in serial occurrences over a period of time.
Growth should be plotted and tracked on the appropriate
growth charts (World Health Organization for ages 0–24
months and Centers For Disease Control and Prevention for
ages 2–20 years). It can also be helpful to assess weight gain
velocity and linear growth velocity (i.e., gains in grams per day
or centimeters per month), and these data can be compared
with standard reference ranges. Weight-for-length and body
mass index (BMI) are also important markers. Monitoring the
weight-for-length or BMI is crucial for identifying the
underweight and trends in weight changes. Since weight and
linear growth can be affected in liver disease, the use of arm
anthropometry should ideally be a component of every nutri-
tion assessment. Mid-arm circumference (MAC) and triceps
skinfold (TSF) thickness are quick, noninvasive measures of
lean body mass and fat reserves, respectively.

Biochemical (See Section on Monitoring)
Clinical
Clinical findings play a large role in the dynamic process of
nutrition assessment and monitoring and, in fact, may repre-
sent the most important component. Clinical findings such as
edema, ascites, hepatomegaly, jaundice, vital signs, and gastro-
intestinal symptoms can shed light on the nutritional status of
patients with ESLD. The skin, hair, and nails of patients should
be reviewed or examined. Assessment of muscle wasting, fat
reserves, bone fractures, bowed legs, and acanthosis should be
considered as well.
Dietary
Lastly, an estimation of dietary and intake history should be
performed including administration, route, frequency, use of
alternative medicine and/or supplements, behavior, knowledge
and beliefs, food security and availability, and physical activity.
Information regarding food allergies and tolerance should also
be obtained. Careful attention should be paid to assessing
formula and enteral and parenteral nutrition intake. Nutritional
needs may vary according to the type and severity of liver disease.
Hepatocellular Disorders
Alcoholic Cirrhosis
Excessive alcohol consumption causes a spectrum of liver dis-
ease ranging from fatty hepatic infiltration to cirrhosis. Though
there is a clear association between alcohol intake and liver
disease, only a small proportion of people progress to cirrhosis.
It is generally accepted that the risk of the development of
cirrhosis increases proportionally with the amount of alcohol
ingested. Typically, cirrhosis occurs in patients who ingest
more than 30 g of alcohol per day for more than 10 years
(standard drinks estimated at 14 g per drink). The highest
risk for the development of cirrhosis is associated with ingest-
ing more than 120 g day 1 (8.5 standard drinks per day).
Alcoholic cirrhosis poses several nutritional challenges
because of its multifactorial nature. Factors that contribute to
malnutrition in this group of patients include anorexia,
decreased energy intake because of substitution of calories
from food for those in alcohol, poor nutrient digestion and
absorption, hypermetabolism, and decreased protein synthesis
and secretion. In addition, ascites can be present and lead to
premature satiety. Also, pancreatic insufficiency may coexist
and further lead to malabsorption.
In addition to the typical deficiencies seen in patients with
cirrhosis (i.e., fat-soluble vitamin deficiencies and protein–
energy malnutrition), individuals with alcoholic cirrhosis are
also predisposed to water-soluble vitamin deficiencies. The
vitamins and minerals most typically found to be suboptimal
in this population include vitamin A, vitamin D, thiamine,
folate, pyridoxine, and zinc. Thiamine deficiency (i.e., beriberi
and Wernicke–Korsakoff syndrome) may occur in alcoholic
cirrhosis due to decreased intake, decreased absorption, and
reduced thiamine storage. Thiamine deficiency is so common
in adults with chronic alcohol intake that it is generally recom-
mended that these patients receive daily supplementation with
thiamine as a preventive measure. In addition, it is important
Cirrhosis 131
to administer thiamine prior to glucose infusions to prevent
Wernicke’s encephalopathy (confusion, ataxia, and coma) in
these patients. Alcohol consumption can also commonly cause
pyridoxine (vitamin B6) deficiency with clinical manifesta-
tions of stomatitis, peripheral neuropathy, and confusion.
These patients also commonly have folate deficiency, resulting
in macrocytic anemia (although liver disease itself can cause
macrocytosis). In general, nutritional therapy involves cessa-
tion of drinking and providing adequate nutrition of proteins,
carbohydrates, and lipids.
Viral Hepatitis
Hepatitis B and hepatitis C can progress to chronic liver disease
and ultimately to cirrhosis, though the prevalence of cirrhosis
in these two populations is highly variable. Neonates who have
acquired hepatitis B infection perinatally have a much higher
chance of developing chronic hepatitis than those who acquire
it later in life. Hepatitis C, however, has a higher rate of devel-
oping into chronic liver disease in adults with approximately
80% of infected individuals developing chronic infection and
up to 50% of those individuals developing cirrhosis within
10–20 years. The nutritional recommendations for this patient
population are similar to the recommendations for other eti-
ologies of cirrhosis.
Autoimmune liver disease
The two main categories of autoimmune liver disease are auto-
immune hepatitis (AIH) and primary biliary cirrhosis (PBC).
PBC will be further discussed in the hepatobiliary section.
Though not all patients with AIH will progress to cirrhosis,
the progression may occur. The management of AIH typically
involves immunosuppressive medications including azathio-
prine and prednisone. Since prednisone is often needed for a
prolonged period of time, nutritional needs may be required to
combat the multiple negative side effects of steroids including
glucose intolerance, body composition changes, growth veloc-
ity disturbances, and osteoporosis, among others.
Metabolic Disorders
Wilson’s Disease
Wilson’s disease is an autosomal recessive inherited disease
involving improper copper excretion resulting in the accumu-
lation of copper in many organs including the brain, kidneys,
liver, and eyes (Kayser–Fleischer rings). In general, nutritional
recommendations for this disease involve avoidance of exces-
sive copper in the diet such as found in liver, mushrooms,
shellfish, nuts, beans, and chocolate. In addition, providers
often use zinc supplementation to help prevent copper absorp-
tion from the gastrointestinal tract and chelating agents such as
penicillamine and triethylenetetramine (trientine). For cases of
severe liver disease, transplantation is sometimes needed
despite dietary restrictions and pharmacological interventions.
Hemochromatosis
This is an autosomal recessive disorder that results in increased
iron absorption from the gut. Excess iron is deposited in many
132 Cirrhosis
organs including the liver and patients typically present in
adulthood with lethargy, abnormal liver function tests, hyper-
pigmented skin, and diabetes. Chronic alcohol use or concom-
itant viral hepatitis can again exacerbate the progression of
liver disease. The treatment for this disease is primarily periodic
phlebotomy. In general, patients should avoid supplements
containing iron, but diets are generally not restricted from
food high in iron, such as red meat, especially in the patients
undergoing therapeutic phlebotomy.
Nonalcoholic Fatty Liver Disease (NAFLD)
NAFLD is defined as hepatic fat accumulation without any
other known causes for secondary fat accumulation (i.e., exces-
sive alcohol consumption). It describes a range of liver diseases
from isolated hepatic steatosis (fatty deposits in the liver with-
out inflammation), to nonalcoholic steatohepatitis (NASH)
(which includes steatosis and inflammation), to hepatic fibro-
sis or cirrhosis and ESLD. The most common risk factors for the
development of NAFLD are central obesity, type 2 diabetes
mellitus, dyslipidemia, and metabolic syndrome. There is an
increasing prevalence of NAFLD over the last few years with it
now being considered the most common form of chronic liver
disease in the Western world. Nutritional therapy is geared
toward weight loss through dietary and lifestyle changes with
a reasonable goal of losing 1–2 lbs per week.
Hepatobiliary Disorders (Including Neonatal
Cholestasis Disorders)
Neonatal Cholestasis
There are many causes of neonatal cholestasis with the most
common being extrahepatic biliary atresia, idiopathic neonatal
hepatitis, alpha-1-antitrypsin deficiency, total parenteral nutri-
tion (TPN)-associated liver disease, cystic fibrosis, intrahepatic
cholestasis syndromes (i.e., Alagille syndrome), infection, meta-
bolic/genetic disorders (i.e., bile acid synthetic defects and galac-
tosemia), and endocrine disorders (i.e., hypothyroidism). For all
of these disorders involving varying degrees of cholestasis, there
is an underlying theme involving malabsorption of fat-soluble
vitamins and steatorrhea. Typically, nutritional management
involves supplementation of vitamins A, D, E, and K. Elemental
formulas are preferred because they are rich in MCT and more
easily digested. MCTs should account for 50% of the fat intake,
and providers should be aware that diets with excessive propor-
tion of total fats from MCTs (>80%) may result in fatty acid
deficiency. Overall, these patients often require energy needs
estimated at 130–150% of predicted needs for age. Risk factors
for poor growth in this population include patients with ESLD,
patients awaiting liver transplant, and patients with complica-
tions such as bleeding varices and ascites.
Biliary Atresia
Biliary atresia is a pediatric disease of unknown etiology that
results in the obliteration of the extrahepatic biliary system. It
causes neonatal cholestasis and ultimately progresses to
hepatic fibrosis and to liver failure if untreated. It remains the
number one cause of pediatric liver transplantation at this time
despite surgical intervention attempts with the Kasai hepato-
portoenterostomy to restore the drainage of bile. As with many
other causes of cholestasis and cirrhosis, severe steatorrhea and
malnutrition are common. Extra attention to nutritional status
should be paid to children with biliary atresia. Studies have
shown that poor nutritional status pretransplant is linked to
prolonged hospital stay, increased risk of mortality, and
increased cost of medical care. Efforts should be made imme-
diately after the Kasai procedure to improve the nutritional
status of this patient population, and clinicians should provide
supplemental nutrition via a nasogastric tube if needed and
even consider parenteral nutrition if enteral nutrition is inad-
equate or poorly tolerated.
Primary Biliary Cirrhosis
Primary biliary cirrhosis (PBC) is an autoimmune process that
destroys the intralobular bile ducts, eventually progressing to
hepatic cirrhosis and ultimately to liver failure. As with other
patients with cholestasis, patients with PBC have decreased
synthesis of bile acids leading to steatorrhea and decreased
absorption of fatty acids. The treatment of PBC is ursodiol,
and nutritional efforts should be made to monitor for fat-
soluble vitamin deficiencies, osteopenia/osteoporosis, and
overall malnutrition. Because PBC often occurs in post-
menopausal women, the incidence of osteoporosis is very
high in this population.
TPN-Associated Liver Disease
TPN-associated liver disease results from chronic intravenous
nutrition, typically in the setting of prolonged enteral fasting.
This condition is most frequently observed in pediatric patients
with short bowel syndrome who are dependent on intravenous
nutrition for extensive periods of time. The cause is multifac-
torial with a large contributor being enteral starvation. When
patients with TPN-associated liver disease have cholestasis,
they are at risk for the fat-soluble vitamin deficiencies as men-
tioned previously. When dependent on intravenous nutrition,
providers must be careful to monitor for deficiencies in vita-
min E and selenium. Conversely, copper, manganese, and
aluminum can accumulate to toxic levels. The treatment of
this disease is aimed largely on prevention. Many providers
limit the amino acid and lipid components when clinically
possible. In addition, though dextrose itself does not promote
liver injury, excess dextrose can lead to excess fatty acid synthe-
sis that causes hepatic steatosis.
Nutritional Therapies
In this section, the various types of nutritional support that can
be given to a patient with cirrhosis will be reviewed in relation-
ship to outcome. The recent Cochrane review of nutritional
support listed in the reference section contains a very detailed
summary of randomized clinical trials addressing these ques-
tions. The types of support include oral supplements, enteral
nutrition, and parenteral nutrition. Outcome has been assessed
as nutritional (weight gain, anthropometric measures, serum
chemistries such as albumin and bilirubin, and nitrogen

balance), clinical (effects on ascites, gastrointestinal bleeding,
and hepatic encephalopathy and rates of infection and post-
operative complications), economic (length of stay in the
intensive care unit and hospital and readmission rates), and
global (mortality). Various means of monitoring will be dis-
cussed as will drug–nutrient interactions.
Types of Support
Oral Nutrition
Frequent feeding can ameliorate the preferential early utiliza-
tion of fat and protein that occurs in patients with cirrhosis.
Late evening meals can positively affect nitrogen balance
compared with an equicaloric diet without a late evening meal.
Oral Supplements
A multitude of trials have been performed in adults with com-
pensated cirrhosis, malnourished cirrhosis, cirrhosis, and
encephalopathy. None of these trials showed an effect on
mortality except for one that actually showed increased fatal
outcome in the supplemented group. Likewise, there was no
difference in the development of encephalopathy, although
one randomized controlled trial did show resolution of hepatic
encephalopathy in patients receiving supplements with
branched-chain amino acids. None showed resolution of asci-
tes although several showed decreased development of ascites.
No effect on serum albumin was seen. Several trials examined
the effects of oral supplements on gastrointestinal bleeding,
and no beneficial effects were observed. Effects of oral supple-
ments on health-related quality of life have been investigated.
In general, no benefits were noted with the exception of the
effects of branched-chain amino acid-rich supplements on
some measures related to hepatic encephalopathy. Likewise,
in malnourished patients with cirrhosis undergoing liver trans-
plantation, the use of oral nutritional supplements was not
associated with effects on serum bilirubin, ascites, hepatic
encephalopathy, postoperative complications, length of stay
in the hospital or intensive care unit, mortality rates, or even
anthropometrics such as triceps skinfold or mid-arm muscle
circumference.
In summary, the administration of oral nutritional supple-
ments to patients with cirrhosis has been associated with few
benefits except for decreased development of ascites and
improvement of chronic hepatic encephalopathy.
Enteral Nutrition
Most trials failed to show a benefit although one was associ-
ated with improved nitrogen balance in medical patients and
another showed reduced postoperative complications in surgi-
cal patients.
Parenteral Nutrition (PN)
Although most studies of PN have failed to show any specific
effect in cirrhotic patients, one showed a reduction in serum
bilirubin and improved nitrogen balance. Another showed a
reduced incidence of postoperative ascites and yet another
Cirrhosis 133
showed that PN was associated with a reduction of postoperative
infections. However, in general, recipients of PN have better
survival versus comparable controls not receiving PN.
Special Nutritional Recommendations
Poor growth secondary to fat malabsorption in cholestasis can
be reversed by oral medium-chain triglycerides (but long-chain
triglycerides must be administered to avoid essential fatty acid
deficiency). Dietary sodium restriction and potassium supple-
mentation should be used to help manage ascites. To prevent
muscle wasting and negative nitrogen balance, 1 g kg 1 day 1
of protein is indicated for adults; 2 g kg 1 day 1 of protein is
recommended for infants and young children. Normalization
of zinc status should be done for patients with hepatic enceph-
alopathy (HE). Protein restriction is rarely necessary short term
and should never be done in the medium term or long term.
Branched-chain amino acid supplements are controversial for
acute HE; they do help with chronic HE.
Cholestatic patients require supplementation of fat-soluble
vitamins. Supplementation of water-miscible versions of vita-
min A is often utilized in clinical practice, but careful monitor-
ing is of paramount importance as toxicity can lead to further
hepatic damage. The recommended oral supplements of vita-
min A range from 5000 to 25 000 IU day 1. However, short-
ages of water-miscible vitamin A supplements may limit
administration. Vitamin D should be supplemented as vitamin
D3 (cholecalciferol). If the serum 25-OH vitamin D is low,
amounts of cholecalciferol three to ten times the DRI might be
necessary. Vitamin E should be supplemented with the water-
miscible form containing D-a-tocopheryl polyethylene glycol
succinate, also known as TPGS. Usually, 25 IU kg 1 day 1 will
suffice. Oral supplements of vitamin K 2.5–5 mg two to seven
times per week should be given. It has been suggested that all
fat-soluble vitamin supplementation be given with TPGS to
enhance absorption.
Monitoring
Monitoring the administration of nutrition to a cirrhotic patient
includes the assessment of weight, length in a growing child,
and anthropometrics, which may be the most reliable non-
invasive way to assess nutritional status. Serum metabolites
include electrolytes, blood urea nitrogen, creatinine, acid–base
status, calcium, magnesium and phosphorus, triglycerides, liver
enzymes, complete blood count/differential, platelets, pro-
thrombin time and partial thromboplastin time, iron indexes,
trace elements especially zinc and selenium, carnitine, and
folate/vitamin B12. Cholestatic cirrhotic patients should be
monitored for deficiencies of vitamins A, D, and E. The use of
triene/tetraene ratio can be used to assess EFA deficiency, while a
lipid panel can be used to assess metabolic syndrome or lipo-
genic changes. Although albumin and prealbumin should be
assessed as indexes of protein malnutrition, the interpretation of
values below normal may be difficult as both of these are
affected by protein nutrition and hepatic synthesis. Similarly,
prolonged prothrombin time may reflect malabsorption of vita-
min K, poor hepatic synthesis, or both. Although ammonia is
classically measured to monitor the risk of hepatic encephalop-
athy and as an indication for pharmacological management,
134 Cirrhosis
this analyte is notoriously subject to artifact. The ratio of plasma
BCAA/aromatic amino acids may be more useful to assess HE.
If nutrients are administered enterally, then gastrointestinal
tube placement and nose care should be monitored as needed
to assess the correct position of tube placement. If nutrients are
administered via an ostomy, then careful site care of the gastro-
stomy/jejunostomy should be done. If parenteral nutrition is
administered via a peripheral or indwelling central venous
catheter, then the line site should be assessed frequently for
signs of infection.
Given all the limitations of monitoring the nutritional sta-
tus of cirrhotic patients, it is useful to understand that the
subjective global assessment of nutritional status, which has
been modified for the cirrhotic patient, is perhaps the most
useful means of assessing the nutritional status of patients with
this condition. It is simple, reproducible, and validated and
includes the history of nutrient intake, physical examination,
and anthropometrics that can be easily performed.
Drug–Nutrient Interactions
Some of these interactions of particular importance in a cir-
rhotic patient are the effects on reduced elimination of potas-
sium by cyclosporine and spironolactone. Cirrhotic patients
are generally total body salt-overloaded, so it is particularly
important to be aware of how much sodium is being delivered
in intravenous lines to support the patient such as saline in
arterial lines and/or peripheral intravenous lines for antibiotic
delivery. Many parenteral medications are available as sodium
salts, further adding to the salt burden of the cirrhotic patient.
Antacids and sucralfate can bind phosphorus in the gut and
lead to hypophosphatemia. Thiazide diuretics may also lead to
phosphaturia and increased excretion of potassium. Hypergly-
cemia may result from cyclosporin A, tacrolimus, and/or cor-
ticosteroids; however, given that marked insulin resistance is
characteristic of cirrhosis, the precise cause of hyperglycemia
in a cirrhotic patient receiving one or more of these drugs may
be difficult to determine. The hepatic metabolism of 25-
hydroxyvitamin D is lowered by the anticonvulsants diphenyl-
hydantoin and phenobarbital.
A number of drugs commonly used in cirrhotic patients are
incompatible with parenteral nutrition solutions, including
amphotericin B, acyclovir, sodium bicarbonate, and ciproflox-
acin. Drugs used to treat HE such as lactulose and neomycin
may result in nutrient malabsorption.
Overall Recommendations
1. Nutritional assessment by anthropometrics should be done
to assess the basis for nutrient recommendations.
2. Given that there is little solid support for specific nutritional
interventions in cirrhotic patients, it is reasonable to provide
nutrients to meet basic requirements in the most cost-
effective manner possible that is easiest for the patient and
provides the best quality of life. Thus, the logical order of
provision of nutrients is oral nutrition with frequent small
meals including a bedtime meal and oral nutritional sup-
plements as necessary to maintain normal vitamin status
and manage chronic HE. If this approach does not work,
then provision of all or at least partial nutritional require-
ments via enteral feeding is recommended with PN reserved
for those who are unable to meet their nutritional needs via
the oral or enteral route. PN should ideally be administered
cyclically as opposed to constant infusion and for as short a
period of time possible, to avoid metabolic and infectious
complications.
3. Monitoring should be done for all cirrhotic patients to
assess the safety of nutritional interventions (weekly or
more frequently for those receiving parenteral nutrition)
and the effect of nutrients on nutritional status (monthly
or every other month) for anthropometrics. For selected
cirrhotic patients, additional studies could be undertaken
to investigate hepatic osteodystrophy (DEXA scan), retinop-
athy (evoked response electroretinography to assess the
functional consequences of vitamin A and E deficiencies),
and HE (ammonia and branched chain/aromatic amino
acids in plasma and proton magnetic resonance spectros-
copy for brain glutamine).
See also: Appetite Control in Humans: A Psychobiological Approach;
Cystic Fibrosis, Nutrition in; Enteral Feeding; Malnutrition: Concept,
Classification and Magnitude; Malnutrition: Prevention and
Management; Parenteral Nutrition; Vitamins: Overview.
Further Reading
American Society for Parenteral and Enteral Nutrition (2002) Guidelines for the use of
parenteral and enteral nutrition in adult and pediatric patients. Journal of Parenteral
and Enteral Nutrition 26: 1SA.
American Society for Parenteral and Enteral Nutrition (2009) Guidelines for the
provision and assessment of nutrition support therapy in the adult critically Ill
patient. Journal of Parenteral and Enteral Nutrition 33: 277.
Baker A, Stevenson R, Dhawan A, Goncalves I, Socha P, and Sokal E (2007 Dec)
Guidelines for nutritional care for infants with cholestatic liver disease before liver
transplantation. Pediatric Transplantation 11(8): 825–834.
Chalasani N, Younossi Z, Lavine J, et al. (2012) The diagnosis and management of non-
alcoholic fatty liver disease: practice guideline by the American Gastroenterological
Association, American Association for the Study of Liver Diseases, and American
College of Gastroenterology. Gastroenterology 142: 1591–1609.
Hasse J, Strong S, Gorman MA, et al. (1993) Subjective global assessment. Alternative
nutrition-assessment technique for liver-transplant candidates. Nutrition
9: 339–343.
Koretz RL, Avenell A, and Lipman TO (2012) Nutritional support for liver disease.
Cochrane Database of Systematic, Reviews 5, CD008344.
Lucey MR, Mathurin P, and Morgan TR (2009) Alcoholic hepatitis. The New England
Journal of Medicine 360(26): 2758–2769.
Mouzaki M, Ng V, Kamath BM, Selzner N, Pencharz P, and Ling SC (2014) Enteral
energy and macronutrients in end stage liver disease. Journal of Parenteral and
Enteral Nutrition 38(6): 673–681. Epub 2014 Feb 14.
Als-Nielsen B, Koretz RL, Kjaergard LL, and Gluud C (2003) Branched chain amino
acids for hepatic encephalopathy. Cochrane Database Systems Review
2, CD001939.
Pediatric Nutrition Care Manual (2014). http://www.nutritioncaremanual.org/&gt
(Accessed 03.2014).
O’Shea RS, Dasarathy S, and McCullough APractice Guideline Committee of the
American Association for the Study of Liver Diseases and the Practice Parameters
Committee of the American College of Gastroenterology (2010) Alcoholic liver
disease. Hepatology 51(1): 307–328.
Shneider BL, Magee JC, Bezerra JA, et al. (2012) Efficacy of fat soluble vitamin
supplementation in infants with biliary atresia. Pediatrics 130(3): 607–614.
Simopoulos A (2013) Dietary omega-3 fatty acid deficiency and high fructose intake in
the development of metabolic syndrome, brain metabolic abnormalities, and non-
alcoholic fatty liver disease. Nutrients 5: 2901–2923.
 Cirrhosis 135

Sultan MI, Leon CDG, and Biank VF (2011) Role of nutrition in pediatric chronic liver Relevant Website
disease. Nutrition in Clinical Practice 26: 401.
Young S, Kwarta E, Azzam R, et al. (2013) Nutrition assessment and support in children http://www.nutritionmd.org/health_care_providers/gastrointestinal/cirrhosis_nutrition.
with end-stage liver disease. Nutrition in Clinical Practice 28(3): 317–329. html – Nutrition MD.
 Citrus Fruits
AC Matheyambath, P Padmanabhan, and G Paliyath, University of Guelph, Guelph, ON, Canada
ã 2016 Elsevier Ltd. All rights reserved.
Citrus Fruits
Citrus fruit is one of the major horticultural crops grown
worldwide, and they are the most traded horticultural com-
modity in the world. The exact place of origin of citrus fruits is
still under debate, but it is believed that it originated from
Southeast Asia and spread to the other parts of the world.
Citrus crop is grown in developed and developing countries
as well. Citrus fruits constitute a crucial source of vitamin C.
Brazil, the Mediterranean countries, China, and the United
States account for about two-thirds of the total citrus produc-
tion. In the last 30 years, there has been a steady increase in the
per capita consumption of citrus fruits worldwide. North
America has the highest per capita consumption of citrus fruits
in the world followed by South America and Europe. Accord-
ing to FAO, fresh citrus fruit consumption is decreasing in the
developed countries while some of the developing countries
are showing an increase in consumption. Great variation exists
in the types of citrus fruits produced and consumed through-
out the various parts of the world. Oranges occupy the major
portion of world citrus production followed by mandarins.
Oranges form the majority of the citrus crop produced in the
United States. About one-third of the citrus fruits produced
globally are used for processing.
Members of Citrus Genus
Citrus is the largest genus of the Rutaceae family. The Citrus
genus is composed of several species and each species has
many varieties. Taxonomic identification is very complicated
and the precise number of species is still unclear because there
are many spontaneous and commercial hybrids. According to
the Swingle system, 16 species were recognized and the Tanaka
system identifies 156 species in the genus. Citrus fruits can be
generally classified into the following categories: sweet oranges
(Citrus sinensis; blood and acidless oranges), mandarins (such
as satsuma (C. unshi), tangerines (C. tangerina), C. reticulata,
and clementines (C. clementina)), sour/bitter oranges (such as
Seville (C. aurantium)), lemons (C. limon), limes
(C. aurantifolia and C. latifolia), grapefruit (C. paradisi) and
pomelo (C. grandis), hybrids (e.g., tangelos, tangors, and lime-
quats), and citrons (C. medica).
Types of Citrus Fruits
Sweet Oranges
Blood, navel, mandarin, and common oranges are the four
groups of sweet oranges. Sweet oranges belong to the species
C. sinensis.
136 Encyclopedia of Food and Health
Common Oranges
They are mostly globose but oval or ellipsoid is also common.
Common Valencia is oblong to spherical. The base of the fruit
is flattened in most varieties. The surface is not only smooth
but also finely pitted. Generally, most sweet oranges have
yellow to yellow-orange rind and flesh. The fruits in this
group are spherical, globose, or ovate. Valencia, Shamouti,
and Sathgudi are some sweet orange types.
Blood or Pigmented Oranges
The blood oranges are characterized by pink to red coloration
of the flesh, juice, and rind. Coloration is due to the presence
of anthocyanin pigments in the rind and flesh of the fruit. Red
color develops with warm days and cool nights, which is the
main feature of the Mediterranean climate. The
common blood oranges are ‘Maltese,’ ‘Moro,’ ‘Sanguinello
Moschato,’ etc.
Navel Oranges
This is an important group of fresh fruit orange varieties due to
its excellent quality. Navel oranges have a navel-like structure
at the stylar end or apex, which is a rudimentary secondary fruit
embedded in the primary fruit. Navel orange fruits are ovate to
oblong in shape. Most navel oranges, particularly the
‘Washington navel’ orange variety, the most widely grown
navel cultivar in the world, are ellipsoid or obovate in shape.
Its fruit’s surface is generally smooth and is moderately pitted
and pebbled. The fruits are mostly large and seedless. Other
common navel oranges are Navelate, Palmer, etc. Orange-
colored fruits are juicy and seedless with aromatic flesh. They
are excellent for fresh fruit market, but do not keep well on the
tree, not adapted to hot arid climate.
Mandarin (C. reticulata Blanco)
Most mandarins are deep orange to reddish orange in color
when fully mature and are easily peelable. Their fruits are small
or large (4–8 cm in diameter) with a low to high collar base
and a deeply depressed apex. Their fruits are globose to oblate,
5–9 cm in diameter, with a thin or thick rind with sunken oil
glands and a relatively smooth or, sometimes rough, surface.
Their fruits are seedless or seeded with nearly 3–7 or more
seeds and have 7–17 segments. Most mandarins lose quality
and the rind ‘puffs’ if not picked when internally ripe.
It is an evergreen tree with many spiny branches tolerant to
drought and cold. Although mandarins prefer subtropical
climate, they can also grow in tropical areas. Mandarin oranges
are classified into five groups: Satsuma, King, Willowleaf (Med-
iterranean), common, and small-fruited. Mandarins are
usually eaten fresh or as desserts as they are easily peelable
and have a distinctive pleasant flavor.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00165-3

Grapefruits (C. paradisi)
Grapefruits (C. paradisi) are considered to have originated in
the West Indies as a hybrid of pomelo or as a mutant. They
have many attributes similar to pomelos. They are one of the
large citrus fruits (although smaller than pomelos) with
diameters of up to 10–15 cm and have commercial possibil-
ities as fresh fruits. They are oblate to spherical in shape,
slightly depressed at the stylar end, and flat at the stem end.
The rind is medium in thickness, yellow or pink-blushed, and
smooth-surfaced. Grapefruits are borne in clusters like grapes
and are seeded to seedless. There are red-fleshed and white
fleshed cultivars of grapefruits. Red fleshness is due to the
accumulation of carotenoid and lycopene pigments. In trop-
ical regions, fruits develop with high juice content, thinner
peel, and lower acidity. Grapefruits can stay on the tree from
September to July in the northern hemisphere. However, it is
not suitable to leave the fruits on the trees for a prolonged
time because granulation may develop and seeds may germi-
nate within the fruit. People in North America, Europe, and
Japan prefer fresh grapefruit.
Pomelo (C. grandis or C. maxima)
Pomelo (C. grandis or C. maxima) originated in southern China
and is found growing wildly in China and in many north-
eastern states of India. Its fruits are the largest among the
commercially grown citrus in the world. The fruit diameter
commonly ranges from 20 to 30 cm. The fruits are globose to
pyriform in shape and are borne singly. Pomelos have a much
bigger fruit with thick peels and are less juicy than grapefruits.
They also have a firm flesh with crisp carpellary membranes
and juice sacs. They are white- or pink-fleshed and are seeded.
Lemon (C. limon (L.) Burm.)
Lemons are believed to have originated in India. The lemon
tree is a small thorny tree that grows well in subtropics and
tropics. The fruit size and shape of the lemon are highly vari-
able from spherical in Baramasi lemon or Eureka to oblong.
The fruit color is green to bright yellow at maturity and is with
or without a collar at the neck. Thickness and smoothness of
rind vary among varieties and lemons are either seedless or
seedy. Some varieties have large and prominent nipple, while
others have very small, inconspicuous nipple. The flesh of
lemon is pale yellow and very acidic. Some varieties of lemon
are quite juicy, while others have less juice. The quality of the
lemon fruit is excellent in semiarid irrigated areas and coastal
areas. In humid tropics, lemon trees produce fruits with coarser
peels. The lemon juice is valued for its tart, tangy, and fresh
flavor and is used as a common food ingredient in many
cuisines. The fruit rind is a source of commercial essential oil
and aroma compounds. Lemon trees are cold-sensitive than
oranges and grapefruits and the fruit can freeze at low temper-
atures owing to its low sugar content. Argentina, Spain, Cali-
fornia, and Italy are the leading producers of lemon. The
lemon fruit structure is similar to other citrus fruits and is
composed of the outer peel and the interior edible pulp. The
outer peel is composed of the exterior flavedo or the epicarp
and the interior white spongy part known as the albedo
Citrus Fruits 137
(mesocarp). The flavedo contains carotenoid pigments, vita-
mins, and essential oils. The albedo is composed of cellulose
and soluble carbohydrates (pectin and protopectin), flavo-
noids, amino acids, and vitamins.
Acid Lime (C. aurantifolia)
Acid lime fruits are high in acid. C. aurantifolia grow through-
out tropical and warm subtropical regions of the world and the
world’s leading producers are Mexico, Florida, the West Indies,
and Egypt. The most common acid lime is the small-fruited
acid lime or C. aurantifolia Swingle. It is also known as the
‘Mexican’ or ‘Key’ lime in the Florida Keys. The fruits are round
or elliptical in shape with a thin or thick rind depending on the
variety. The fruits have very small neck, a flat base, and a small
nipple at the apex. The highly acidic flesh is juicy with a distinct
aroma and flavor. The fruits are mostly seeded but seedless
varieties are also commercially available. The fruits of
C. latifolia Tan. are large (80–100 g) and are oblong in shape.
These limes are also known as Tahitian or Persian lime. Lime
trees are small shrubby and spiny, and they are cold-sensitive.
The rind surface is greenish yellow to yellow at maturity and
the tender flesh is juicy.
Sweet Lime (C. limettioides Tan.)
Sweet lime is also known as Indian sweet lime or Palestine
lime. It is mainly utilized for its medicinal properties. The
medium-sized fruit is pale yellow to orange yellow at maturity.
The fruit shape is round to oblong and the juice is nonacidic.
These limes are not commonly processed.
Citron (C. medica L.)
Citrons are grown in India. Citron fruits are long-oval or ellip-
soidal to obovate in shape like some of the lemons. Their fruits
are quite large with a yellow color and a very thick, rough, and
bumpy peel at maturity and are also very seeded.
Calamondin
Calamondin is commonly grown in southern China, Taiwan,
Japan, the Philippines, and northern India. It is a small
mandarin-like fruit of 3–3.25 cm in diameter, weighing
20–30 g. It has a smooth orange-colored peel that is very thin
(1–2 mm). Its fruit has an orange-colored acidic flesh with
seeds, but the peel is sweet and edible. It is commonly used
for making marmalade and for culinary purposes.
Citrus Fruit Development
Citrus fruits follow a typical sigmoid pattern of growth and
development, which is divided into three stages. The initial
phase, or phase I, is of slow growth including the period
between anthesis and June drop. Phase II is the rapid growth
period in which the fruit experiences a huge increase in size by
cell enlargement during 4–6 months. In the final phase, or
phase III or ripening period, growth is mostly arrested and
fruits undergo a nonclimacteric process. Citrus fruits are
138 Citrus Fruits
nonclimacteric fruits and hence there is no remarkable increase
of respiration rate and ethylene production during ripening as
in climacteric fruits such as mango or banana. Being non-
climacteric, citrus fruits do not ripen after harvest. Proper
handling of citrus fruits is needed during harvesting, storage,
transportation, and marketing to minimize wounding, stimu-
lation of respiration, and ethylene production, which can
induce physiological disorders and fungal rot. Citrus fruits
produce a very low quantity of ethylene after harvest and
there is no associated rise in respiration. However, fruits
respond to exogenous ethylene supply by an increase in
respiration, chlorophyll loss, calyx drying, and abscission. Dur-
ing ripening, citrus fruits change color from green to yellow or
orange or orange-red according to the variety. This is called
natural degreening, or natural color development. In some
citrus fruits, if held on the tree beyond maturity, the yellow-
orange color again changes to green, which is called regreening.
Regreened fruit, although internally mature, loses marketabil-
ity. The regreening process can occur on the tree and also after
harvest. In most citrus fruits, regreening occurs when the fruit is
on the tree, but in pomelo, regreening has been observed in
harvested fruit stored in natural light or fluorescent light.
Fruit Morphology
Citrus fruits are classified as a hesperidium. Hesperidium is a
modified berry resulting from a single ovary. The fruit consists
of 8–16 carpels that form the core of the fruit or segments that
contain the seeds and juice. Citrus fruits are characterized by
the presence of an outer rind or skin. The rind or peel of citrus
fruits is divided into an exocarp or flavedo, which is the outer,
colored part, and the mesocarp or albedo, which is the inner
colorless (white) or sometimes tinted part. The flavedo consists
of the epicarp proper, hypodermis, outer mesocarp, and oil
glands. Above the epicarp is a multilayered protective skin or
cuticle. The edible pulp of a mature citrus fruit is divided into
segments with or without seeds or juice sacs by a thick film or
endocarp surrounding the soft central axis. Each segment is
surrounded by a continuous endocarp membrane. In the seg-
ments, juice is contained in closely packed, club-shaped multi-
cellular sacs, also called juice vesicles, which completely fill the
segments. A thin wall called the carpellary septum surrounds
the segments. Each juice sac also has a very minute oil gland at
the center. The seeds (ovules) are also attached to segment
walls by means of axial placentation.
The pomological and organoleptic attributes of citrus fruits
are quite diverse. The fruit diameter ranges from a few
centimeters in Mandarins to more than 25 cm for some
pomelos (C. grandis). The fruit’s shape also varies: fruits are
oblate in tangerines, mandarins, and grapefruits, spherical or
oval in sweet oranges, oblong in lemons (C. limon) and citrons
(C. medica), and spherical in limes (C. aurantifolia). The fruits
usually have 8–16 segments with or without seeds. Albedo
forms the major portion of the citron fruits, while it is absent
in kumquats and poorly developed in mandarins. The fruit
pulp color depends on the presence or absence and abundance
of carotenoids and anthocyanins in the pulp, and it can
be pale, yellow, orange, or red. The size and shape of the
seeds are also variable among species. Navel oranges and
Tahiti lime (C. latifolia) are seedless, while grapefruit and
pomelo have 30–50 seeds. The fruit flavors and essential oils
are diverse both qualitatively and quantitatively.
Methods of Consumption
Citrus fruits are consumed fresh or processed into various
products. Citrus fruits including oranges, mandarins, tanger-
ines, and grapefruits are peeled and eaten fresh or as desserts.
A major portion of citrus fruits produced are utilized for pro-
cessing. The fresh fruit segments are also added to salads and
desserts and on cakes. The most common citrus fruit product is
fresh or frozen orange juice concentrate made from freshly
squeezed and filtered orange juice. Additionally, citrus fruits
are also made into marmalade, jams, jellies, candies, or wine.
The dried and pulverized fruits are used for preparing confec-
tionaries and flavoring baked products. Fresh or dried peel of
citrus fruits has a variety of uses. The grated outermost layer of
the rind, zest, is used in cooking and baking. The pulp,
obtained after the extraction of orange juice, is dried and
used as an emulsifier and binder in the food and beverage
industries. Essential oils extracted from the peel of citrus fruits
are used commercially for flavoring drinks, ice cream, pudding,
desserts, chewing gum, sweetmeats, ice cream, sherbet, and
other products. They are also a used by the pharmaceutical
industry for the preparation of drugs, soaps, perfumes,
cosmetics, and home cleaning products. Dried orange blos-
soms and leaves are used to make herbal teas. Fresh juices
from limes and lemons are made into refreshing drinks. Lime
and lemon juices are also suitable for garnishing and for
flavoring dishes and curries. The fruit is also made into pickles,
salted preserves, and dried limes and processed into beverages,
jams, jellies, and marmalade. Essential oils are also extracted
from the peel and are extensively used in flavoring soft drinks,
confectionery, chewing gum, sweets, ice cream, sherbet, and
other food products.
Biochemical Composition of Fruits
Sugars and Acids
The total soluble solids form 10–20% of the fruit fresh weight
and are mainly composed of carbohydrates (70–80%) and
small quantities of organic acid, proteins, lipids, and minerals.
The insoluble portion mainly contains cell structure polysac-
charides. The total solid contents in citrus fruit increase with
ripeness due to an increase in sugar content. Sucrose, glucose,
and fructose constitute the major fruit carbohydrate compo-
nents. In mandarins and tangerines, sucrose, glucose, and
fructose occur in a general ratio of 2:1:1. Sugars are prominent
in tangerine, oranges, and grapefruits, while limes and lemons
are rich in citric acid. Additionally, trace amounts of mannose,
maltose, heptuloses, and galactose have also been identified in
citrus fruits. Sucrose is the major nonreducing sugar in citrus
fruits. Sucrose, in very small amounts, is present in limes and
lemons. Arabinose, a pentose sugar, has been identified in lime
and grapefruit. Starch content in citrus fruits is negligible.

Organic acid contributes to the acidic properties of citric
fruits. Citric acid forms the major acid in citrus fruits. Malic
acid forms the second most acidic component though present
in much lower amounts than citric acid. Other organic acids
including succinic, lactic, and oxalic have also been detected in
fruits. Organic acids exist in free form or in combined form in
salts (such as citrates and malates), esters, or glycosides. The
titratable acidity or the free acidity of the citrus fruit juice is
mainly due to citric acid contents. The total acidity represents
the sum of all acids in free and combined forms. Citric acid
is the predominant organic acid in juices, while malic, malo-
nic, oxalic, and quinic acids form the major citrus peel organic
acids. Acidity varies with fruit maturity and citrus species. It
ranges from 0.5% to 1.5% in orange juice, 0.8% to 2.5% in
grapefruit, and 4% to 8% in lemons and limes. In many
oranges and grapefruit varieties, acidity declines with fruit
ripeness due to a decrease in citric acid content. In lemons, a
decrease of pH with an increase in acidity occurs with fruit
ripeness. Citrus fruits are very low in fat content and are also
not a good source of proteins.
Nutritional Value of Citrus Fruits
Citrus fruits have long been valued as important sources of
vitamin C. Besides being deficient in cholesterol, citrus fruits
also contain an impressive list of health-benefiting nutrients
including essential minerals, vitamins, and dietary fibers.
Citrus fruits are principal sources of many bioactive
phytochemicals with disease-preventive properties including
flavonoids and limonoids.
Minerals
Citrus fruits are good sources of dietary potassium and are
relatively low in sodium (3–4 mg per 178 ml of orange
juice). The ratio of sodium and potassium plays a role in
the maintenance of electrolyte balance. Citric acid in citrus
fruit can help in the absorption of calcium and other minerals
by acting as a chelator.
Vitamin C
Among vegetables and fruits, citrus fruits are particularly
popular as excellent dietary sources of vitamin C and can
supply vitamin C content ranging from 23 to 83 mg per
100 g fresh weight on an average. Consumption of citrus fruits
in moderate amounts may be the best way to meet the 100% of
the RDA for vitamin C. Studies showed that intake of orange
juice (500 ml day 1) for two weeks (250 mg ascorbic acid per
day) resulted in increased plasma vitamin C concentrations by
40–64% and reduced oxidative markers in adults. A reduction
in plasma lipid peroxidation was observed in healthy adults
who consumed orange juice (8 oz or 236 ml) daily (70 mg
vitamin C) for 2 weeks.
b-Carotene
The only fat-soluble vitamin occurring in citrus fruits in sub-
stantial quantity is vitamin A in the form of provitamin A
Citrus Fruits 139
carotenoids. Carotenes and b-cryptoxanthin form the major
vitamin A precursors in citrus fruits. b-Cryptoxanthin is the
major vitamin A precursor in tangerines, mandarins, and
oranges, while a- and b-carotenes represent a minor portion
of carotenoids in some oranges. Among various foods, citrus
fruits including oranges and tangerines are the most concen-
trated dietary sources of b-cryptoxanthin. The content of pro-
vitamin A carotenoids varies greatly among different citrus
fruits.
Folic Acid
Citrus fruits and their juices are good sources of natural folates,
and according to a study in Europe, folate was 100% bioavail-
able from orange juices and is highly stable. There is strong
scientific evidence supporting a positive relationship between
dietary folate consumption and the prevention of neural tube
defects in infants. Citrus fruits (100 g) can provide up to
10–20% of the RDA for adults and children less than 9 years
of age, complementing to the dietary folate requirement.
Another study reported that the consumption of orange juice
(750 ml) daily for four weeks increased the plasma folate
concentrations in adults by 18%.
Dietary Fiber
Cellulose, hemicelluloses, and pectins in citrus fruits are a
source of dietary fiber. Pectin is the predominant soluble die-
tary fiber component of citrus fruits constituting about
65–70% of the total fiber content. Pectin occurs both in the
edible portions of fruit and in the inedible residues such as
peel, rag, and core. Consumption of fresh citrus fruits such as
oranges and grapefruits can contribute significant quantities of
pectin to the human diet. Insoluble fiber in citrus fruits is
composed of polysaccharides and they play an important role
in human diet in preventing digestive disorders because of
their water-holding capacity.
Limonoids
Limonoids are one of the most health-benefiting bioactive
components of citrus fruits owing to their versatile health-
promoting and disease-preventing properties. Citrus
limonoids were considered as the culprits causing delayed
bitterness of the juices at room temperatures lowering the
juice quality. Limonoids are highly oxygenated and modified
triterpenes classified as tetranortriterpenoids. The term ‘limo-
noid’ is derived from limonin, the first limonoid component
identified as the bitter component of citrus seeds. Several other
limonoid compounds such as obacunone, nomilin, obacunoic
acid, isolimonic acid, deacetylnomilin, ichigan, isoobacunoic,
and dictomnolide were also identified. Limonin and nomilin
are the predominant limonoids of citrus fruit. Limonoids are
grouped into aglycones and glucosides. Aglycones are bitter
limonoid compounds in the peels of citrus fruits, while gluco-
sides are tasteless components. Both aglycones and glucosides
are present in citrus seeds, but fruit tissue contains only
glucosides. More than 50 limonoid aglycones and glucosides
have been identified from various Citrus species. Evidence from
several in vivo, in vitro, and animal studies suggests that
140 Citrus Fruits
limonoids have anticancer properties and showed potent cyto-
toxic activities. In studies conducted using animal models and
cell lines, limonoids have been shown to inhibit proliferation
of the cancers of the stomach, colon, breast, skin, and pancreas.
Limonoids including limonin and nomilin were also found to
have antibacterial and antiviral properties. Studies also indi-
cated that the anticancer properties of limonoids can be corre-
lated to the induction of glutathione S-transferase, a major
detoxifying enzyme system.
Flavonoids
Citrus fruits are known to be rich sources of polyphenolics
such as flavonoids and phenolic acids. More than 60 types of
flavonoids have been identified in citrus fruits belonging
mostly to 4 different flavonoid groups: flavones, flavanones,
flavonols, and anthocyanins. In the genus Citrus, flavanones
are more abundant in high concentrations than flavones. Fla-
vanones are concentrated in albedo and in the membranes
than in the juice sacs. Hesperidin, naringin, and neohesperidin
are the major flavonoids in citrus fruits. Citrus flavanones
occur in the form of aglycones or glycosides. Hesperidin is
the prominent flavonoid in orange and it is not bitter, but
less soluble in water. Hesperidin also occurs in mandarins,
lemons, and limes. Naringin, the major flavonoid in grape-
fruits and shaddock, has a bitter taste and it is soluble in water.
Seeds and peels of citrus fruits are also rich in phenolic
compounds such as phenolic acid and flavonoids. Naringin,
neohesperidin, and neoeriocitrin are the main neohesperido-
side flavanones present in bergamot, grapefruit, and bitter
orange juices. Bergamot, orange, mandarin, and lemon juices
contain the rutinoside flavanones: hesperidin, narirutin, and
didymin. Caffeic, chlorogenic, ferulic, sinapic, and p-coumaric
acids are the most abundant phenolic acids present in citruses.
Citrus flavonoids are known for their powerful free radical
scavenging and for their anti-inflammatory activities. The anti-
inflammatory properties of citrus flavonoids, hesperidin and
its flavone analog diosmin, are due to their inhibition of the
activities of proinflammatory mediators such as prostaglandins
E2 and F2 and thromboxane A2. In vitro studies have also
shown that citrus flavonoids can inhibit the reactions catalyzed
by cyclooxygenase, lipoxygenase, and phospholipase A2. Cit-
rus flavonoids have also been shown to possess platelet anti-
adhesive and antiaggregation properties. Epidemiological
studies have indicated an inverse relationship between regular
consumption of citrus fruits and reduced risk of developing
cardiovascular diseases, atherosclerosis, and inflammation.
Flavonoids also exhibit antibacterial and antiviral activities.
Evidences from a number of studies have demonstrated that
citrus flavonoids or the purified single citrus flavonoid com-
ponents (hesperidin, naringin, naringenin, etc.) possess
antiproliferative activities and are capable of inhibiting the
proliferation of many kinds of cancer cell lines including
melanoma, colon, breast, squamous, leukemia, lung, prostate,
colorectal, and hepatomas. Although the exact mechanisms
explaining the anticancer properties of citrus flavonoids are
still largely unclear, a number of mechanisms have been
proposed. Flavonoids have been suggested to exert multiple
actions and are involved in the inhibition of metastasis, inhi-
bition of tumor progression, inhibition of angiogenesis in
tumor cells, arrest of cell cycle progression, protection of
DNA against oxidative damage, inactivation of carcinogens,
signal transduction, etc.
See also: Cancer: Diet in Cancer Prevention; Vitamins: Overview.
Further Reading
Bayazit V and Konar V (2010) Biochemical and physiological evaluations of limonoids
as potential cancer destroyers. Journal of Animal and Veterinary Advances
9: 1099–1107.
Benavante-Garcia O and Castillo J (2008) Update on uses and properties of Citrus
flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory
activity. Journal of Agricultural and Food Chemistry 56: 6185–6205.
Gattuso G, Barreca D, Gargiulli C, Leuzzi U, and Carristi C (2007) Flavonoid
composition of citrus juices. Molecules 12: 1641–1673.
Liu YQ, Heying E, and Tanumihardjo SA (2012) History, global importance and
nutritional importance of citrus fruits. Comprehensive Reviews in Food Science and
Food Safety 11: 530–545.
Peterson JJ, Dwyer JT, Beecher GR, Bhagavat SA, Gebhardt SE, Haytowitz DB, and
Holden JM (2006) Flavonones in oranges, tangerines (mandarins), tangors, and
tangelos: a compilation and review of the data from the analytical literature. Journal
of Food Composition and Analysis 19: S66–S73.
Silalahi J (2002) Anticancer and health protective properties of citrus fruit components.
Asia Pacific Journal of Clinical Nutrition 11: 79–84.
Tripolis E, La Guardia M, Giammanco S, Di Majo D, and Giammanco M (2007) Citrus
flavonoids: molecular structure, biological activity and nutritional properties: a
review. Food Chemistry 104: 466–479.
Tundis R, Loizzo MR, and Menichini F (2014) An overview on chemical aspects and
potential health benefits of limonoids and their derivatives. Critical Reviews in Food
Science and Nutrition 54: 225–250.
Turner T and Burri BJ (2013) Potential nutritional benefits of current citrus
consumption. Agriculture 3: 170–187.
Relevant Website
http://apps.fas.usda.gov/psdonline/circulars/citrus.pdf – The United State Department
of Agriculture, Foreign Agriculture Service.
 Clostridium botulinum
A Harris, The Food and Environment Research Agency (Fera), York, UK
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Clostridium botulinum is an anaerobic spore-forming gram-
positive bacillus, which is ubiquitous in the environment. C.
botulinum and in rare cases C. butyricum and C. baratii can
produce neurotoxins that are among the most potent known
to man. C. botulinum produces eight botulinal neurotoxins
(BoNTs), types A–G. Human botulism is usually associated
with types A, B, and E and rarely with type F, while animal
botulism is usually associated with types C and D. Type G has
been reassigned as C. argentinense, which has been associated
with sudden death but not neuroparalytic illness. Disease from
type G is unknown in animals.
C. botulinum can be divided into four subgroups I–IV, dif-
ferentiated by their biochemical activities. Group I is proteo-
lytic, grows at temperatures between 10 and 45
C, and can
produce toxin types A, B, and F. Group II is nonproteolytic,
grows at temperatures between 3 and 45
C, and can produce
toxin types B, E, and F. Those in group II are also termed
psychrotrophic because of their ability to grow at temperatures
below 5
C. Groups I and II are the main groups associated
with human illness, and those in group I account for almost all
reported cases of infant botulism. Group III is nonproteolytic
and produces toxin types C and D; and group IV can be weakly
proteolytic and produces only toxin type G.
Types of Botulism
There are three main types of botulism, namely, foodborne,
infant, and wound. Another two clinical categories are
recognized – adult intestinal toxemia and iatrogenic botulism.
Foodborne botulism is the most common form of botulism
and is caused from ingestion of foods containing C. botulinum
toxin. In the case of foodborne botulism, contaminated prod-
ucts may be widely consumed, thus exposing a large number of
people to the hazard and may subsequently represent a med-
ical and public health emergency.
Infant botulism affects infants less than 1 year of age and is
caused when infants ingest C. botulinum spores, which then
germinate and produce toxin in the intestine. The spores may
come from the environment or from foodstuffs. Honey has
been previously implicated in the disease and is no longer
recommended for infants less than 12 months. Almost all
cases of infant botulism have been due to types A and B.
Wound botulism occurs when C. botulinum infects a wound
and subsequently produces toxin. It has been associated with
major soil contamination through compound fractures or
severe traumas/lacerations. The incidence of wound botulism
has increased considerably in recent years, particularly among
injectors of black tar heroin.
Adult intestinal toxemia is the colonization of the intestine
with C. botulinum following ingestion of spores and is
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00172-0
analogous to infant botulism. Germination, vegetation, and
toxin production by C. botulinum are not usually supported in
the normal adult intestine; however, in a small number of
adults who have a bowel abnormality or bowel disease or are
undergoing antimicrobial therapy, colonization can occur.
Iatrogenic botulism is the accidental overdose of toxin (e.g.,
via inhalation by laboratory workers or via injection for ther-
apeutic or cosmetic purposes).
Toxin Action
All of the toxins produced are large single polypeptides
(130–150 kDa) of a similar structure. Enzymatic cleavage pro-
duces a 100 kDa heavy chain and a 50 kDa light chain linked
via a single disulfide bond. In proteolytic strains, the enzymatic
cleavage is caused by proteases produced by the cell, while in
nonproteolytic strains, an exogenous protease such as trypsin is
involved.
The toxin either enters the blood stream directly or is
absorbed intact from the gastrointestinal tract. The toxin
binds via gangliosides to high-affinity presynaptic receptors.
It is then transported into the nerve cell through a receptor-
mediated endocytosis process common with dichain toxins.
The toxin acts by blocking the normal release of the neuro-
transmitter acetylcholine that under normal circumstances trig-
gers contractions of the skeletal muscle. The toxin binds
irreversibly, and the recovery of function depends on ultra-
terminal sprouting of the nerve to form new motor endplates.
Each of the seven toxins (A–F) has different specific toxic-
ities and durations of persistence within the nerve cells.
Symptoms
C. botulinum intoxication in adults is characterized by a des-
cending symmetrical flaccid paralysis. Early symptoms include
blurred vision, slurred speech, dry mouth, difficulty in
swallowing, and muscle weakness. Botulism, particularly the
early symptoms, may be confused with a number of other
conditions including poliomyelitis, viral encephalitis and
meningitis, myasthenia gravis, Guillain–Barré syndrome, tick
paralysis, stroke syndromes, Eaton–Lambert syndrome, alco-
hol intoxication, drug overdose, antimicrobial-associated
paralysis, and atropine, shellfish, or mushroom poisoning.
Early symptoms may vary depending on the route of infec-
tion/intoxication. Foodborne botulism has an incubation
period ranging from 2 h to 8 days with the majority of cases
having an onset 12–72 h postexposure. Nausea, vomiting,
abdominal cramps, and diarrhea may precede the neurological
symptoms described earlier.
The incubation period for wound botulism is considerably
longer than for foodborne botulism and may range from 4 to
141
142 Clostridium botulinum
18 days. This is due to the organism first causing infection and
then the subsequent in vivo production of toxin in the wound.
Neurological symptoms are as described earlier and may
include the presence of a fever.
The incubation period in infant botulism is difficult to
ascertain but can be between 3 and 30 days. C. botulinum
intoxication in infants is characterized by ‘floppy baby
syndrome.’ Infants may be constipated and have feeding diffi-
culties, poor sucking reflex, lethargy, loss of head control, and a
feeble cry. In the adult version of intestinal toxemia, clinical
manifestations are similar to those observed with foodborne
botulism and may include the early gastrointestinal symptoms.
In comparison to foodborne botulism, the onset is generally
gradual and less dramatic.
In cases of iatrogenic botulism, the incubation period for
inhalational exposure is similar to that of foodborne botulism
with incubation periods ranging from 24–36 h to several days
postexposure. Clinical symptoms are as described earlier while
lacking any gastrointestinal symptoms.
In cases of overdose by injection, incubation periods vary
depending on dosage. The doses usually recommended for
cosmetic purposes are too low to cause disease. However,
higher doses injected for treatment of muscle movement dis-
orders have caused systemic botulism like symptoms in
patients. Patients who received injections of botulinum toxin
at a much higher dose than that recommended for cosmetic
purposes have resulted in symptoms of severe botulism and
long-term recovery.
In the absence of medical intervention, mortality rates as
high as 50–60% have occurred. Mortality rates vary based on
the patient age and type of botulism and are generally lower in
those aged <20 years. With improvements in critical care
management, fatality rates have dropped to ca. 16%, and
where death does occur, it is often due to delayed diagnosis/
treatment. Death is usually via respiratory failure due to paral-
ysis or nosocomial infections (particularly pneumonia) result-
ing from long-term hospitalization. Mortality rates for
foodborne botulism are 5–10%, 15–17% for wound botulism,
and <1% for infant botulism.
Recovery can take weeks to months and full neurological
recovery from weakness and fatigue may require as long as 1
year. In 95% of cases, the nerve terminals regenerate and
recovery is complete. Extended outpatient rehabilitation is
often required.
Complications
Although there are no additional specific complications
resulting from botulism intoxication, the potential complica-
tions of prolonged paralysis, assisted ventilation, and
nutritional support are significant. Nosocomial infections
may include pneumonia; urinary tract infections (from
indwelling Foley catheters); stress ulcers and sores; thrombo-
phlebitis, cellulitis, and line infections due to peripheral and
central intravenous catheters for prolonged periods; fungal
infections; and also deep vein thrombosis and pulmonary
embolism due to patients being bedridden for weeks to
months.
Infectious Dose
The estimated lethal doses for purified crystalline botulinum
toxin type A for a 70 kg man are 0.09–0.15 mg when introduced
intravenously, 0.7–0.9 mg through inhalation, and 70 mg
orally. The precise human toxicities for the remaining BoNTs
are uncertain. All seven toxins have been shown to cause
inhalational botulism in primates and therefore have the
potential to cause human botulism if a significant exposure
occurs. It is thought that 1 g of pure toxin dispersed in animal
feed would be enough to kill 400 000 adult cows. Type A toxin
produces a more severe disease than types B and E because of
differences in amount of ingested toxin, absorption, or recep-
tor affinity. Type A intoxication results in respiratory failure
occurring more rapidly and more severely than with the other
types of botulism. Mortality rates are lower in type B disease
than with type A or E, and it has been shown generally that the
longer the incubation period, the better the prognosis.
Diagnosis/Confirmation
The early diagnosis of botulinum intoxication is paramount as
antitoxin therapy is most effective when administered early.
Initial diagnosis is based on clinical history, physical examina-
tion, epidemiological history, and electromyography results.
Epidemiological histories should include risk factors that may
support the diagnosis including black tar heroin injection
(wound), laboratory work with botulinum toxins or receiving
nonapproved neurotoxin preparations for therapeutic or cos-
metic purposes (iatrogenic), and recent consumption of a
home-canned product (foodborne).
Diagnosis is confirmed by demonstration of botulinum
toxin or the spores in the suspected food or patient specimens
(vomit, feces, serum, gastric aspirate, and wound). It has been
shown that the toxin may routinely be present in the serum
7–9 days after exposure and in some patients for up to 28 days
postexposure.
Infant botulism diagnosis is made on clinical symptoms
and confirmed by recovery of the organism or by detection of
the toxin in stools.
In cases of inhalational botulism, diagnosis is more difficult
as often toxin is not identifiable in the serum/stool.
The definitive diagnosis is made by the mouse bioassay,
and positive in vitro assays are confirmed using the mouse
bioassay.
Treatment
Treatment of botulism required intensive hospital care and
may include where necessary mechanical ventilation and
administration of antitoxin. The antitoxin is of equine origin
and may carry a significant risk of serum sickness, and skin
sensitivity testing should always be undertaken before admin-
istration. Hypersensitivity is in the region of 10–20%.
Control of the airway and ensuring adequate ventilation are
of primary importance, and in some cases, endotracheal intu-
bation may be required.

If exposure is known to have occurred within several
hours, then induced vomiting and gastric lavage should be
commenced. Even after several days, an emetic agent may be
advisable to help eliminate any unabsorbed toxin still
present.
Several forms of antitoxin are available and some agencies
(e.g., Centers for Disease Control, the United States, and Public
Health England, the United Kingdom) recommend the admin-
istration of antitoxin based on clinical diagnosis without wait-
ing for laboratory confirmation. The antitoxin neutralizes toxin
not yet bound to nerve terminals but does not neutralize toxin
already bound.
Additional treatment for wound botulism includes debride-
ment, drainage, and irrigation of the wound. As toxin produc-
tion may continue until the infection with C. botulinum is
eliminated, the administration of antibiotics is also likely.
In March 2013, the Food and Drug Administration (FDA),
the United States, approved the first botulism antitoxin (BAT)
that can neutralize all seven known botulinum nerve toxin
serotypes. The BAT heptavalent (A, B, C, D, E, F, and G)
(equine) is derived from horse plasma and is now the only
drug available for treating adult botulism in the United States.
BAT has superseded the use of a licensed bivalent botulinum
antitoxin AB and an investigational monovalent botulinum
antitoxin E. It is also the only available drug for treating infant
botulism that is not caused by nerve toxin type A or B.
In cases of infant botulism, a human-derived botulinum
immune globulin (BabyBIG) is available. BabyBIG is obtained
from the plasma of immunized adults who have subsequently
developed high titers of neutralizing antibodies against BoNTs
A and B. BabyBIG is derived of human origin and therefore
does not have the high anaphylaxis risk inherent with equine
sources or the risk of lifelong equine antigen hypersensitivity.
Although supportive intensive care including mechanical ven-
tilation may be required, the use of BabyBIG can significantly
reduce the length of hospital stay for infants.
Prevalence in the Environment
C. botulinum is ubiquitous in the environment and can be
found in soil; dust; marine and freshwater sediments; intestinal
tracts of animals, birds, and fish; vegetables; fruits; leaves;
silage; and animal manure.
There is a geographic predilection by C. botulinum type
with type A spores prevalent in western United States, Bra-
zil, Argentina, and China. Proteolytic type B spores are
primarily found in eastern United States and nonproteolytic
type B spores in the United Kingdom and continental
Europe. Type E spores predominate in aquatic environ-
ments and are found in many northern regions such as
Alaska, northern Canada, Scandinavia, Russia, and Japan.
Types C and D are more associated with the warmer cli-
mates of Indonesia, Australia, and South Africa with type C
more prevalent in some aquatic environments occupied by
waterbirds. Type F, which is found more rarely, has been
detected in the soils/sediments of Brazil and Paraguay and
in aquatic environments. Type G has been isolated from
soil in Argentina and Switzerland.
Clostridium botulinum 143
Prevalence in Foods
Because of the prevalence of C. botulinum in the environment,
there is potential for a wide range of raw food materials to carry
spores. In many cases, the common food vehicles associated
with C. botulinum are characteristic of the country or culture
and the prevalence of the C. botulinum type found in that
environment. In certain parts of the United States, China,
Spain, and Italy, consumption of vegetables contaminated
with type A is the most common cause of botulism, while in
Alaska, Japan, Canada, and Scandinavia, cases are usually asso-
ciated with fish/aquatic products contaminated with type E. In
central continental Europe, cases are typically from home-
cured meats and usually associated with type B spores. In
cases of infant botulism, honey has become a recognized
source for C. botulinum spores.
Group I strains are frequently related to insufficiently pro-
cessed home-preserved foods such as canned vegetables and
cured meats, whereas group II strains are more of a risk in
foods processed with mild heat treatments and subject to
refrigerated storage (e.g., REPFEDS (refrigerated processed
foods of extended durability)).
Commercially prepared foods are rarely implicated in bot-
ulinum outbreaks due to stringent controls, but when a failure
does occur, mass intoxication may result. In the United
Kingdom in 1989, a major outbreak occurred resulting in 27
people affected with one fatality. The outbreak was due to a
yogurt containing hazelnut puree, which had been sweetened
with aspartame rather than sugar. The processing of the con-
serve had been inadequate to destroy C. botulinum spores. In
the United States in 2007, eight people contracted foodborne
botulism after eating commercially produced canned food
products (including hot dog chili sauce), which had been
underprocessed.
The majority of botulism cases today are associated with
low-acid home-preserved foods particularly vegetables that
have a pH > 4.6, for example, peppers, garlic, and carrots. In
2006 in Thailand, 209 people were affected with 42 people
developing respiratory failure, neuromuscular failure, and
autonomic nervous system failure. The outbreak was due to
ingestion of pickled home-canned bamboo shoots.
Global Incidence
In 2010, in 29 of the European Union (EU) and European
Economic Area (EEA) countries (all except Liechtenstein), 104
cases of botulism were confirmed. This was a decrease of 21%
when compared to 2009 when 132 cases were confirmed.
Poland, Romania, Italy, and France accounted for 80% of the
confirmed cases. The EU trend has been stable during 2006–10
with the confirmed case rate ranging from 0.02 to 0.03 cases
per 100 000 population (European Centre for Disease Preven-
tion and Control, Annual Epidemiological Report 2012). Pre-
vious data from the same source indicate similar numbers of
cases reported between 2005 and 2010 with 152 cases reported
in 2005, 157 cases (109 confirmed) in 2006, 171 cases (129
confirmed) in 2007, and 151 cases (112 confirmed) in 2008.
Between 1995 and 2004, there were 2388 reported cases
144 Clostridium botulinum
although not all countries reported for all years. Incidence in
Europe (50 countries) is considerably higher than in just the
EU/EAA (31 countries).
In the United States, a total of 112 laboratory-confirmed
cases of botulism were reported to the CDC in 2010. Of these,
foodborne botulism accounted for 9 (8%), infant botulism for
85 (76%), wound botulism for 17 (15%), and botulism of
unknown or other etiology for 1 (<1%) cases (Centers for
Disease Control and Prevention, Botulism Annual Summary,
2010). Previous data from the same source indicate similar
numbers of cases reported between 2005 and 2010 with 145
cases in 2005, 170 cases in 2006, 144 cases in 2007, 153 cases
in 2008, and 121 cases in 2009. In general, infant botulism was
the predominant cause each year.
Between 1976 and 2007, a total of more than 3000 cases of
infant botulism have been globally identified, and as of 2009,
infant botulism has been documented in 26 countries repre-
senting 4 continents.
In England and Wales, wound botulism accounts for the
most cases of botulism with 144 cases between 2000 and 2010.
This compares to four cases of foodborne botulism and six
cases of infant botulism during the same time period. In the
United States between 2005 and 2010, there were 158 cases of
wound botulism compared to 573 cases of infant botulism and
101 cases of foodborne botulism. Thirteen cases were of
unknown or other etiology.
Survival, Prevention, and Control
In order for foodborne botulism to occur, the food must
initially be contaminated with C. botulinum spores. The envi-
ronment provided by the food must be anaerobic and non-
acidic with low sugar and salt to enable the germination of the
spores into cells capable of growth and toxin production. The
food must then be consumed without sufficient prior heating
to destroy the toxins (85
C for >5 min).
To control and prevent botulism, the processing and sub-
sequent storage of the food must be such that it results in either
the destruction of the spores or the prevention of their germi-
nation, growth, and subsequent toxin production.
C. botulinum produces spores that can survive in a dry state
for decades. The spores are highly resistant to heat, desiccation,
UV light, and alcohols. They may survive several hours at
100
C but will not survive exposure to moist heat at 120
C
for 30 min. Spores of the nonproteolytic group II types are
more heat-sensitive than the group I types (proteolytic) but
can still withstand many of the mild pasteurization techniques
currently in use (70–95
C). In contrast, the toxins are heat-
labile and are inactivated by heating at 85
C for  5 min.
Processing/Storage/Shelf Life
The proteolytic group I types cannot grow below temperatures
of 10
C, so are not of concern in cold-distributed foods. These
group I types when they grow often cause food spoilage that
may indicate to consumers the defective quality of the food.
However, the potency of the toxin is such that even tasting a
product spoiled by C. botulinum may cause intoxication. A case
in 2002 occurred where an individual tasted a mouthful of
foil-wrapped baked potatoes but did not swallow it due to the
spoiled taste. He had however consumed sufficient toxin for
botulism to occur and spent more than 6 months in hospital
recovering.
The strains of group II are nonproteolytic and can grow at
temperatures as low as 3
C. These strains may show no out-
ward signs of obvious spoilage of the foodstuff and are more of
concern in products that are minimally processed and have
refrigerated storage, for example, REPFEDS.
The increased consumer demand for fresher less preserved
foods has led to the use of milder heat treatments combined
with other food preservation hurdles. This includes chilled
storage, a controlled shelf life, and occasionally intrinsic pre-
servatives (e.g., reduced pH). Hermetic sealing may be used to
create anaerobic conditions that may extend the shelf life, but
this may also create conditions favorable for the growth of
C. botulinum in cases of product abuse. Product abuse (i.e.,
where there is failure to store a product at a low temperature
or to consume within a certain timeframe) may lead to
increased consumer risk from group II spores, which can
grow and produce toxin at temperatures as low as 3
C.
C. botulinum spores are resistant to pressure but can be
destroyed by combinations of high-pressure and high-
temperature treatments. The spores and toxins of all strains
are also relatively resistant to the freezing temperatures used
for food storage.
Heat Resistance
The proteolytic strains of C. botulinum (group I) produce spores
that are highly heat-resistant, and these are the principal con-
cern for the safe production of low-acid canned foods. As such,
a universal botulinum cook is used in the canning of low-acid
foods (pH >4.6). A botulinum cook results in a 12 log reduc-
tion of spores at 121
C for 3 min or an equivalent time/
temperature combination. High-acid foods (<4.6) do not sup-
port the germination and growth of group I spores, and for
these products, the botulinum cook is not required.
The D-value or decimal reduction time is the time it takes at
a given temperature to reduce a population by 90%. Group I
spores are considerably more heat-resistant than group II
spores although these still show moderate heat resistance.
Group I spores have a D121
C of 0.21 min and a D100
C of
25 min, while group II spores have a D121
C ¼ < 0.001 min
and a D100
C of <0.1 min. It has been shown that heating
group II spores for 5–10 min at 80–85
C can inactivate their
spore germination system due to sublethal injury but that in
the presence of lysozyme, germination can still be induced and
the heat resistance increased by up to two orders of magnitude.
pH/Sodium Chloride Concentration
The growth of proteolytic C. botulinum strains is inhibited at
pH <4.6 or by 10% sodium chloride (NaCl), with the mini-
mum water activity to allow growth being 0.96 where NaCl is
used as the humectant (Table 1). Growth and toxin production
of nonproteolytic C. botulinum strains are inhibited at pH <5 or
in the presence of >5% NaCl concentration. For these strains,
the minimum water activity to allow growth is 0.97 where
NaCl is the humectant. It should be noted that water activity
 Clostridium botulinum 145

Table 1 Characteristics of C. botulinum groups I and II Therapeutic/Cosmetic Use of Botulinum Toxin

C. botulinum group I C. botulinum group II
Despite the potency of C. botulinum neurotoxins and their
Toxin types A, B, F B, E, F involvement in serious disease, their use for therapeutic and
Proteolytic Yes No cosmetic purposes (particularly of BoNT type A) is increasing.
Minimum temperature 10
C 3
C The use of neurotoxins for cosmetic purposes such as reducing
Optimum temperature 35–40
C 18–25
C the appearance of wrinkles and frown lines is well established
Minimum pH 4.3–4.6 5 but offers only temporary improvement. C. botulinum neuro-
NaCl 10% 5% toxin is used in a wide variety of medical conditions associated
Minimum awa 0.94 0.97 with muscular hyperactivity, glandular hypersecretions, and
D121
C spores 0.21 min <0.001 min
pain. These include focal dystonias, spasticity, nondystonic
D100
C spores 25 min <0.1 min
disorders, strabismus, chronic pain and disorders of localized
a
May vary, depending on humectant. muscle spasms, smooth muscle hyperactive disorders, and
sweating and salivary and allergy disorders. The use of neuro-
values may vary depending on the humectant used with glyc- toxin does not correct the underlying condition but improves
erol permitting growth at lower aw values. Humectants (salts or clinical symptoms for a time. The duration and quality of
sugars) absorb moisture making water less available for micro- improvement vary depending on the dose, dilution, and
bial growth. The toxins of both groups I and II are stable at method of injection. The effects generally last approximately
low pH. 3 months after which further injections will be needed. There is
a risk of the patient developing resistance, but this has been
reduced by giving the lowest effective dose and ensuring long
Disinfectants periods between injections.
C. botulinum spores are readily killed by chlorine-based disin-
fectants and exposure to formaldehyde. The spores are sensi-
tive to most disinfectants authorized in the food processing See also: Canning: Process of Canning; Chilled Foods: Modified
sector as long as the correct time/concentration combinations Atmosphere Packaging; Chilled Foods: Packaging Under Vacuum;
are used. Clostridium: Occurrence and Detection of Clostridium botulinum and
Botulinum Neurotoxin; Minimally Processed Foods.

Detection

A variety of methods exist for the detection of C. botulinum

toxins and may include ELISA (enzyme-linked immunosor-
bent assay), PCR (polymerase chain reaction), endopeptidase
assays, and lateral flow immunoassays, among others. These
methods have been used as screening tools and may provide a
rapid result in some matrices. However, the gold standard for
the detection of toxin still remains the mouse bioassay. This
involves the extraction of the toxin (if required, e.g., from
foodstuffs), trypsinization if required (necessary for non-
proteolytic strains), and then intraperitoneal inoculation of
laboratory mice. Mice are subsequently observed for signs of
botulism, which may include ruffled fur, labored breathing,
Cheyne–Stokes respiration, and pinching of waist resulting in a
‘wasp waist.’ The mice are observed for 3 days, but generally,
symptoms of botulism will be observed within the first 24 h.
The toxin type may be determined by neutralization tests with
specific antitoxins. The mouse bioassay is very sensitive and is
measured as MLD (minimum lethal dose) with one mouse
LD50 corresponding to 5–10 pg and a detection limit of
20–30 pg ml1. Detection of C. botulinum spores usually
results from providing a suitable environment, which enables
the growth and toxin production of the organism. Detection is
then confirmed by the presence of toxin as described earlier.
Further Reading
Advisory Committee on the Microbiological Safety of Foods (ACMSF). (1992). Report
on vacuum packaging and associated processes. London. Her Majesty’s Stationary
Office.
Brook I (2006) Botulism: the challenge of diagnosis and treatment. Reviews in
Neurological Diseases 3(4): 182–189.
Cai S (2007) Botulism diagnostics: from clinical symptoms to in vitro assays. Critical
Reviews in Microbiology 33(2): 109–125.
Dembeck ZF, Smith LS, and Rusnak JM (2007) Botulism: cause, effects, diagnosis,
clinical and laboratory identification, and treatment modalities. Disaster Medicine
and Public Health Preparedness 1: 122–134.
Hauschild AHW and Dodds KL (1993) Clostridium botulinum, ecology and control in
foods. New York: Marcel Dekker.
Hogg R (2009) Botulism update and historical review. Government Veterinary Journal
20(1): 5–13.
Peck MW (2006) Clostridium botulinum and the safety of minimally heated, chilled
foods: an emerging issue? Journal of Applied Microbiology 101: 556–570.
Zhang JC (2010) Botulism, where are we now? Clinical Toxicology 48: 867–879.
Relevant Websites
http://wwwnc.cdc.gov/eid/article/10/9/03-0745.htm – CDC.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/default.htm –
FDA.
http://emedicine.medscape.com/article/213311-overview – Botulism - Medscape.
 Clostridium: Occurrence and Detection of Clostridium perfringens
R Labbé, University of Massachusetts, Amherst, MA, USA
V Juneja, Eastern Regional Research Center, Wyndmoor, PA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Background
Clostridium perfringens was recognized as a potential cause of
foodborne illness in the 1950s following the isolation of a
large number of the organism from suspect foods involved in
foodborne illness. In the United States, it is the second leading
cause of bacterial foodborne illness. It was eventually realized
that the responsible enterotoxin was produced following the
ingestion of a large number of cells from outbreak foods. The
cells sporulate in the small intestine and release the entero-
toxin. The enterotoxin was subsequently isolated in the early
1970s. Extensive work has been conducted on the mode of
action of the toxin using animal models and in cell culture.
C. perfringens is divided into types A–E. Types B–E are
typically associated with domestic animals and are responsible
for a variety of veterinary illnesses. Type A is associated with
foodborne illness and is part of the microflora of soil.
Extensive surveys of the incidence of C. perfringens in retail
foods were conducted beginning in the mid-twentieth century
after realization of its role in foodborne illness. Specialized
selective and presumptive media have been developed and
modified to take advantage of the special ability of
C. perfringens for rapid growth at elevated temperature, includ-
ing in the presence of specific antibiotics.
The Organism
C. perfringens is classified into five types, A–E, depending on
their ability to produce four toxins, alpha, beta, epsilon, and
iota. Food poisoning is caused by type A, while a more serious
gastrointestinal illness, necrotic enteritis, is due to type C but is
rare in industrialized countries.
Foodborne illness caused by type A C. perfringens is due to
the production of an enterotoxin during sporulation in the
small intestine following consumption of a large number of
vegetative cells in temperature-abused foods. The enterotoxin
is encoded by a gene (cpe) located on either the chromosome
or large plasmids. In the majority of outbreaks, the responsible
isolates carry the enterotoxin gene on the chromosome.
C. perfringens is an anaerobe, that is, requires the absence of
oxygen for rapid growth. This is readily achieved during cook-
ing procedures in which high temperatures drive off oxygen. In
the vegetative (growth) state, the organism possesses the short-
est generation time of any bacterium, able to divide in
<10 min in protein foods under optimal conditions such as
those that may be found following the cooling of large por-
tions of meat, poultry, and gravies.
C. perfringens is a spore-forming organism. Under conditions
of limited nutrients, vegetative cells (i.e., those in the growth
phase) can enter the spore state, which is a cell from highly
resistant to physical insults such as radiation, high temperatures,
146 Encyclopedia of Food and Health
pasteurization, and sanitizing agents. However, the spores of
this organism are inactivated by canning procedures. In the
natural environment and in nonoutbreak foods, the organism
is often found in the spore state. When placed in conditions of
proper nutrients (germinants), such as protein foods, spores can
germinate and resume vegetative cell growth.
Occurrence
Reservoirs
The environment
Type A strains have been isolated from most soil samples
examined, at levels of up to 103–104 g. However, soil is not
considered a major reservoir as the level of enterotoxin-positive
isolates is low.
Feces
C. perfringens is present in the intestinal contents of virtually all
animals including most humans examined with great variation
in number and between species. Levels may also vary over
time. Median counts in healthy individuals range from 103 to
106 g and are higher in neonates and elderly than adults. There
is some uncertainty as to whether humans are reservoirs of
foodborne, enterotoxigenic C. perfringens.
The widespread presence of C. perfringens spores in fecal
samples has led to the use of C. perfringens as an indicator of
fecal pollution of water by certain governmental jurisdictions
on several continents. As well, the World Health Organization
has also recommended it as a useful indicator of fecal pollu-
tion. C. perfringens spores were also found to be good indica-
tors of the distribution of sewage sludge on the ocean floor
where low temperature and high pressure are less suitable for
common indicators of fecal pollution such as coliforms and
fecal streptococci.
Food/food animals
Following the identification of C. perfringens as an etiological
agent of foodborne illness, many surveys of the incidence of
the organism in foods were conducted. Early surveys revealed
an incidence of approximately 50% (range of 30–80%) of raw
or frozen meat and poultry items containing generic (i.e.,
enterotoxin-positive and enterotoxin-negative) C. perfringens
(Table 1). Typical ranges in early surveys were at levels up to
102 g in meat and poultry, the commodities typically associ-
ated with foodborne outbreaks. Certain foods, such as spices,
were found to contain the organism at higher levels, even up to
103 g. More recent surveys have employed techniques that
allow the determination of the presence of the enterotoxin
gene. This revealed that enterotoxigenic C. perfringens is present
at low levels in retail foods (Table 2).
http://dx.doi.org/10.1016/B978-0-12-384947-2.00169-0
 Clostridium: Occurrence and Detection of Clostridium perfringens 147

Table 1 Levels of generic C. perfringens in retail foods home and institutional settings, the temperatures recom-
mended by governmental agencies for holding of cooked
% positive Levels (per gm or cm2) foods during serving or holding should be followed. These
Beef, pork, poultry 52–94 1–100 are 5–60  C (40–140  F).
Pork sausage 39 5–93
Ground beef 50 5–100
Pork carcasses 66 20/100 cm2 Detection
‘Raw meat’ 70 <3 Detection of Viable C. perfringens
Raw fruits and vegetables 4 10–100
Spices/herbs 5–100 10–10 000 Methods for detection of C. perfringens depend on the level of
Frozen foods 3 10–20 expected cells. The levels in retail foods are expected to be low
and the standard technique involves the most probable
Compiled from Guran and Oksuztepe; Juneja et al.; Lindström et al.
number (MPN) method (see Section ‘Most Probable
Number’), while the cell number in outbreak foods is much
Table 2 Levels of enterotoxigenic C. perfringens isolates from higher and requires the use of specially developed selective and
selected sources differential media.
Source % enterotoxin-positive
Criteria for Outbreak
Soil 7
Healthy adults 8a The typical criteria for implicating the role of C. perfringens in
Meat, fish, poultry, retail 0.1–1.7 outbreak foods involve either (a) detection of enterotoxin-
‘Raw meat,’ retail 8–12 positive spores (>106 g) in feces of ill individuals, (b) detection
Intestine, food animals 10–40 of elevated (>105 g) numbers of vegetative cells in outbreak
a foods, or (c) detection of enterotoxin in feces of ill individuals.
Compiled from Carmen et al.; Guran and Oksuztepe; Lindström et al.; Li et al.; Miki
et al.
Most Probable Number
Taken together, and in spite of many surveys of retail food, An MPN procedure is used when a low number of C. perfringens
food animals, humans, and the environment, the natural are expected, for example, in routine surveys of retail foods. For
reservoir of foodborne, enterotoxigenic C. perfringens remains this purpose, iron milk medium containing 2% iron powder is
unknown. It is now generally accepted that <10% of global used and is inexpensive and simple to prepare. Selection is based
isolates carry the enterotoxin gene (Table 2). on the appearance of a ‘stormy fermentation.’ In the reaction,
casein is precipitated by acid from the fermented lactose
Entry and Growth in Outbreak Foods followed by fractionation of the curd into a spongy appearance
when the milk is incubated at 45  C. If results are read after 18 h,
High-protein foods, such as meat and poultry, are typically confirmatory tests are not necessary.
involved in the outbreaks of foodborne illness. These have
been prepared in advance often in large amounts in settings
such as cafeterias, restaurants, and caterer preparation sites. Examination of Outbreak Foods
Spores of the organism are able to survive the heating steps Many media for enumeration of C. perfringens from outbreak
associated with food preparation. Such spores can then germi- foods have been developed over the years. The currently
nate and grow during slow cooling procedures. Because out- recommended medium in North America and elsewhere is
breaks of C. perfringens typically occur in institutionalized tryptose-sulfite-cycloserine (TSC) agar. Following anaerobic
settings, the average outbreak size consists of several dozen incubation at 37  C, C. perfringens colonies appear as black
cases. However, the relatively mild symptoms of most cases due to the reduction of sulfite to sulfide and reaction of the
result in the involvement of public health officials when only a sulfide with iron in the medium to form the black iron sulfite
large number of people have become ill. precipitate. Because certain other clostridia may produce black
colonies on this medium, confirmation of a selected number
Factors Affecting Growth of isolates is necessary (see ‘Confirmatory Tests’ later).

The temperature limits for growth of C. perfringens are between

15  C (60  F) and 50  C (122  F) with an optimum at about
45  C (112  F). Deficiencies in cooling are involved in virtually
all cases of foodborne illness caused by C. perfringens. Specifi-
cally, slow or improper cooling allows the rapid growth of the
organism between 37 and 50  C. This highlights the need to
reduce the size of portions of meat or the volumes of gravies
after cooking as spores can survive the internal temperatures.
The potentially rapid growth of C. perfringens during cooling
has led to governmental regulations regarding the cooling of
commercially produced, processed meat and poultry. For
Confirmatory Tests
Confirmatory tests can rule out the possible (though unlikely)
presence of other sulfite-reducing clostridia. The media for this
purpose are tubes of motility nitrate (MN) and lactose gelatin
(LG). The use of TSC agar together with the LG and MN has
been adopted as official first action by AOAC (Association of
Official Analytical Chemists) International. Details of the lab-
oratory procedures are described as standard methods by the
US Food and Drug Administration and by the International
Organization for Standardization. Commercial tests kits for
148 Clostridium: Occurrence and Detection of Clostridium perfringens
identification of clostridia are also available. Their use has been
described in publications from professional organizations such
as the American Society for Microbiology.
Enterotoxin Detection
Detection of C. perfringens enterotoxin in feces is a method of
confirming the role of C. perfringens in foodborne illness. The
two available commercial assays are an enzyme-linked immu-
nosorbent assay and a reverse passive latex agglutination.
Stools of healthy individuals contain undetectable levels of
the enterotoxin. The commercial kits for each technique,
respectively, are available from TECHLAB Inc. and Oxoid Inc.
Enterotoxin Gene
As shown in Table 1 versus Table 2, the ability of C. perfringens
to produce enterotoxin is limited to a small percentage of iso-
lates. To confirm the role of the organism in foodborne out-
breaks by the presence of a large number of viable cells or
spores, it is necessary to demonstrate that suspect isolates
(colonies) possess cpe. This is performed using the polymerase
chain reaction (PCR) assay. The procedure amplifies cpe from
lysed cells (i.e., isolates confirmed as C. perfringens) allowing
the determination of its presence. PCR protocols have been
developed to determine the location of cpe, that is, whether on
the chromosome or on a plasmid, though this distinction is
not routinely done or required in investigations of outbreaks.
Possible Medical Applications of C. perfringens Enterotoxin
As part of its biological activity in cases of foodborne illness,
C. perfringens enterotoxin binds to membrane proteins called
claudins. Certain solid cancer cell lines often overexpress
membrane-associated claudin molecules. This has led to a
possible use of C. perfringens enterotoxin as an anticancer
agent. In particular, tumor necrosis and shrinkage against
xenografts of human pancreatic cancer tumors growing on
the back of mice have been demonstrated. Current research
involves limiting immune responses to the enterotoxin and
employing particular enterotoxin fragments. Its potential med-
ical application is an exciting possibility for this toxin.
See also: Clostridium: Food Poisoning by Clostridium perfringens;
Clostridium: Occurrence and Detection of Clostridium botulinum and
Botulinum Neurotoxin; Food Poisoning: Tracing Origins and Testing;
Foodborne Pathogens.
Further Reading
Baez L and Juneja V (1995) Detection of enterotoxigenic Clostridium perfringens in
raw beef by polymerase chain reaction. Journal of Food Protection
58: 154–159.
Carman R, Sayeed S, Li J, Genheimer C, Hiltonsmith M, Wilkins T, and McClane B
(2008) Clostridium perfringens toxin types in the feces of healthy North Americans.
Anaerobe 14: 102–108.
Garcia L and LSG Associates (2010) Anaerobic bacteriology, Clinical microbiology
procedures handbook, 3rd ed. Washington, DC: ASM Press.
Guran H and Oksuztepe G (2013) Detection and typing of Clostridium
perfringens from retail chicken meat parts. Letters in Applied Microbiology
57: 77–82.
International Organization for Standardization (2004) Microbiology of food and animal
feeding stuffs – horizontal method for the enumeration of Clostridium perfringens –
colony count technique. Geneva: International Organization for Standardization, ISO
7937:2004.
Juneja V, Novak J, and Labbe R (2010) Clostridium perfringens. In: Juneja V and
Sofos J (eds.) Pathogens and toxins in foods, pp. 53–70. Washington, DC: ASM
Press.
Juneja V, Baker D, Thippareddi H, Synder OP, and Mohr T (2013) Growth potential of
Clostridium perfringens from spores in acidified beef, pork, and poultry products
during chilling. Journal of Food Protection 76: 65–71.
Labbe R and Grant K (2011) Clostridium perfringens in food service. In: Hoofar J (ed.)
Rapid detection, characterization, and enumeration of foodborne pathogens,
pp. 381–391. Washington, DC: ASM Press.
Li J, Sayeed S, and McClane B (2007) Prevalence of enterotoxigenic Clostridium
perfringens isolates in Pittsburgh (Pennsylvania) area soils and home kitchens.
Applied and Environmental Microbiology 73: 7218–7224.
Lindstrom M, Heikinheimo A, Lahti P, and Korkeala H (2011) Novel insights into the
epidemiology of Clostridium perfringens type A food poisoning. Food Microbiology
28: 192–198.
McClane B, Robertson S, and Li J (2013) Clostridium perfringens. In: Doyle M and
Buchanan R (eds.) Food microbiology, fundamentals and frontiers, pp. 464–491.
Washington, DC: ASM Press.
Miki Y, Miyamoto K, Kaneko-Hirano I, Fujiuchi K, and Akimoto S (2008) Prevalence and
characterization of enterotoxin gene-carrying Clostridium perfringens isolates from
retail meat products in Japan. Applied and Environmental Microbiology
74: 5366–5372.
Miyamoto K, Wen Q, and McClane B (2004) Multiplex PCR genotyping assay that
distinguishes between isolates of Clostridium perfringens type A carrying a
chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like
sequence or a plasmid cpe locus with an IS1151 sequence. Journal of Clinical
Microbiology 41: 1551–1558.
U.S. Department of Agriculture (1999) Food Safety and Inspection Service Performance
standards for the production of certain meats and poultry products. Federal Register
64: 732–749.
Veshnyakova A, Protz J, Rossa J, et al. (2010) On the interaction of Clostridium
perfringens enterotoxin with claudins. Toxins 2: 1336–1356.
Relevant Websites
http://www.foodsafety.gov/poisoning/causes/bacteriaviruses/cperfringens/ –
Foodsafety.
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/
BacteriologicalAnalyticalManualBAM/default.htm – Food and Drug Administration.
 Clostridium: Food Poisoning by Clostridium perfringens
K Miyamoto and M Nagahama, Tokushima Bunri University, Tokushima, Japan
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Clostridium perfringens was initially identified as a cause of food
poisoning in the 1940s. This bacterium then became known as
an important cause of foodborne disease. Many cases of food
poisoning due to enterotoxigenic C. perfringens are now
reported every year, and C. perfringens food poisoning ranks
among the most common foodborne diseases in industrialized
countries. The most important virulence factor is the toxin
known as Clostridium perfringens enterotoxin (CPE), while the
biological properties of this bacterium also contribute to the
induction of foodborne disease.
In this article, we described the basic features of
C. perfringens food poisoning, with a focus on the genetic and
biological characteristics of CPE-producing strains, and also
introduced novel insights into the molecular mechanism of
CPE cytotoxicity.
Characteristics of the Organism
C. perfringens is a Gram-positive, rod-shaped, encapsulated,
nonmotile bacterium that causes a broad spectrum of human
and veterinary diseases. The virulence of C. perfringens mostly
attributes its toxin-producing ability; that is, more than 16
different toxins have been identified from human and veteri-
nary disease isolates. Of these toxins, the human gastrointesti-
nal (GI) virulence of C. perfringens is known to depend on three
toxins, CPE, beta-toxin (the main toxin responsible for necro-
tizing enteritis in humans), and also possibly beta2-toxin (an
additional toxin for human colitis by CPE-producing type A
strain). While toxin-producing ability is crucial for human GI
diseases, C. perfringens food poisoning isolates have several
other biological abilities: a rapid doubling time (<10 min for
vegetative cells), the ability of vegetative cells to grow in rela-
tively high optimal growth temperatures (43–45
C), the abil-
ity to form environmental stress-resistant spores, and tolerance
to air exposure in spite of being classified as an anaerobic
bacterium. These properties also play important roles in the
induction of C. perfringens foodborne illness.
Based on the production of an arsenal of four major toxins
(alpha, beta, epsilon, and iota), C. perfringens isolates are clas-
sified as type A–E. Toxin typing of C. perfringens strains was
traditionally identified by toxin–antitoxin serum neutraliza-
tion tests in mice. The recent development of PCR-based
schemes permitting toxin genotyping has simplified toxin typ-
ing of C. perfringens isolates. Food poisoning isolates are most
frequently identified as type A, while the most important toxin
gene, the cpe gene, is rarely found in type C, D, and novel E
strains. These type C to E strains have not yet been identified as
causative agents of C. perfringens food poisoning outbreaks,
even though type C to E strains sometimes express an entero-
toxin with very similar or almost identical physical, serological,
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00171-9
and biological properties to CPE produced by the type A strain.
Of these cpe-carrying type A–E strains, the CPE-producing type
A strain is more widely distributed than the other types of CPE-
producing strains. On the other hand, a cpe-positive type C
isolate was found in some cases of necrotic enteritis (also
known as Darmbrand or pigbel), while the cpe-positive type
C strain may have accidentally been isolated from a patient
with type A food poisoning. The cpe gene is present on a
conjugative transferable large plasmid in most, if not all, type
C, D, and E strains. However, genetic organization of the cpe
region of type C, D, and E strains differs from that of cpe-
positive type A strains.
Factors Contributing to C. perfringens Type A
Foodborne Illness
A unique toxin, CPE, plays a major role in C. perfringens type A
food poisoning. A large number of epidemiological studies
confirmed the importance of CPE in GI virulence. First, most
type A C. perfringens isolates from food poisoning outbreaks
produce CPE. Second, a strong positive correlation exists
between illness and the presence of CPE, the level of which
in a patient’s feces is known to cause serious intestinal effects in
experimental animals. And third, human volunteers fed highly
purified CPE will develop all of symptoms characteristic of
C. perfringens type A food poisoning.
Animal model experiments revealed that CPE is active on
the GI tract; that is, all regions of the small intestine are
sensitive to treatment with CPE. In rabbit ileal loops, purified
CPE causes the rapid loss of fluid and electrolytes from the GI
tract. These effects on rabbit ileal loops can be neutralized with
CPE-specific antiserums. Moreover, the ability of CPE-positive
C. perfringens food poisoning isolates to produce both fluid
accumulation and histopathologic damage in rabbit ileal loops
is remarkably higher than that of CPE-negative isolates or that
of a cpe knockout mutant of the food poisoning strain.
The importance of CPE to human GI virulence has been
supported by the finding that CPE-producing type A
C. perfringens strains (most isolates harbor the cpe gene on a
plasmid) are also isolated from nonfoodborne human GI dis-
eases, including antibiotic-associated diarrhea, sporadic diar-
rhea, and nosocomial diarrheal outbreak.
The Biochemistry of CPE
CPE is a single polypeptide of 35 317 Da composing 319
amino acids with an isoelectric point of 4.3. This toxin is not
a heat-stable enterotoxin; its activity is inactivated by heating
for 5 min at 60
C. It is also sensitive to pH extremes but
resistant to some proteases, such as trypsin and chymotrypsin.
This toxin is not a secreted toxin because the toxin gene (cpe)
149
150 Clostridium: Food Poisoning by Clostridium perfringens
does not encode the 50 signal peptide, which is often associated
with secreted toxins.
Native CPE crystal structure analysis revealed CPE to be a
three-domain protein with an elongated architecture of
beta-sheets. These features of CPE are common among
pore-forming toxins, such as C. perfringens epsilon-toxin and
Laetiporus sulphureus hemolytic pore-forming lectin. However,
the domain arrangement of CPE is in the opposite order;
domain I is composed of the C-terminal region of CPE,
whereas domains II and III are formed by the N-terminal
region. The C-terminal half of the toxin (domain I) is respon-
sible for specific receptor-binding activity. CPE domain II con-
tains a region that appears to be a transmembrane stem, and
this region is crucial for pore formation and cell killing. The
CPE region residing in domain II also promotes CPE hexamer
formation (450 kDa SDS (sodium dodecyl sulfate)-resistant
large complex, described later). Domain III may undergo struc-
tural changes during prepore to pore transition. The
N-terminal 37 amino acids of CPE lack a definable structure
and are not necessary for toxicity.
Genetics of CPE
Cpe ORF nucleic acid sequences are highly conserved among
CPE-positive type A isolates; that is, CPE must have a highly
conserved amino acid sequence. Interestingly, the cpe gene
appears to be harbored on either the chromosome or the
large plasmids with a single copy. In the chromosomal cpe
strain, the cpe gene is flanked by insertion sequence (IS) ele-
ments (IS1469 and IS1470), which is associated with the puta-
tive 6.3-kb cpe-containing transposon, Tn5555. On the cpe
region of cpe-carrying plasmids, the cpe gene is resided by
upstream IS1469 and by downstream IS1151 or IS1470-like
elements in almost all type A C. perfringens isolates; that is,
based on gene arrangement differences in the cpe region, most,
but not all, CPE-producing type A strains are divided into three
cpe genotypes: one type of the chromosomal cpe strain and two
types of the plasmid cpe strain. Similar to other toxin genes
such as the epsilon-toxin gene, the cpe gene is harbored on
conjugatively transferable plasmids. Collectively, the cpe region
in both chromosomal and plasmid cpe strains is on a transpo-
son, a mobile genetic element; that is, these IS elements may be
related to mobilization or transfer of the cpe gene.
Synthesis and Release of CPE
Although CPE-producing C. perfringens isolates are divided
into three cpe genotypes, the amount of CPE expressed by an
isolate is not affected by whether that isolate carries the cpe
gene on the chromosome or a large plasmid. CPE synthesis
begins shortly after the induction of sporulation and progres-
sively increases for the next 6–8 h. CPE expression is regulated
at the transcriptional level, with significant amounts of cpe
mRNA being produced during sporulation; however, cpe
mRNA is not produced during the vegetative growth of
C. perfringens. This phenomenon is strongly supported by the
findings of genetic analyses as follows: expression of the cpe
gene in type A strains is strictly regulated by sporulation-
associated alternative sigma factors, including SigF, SigK, and
SigE. SigK and SigE (SigE and SigK are sporulation-associated
sigma factors active in mother cells during the sporulation of
Bacillus subtilis) directly induce the transcription of cpe mRNA
by binding SigK- or SigE-dependent promoters (P1, P2, and
P3) located upstream of the cpe ORF. The abundant CPE
expression during sporulation also depends on the exceptional
stability of cpe mRNA (the functional half-life of cpe mRNA is
approximately 60 min).
Unlike most C. perfringens toxins, CPE is not actively
secreted; following its synthesis, the CPE protein accumulates
in the cytoplasm of the mother cell as a cytoplasmic CPE-
containing paracrystalline inclusion body. When sporulation
events have been completed, the mother cell lyses with the
simultaneous release of CPE into the intestinal lumen, in
which then CPE acts as an enterotoxin.
Effects of CPE on the GI Tract
C. perfringens food poisoning is basically a self-limited illness
in humans. Therefore, histopathologic studies have seldomly
been conducted on humans. Instead, the actions of CPE have
been examined using animal models. Using an in vivo rat or
rabbit ileal loop model, CPE causes the loss of fluid and
electrolytes from the GI tract and then produces extensive
histopathologic damage to the small intestine; that is, the
target organ for CPE is believed to be the small intestine, with
the ileum being particularly sensitive to this toxin. CPE-
induced histopathologic damage is initially limited to the
villus tips of the small intestine; however, the entire small
intestinal villus eventually suffers extensive damage over time:
desquamation of the villus epithelium, inflammatory cell infil-
tration (mostly lymphocytes), and slight hyperemia in the
mucosa. This CPE-induced tissue damage plays a major role
in initiating CPE-induced fluid/electrolyte intestinal transport
alterations; that is, the onset of fluid transport changes closely
coincides with the development of tissue damage, and CPE
levels that produce tissue damage can induce intestinal fluid
and electrolyte transport alterations. CPE-induced intestinal
damage can develop within 15–30 min of toxin treatment in
the rabbit ileum.
The cellular actions of CPE were extensively investigated
using a cultured intestinal epithelial cell line. These studies
speculated that CPE would act in a multistep process. The
current model for the actions of CPE begins with binding to
the toxin-specific receptors, claudin-3 and claudin-4, in a
temperature-sensitive manner. Claudins are a 24-member fam-
ily of 20 to 25 kDa proteins and are the most important
proteins in epithelial tight junctions. These tight junction pro-
teins are predicted to consist of four transmembrane domains,
two extracellular loops, and cytoplasmic tail-mediating signal
cascades. CPE binding to receptor claudin proteins is mediated
by the second extracellular loop (at a helix–turn–helix motif)
of the tight junction protein. CPE can bind to claudin-3,
claudin-4, claudin-6, claudin-7, claudin-8, and claudin-14
(the contribution of claudins-8 and claudin-14 to CPE cyto-
toxicity is less than that of claudin-4), but not to claudin-1,
claudin-2, claudin-5, or claudin-10. CPE–claudin binding rap-
idly results in the formation of an 90 kDa small complex,
which contains claudin families and claudin receptors
along with claudins incapable of binding CPE (nonreceptor
claudin(s)).
 Clostridium: Food Poisoning by Clostridium perfringens
Within as little as 5 min of CPE binding, six small complexes
are then thought to oligomerize into a stable large complex
(450 kDa), which initially assembles as a prepore and rapidly
inserts into membranes to form an active pore. The formation of
active pores causes the loss of normal plasma membrane per-
meability properties. These CPE-induced permeability alter-
ations are initially restricted to small molecules of <200 Da,
for example, calcium ions and amino acids. As a result of these
events, CPE-treated cells die from classical caspase 3-mediated
apoptosis at moderate levels of CPE, whereas cells die from
oncosis at higher levels of CPE. Thus, large-complex formation
is a necessary step in CPE-induced cytotoxicity.
CPE pore formation also leads to morphological damage,
which allows the formation of a bigger large complex
(650 kDa). This 650 kDa large complex contains another
65 kDa tight junction protein, occludin. This event induces
the further disruption of tight junctions.
Collectively, the involvement of tight junction proteins in
the actions of CPE causes intestinal paracellular permeability
changes. The overall sequence of events on CPE-treated culture
cells is believed to lead to CPE’s effects on the small intestine
both in vivo and in vitro.
Tolerance of C. perfringens to Environmental Stress
Besides its toxin-producing ability, C. perfringens type A food-
borne illness is commonly the result of inadequate handing of
food during cooking, cooling, or holding. The biological prop-
erties of C. perfringens vegetative cells and spores in these settings
contribute to its ability to cause food poisoning by enabling its
survival and growth in cooked food; for example, resistance to
heat, low temperatures, NaCl, and nitrates (typically used to
process and protect foods). Of these tolerant properties to envi-
ronmental stresses during cooking, extremely high heat toler-
ance has been a well-established property of C. perfringens
vegetative cells and spores. The resistance of C. perfringens spores
to heat is influenced by both environmental and genetic factors.
Regarding environmental factors, the spores of C. perfringens
food poisoning strains can survive boiling in a protective
meat-based medium (e.g., cooked-meat medium). Incomplete
heating can also induce the germination of C. perfringens spores.
These resistant properties of spores are genetically based on the
products of ssp genes, which encode a/b-type small acid-soluble
proteins. The three previously identified ssp genes (ssp1, ssp2,
and ssp3) in C. perfringens strains share identical sequences and
are also expressed at similar levels in several C. perfringens iso-
lates, including strains that produce heat-resistant or less heat-
resistant spores. The novel ssp gene, ssp4, was recently identified.
One of the ssp4 gene products, the Asp Ssp4 variant, is harbored
in most chromosomal cpe strains, and this variant mostly con-
tributes to the extreme heat resistance of spores made by food
poisoning strains with the chromosomal cpe gene. This gene
product also appears to contribute to sodium nitrate resistance.
Pathogenesis of C. perfringens Type A Food Poisoning
Inappropriate cooking and preservation cause the rapid prolif-
eration of the vegetative cells of enterotoxigenic C. perfringens
in foods. While many ingested C. perfringens vegetative cells are
151
likely killed due to the acidity of the stomach, some vegetative
cells that survive pass through the stomach and remain viable
in a sufficiently contaminated food vehicle (i.e., food contain-
ing 106–107 C. perfringens vegetative cells per gram). These
surviving vegetative cells multiply and sporulate in the small
intestine; hence, this illness is considered to be an infection,
not an intoxication. A low-molecular-weight (1000–5000)
sporulation factor, produced by both CPE-negative and CPE-
positive vegetative cells, may promote in vivo sporulation. CPE
is expressed during sporulation and then accumulates in the
mother cells of C. perfringens. After mother cells lyse, CPE is
released into the intestinal lumen, in which the CPE quickly
binds to intestinal epithelial cells and exerts its action. These
events induce fluid and electrolytes losses and also morpho-
logical damage to intestinal epithelial cells.
Epidemiology
C. perfringens type A food poisoning annually ranks among the
most common foodborne diseases in the United States,
Europe, and Japan. Identified outbreaks of C. perfringens type
A food poisoning commonly involve large outbreaks (the aver-
age outbreak size is 50 to 100 in United States) and often
occur in institutions, such as hospitals, school cafeterias,
prisons, and nursing homes. This epidemiological feature has
largely been attributed to at least four factors. First, large
amounts of food are prepared at once in institutions, which
is a factor that increases the risk of contamination. Second, the
preparation of large amounts of food is associated with insuf-
ficient heating, especially the ‘core’ region of food. Third, large
amounts of food are often prepared in advance and held for
later serving. These conditions appear to facilitate the growth
of heat-tolerant bacterium in prepared food. Fourth, relatively
mild and nondistinguishing symptoms develop in most cases
of C. perfringens type A food poisoning; in other words,
C. perfringens food poisoning with a small number of cases is
not recognized or reported. Therefore, the true prevalence and
impact of this foodborne disease appear to be significantly
understated.
Molecular Characteristics of Food Poisoning Strains
Isolates from food poisoning outbreaks are mainly classified
into three cpe genotypes based on the gene arrangement of the
cpe region, as described earlier. Plasmid cpe strains have been
identified as the pathogen in human GI diseases such as
antibiotic-associated diarrhea, nosocomial diarrhea, and spo-
radic diarrhea and also as isolates in healthy human feces and
environmental samples. Studies using recently developed PCR
assays, which allow these ‘cpe genotypes’ to be differentiated
from each other, revealed that the plasmid cpe strain can
induce C. perfringens type A foodborne outbreaks, similar to
the chromosomal cpe-positive strains; isolates from approxi-
mately two-thirds of outbreak cases harbored the cpe gene on
the chromosome, and plasmid cpe isolates were the causative
strain in another one-third of cases. However, as other cpe-
negative environmental strains, most of these plasmid cpe
strains produce spores that are less resistant to high and low
temperatures, NaCl, and nitrates, than the chromosomal food
152 Clostridium: Food Poisoning by Clostridium perfringens
poisoning strains. These properties are based on the genetic
background of the plasmid cpe strains; plasmid cpe strains carry
the ssp4 gene, which encodes the less heat-resistant Ssp4 pro-
tein the Gly Ssp4 variant.
Source of Enterotoxigenic C. perfringens Strains in Food
Poisoning Outbreaks
While common food vehicles for C. perfringens type A food-
borne illness were initially thought to be meat and meat prod-
ucts (notably beef and poultry and gravies) in the United
States, various foods have also been identified as a contami-
nated food in C. perfringens type A food poisoning outbreaks.
In general, C. perfringens is ubiquitous in nature; it is present in
soil (at levels of 103–104 CFU g1), foods (e.g., approximately
50% of raw or frozen meat contains some level of
C. perfringens), dust, and the intestinal tract of humans and
domestic animals (e.g., human feces typically contain
104–106 CFU g1).
In independent surveys investigating various kinds of envi-
ronmental samples, some surveys identified both types (chro-
mosomal or plasmid-borne) of cpe-positive isolates, while
other surveys failed to detect cpe-positive strains in environ-
mental samples. The reasons for the difficulty in detecting CPE-
producing C. perfringens in environmental samples are as
follows:
(1) Very low number of C. perfringens is present in retail food
samples, while the C. perfringens strain is frequently pre-
sent in retail food.
(2) Only a small subset of food isolates (less than 5%) can
produce CPE, maybe even in putative reservoir(s).
(3) CPE is produced only during sporulation; however, it is
sometimes difficult to induce the sporulation of isolates
using several kinds of sporulation-specified media (e.g.,
Duncan–Strong medium).
Results from the limited number of surveys identifying cpe-
positive strains in investigated samples revealed that
C. perfringens strains carrying the cpe gene on a large plasmid
are likely to be the main population in the environment, while
the three cpe genotypes of the C. perfringens strain are broadly
distributed. Therefore, foods could be accidentally contami-
nated with cpe-positive strains from the environment in any
steps during food processing (Figure 1).
Interestingly, recent studies using molecular assays (multi-
locus sequence typing and microarray assays) revealed the
genetic characteristics of chromosomal and plasmid cpe strains.
The multilocus sequence typing (MLST) procedure has charac-
terized isolates using the DNA sequences of approximately
450–500 bp internal fragments of multiple housekeeping
genes. The different sequences present within isolates of each
housekeeping gene have been assigned as distinct alleles, and,
for each isolate, the alleles at each of the loci define the allelic
profile or sequence type (ST). Using MLST assays, chromo-
somal cpe strains construct a distinct ST from plasmid cpe
strains, cpe-negative strain, and type B to E veterinary strains.
Another molecular assay is a microarray assay, in which the
property of nucleic acid sequences specifically pairs with each
other between complementary nucleotide base pairs. With a
FOODS
Inadequate
cooking
CPE-producing
strain
Cooked food
Clostridium perfringens pool
containing spores
Inadequate
preserving
before serving
Cooked food
containing a large
number of CPE-
producing strain
Food poisoning
outbreak
Figure 1 Events leading to C. perfringens food poisoning outbreaks,
when accidentally contaminated with CPE-producing strain. Foods
are more frequently contaminated with non-CPE-producing strains.
completely sequenced C. perfringens food poisoning strain
genome-based DNA microarray assay, type A chromosomal
cpe strains have different genetic backgrounds from type A
plasmid cpe strains and cpe-negative type A strains; that is, the
gene clusters of myo-inositol, ethanolamine, and biotin syn-
thesis are absent in the variable region of chromosomal cpe
strains, whereas the gene cluster of cellobiose metabolism is
present. Combined with the results of MLST analysis for chro-
mosomal cpe strains, chromosomal cpe strains and plasmid cpe
strains are likely to have different habitats in the environment;
plasmid cpe strains might have adapted to the mammalian
intestine environment, while chromosomal cpe strain might
be present in environments with plant materials.
The principal reservoir(s) of C. perfringens type A food
poisoning strains has not been examined in detail. The devel-
opment of a method(s) capable of detecting low numbers of
bacterial cells is required to further investigate the source of
CPE-producing C. perfringens in environmental samples
because contaminated samples commonly contain a low num-
ber of CPE-producing C. perfringens with a small subset of total
C. perfringens isolates. Unfortunately, the current standard pro-
cedures established to diagnose C. perfringens food poisoning
are only able to detect large numbers of cpe-positive strains in
samples. Several assays based on the methods of ‘microbial
source tracking’ might be useful for detecting low numbers of
vegetative cells and/or spores in environmental samples.
In the future, epidemiological surveys using assays, which
can differentiate between the genetic backgrounds of cpe-
positive and cpe-negative strains and are based on microbial
 Clostridium: Food Poisoning by Clostridium perfringens
source tracking, help to understand the cpe-positive
C. perfringens habitat in the environment, and based on these
epidemiological findings, candidate(s) of native reservoirs of
chromosomal and/or plasmid cpe-positive C. perfringens strains
would be identified.
Clinical Features
Food poisoning outbreaks due to type A enterotoxigenic
C. perfringens typically involve a large number of cases. The
primary clinical symptoms associated with food poisoning are
moderately severe abdominal cramps, nausea, and watery diar-
rhea; vomiting and fever are rare. Symptoms of food poisoning
by type A C. perfringens strains develop 8–24 h after the inges-
tion of food heavily contaminated with the organism. The
illness is generally self-limited but typically lasts 12–24 h.
Mild symptoms may last 1 or 2 weeks in some cases.
Fatalities are very rare, occurring in <0.05% of cases. While
everyone is susceptible to C. perfringens type A food poisoning,
fatalities are commonly caused by dehydration and occur
among the very young, very old, debilitated, and chronically
ill individuals. Three foodborne outbreaks of CPE-producing
strains with fatalities have recently been reported, and isolates
in these outbreaks were identified using PCR assays as a type A
strain harboring the cpe gene on the chromosome. Of these
outbreaks, fatalities were attributed to necrotizing colitis,
which has features similar to those of necrotizing enteritis
caused by the C. perfringens type C strain. These fatal cases
had nausea and vomiting in addition to abdominal cramps
and watery diarrhea.
Diagnosis
A diarrheal disease is difficult to identify as a foodborne illness
due to enterotoxigenic C. perfringens in a clinical setting. For
example, the clinical symptoms and degree of severity of food-
borne diseases by C. perfringens and B. cereus are very similar.
Therefore, a laboratory investigation must be performed to
identify a diarrheal disease as food poisoning caused by CPE-
producing C. perfringens.
The most conventional diagnostic criterion to identify
C. perfringens type A food poisoning outbreaks is the detection
of CPE in the feces of patients. However, the usefulness of fecal
CPE detection approaches for identifying C. perfringens food
poisoning outbreaks is limited because fecal samples from
suspected cases must be collected soon after the onset of food
poisoning symptoms to ensure meaningful results. Serological
assays such as the reversed-passive latex agglutination assay
and enzyme-linked immunosorbent assay are used to detect
CPE in properly collected fecal samples. The detection of CPE
in the feces of several patients, who exhibit a common clinical
features (common food consumption, typical incubation time,
and characteristic symptoms), provides compelling evidence
for the occurrence of C. perfringens type A food poisoning,
while nosocomial type A C. perfringens outbreaks rarely
occurred via contaminated environments, mainly lavatory
equipment.
153
Further laboratory analyses including bacterial isolation
and species identification are reliable approaches to identify
C. perfringens type A food poisoning. For example, in the
United States, bacteriologic criteria used by the Centers for
Disease Control and Prevention (CDC) to identify an outbreak
include demonstrating the presence of either (i) 105
C. perfringens organisms per gram of stools from two or more
patients or (ii) 105 C. perfringens organisms per gram of epide-
miologically implicated food.
Only demonstrating the presence of C. perfringens in suspected
food or the feces of patients is insufficient for the unequivocal
identification of C. perfringens type A food poisoning because
C. perfringens is ubiquitous and is found in raw food and the
GI tracts of healthy individuals. Therefore, to identify food poi-
soning outbreaks caused by type A C. perfringens, isolates from
suspected cases should be identified as the type A CPE-producing
C. perfringens strain. Culture samples using spore-formation
medium (such as Duncan–Strong medium) are used to investi-
gate the ability of foodborne disease isolates to produce CPE.
However, a definite protocol for CPE expression by food poison-
ing isolates has not yet been established. Moreover, the in vitro
sporulation of food poisoning isolates is often difficult to achieve
under laboratory conditions.
Instead of investigating CPE-producing ability, molecular
assays, for example, simple PCR assays, are available to specif-
ically detect the cpe gene. The cpe gene must be functional in
most, if not all, cpe-positive strains because the cpe region in the
cpe-positive C. perfringens strains examined, which can produce
CPE during in vitro sporulation, has almost identical
sequences, such as the ORF sequence of the cpe gene, upstream
sequence of the cpe ORF including SigK- and SigE-dependent
promoters, and ribosome-binding sites. Developed PCR assays
can relatively easily detect the presence of the cpe gene carried
by C. perfringens strains in properly collected contaminated
food samples and/or fecal samples from patients with
C. perfringens type A food poisoning. However, many protocols
for the cpe-detecting PCR assay also detect the cpe gene in type
C and type D C. perfringens and the cpe pseudogene in type E
strain; that is, PCR assays detecting the cpe gene must be mean-
ingful when combined with PCR assays identifying the
C. perfringens toxin genotype.
CPE-positive strains must be present, even at very low fre-
quencies, in various foods, healthy human feces, and the envi-
ronment. Therefore, similarities must be investigated between
isolates from feces and suspected food to more strictly
diagnose cases and identify contaminated food in foodborne
illnesses. To investigate similarities or differences between
cpe-positive isolates in these samples, identifying the serotype
and/or genotype (such as the pulsed-field gel electrophoresis
type) of CPE-producing isolates can be an alternative and
supplemental approach for diagnosing C. perfringens type A
food poisoning outbreaks.
Treatment and Prevention
The clinical symptoms of C. perfringens type A food poisoning
are commonly mild and the clinical course is self-limited.
Antibiotic treatment is typically not required. Rehydration
154 Clostridium: Food Poisoning by Clostridium perfringens
drinks containing important electrolytes are useful for manag-
ing the dehydration caused by diarrhea.
CPE-producing C. perfringens strains are broadly distributed
in the environment, including foods, soil, and kitchen surfaces.
Therefore, contamination events to food may occur during the
cooking process (food ingredients, cooking, and preserving)
(Figure 1). For this reason, completely protecting against C. per-
fringens contamination to food(s) is likely to be difficult. For-
tunately, a large number of CPE-producing bacterial cells are
needed to induce C. perfringens food poisoning, which is
because many ingested C. perfringens vegetative cells are killed
when exposed to the acidity of the stomach. Collectively, the
most important factors preventing and controlling
C. perfringens type A foodborne illness are ‘careful cooking’
and ‘careful storage,’ which prohibit the vegetative growth of
C. perfringens in cooked foods. Therefore, reducing the total
amount or size of food for heating and cooling (it is difficult to
archive high internal temperatures in large amounts of food),
appropriate storage at less than 10
C for cooked foods before
serving (growth rates of C. perfringens vegetative cells rapidly
decrease at temperatures below 15
C, with no growth occur-
ring at 6
C), and the immediate consumption of served foods
as soon as possible are the best ways to prevent C. perfringens
food poisoning.
Biologically, the growth of C. perfringens strains is affected
by water activity (aw), reduction potential (Eh), pH (optimal
growth at pH 6 to 7), and chemical preservatives (e.g., NaCl).
These preservation factors also control the growth of
C. perfringens vegetative cells and inhibit the outgrowth of
germinating C. perfringens spores in food.
See also: Clostridium: Occurrence and Detection of Clostridium
perfringens; Diarrheal Diseases; Food Poisoning: Classification; Food
Poisoning: Epidemiology; Food Poisoning: Tracing Origins and
Testing; Foodborne Pathogens.
References
Books
Durre P (ed.) (2005) Handbook on clostridia. Boca Raton, FL: CRC Press Inc.
Doyle MP and Buchanan RL (eds.) (2013) Food microbiology: fundamentals and
frontier, 4th ed. Washington, DC: ASM Press.
Falkow S, Dworkin M, Rosenburg E, Schleifer H, and Stackbrandt E (eds.) (2006) The
prokaryotes. New York: Springer.
Rood JI, McClane BA, Songer JG, and Titball RW (eds.) (1997) The clostridia:
molecular biology and pathogenesis. San Diego, CA: Academic Press Inc.
Santo Domingo JW and Sadowsky MJ (eds.) (2007) Microbial source tracking.
Washington, DC: ASM Press.
Review articles
Gao Z and McClane BA (2012) Use of Clostridium perfringens enterotoxin and the
enterotoxin receptor-binding domain (C-CPE) for cancer treatment: opportunities
and challenges. Journal of Toxicology 2012: 981626.
Li J, Adams V, Bannam TL, et al. (2013) Toxin plasmids of Clostridium perfringens.
Microbiology and Molecular Biology Reviews 77: 208–233.
Lindstrom M, Heikinheimo A, Lathi P, and Korkeala H (2011) Novel insights into the
epidemiology of Clostridium perfringens type A food poisoning. Food Microbiology
28: 192–198.
Miyamoto K, Li J, and McClane BA (2012) Enterotoxigenic Clostridium perfringens:
detection and identification. Microbes and Environments 27: 343–349.
Petit L, Gibert M, and Popoff MR (1999) Clostridium perfringens: toxinotype and
genotype. Trends in Microbiology 7: 104–110.
Articles in edited books
Hobbs BC (1979) Clostridium perfringens gastroenteritis. In: Riemann H and Bryan FL
(eds.) Foodborne infections and intoxications, 2nd ed., pp. 131–167. New York:
Academic Press, Inc.
Labbe RG (1989) Clostridium perfringens. In: Doyle MP (ed.) Foodborne bacterial
pathogens, pp. 192–234. New York: Marcel Decker, Inc.
MacDonel JL (1986) Toxins of Clostridium perfringens types A, B, C, D, and E.
In: Dorner F and Drews H (eds.) Pharmacology of bacterial toxins, pp. 477–517.
Oxford, UK: Pergamon Press.
McClane BA (2007) Clostridium perfringens. In: Doyle MP and Beuchat LR (eds.) Food
microbiology: fundamentals and frontier, 3rd ed. Washington, DC: ASM Press.
 Clostridium: Occurrence and Detection of Clostridium botulinum
and Botulinum Neurotoxin
JW Austin, Microbiology Research Division, Food Directorate, Health Canada, Ottawa, ON, Canada
Crown Copyright ã 2016 Published by Elsevier Ltd. All rights reserved.
Introduction
Botulinum neurotoxin (BoNT)-producing clostridia normally
called Clostridium (C.) botulinum are, from the genetic and
physiological points of view, a diverse taxonomic group.
The group comprises Gram-positive, anaerobic, rod-shaped,
spore-forming bacteria that produce the most toxic biological
substance known, botulinum neurotoxin (BoNT). Foodborne
botulism is a neuroparalytic disease resulting from neurotoxin-
induced inhibition of skeletal and autonomic peripheral cho-
linergic nerve terminals. It results from consumption of food in
which C. botulinum has grown and produced botulinum toxin.
Without prompt diagnosis and treatment, the resulting cranial
neuropathy and symmetrical, descending flaccid paralysis may
progress to respiratory failure and death.
BoNTs are 150 kDa proteins produced by C. botulinum or
neurotoxigenic strains of C. butyricum or C. baratii. Maximal
potency of BoNTs is only obtained after cleavage of the
150 kDa polypeptide into a 100 kDa heavy chain (HC) and a
50 kDa light chain (LC) linked by a single disulfide bond. The
HC binds to receptors on neurons and enables the internaliza-
tion of the LC, a zinc metalloprotease, into presynaptic neu-
rons at the neuromuscular junction. The internalized LC
cleaves one of the soluble N-ethylmaleimide-sensitive attach-
ment receptor (SNARE) proteins. The LC of BoNT type A
cleaves synaptosomal-associated protein 25 (SNAP-25),
whereas the LC of BoNT type B cleaves synaptobrevin-2. Cleav-
age of these proteins prevents the docking of small synaptic
vesicles with the presynaptic membrane, preventing the release
of acetylcholine into the neuromuscular junction and resulting
in flaccid paralysis of the corresponding muscle.
Seven serologically distinct BoNTs (A–G) can be distin-
guished based on neutralization of toxicity with specific anti-
sera. Recently, a strain of C. botulinum producing BoNT type B
and another BoNT that is not neutralized by antitoxins to
BoNTs A–G has been isolated from a case of infant botulism.
It has been proposed that this novel neurotoxin is an eighth
serotype – BoNT type H. While sequence data have not yet
been made available, genomic studies have indicated that
BoNT type H differs substantially from the other seven BoNT
serotypes. Human botulism, including foodborne, wound,
and infant botulism, is associated with types A, B, E, and,
very rarely, F. The majority of cases of animal botulism are
caused by type C; cattle and sheep are also particularly suscep-
tible to botulism caused by type D. To date, there is no direct
evidence linking type G to disease.
Based on physiological differences, and now supported by
whole genome sequencing, C. botulinum is divided into four
groups: (I) all type A and proteolytic strains of types B and F,
(II) all type E and nonproteolytic strains of types B and F, (III)
type C and D strains, and (IV) type G strains. Strains producing
two toxin types have been reported, as have those producing
only one type of toxin but carrying a silent gene for a different
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00170-7
toxin type. Groups I and II are commonly referred to as
‘proteolytic’ and ‘nonproteolytic’ strains, respectively. Proteo-
lytic strains are relatively resistant to heat and preservatives
such as salt, acidulants, and nitrite. They are therefore the
most likely forms involved in outbreaks from underprocessed
canned foods or from salted, pickled, and otherwise cured
products. Exceptions are some dry- and brine-cured hams
and pickled fish where C. botulinum may develop before the
diffusion of the main ingredients conferring safety (e.g., salt
and acid) is complete. With few exceptions, infant botulism is
caused by proteolytic strains of C. botulinum. As a result of their
sensitivity to heat and ability to grow at refrigeration tempera-
tures, group II, or nonproteolytic, strains present a risk of
botulism from minimally processed packaged foods that have
extended shelf lives at refrigeration temperatures. In a few
exceptional cases, other clostridia (C. baratii producing type F
toxin and C. butyricum producing type E toxin) have been
implicated in foodborne botulism incidents. Type E is the
prevailing form of C. botulinum in aquatic environments and
the most likely form of botulism from preserved fish. Type E is
also the main cause of botulism involving the indigenous
population of Canada and Alaska. The emphasis here will be
on groups I and II since they are involved in human illness.
Strains belonging to group III are involved with animal botu-
lism. This article discusses the distribution of botulinum spores
in the environment and in foods and detection of the organism
and its neurotoxin in foods.
Presence of C. botulinum in the Environment
Spores of C. botulinum are commonly present in soils and
sediments, but their numbers and types vary, depending on
the location. The possibility of contamination of food with
C. botulinum depends on the distribution and incidence of
spores in the environment where the food originates.
C. botulinum spores are widely distributed in North
America, but the spore load varies considerably, as does the
predominating type. Soils in the United States west of the rise
of the Rocky Mountains usually contain type A spores, while
type B spores predominate in the Eastern United States. Most
US type B strains are proteolytic. C. botulinum type E is found in
damp to wet locations. In the region around the Great Lakes,
and particularly around Green Bay of Lake Michigan, high
numbers of type E are found in shoreline and sediment sam-
ples. Type E is also found in the coastal areas of Washington
and Alaska. The distribution of types on the Pacific coast
changes with latitude; south of 36N, the prevalent types shift
from E to A and B.
Type B predominates in the terrestrial environments of
Britain, Ireland, Iceland, Denmark, and Switzerland. It is asso-
ciated with most botulism outbreaks in Spain, Portugal, Italy,
France, Belgium, Germany, and Poland, indicating wide
155
156 Clostridium: Occurrence and Detection of Clostridium botulinum and Botulinum Neurotoxin
distribution of this type in the European environment. Type B
also predominates in the aquatic environments of the United
Kingdom. Most European type B strains are nonproteolytic.
C. botulinum type E predominates in marine and freshwater
environments in temperate to subarctic regions. High inci-
dences of C. botulinum type E have been recorded from the
Nunavik region of Quebec, the northwest coast of Alaska, the
Great Lakes, and lakes and rivers of North Japan. High levels of
type E spores were found in Scandinavian waters, particularly
in the Kattegat, between Denmark and Sweden, and in the
Baltic Sea. The presence of high numbers of C. botulinum type
E in these regions suggests a terrestrial origin of the organism
and passive accumulation from land drainage. Type E spores
also predominate in the Ukraine and most parts of Russia.
High numbers of type E botulism outbreaks associated with
fish caught from Lake Baikal suggest a high prevalence of
C. botulinum type E in the Irkutsk region of Russia. In general,
surveys of Asia report lower numbers, with the exceptions of a
high incidence of type E spores around the Caspian Sea and a
high incidence of all types in the Xinjiang district of China.
Fewer surveys have been carried out in the southern hemi-
sphere. Spores of all types have been detected in South
America. In a study of over 2000 Argentine soil samples,
23.5% tested positive for BoNT-producing clostridia, with
type A being the most frequently detected. C. botulinum type
E has been found in fish, oyster, and shrimp samples taken off
the Brazilian coast.
As a result of its role in outbreaks of avian botulism across
the world, the distribution of C. botulinum type C has been
widely studied. Based on both surveys and recorded cases of
animal botulism, C. botulinum type C has been found in several
countries including Australia, Japan, Korea, Canada, the
United States, Costa Rica, Spain, Sweden, Finland, Germany,
Ireland, Italy, Norway, Brazil, South Africa, and Turkey.
Genetic recombination between C. botulinum type C and type
D strains has resulted in C. botulinum strains producing a
mosaic C/D type toxin. Chimeric type C/D toxin is more lethal
to avian species than either type C or type D. Type C botulism
has become an emerging and serious problem in poultry flocks
in Sweden, causing 13 separate outbreaks in 2008. Recent
molecular typing evidence indicates the type C/D strain
responsible for avian botulism outbreaks in waterbirds in
Spain shares a very high level of genetic similarity with the
strains responsible for the Scandinavian outbreaks in poultry
flocks. C. botulinum type D is the cause of botulism outbreaks
in cattle in several parts of the world including parts of the
United States, Canada, Australia, Israel, Turkey, Germany,
South Africa, and the Netherlands.
In summary, type A spores predominate in soils in the
Western United States, China, Brazil, and Argentina, and type
B spores in the Eastern United States, the United Kingdom, and
much of continental Europe. However, most US type B strains
are proteolytic, while most European strains are nonproteolytic.
Type E is the predominant type in northern regions and in most
temperate aquatic regions and their surroundings. Types C and
D are found more frequently in tropical environments. The
reasons for this distribution pattern are not well understood.
Type A appears to favor neutral to alkaline soils with low organic
content, consistent with its virtual absence in the highly culti-
vated soils of the Eastern United States and Europe. Type B
strains predominate in heavily farmed soils in Argentina,
Denmark, and the United States. Type E is psychrotolerant,
which undoubtedly plays a role in its prevalence in the north
and in many aquatic environments. Based on outbreaks in wild
and domestic animals, C. botulinum types C and D can be found
over most continents.
Presence of C. botulinum in Foods
Spores of C. botulinum are widespread in nature and are found
in soils, aquatic sediments, and the gastrointestinal tracts of
animals. Contamination of food with C. botulinum often
occurs during growth or harvesting and is most likely to
occur when a product originates in an environment with a
high incidence of spores. However, contamination can also
occur during or after processing. There have been considerably
fewer surveys of foods for contamination with C. botulinum
than environmental surveys, and they have focussed primarily
on fish, meats, and infant foods, particularly honey.
Fish may become contaminated with spores of C. botulinum
in their environment or during processing and handling. The
presence of C. botulinum, mostly type E, in fish is readily
demonstrated, although the incidence is lower than in envi-
ronmental surveys. For example, 20% of the fish caught in the
Baltic Sea and Finnish freshwaters tested positive for
C. botulinum type E, while 7% of vacuum-packed hot-smoked
products marketed in Finland contained spores of the organ-
ism. Other studies have indicated a range from 0% to 100% of
fishery products testing positive for C. botulinum type E. Most
fish-borne botulism outbreaks recorded in Canada, the United
States, Russia, Europe, Japan, Egypt, and Iran have been linked
to the consumption of smoked, salt-dried, canned, or fermen-
ted fish usually eaten without further cooking.
The level of contamination of meats is generally very low,
relative to the incidence on fish and fishery products. The few
studies indicate that the incidence of C. botulinum in meat
samples is often less than 10%, and several studies have not
been able to detect C. botulinum in meats. The concentration of
spores in meat is typically <1 spore per kg, with the exception
of raw pork and vacuum-packed bacon in the United
Kingdom, where some samples had up to 7 spores per kg.
Survey results reveal that nearly all toxin types identified from
cured meat, raw pork, vacuum-packed bacon, and liver sausage
were either type A or type B. C. botulinum types A and B are the
two serotypes most often associated with botulism outbreaks
from meat products. The presence of low numbers of
C. botulinum in pig and cattle’s feces indicates carriage of
C. botulinum by healthy animals and suggests contamination
of carcasses with C. botulinum during carcass dressing.
C. botulinum types A or B may be present on vegetables,
particularly those harvested from the soil. C. botulinum is com-
mon in soil and organic fertilizers, usually at low concentra-
tions. However, one survey reported 25 000 spores per kg in
soil in China. One product of particular concern is cultivated
mushrooms, in which up to 2100 type B spores per kg have
been detected. As a result of the relatively frequent occurrence
of botulism outbreaks resulting from canned vegetables in
California in the early part of the twentieth century, an exten-
sive survey of vegetables from markets and gardens was
 Clostridium: Occurrence and Detection of Clostridium botulinum and Botulinum Neurotoxin
conducted. Fruits and vegetables, including asparagus, beans,
carrots, celery, corn, lima beans, olives, potatoes, turnips, apri-
cots, cherries, and peaches, all tested positive for C. botulinum,
primarily type A. Improperly processed or temperature-abused
vegetable products, including baked potato, potato salad, car-
rot juice, olives, mushrooms, bean curd, and onions, have all
been implicated in foodborne botulism outbreaks. Home-
canned vegetables including beans, carrots, asparagus, and
vegetable products stored in oil including garlic in oil and
eggplant in oil present an especially high risk of foodborne
botulism.
Spores in honey and other infant foods pose a unique
hazard because, in some infants, the spores are able to colonize
the intestines, produce toxin, and cause infant botulism. By
1984, honey had been implicated as the likely source of botu-
linum spores in 20 cases of infant botulism in California.
Surveys for the presence of C. botulinum spores in honey sug-
gest that the incidence ranges from 2% to 26% of samples
depending upon the geographic source of honey, with spore
levels in random samples of honey in the order of 1–10 spores
per kg. However, in honey samples associated with infant
botulism, the level is approximately 104 spores per kg. The
pasteurization process applied to honey is used to inactivate
osmophilic yeasts and prevent granulation, but is not sufficient
to inactivate spores of C. botulinum. Other infant foods have
also been examined. While C. botulinum has been detected in
samples of corn syrup and rice cereal, exposure of infants to
botulinum spores via these foods seems to be minimal as the
spore levels are low and spores are unlikely to multiply during
manufacture and storage. Due to the risk for infant botulism,
health authorities in several countries recommend that honey
should not be given to infants less than 1 year of age.
Dairy products are rarely vehicles for botulism outbreaks.
Six reported outbreaks of botulism caused by cheeses, primar-
ily cottage cheese, occurred in California and New York
between 1912 and 1951. One of these outbreaks occurred in
Albany, New York, in 1914 and resulted in the death of three
persons and was one of the first reported outbreaks caused by
C. botulinum type B. In October 1993, eight cases resulted after
eating a potato stuffed with meat and a commercial cheese
sauce. In August 1996, an outbreak of botulism in Italy affected
eight people after consumption of mascarpone cheese, either
alone or as a component of the desert tiramisu.
Detection of C. botulinum
Safety Precautions When Working with C. botulinum or BoNT
The extreme toxicity of BoNT requires that specific safety pro-
cedures be followed when working with cultures containing
C. botulinum and BoNT. With the rare exception of wound
botulism, C. botulinum is noninfectious in healthy adults. As
a result of its noninfectious nature, C. botulinum is a contain-
ment level 2 organism. In Canada, procedures generating sig-
nificant quantities of toxin, or aerosol formation of toxin,
require BSL3 laboratory facilities. All personnel engaged in
handling toxic materials must be fully informed about the
hazards, and all materials to be autoclaved should be con-
tained in stainless steel boxes with handles. Therapeutic anti-
sera must be available in case of accidental intoxication.
157
Potentially toxic materials should always be contained in
unbreakable, leak-proof trays or boxes. This is particularly
important in the incubation or manipulation of botulinal
cultures, which may contain in excess of 105 mouse LD per
ml. If toxin should be spilled, it must be inactivated immedi-
ately with 0.1 N NaOH.
Traditional Culture Methods
Prevalence rates for C. botulinum in foods are not readily avail-
able, mainly because of the cost and difficulties of detecting the
organism in foods. C. botulinum is traditionally detected by
enriching food or environmental samples for spores, followed
by broth culture and detection of BoNT in the culture superna-
tant. Common enrichment media for detecting viable
C. botulinum are cooked meat medium (CMM), CMM glucose,
chopped meat glucose starch medium, and tryptone–peptone–
glucose–yeast extract (TPGY) broth to which trypsin may be
added. Trypsin is necessary to activate the toxin produced by
group II organisms and may also inactivate potential inhibitors
of C. botulinum such as bacteriocins in mixed cultures. While
foods may be inoculated directly, the sediments of centrifuged
samples are preferred because potential growth inhibitors are
removed. At least two tubes of media are inoculated. One is
heated at 75–80 or 60
C, depending on whether the suspected
type belongs to group I or II, to select for spores. Alternatively,
spores of group II may be selected by holding samples in 50%
alcohol for 1 h before inoculation. The other tube is incubated
without any heating to allow development of vegetative
C. botulinum cells in cases where few or no spores are suspected.
Adding lysozyme to the medium may increase recovery of heat-
injured spores. Group I strains grow optimally at 37
C, while
group II strains have an optimal growth temperature of 30
C.
C. botulinum is identified after incubation of the enrichment
medium by toxin analysis of the supernatant fluid. Botulinal
toxin is detected by injecting serum or extracts from foods and
clinical specimens into mice, observing these for lethality, and
neutralizing the toxin with specific antisera.
It is not necessary to isolate C. botulinum in pure culture
from foods in order to demonstrate its presence. If the neuro-
toxin is present in the nonselective culture after incubation, the
toxin-producing organism must have been originally present.
If an enrichment culture tests positive for either C. botulinum
type B or F, the organism must be isolated to determine
whether the strains belong to group I or group II.
C. botulinum can be isolated by streaking toxic enrichment
cultures onto C. botulinum isolation (CBI) agar. CBI agar is
supplemented with cycloserine, sulfamethoxazole, and tri-
methoprim to inhibit background flora and egg yolk for detec-
tion of lipase-positive colonies. Suspect lipase-positive
colonies showing spreading and an irregular edge may be
picked and restreaked for isolation. Many other common clos-
tridia, such as C. sporogenes, produce a lipase reaction on agar
plates containing egg yolk. Isolates are grown in TPGY or CMM
broth, and toxicity of the culture supernatant confirms identity
of the isolate as a neurotoxigenic clostridium. It is important to
note that C. butyricum is lipase-negative; therefore, C. butyricum
strains producing type E toxin will appear as lipase-negative
colonies on CBI agar. Colony immunoblotting to detect
158 Clostridium: Occurrence and Detection of Clostridium botulinum and Botulinum Neurotoxin
individual botulinum toxin-producing colonies on an agar
plate is a sensitive method for isolation from enrichment cul-
tures with a high background flora.
As an alternative to testing enrichment cultures, or pure
cultures, with the mouse assay to detect BoNT, polymerase
chain reaction (PCR) can be used to detect the presence of
genes encoding BoNT. Multiplex PCR assays (both conven-
tional and real-time-PCR) have been developed to allow simul-
taneous detection of botA, botB, botE, and botF genes in a single
reaction. These multiplex PCR reactions allow screening of
many samples at once, with the advantages of reduced time
to detection without a requirement for use of live animals.
Application of multiplex PCR to isolated colonies also allows
identification of bot-containing colonies, facilitating isolation
of botulinogenic clostridia. As PCR does not detect botulinum
toxin, the possibility exists that clostridial strains with silent
BoNT genes may be detected, resulting in a false-positive result.
PCR has also been used for molecular characterization of
C. botulinum based on the sequences of the neurotoxin and
flagellin genes.
When C. botulinum is present in food or environmental
samples, it is typically present in low concentrations (<1 to
1000 spores per kg). Quantitative estimates of such low num-
bers require the use of broth cultures and most probable num-
ber (MPN) analysis. Depending on the precision required,
three or five tube MPN tests are performed in a similar manner
described earlier for the detection of C. botulinum. Toxicity
testing for the large number of samples involved in MPN
analysis is facilitated by using an in vitro assay, in place of the
standard mouse bioassay.
Detection of BoNT
BoNTs are extraordinarily potent with the parenteral human
lethal dose estimated to be 0.1–1 ng kg1 and the oral lethal
dose estimated at 1 mg kg1, thereby requiring an extremely
sensitive assay for detection.
Mouse Bioassay
BoNTs are recognized by their lethal action in mice and neu-
tralization with specific antisera. The sample, or an extract
prepared by homogenizing it in gelatin phosphate buffer, is
clarified by centrifugation and filter sterilized. Trypsin treat-
ment may be required to activate low levels of toxin from
nonproteolytic strains. The prepared sample is injected intra-
peritoneally into mice with and without neutralization with
antitoxin. Typical symptoms of botulism are ruffled fur,
pinched waist, labored breathing, limb paresis, and general
paralysis before death. The time required to onset of symptoms
is dependent upon the concentration of toxin in the extract,
with symptoms typically occurring within the first 24 h post-
injection. Definitive results are obtained if mice injected with
untreated sample display symptoms within 72 h, while mice
injected with neutralized sample do not display symptoms.
The use of an end point earlier than death is encouraged, as
ruffled fur, pinched waist, and labored breathing are typical
and easily observable signs of botulinum toxin. The mouse
bioassay has several advantages including high sensitivity
(approximately 10 pg of botulinum toxin) and is sufficiently
robust to detect botulinum toxins in a wide range of sample
matrices including feces, serum, gastric liquid, a wide range of
food samples, and supernatants of bacterial cultures. In addi-
tion, the mouse bioassay is the only assay capable of detecting
all serotypes, including any possible new serotypes, of BoNTs.
In addition to the obvious ethical objection of sacrificing ani-
mals, the mouse bioassay has the disadvantages of being slow,
expensive, and low throughput.
A refinement of the typical mouse bioassay, nonlethal
mouse assays involve subcutaneous injection of mice and
observation for flaccid paralysis of muscle near the injection
site. Subcutaneous injection at the inguinocrural region in
mice has been demonstrated to yield sensitivity results compa-
rable to the typical mouse assay. A nonlethal mouse toe-spread
reflex model has been used to detect botulinum toxins in
buffer, serum, and milk samples at sensitivities similar to the
typical mouse bioassay. Preincubation of samples with specific
antitoxins prevents development of paralysis, allowing identi-
fication of toxin serotype with these methods. Ex vivo assays,
using rat or mouse phrenic nerve diaphragm, and the rat
intercostal muscle strips assay allow several tests from tissues
of a single animal.
Immunologic Assays
The earliest immunologic assays used to detect botulinum
toxins included passive agglutination and immunodiffusion
assays. Enzyme-linked immunosorbent assays (ELISAs) were
first developed for BoNT in the late 1970s and were capable of
detecting approximately 400 mouse lethal doses (1 MLD is
approximately 10 pg of neurotoxin). Improvements in ELISA
technology, such as use of capture antibodies in a sandwich
ELISA and generation of specific monoclonal antibodies for
capture and detection, have increased the sensitivity of ELISA
detection of botulinum toxins to less than one mouse lethal
dose per ml of food matrix. Traditional ELISAs used alkaline
phosphatase or horseradish peroxidase to cleave a chromo-
genic substrate. Reporters using signal amplification or chemi-
luminescence have further increased the sensitivities of
immunoassays. Lateral flow assays are rapid and easy to per-
form with minimum requirements for laboratory equipment
or skills. The sensitivity of lateral flow assays is in the range of
5–20 ng, approximately 1000 times less sensitive than the
mouse bioassay. Some commercial lateral flow assays have
been reported to be limited by matrix effects and have been
recommended only for rapid detection of botulinum toxin in
bacterial cultures.
Endopeptidase Assays
Development of in vitro assays to detect botulinum toxins has
accelerated as a result of increased investment to defend against
biological threat agents, and also assays are used for potency
testing for lot release of therapeutic BoNT (e.g., Botox, Dysport,
 Clostridium: Occurrence and Detection of Clostridium botulinum and Botulinum Neurotoxin
or Xeomin). The discovery that botulinum toxin is a specific
endopeptidase has led to the development of several assays
that rely on detection of the endopeptidase activity of the LC.
These assays typically employ a peptide containing a sequence
from one of the substrate proteins – SNAP-25, synaptobrevin,
or syntaxin. Cleavage of the peptide is measured by several
methods including generation of a bioluminescent signal
through cleavage of a SNAP-25 luciferase fusion protein,
changes in fluorescence emission ratios using fluorescence
resonance energy transfer (FRET), and detection of cleavage
products using mass spectrometry. Most of the assays relying
on endopeptidase activity require an initial step to remove
interfering substances and concentration of the neurotoxin.
This step often employs immunomagnetic ‘pulldown’ using
magnetic beads coated with antibody to the neurotoxin HC.
Endopeptidase assays have the advantage over immunoassays
of detecting only botulinum toxins with endopeptidase activ-
ity, and not inactive toxins.
Cell-Based Assays
Cell-based assays take detection of biological activity even
further by providing a model for BoNT detection that provides
information on the activity of the BoNT preparation. This
includes cell surface binding, endocytosis, translocation of
the LC into the cellular cytosol, and enzymatic activity of the
LC on SNARE substrates. This makes cell-based assays ideal for
high-throughput screening for botulinum toxin inhibitors. In
addition, cell-based assays may present a more accurate indi-
cation of the biological activity of therapeutic preparations of
BoNTs. The drawbacks of cell-based assays are their limited
sensitivity in comparison to the mouse assay and in vitro endo-
peptidase assays, and the difficulty in obtaining quantitative
results. Cell-based assays also require the maintenance of cell
cultures and take much longer to obtain results, when com-
pared to other in vitro assays.
See also: Clostridium botulinum; Clostridium: Food Poisoning by
Clostridium perfringens; Clostridium: Occurrence and Detection of
Clostridium perfringens.
159
Further Reading
Capek P and Dickerson TJ (2010) Sensing the deadliest toxin: technologies for
botulinum neurotoxin detection. Toxins 2: 24–53.
Dunning FM, Ruge DR, Piazza TM, Stanker LH, Zeytin FN, and Tucker WC (2012)
Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in
complex matrices with picomolar to femtomolar sensitivity. Applied and
Environmental Microbiology 78: 7687–7697.
Hill KK, Smith TJ, Helma CH, et al. (2007) Genetic diversity among botulinum
neurotoxin-producing clostridial strains. Journal of Bacteriology 189: 818–832.
Leclair D, Farber JM, Doidge B, et al. (2013) Distribution of Clostridium botulinum type
E strains in Nunavik, Northern Quebec, Canada. Applied and Environmental
Microbiology 79: 646–654.
Lindstrom M and Korkeala H (2006) Laboratory diagnostics of botulism. Clinical
Microbiology Reviews 19: 298–314.
Maslanka SE, Lúquez C, Raphael B, Dykes JK, and Joseph LA (2011) Utility of
botulinum toxin ELISA A, B, E, F kits for clinical laboratory investigations of human
botulism. Botulinum Journal 2: 72–92.
Peck MW, Stringer SC, and Carter AT (2011) Clostridium botulinum in the post-
genomic era. Food Microbiology 28: 183–191.
Pellett S (2013) Progress in cell based assays for botulinum neurotoxin detection.
Current Topics in Microbiology and Immunology 364: 257–285.
Rossetto O, Megighian A, Scorzeto M, and Motecucco C (2013) Botulinum neurotoxins.
Toxicon 67: 31–36.
Sebaihia M, Peck MW, Minton NP, et al. (2007) Genome sequence of a proteolytic
(Group I) Clostridium botulinum strain Hall A and comparative analysis of the
clostridial genomes. Genome Research 17: 1082–1092.
Sevenier V, Delannoy S, André S, Fach P, and Remize F (2012) Prevalence of
Clostridium botulinum and thermophilic heat-resistant spores in raw carrots and
green beans used in French canning industry. International Journal of Food
Microbiology 155: 263–268.
Shapiro RL, Hatheway C, and Swerdlow DL (1998) Botulism in the United States: a
clinical and epidemiologic review. Annals of Internal Medicine 129: 221–228.
Singh AK, Stanker LH, and Sharma SK (2012) Botulinum neurotoxin: where are we with
detection technologies? Critical Reviews in Microbiology 39: 43–56.
Wang D, Baudys J, Ye Y, et al. (2012) Improved detection of botulinum neurotoxin
serotype A by Endopep-MS through peptide substrate modification. Analytical
Biochemistry 432: 115–123.
Zhang Y, Lou J, Jenko KL, Marks JD, and Varnum SM (2012) Simultaneous and
sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked
immunosorbent assay-based protein antibody microarrays. Analytical Biochemistry
430: 185–192.
Relevant Websites
http://healthycanadians.gc.ca/eating-nutrition/poisoning-intoxication/botulism-
botulisme-eng.php?_ga¼1.161587964.1921413621.1402510148 – Botulism
(Clostridium botulinum).
http://www.hc-sc.gc.ca/fn-an/legislation/guide-ld/botulism-botulisme-prof-eng.php –
Botulism – Guide for Healthcare Professionals.
http://www.hc-sc.gc.ca/sr-sr/activ/micro/botulism-eng.php – Botulism Reference
Service for Canada.
 Cobalamin (Vitamin B12): Metabolism and Disorders
E Andrès, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; Faculty of Medicine, Strasbourg, France
N Dali-Youcef, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; Faculty of Medicine, Strasbourg, France;
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Vitamin B12 is the largest and most complex of all the vita-
mins. The name vitamin B12 is generic for a specific group of
cobalt-containing corrinoids, also known as cobalamins, with
biological activity in humans. Cobalt gives this water-soluble
vitamin its red color. The main cobalamins in humans and
animals are hydroxocobalamin, adenosylcobalamin, and
methylcobalamin, the last two being the active coenzyme
forms.
Although vitamin B12 was isolated almost 60 years ago, its
metabolism remains incompletely defined. In practice, cobal-
amin metabolism is complex and requires many processes and
steps, any one of which, if not present, may lead to cobalamin
deficiency. Table 1 enumerates the elements establishing the
definition of cobalamin deficiency.
This article summarizes the current knowledge on cobala-
min metabolism and disorders.
Vitamin B12 Ingestion and Related Disorders
Vitamin B12 is produced exclusively by microbial synthesis in
the digestive tract of animals. Therefore, animal protein prod-
ucts are the source of vitamin B12 in the human diet, in
particular organ meats (liver and kidney). Other good sources
are fish, eggs, and dairy products. In foods, hydroxo-, methyl-,
and 50 -deoxyadenosyl cobalamins are the main cobalamins
present.
A typical Western diet contributes 3–30 mg of cobalamin
per day. The recommended dietary allowance set by the Food
and Nutrition Board of the Institute of Medicine (the United
States) is 2.4 mg day1 for adults and 2.6–2.8 mg day1 during
pregnancy.
Table 2 describes through a synthetic view the different
stages of vitamin B12 metabolism used in clinical practice
and the corresponding causes of cobalamin deficiency. In clin-
ical practice, vitamin B12 deficiency caused by dietary defi-
ciency is rare.
The dietary causes of deficiency are limited to elderly people
who are already malnourished, such as elderly patients living
in institutions (they may consume inadequate amounts of
vitamin B12-containing foods) or in psychiatric hospitals
(strict vegetarians). Studies focussing on elderly people, partic-
ularly those who are in institutions or who are sick and mal-
nourished, have suggested a cobalamin deficiency prevalence
of 30–40%. The Framingham Study demonstrated a prevalence
of 12% among elderly people living in the community. Using a
stringent definition, a prevalence of 5% had been reported in a
group of patients followed or hospitalized in a tertiary refer-
ence hospital.
160 Encyclopedia of Food and Health
A dietary cause of cobalamin deficit or deficiency was
also found in newborns from strictly vegetarian pregnant
women.
Vitamin B12 Digestion and Related Disorders
Dietary vitamin B12, which is bound to proteins in food, is
released in the acidic environment of the stomach where it is
rapidly complexed to the binding protein and transporter hap-
tocorrin (HC), also referred to as the R-binder or transcobala-
min I. About 80% of circulating cobalamin is bound to HC,
and serum cobalamin levels have been correlated with serum
HC concentrations. Although some unexplained low serum
cobalamin concentrations were reported to be caused by mild
to severe HC deficiencies, these abnormalities were not accom-
panied by pernicious anemia and are not thought to cause
functional cobalamin deficiency.
Cobalamin continues its route in the gastrointestinal
tract and dissociates from HC under the action of pancreatic
proteases. Then, cobalamin is associated in the intestine
with the intrinsic factor (IF, also known as the S-binder).
This complex is essential for the ileal absorption of cobal-
amin (Table 2).
Digestion disorders related to vitamin B12 deficiency are
mainly represented by food-cobalamin malabsorption (FCM)
syndrome. This syndrome is characterized by the inability of
the body to release cobalamin from food or intestinal transport
proteins, particularly in the presence of hypochlorhydria
(therefore, the appropriated denomination is ‘maldigestion’).
The principal characteristics of this syndrome are listed in
Table 3.
FCM accounted for at least half of subtle or clinical docu-
mented cobalamin deficiency in elderly patients (60–70% in
our experience). FCM is caused primarily by atrophic gastritis,
related or not to chronic carriage of Helicobacter pylori. Other
factors that mainly contribute to FCM are long-term ingestion
of antacids, such as H2 receptor antagonists and proton pump
inhibitors, particularly among patients with Zollinger–Ellison
syndrome, and biguanides (metformin). In addition, other
FCM inducers include chronic alcoholism, surgery or gastric
reconstruction (e.g., bypass surgery for obesity), and partial
exocrine pancreatic failure.
It is to note that in the case of FCM, patients can absorb
‘unbound’ cobalamin through IF or passive diffusion mecha-
nisms. Thus, the recognition of the syndrome permits new
developments of oral vitamin B12 therapy.
At this level, it is also important to note that several homo-
zygous nonsense and missense mutations in the gene encoding
the gastric IF have been reported to cause hereditary juvenile
cobalamin deficiency.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00174-4

Vitamin B12 Absorption and Related Disorders
Absorption depends mainly on IF, which is secreted by the
gastric mucosa. IF binds cobalamin forming a complex that is
absorbed by the terminal ileum (Table 2). This complex is
located at the apical side of brush border membranes (BBMs)
of polarized epithelia, such as the intestinal apical BBM. It
consists of the IF–vitamin B12 receptor named cubilin, a
460 kDa peripheral membrane glycoprotein, encoded by the
CUBN gene, which was mapped to chromosomal region
10p12.33-p13, and the 48 kDa amnionless protein encoded
Table 1 Definitions of vitamin B12 (cobalamin) deficiency
• Serum cobalamin levels <150 pmol l1 and clinical features and/or
hematologic anomalies related to cobalamin deficiency
• Serum cobalamin levels <150 pmol l1 (<200 pg ml1) on two
separate occasions
• Serum cobalamin levels <150 pmol l1 and total serum
homocysteine levels >13 mmol l1 or methylmalonic acid levels
>0.4 mmol l1 (in the absence of renal failure and folate and vitamin
B6 deficiencies)
• Low serum holotranscobalamin levels <35 pmol l1
Table 2 Stages of vitamin B12 metabolism and corresponding causes of vitamin B12 deficiency
Stages and actors in vitamin B12 metabolism
Intake solely through food
Digestion brings into play the following:
• Haptocorrin
• Gastric secretions (hydrochloric acid and pepsin)
• Intrinsic factor
• Pancreatic and biliary secretions
• Enterohepatic cycle
Absorption brings into play the following:
• Intrinsic factor
• Cubilin, amnionless
• Calcium and energy
Transport by transcobalamins
Intracellular metabolism based on various intracellular enzymes
Table 3 Main characteristics of the food-cobalamin malabsorption syndrome
Characteristics of food-cobalamin malabsorption
• Low serum vitamin B12 (cobalamin) levels
• Normal results of Schilling test using free cyanocobalamin
labeled with cobalt-58 or abnormal results of derived Schilling
testa
• No anti-intrinsic factor antibodies
• No dietary vitamin B12 deficiency
a
Derived Schilling tests use food-bound cobalamin (e.g., egg yolk, chicken, and fish proteins).
Cobalamin (Vitamin B12): Metabolism and Disorders 161
by the AMN gene, a gene essential for mouse gastrulation and
localized on human chromosome 14.
The human megalin/gp330/LRP2 receptor, encoded by the
LRP2 gene located on chromosome 2q24-q31, is a giant endo-
cytic receptor (600 kDa) of the low-density lipoprotein
receptor family that was strongly suggested to play an impor-
tant role in the stability of the cubilin–AMN complex.
The mechanism of vitamin B12 absorption through the
complex IF–cubilin–AMN is Ca2þ-dependent.
Mutations in CUBN 7 were reported to cause hereditary
megaloblastic anemia 1 (MGA1). MGA1 is a rare autosomal
recessive disorder affecting human subjects with neurological
symptoms and juvenile MGA.
Two principal mutations were identified in Finnish patients
(FM): a mutation named FM1 changing a highly conserved
proline to leucine (P1297L) in CUB domain 8, suggesting
that this proline is functionally crucial in cubilin, and one
point mutation FM2 in the intron interrupting CUB domain
6, which produced a truncated cubilin. Interestingly, a normal-
size cubilin protein was identified in urine samples from
homozygous FM1 patients, whereas a complete absence of
the protein was reported in a patient homozygous for the
FM2 mutation. Other mutations were also uncovered but
were subsequently identified as polymorphisms after their
Causes of vitamin B12 deficiency
• Strict vegetarianism and patients who are sick in institutions or in psychiatric
hospitals
• Gastrectomies
• Pernicious anemia
• Food-cobalamin malabsorption
• Ileal resections and malabsorption
• Pernicious anemia
• Food-cobalamin malabsorption
• Congenital deficiency in transcobalamin II
• Congenital deficiency in various intracellular enzymes
Associated conditions or agents
• Gastric disease: atrophic gastritis, type A atrophic gastritis, gastric disease
associated with Helicobacter pylori infection, partial gastrectomy, gastric
bypass, vagotomy
• Pancreatic insufficiency: alcohol abuse
• Gastric or intestinal bacterial overgrowth: achlorhydria, tropical sprue,
Ogilvie’s syndrome, HIV
• Drugs: antacids (H2 receptor antagonists and proton pump inhibitors) or
biguanides (metformin)
• Alcohol abuse
• Aging or idiopathic
162 Cobalamin (Vitamin B12): Metabolism and Disorders
detection in normal individuals in the general population.
The cubilin P1297L mutation associated with hereditary
MGA1 was reported to cause impaired recognition of the
cobalamin–IF complex by cubilin.
In addition, a mutation in AMN was reported in recessive
hereditary MGA1 and was demonstrated to be essential for a
functional cobalamin–IF receptor. This study demonstrated
that homozygous mutations affecting exons 1–4 of the
human AMN gene translated into selective malabsorption of
vitamin B12, a phenotype associated with hereditary MGA1.
The essential AMN–cubilin interaction was confirmed in vitro,
thereby explaining the molecular basis of intestinal cobalamin
malabsorption syndrome.
In adults, cobalamin deficiency is classically caused by per-
nicious anemia or Biermer’s disease or Addison’s disease. It is
an autoimmune disease characterized by the destruction of the
gastric mucosa, especially the fundus, associated with a primar-
ily cell-mediated autoimmune process and the presence of
various antibodies anti-IF and gastric parietal anticell anti-
bodies that target the Hþ/K þ-ATPase a and ß subunits.
Pernicious anemia is the leading cause of cobalamin defi-
ciency and accounted for 40–50% of cases in adults. In the
general population, the prevalence of pernicious anemia is
0.1%; in subjects over the age of 60, it reaches 1.9%. The
principal characteristics of pernicious anemia have been
reported in detail in several reviews and are listed in Table 4.
In practice, the diagnosis of pernicious anemia is based on the
presence of IF antibodies in serum (specificity, >98%, and
sensitivity, around 50%) or biopsy-proved autoimmune atro-
phic gastritis. The presence of H. pylori infection in gastric
biopsies is an exclusion factor.
Genetic susceptibility to pernicious anemia appears to be
genetically determined, although the mode of inheritance
remains unknown. Evidence for the role of genetic factors
includes familial co-occurrence of pernicious anemia and its
association with other autoimmune diseases. Thus, a certain
number of autoimmune diseases occur at a higher frequency in
patients with pernicious anemia – around 30% – or among
family members of pernicious anemia patients. They can pre-
cede the disease or occur after its onset. The association
with autoimmune diseases such as type 1 diabetes (insulin-
dependent), autoimmune thyroiditis (particularly Hashimo-
to’s thyroiditis), and vitiligo is common. Other associations
have also been frequently described, for example, Sjögren’s
syndrome, celiac disease, and Addison’s disease (adrenal
insufficiency). Cases of multiple autoimmune syndrome
including pernicious anemia have also been documented.
Table 4 Main characteristics of pernicious anemia
Characteristics of pernicious anemia
• Low serum vitamin B12 (cobalamin) levels
• Hematologic abnormalities or neurological manifestations
• Abnormal results of Schilling test using free cyanocobalamin labeled
with cobalt-58
• Presence of anti-intrinsic factor antibodies
• Autoimmune gastritis (Helicobacter pylori-negative)
• Associated autoimmune diseases (Sjögren’s syndrome, Hashimoto’s
thyroiditis, type 1 diabetes mellitus, etc.)
Since the 1980s, the malabsorption of cobalamin has
become rare, owing mainly to the decreasing frequency of
gastrectomy and terminal small intestine surgical resection.
Several disorders commonly seen in practice might, however,
be associated with cobalamin malabsorption. These disorders
include exocrine pancreas’ function deficiency following
chronic pancreatitis (usually alcoholic), lymphomas or
tuberculosis (of the intestine), celiac disease, Crohn’s disease,
Whipple’s disease, and uncommon celiac disease.
Vitamin B12 in Blood and Tissues and
Related Disorders
After cobalamin is absorbed at the BBM–blood barrier, it dis-
sociates from the IF and reaches the systemic circulation where
it associates with transcobalamin II (TCII) (Table 2).
The kidney represents an essential organ where the body’s
vitamin B12 stores are maintained, and studies demonstrated
that the kidney regulates plasma vitamin B12 levels by main-
taining a pool of unbound cobalamin that can be released in
the case of vitamin B12 deficiency.
The tissular cobalamin–TCII complex uptake is achieved
through megalin (LRP2) and transcobalamin II receptor
(TCII-R)-mediated endocytosis, which plays a crucial role in
cobalamin homeostasis. It is worth mentioning that TCII is
responsible for the cellular uptake of vitamin B12 in most
tissues and that TC deficiency is associated with severe MGA.
Following cobalamin–TCII cellular uptake, TCII undergoes
lysosomal digestion, which allows cobalamin separation
from TCII and its cytoplasmic transfer.
It has been estimated that there is a delay of ranging from 5
to 10 years between the onset of cobalamin deficiency and the
appearance of clinical manifestations, due to important hepatic
stores (> 1.5 mg) and the enterohepatic cycle. The average vita-
min B12 content is 1.0 mg in healthy adults, with 20–30 mg
found in the kidneys, heart, spleen, and brain. Estimates of total
vitamin B12 body content for adults range from 0.6 to 3.9 mg
with mean values of 2–3 mg. The normal range of vitamin B12
plasma concentrations is 150–750 pg ml1, with peak levels
achieved 8–12 h after ingestion.
Part of the unbound cobalamin serves as a cofactor
for methionine synthase-mediated homocysteine catabolism
into methionine and methylenetetrahydrofolate reductase
(MTHFR)-mediated formation of the vitamin B9 (folate) bio-
logically active form, tetrahydrofolate, which is then involved
in the synthesis of purines and pyrimidines. The other part of
free vitamin B12 is transferred to the mitochondria where
it is transformed into adenosyl-B12, an important cofactor in
the methylmalonyl-coenzyme A mutase-mediated formation
of succinyl-CoA from methylmalonyl-CoA, the product of
odd-chain fatty acid and some amino acid catabolisms.
Biochemically, cobalamin deficiency will cause homocyste-
ine accumulation, increased methylmalonyl-CoA levels, and
decreased MTHFR activity. These changes translate into several
abnormalities including folate deficiency and subsequent inhi-
bition of purine and pyrimidine formation essential for RNA
and DNA syntheses.
The clinical manifestations of these metabolic abnormalities
are MGA, neurological defects, malformations of neurological
 Cobalamin (Vitamin B12): Metabolism and Disorders 163

structures, increased cardiovascular thrombotic risk and renal
disease, and methylmalonic acidemia.
Functional cobalamin deficiency can also be caused by
defects in the intracellular processing of cobalamin such as
abnormal lysosomal digestion of the TCII–cobalamin com-
plex, and subsequent defective lysosomal release of cobalamin,
and abnormalities in intracytoplasmic cobalamin metabolism
with all the consequences on biochemical reactions in which
cobalamin acts as an important cofactor.
Clinical Manifestations of Vitamin B12 Deficiency
Clinical manifestations related to vitamin B12 deficiency are
highly polymorphic and of varying severity, ranging from
milder conditions, such as fatigue, common sensory neuropa-
thy, atrophic glossitis (Hunter’s glossitis), and isolated macro-
cytosis or neutrophil hypersegmentation, to severe disorders,
including combined sclerosis of the spinal cord, hemolytic
anemia, and even pancytopenia.
These later years, the use of strict criteria to define vitamin
B12 deficiency (see Table 1) has resulted in more recognition
of previously unknown or atypical clinical presentations of
cobalamin deficiency. The established manifestations of cobal-
amin deficiency are described in Table 5.
Vitamin B12 Deficiency Therapy
The treatment of established vitamin B12 deficiency is based
upon the administration of intramuscular cyanocobalamin. Its
efficacy on the resolution of clinical signs and symptoms has
been proved.
The classic treatment for vitamin B12 deficiency is based on
parenteral administration – in most countries as intramuscular
injections – in the form of cyanocobalamin and, more rarely,
hydroxo- or methylcobalamin. Hydroxocobalamin may have
several advantages due to a better tissue retention and storage.
However, the management concerning both the dose and
schedule of administration varies considerably between
countries.
In the United States and United Kingdom, doses ranging
from 100 to 1000 mg per month (or every 2–3 months when
hydroxocobalamin is given) are used for the duration of the
patient’s life. In France, treatment involves the administration of
1000 mg of cyanocobalamin per day for 1 week, followed by
1000 mg per week for 1 month, followed by 1000 mg per month,
again, normally for the remainder of the patient’s lifetime.
Likewise, oral cyanocobalamin given at treatment doses has
been shown to exhibit a similar efficacy to intramuscular cya-
nocobalamin, resulting in significantly increased levels of
serum vitamin B12 and improvements in hematologic param-
eters. In fact, about 1–5% of free cobalamin (or crystalline
cobalamin) is absorbed along the entire intestine by passive
diffusion. A systematic review carried out under the auspices of
the Cochrane Metabolic and Endocrine Disorders Review Group
supports the efficacy of oral cobalamin therapy, with a daily
dose of 2000 mg initially and then 1000 mg weekly of vitamin
B12. However, the efficacy of oral cobalamin in the treatment
of severe neurological diseases has not yet been sufficiently
documented. Therefore, in these patients, cobalamin must
still be administered via the parenteral route.
Our working group has developed an effective oral treat-
ment for FCM and pernicious anemia using crystalline cobala-
min (cyanocobalamin). Our principal studies of oral
cobalamin treatment (open, not randomized studies) are
described in Table 6. These other routes of administration
have been proposed as a way of avoiding the discomfort,
inconvenience, and cost of monthly injections.
Since the 1990s, at least half of these patients were treated
with oral cyanocobalamin, with a dose between 125 and
2000 mg day1. All of the patients who were treated orally
corrected their vitamin B12 levels, and at least 80% corrected
their hematologic abnormalities. Moreover, half of the patients
experienced a clinical improvement on oral treatment. Table 7
shows our ‘façon de faire’ (take home recommendations). The
following can be proposed: ongoing supplementation is
needed until any associated disorders are corrected (e.g., by

Table 5 Main clinical features of vitamin B12 deficiency

Hematologic manifestations Neuropsychiatric manifestations Digestive manifestations Other manifestations

• Frequent: macrocytosis,
neutrophil hypersegmentation,
regenerative macrocytic
anemia, medullary
megaloblastosis (‘blue spinal
cord’)
• Rare: isolated
thrombocytopenia and
neutropenia, pancytopenia
• Very rare: hemolytic anemia,
thrombotic microangiopathy
(presence of schistocytes)
• Frequent: polyneuritis (especially
sensitive), ataxia, Babinski’s
phenomenon
• Classic: combined sclerosis of the
spinal cord
• Rare: cerebellar syndromes
affecting the cranial nerves
including optic neuritis, optic
atrophy, urinary and/or fecal
incontinence
• Under study: changes in the
higher functions, dementia, stroke
and atherosclerosis
(hyperhomocysteinemia),
parkinsonian syndromes,
depression, multiple sclerosis,
autism
• Classic: Hunter’s • Frequent: Tiredness, loss of
glossitis, jaundice, LDH appetite
and bilirubin elevation • Under study: atrophy of the
(‘intramedullary vaginal mucosa and chronic
destruction’) vaginal and urinary infections
• Debatable: abdominal (especially mycosis), hypofertility
pain, dyspepsia, and repeated miscarriages,
nausea, vomiting, venous thromboembolic disease,
diarrhea, disturbances angina (hyperhomocysteinemia)
in intestinal functioning
• Rare: resistant and
recurring
mucocutaneous ulcers
164 Cobalamin (Vitamin B12): Metabolism and Disorders

Table 6 Experience of oral vitamin B12 therapy in the University Hospital of Strasbourg, Strasbourg, France

Study characteristics (number of patients) Therapeutic modalities Results

Open prospective study of well-documented
vitamin B12 deficiency related to food-
cobalamin malabsorption (n ¼ 10)
Open prospective study of low vitamin B12
levels not related to pernicious anemia
(n ¼ 20)
Open prospective study of well-documented
vitamin B12 deficiency related to food-
cobalamin malabsorption (n ¼ 30)
Open prospective study of low vitamin B12
levels not related to pernicious anemia
(n ¼ 30)
Open prospective study of low vitamin B12
levels related to pernicious anemia (n ¼ 10)
Oral crystalline cyanocobalamin:
650 mg day1, during at least 3
months
Oral crystalline cyanocobalamin:
1000 mg day1, for at least 1
week
Oral crystalline cyanocobalamin:
between 1000 and 250 mg day1,
during 1 month
Oral crystalline cyanocobalamin:
between 1000 and 125 mg day1
during at least 1 week
Oral crystalline cyanocobalamin:
1000 mg day1, during at least 3
months
• Normalization of serum vitamin B12 levels in 80% of the
patients
• Significant increase in hemoglobin (Hb) levels (mean of
1.9 g dl1) and decrease in mean erythrocyte cell
volume (ECV) (mean of 7.8 fl)
• Improvement of clinical abnormalities in 20% of the
patients
• No adverse effect
• Normalization of serum vitamin B12 levels in 85% of the
patients
• No adverse effect
• Normalization of serum vitamin B12 levels in 87% of the
patients
• Significant increase in Hb levels (mean of 0.6 g dl1) and
decrease in ECV (mean of 3 fl); normalization of Hb
levels and ECV in 54% and 100% of the patients,
respectively
• Dose effect – effectiveness dose of vitamin
B12  500 mg day1
• No adverse effect
• Normalization of serum vitamin B12 levels in all patients
with at least a dose of vitamin 250 mg day1
• Dose effect – effectiveness dose of vitamin
B12  500 mg day1
• No adverse effect
• Significant increase in serum vitamin B12 levels in 90%
of the patients (mean of 117.4 pg ml1)
• Significant increase in Hb levels (mean of 2.45 g dl1)
and decrease in ECV (mean of 10.4 fl)
• Improvement of clinical abnormalities in 30% of the
patients

Table 7 Expert opinion recommendations for oral vitamin B12 treatment

Pernicious anemia Intake deficiency and food-cobalamin malabsorption

Parenteral administration Cyanocobalamin: Cyanocobalamin:

(intramuscular) • 1000 mg day1 for 1 week • 1000 mg day1 for 1 week
• 1000 mg per week for 1 month • 1000 mg per week for 1 month
• 1000 mg per each month, for life • 1000 mg per 1 or 3 months, until the cobalamin
deficiency cause is corrected
(1000–2000 mg day1 for at least 1–3 months
in the case of severe neurological manifestations) (1000 mg day1 for at least 1–3 months
in the case of severe neurological manifestations)
Oral administration Cyanocobalamin: Cyanocobalamin:
• 1000 mg day1 for lifea • 1000 mg day1 for11 month
• 125–1000 mg day , until thea cobalamin
deficiency cause is corrected
a
The effect of oral cobalamin treatment in patients presenting with severe neurological manifestations has not yet been adequately documented.

halting the ingestion of the offending medication or chronic deficiency was supported by a grant of the Fondation de France
alcoholism or by treating H. pylori infection or pancreatic exo- (Prix Robert et Jacqueline Zittoun, 2004).
crine failure). This may result in lifelong administration or,
when applicable, sequential administration.

Acknowledgments
See also: Anemia: Causes and Prevalence; Anemia: Prevention and
We are indebted to Professor Marc Imler and Jean-Louis Dietary Strategies; Bioavailability of Nutrients; Malnutrition:
Schlienger who initiated this work. The research on cobalamin Prevention and Management; Vegetarian Diets; Vitamins: Overview.

Further Reading
Andrès E (2011) Signs and symptoms of vitamin B12 (cobalamin) deficiency: a critical
review of the literature. In: Hermann W and Obeid R (eds.) Vitamins for prevention of
human diseases, pp. 242–253. Berlin: Walter De Gruyter.
Andrès E, Loukili NH, Noel E, et al. (2004) Vitamin B12 (cobalamin) deficiency in
elderly patients. CMAJ 171: 251–259.
Andrès E, Fothergill H, and Mecili M (2010) Efficacy of oral cobalamin (vitamin B12)
therapy. Expert Opinion on Pharmacotherapy 11: 249–256.
Carmel R (2000) Current concepts in cobalamin deficiency. Annual Review of Medicine
51: 357–375.
Dali-Youcef N and Andres E (2009) An update on cobalamin deficiency in adults. QJM
102: 17–28.
Fowler B (1998) Genetic defects of folate and cobalamin metabolism. European Journal
of Pediatrics 157(Suppl. 2): S60–S66.
Hvas AM and Nexo E (2006) Diagnosis and treatment of vitamin B12 deficiency—an
update. Haematologica 91: 1506–1512.
Cobalamin (Vitamin B12): Metabolism and Disorders 165
Kuzminski AM, Del Giacco EJ, Allen RH, et al. (1998) Effective treatment of cobalamin
deficiency with oral cobalamin. Blood 92: 1191–1198.
Nicolas JP and Gueant JL (1994) Absorption, distribution and excretion of vitamin B12.
Annales de gastroentérologie et d’hépatologie 30: 270–276, 281; discussion 281–282.
Solomon LR (2007) Disorders of cobalamin (vitamin B12) metabolism: emerging
concepts in pathophysiology, diagnosis and treatment. Blood Reviews 21: 113–130.
Stabler SP (2013) Clinical practice. Vitamin B12 deficiency. New England Journal of
Medicine 368: 149–160.
Stabler SP, Allen RH, Savage DG, and Lindenbaum J (1990) Clinical spectrum and
diagnosis of cobalamin deficiency. Blood 76: 871–881.
Toh BH and Alderuccio F (2004) Pernicious anaemia. Autoimmunity
37: 357–361.
Vidal-Alaball J, Butler CC, Cannings-John R, et al. (2005) Oral vitamin B12 versus
intramuscular vitamin B12 for vitamin B12 deficiency. Cochrane Database of
Systematic Reviews, CD004655.
Wickramasinghe SN (2006) Diagnosis of megaloblastic anaemias. Blood Reviews
20: 299–318.
 Cobalt: Properties and Determination
F Cámara-Martos and R Moreno-Rojas, Universidad de Córdoba, Córdoba, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Cobalt Properties
Cobalt is the 27th element of the periodic table (atomic num-
ber 27). It was discovered by Swedish chemist Georg Brandt
about 1730. Its symbol is Co, and it is included in group 9 (or
VIIB) in the periodic table, thus corresponding to the transition
metals. It is a brittle, hard, silver-gray metal with magnetic
properties similar to those of iron (ferromagnetic). It is alloyed
with aluminum and nickel to make particularly powerful mag-
nets. The abundance of cobalt in the Earth’s crust is 0.003%.
It has a fusion point of 1493
C, a boiling point of 3100
C,
an atomic weight of 58.93320, and a relative density of
8.86 g cm3.
Cobalt has an electronic structure of [Ar] 4s23d7, and its
most common oxidation states are þ2 and þ3. Nevertheless,
some compounds with oxidation states ranging from 3 to þ 4
are also known. At temperatures below 417
C, cobalt exhibits
a hexagonal close-packed structure (e-cobalt). Between 417
C
and its melting point of 1493
C, cobalt has a face-centered
cubic structure (a-cobalt).
Cobalt occurs in nature in a fairly widespread but dispersed
form, being detectable in trace quantities in many rocks, soils,
and manganese-rich marine nodules. The currently exploited
sources of cobalt are mainly those where it originates as a by-
product of more valuable metals, particularly copper, nickel,
zinc, lead, and platinum. It is distributed in minerals such as
erythrite or red cobalt [Co3(AsO4)28H2O], cobaltite (CoAsS),
skutterudite [(Co,Ni)As3], carrollite [CuCo2S4], linnaeite
[Co3S4 (þNi,Cu,Fe)], cattierite [CoS2], nickel cattierite [(Co,
Ni)S2], heterogenite [2Co2O3CuO6H2O], or asbolane (mixed
manganese–iron oxide with cobalt). In seawater, the metal is
present primarily as the cobalt ion and its chloro, sulfate, and
carbonate complexes. In shallow waters, up to 98% of the metal
can be found in the sediments and in suspended particulate
matter. There are several cobalt isotopes with different half-lives
among which are 56Co (77.3 days), 57Co (271.8 days), 58Co
(70.9 days), 59Co (stable), 60Co (5.3 years), and 61Co (1.7 h).
The main use of cobalt compounds remained as a coloring
agent (a rich blue color) to glass, enamels, and porcelain, right
up to the twentieth century. It is also employed to make heat-
resistant superalloys. Some alloys of cobalt are used in jet
turbines and gas turbine generators, where high-temperature
strength is important. Cobalt compounds are also important
for nonmetallurgical applications, such as catalysts for the
petroleum and chemical industries, a drying agent, or in
lithium-ion battery electrodes. Cobalt salts of the higher
carboxylic acids (cobalt soaps) are used to accelerate drying in
oil-based paints, varnishes, and inks. As a ferromagnetic mate-
rial, it forms permanent magnets and it can be used as an
electromagnet. It has also been used in human medicine in
the treatment of certain iron-resistant anemia. Finally, radioac-
tive 60Co is used to treat cancer as a medical device for the
precise treatment of brain tumors and deformities of blood
166 Encyclopedia of Food and Health
vessels. Cobalt–chromium alloys are also used as biomaterials
because of its high degree of biocompatibility and the low
volume of attrition produced at the articulating surfaces.
Cobalt is essential to hematopoiesis by virtue of the fact that
it is the metal confined by the corrin ring of vitamin B12. The
ring consists of four pyrrole subunits, linked together to form a
macrocyclic ring. It is thus like a porphyrin, but with one of the
bridging methylene groups removed. The nitrogen of each
pyrrole is coordinated to the central cobalt atom. In ruminants,
bacteria present in their digestive tracts are able to convert
cobalt ions to vitamin B12. It is also associated with glycylg-
lycine dipeptidase enzyme. In the presence of hydrogen perox-
ide, cobalt ions are able to produce hydroxyl radicals through a
Fenton-like mechanism. Good food sources of cobalt include
meat muscles, fish, nuts, oats, and green leafy vegetables such
as broccoli and spinach. The bioavailability in vivo of cobalt
ions is relatively limited, because these cations precipitate in
the presence of physiological concentrations of phosphates
and nonspecifically bind to proteins such as albumin. Dietary
factors that seem to have the greatest effect on the ruminal
production of vitamin B12 are cobalt concentration, roughage
content of the diet, and total feed intake.
Cobalt Analysis
Sample Treatment
Most of the methods used for cobalt determination require the
availability of a treated sample solution. Previously, the sample
must be subjected to a treatment process where the organic
matter and other matrix components are destroyed and metal
ions for further analysis are released. During this treatment, the
risk of sample contamination or analyte losses is high.
A common practice is the incineration of the sample in a
muffle furnace using a time–temperature-controlled program.
This process is defined as dry mineralization. Process efficiency
and analyte retention mainly depend on the food matrix and
the time–temperature program applied. The ash residue is
usually dissolved with acids (HCl, HNO3, etc.) after a blanch-
ing step.
An alternative to the earlier technique is wet mineralization.
This method is based on the oxidizing action of strong acids
either alone or in acid mixtures that sometimes include hydro-
gen peroxide. These reagents have the capacity to release cobalt
ions and other trace elements keeping them in solution.
Dry and wet mineralization procedures are often tedious
and time-consuming. Another drawback of the earlier miner-
alization methods lies on the high risk of volatilization of
certain mineral elements such as selenium, mercury, or arsenic,
thus causing analyte losses. For cobalt determination, this
problem is not initially present unless the final objective of
the applied technique is oriented to determine these volatile
elements simultaneously.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00175-6
 Cobalt: Properties and Determination 167

The microwave digestion procedure could be a faster and more
efficient alternative. In this technique, samples are introduced in
microwave ovens using pressure vessels. It also allows the use of a
smaller volume of reagent. The most significant disadvantage is
the risk of explosion due to increase in pressure caused by the
destruction of organic matter. The pressure vessels are placed on a
turntable to avoid problems arising from lack of homogeneity in
the distribution of radiation within the microwave oven.
Despite the discrepancies earlier discussed between these
treatment processes, usually, the comparison of dry and wet
mineralization and microwave digestion methods did not
show statistically significant differences in relation to accuracy
and recovery percentage of mineral elements determined.
Preconcentration Methods
In some cases, the cobalt concentration present in food and
biological samples could be below the detection limit of the
analytic technique used. Another possibility is a strong inter-
fering effect with the food matrix, which impairs the measure-
ment procedure. In these cases, preconcentration and/or
separation methods prior to cobalt analysis are needed to
improve sensitivity and selectivity. The detection limit of the
analyte can be substantially improved if the final volume is
reduced. Several procedures, such as solid-phase extraction
(SPE), liquid–liquid extraction (LLE), cloud point extraction
(CPE), coprecipitation, and membrane filtration, have been
proposed and applied for the enrichment and separation of
cobalt from a sample matrix. Table 1 summarizes some
published papers on cobalt preconcentration techniques
in foods.
SPE is an advantageous separation and preconcentration
technique for trace metal ions given its simplicity, flexibility,
and high enrichment factor. The general procedure is to use
a solid phase that separates the analyte of interest from inter-
fering substances of the food matrix. With this purpose, the
original sample, previously dissolved or suspended in a
liquid phase, is poured on the solid phase on which the
analytes are retained. Subsequently, another liquid phase is
necessary to elute the retained analytes. Several solid-phase
materials have been employed for the preconcentration of
cobalt ions. These include chemically modified silica gel
with aminothioamidoanthraquinone, cellulose functiona-
lized with 8-hydroxyquinoline, silica gel–polyethylene glycol,
b-cyclodextrin cross-linked polymer, Aliquat 336 chloride
immobilized in poly(vinyl chloride), glycerol-bonded silica
gel, and activated carbon modified by dithiooxamide. Carbon
nanotubes have been currently proposed as a novel solid-
phase extractant for cobalt determination at trace levels. The
hexagonal arrangements of the carbon atoms in graphite
sheets, attached to their high surface area, make them a prom-
ising solid sorbent for preconcentration procedures. Process
parameters such as pH, sample flow rate, and sample volume
can influence on the adsorption efficiency as previously stud-
ied. Thus, it is shown that the acidity of the solution affects the
adsorption process because protons in acid solution can pro-
tonate the binding site of the chelating molecules, and hydrox-
ides in basic solution may form complexes causing metal

Table 1 Preconcentration and separation techniques for cobalt analysis

Investigation Reference

Separation of cobalt(II) from nickel(II) by solid-phase extraction (SPE) into
Aliquat 336 chloride immobilized in poly(vinyl chloride)
Simultaneous coprecipitation of lead, cobalt, copper, cadmium, iron, and nickel
in food samples with zirconium(IV) hydroxide prior to their flame atomic
absorption spectrometric determination
Acid extraction and cloud point preconcentration as sample preparation
strategies for cobalt determination in biological materials by thermospray
flame furnace atomic absorption spectrometry
Cloud point extraction for cobalt preconcentration with on-line phase separation
in a knotted reactor followed by ETAAS determination in drinking waters
Solid phase extraction of Co ions using L-tyrosine immobilized on multiwall
carbon nanotubes
Miniaturized preconcentration methods based on liquid–liquid extraction and
their application in inorganic ultratrace analysis and speciation: A review
Silica gel–polyethylene glycol as a new adsorbent for solid phase extraction of
cobalt and nickel and determination by flame atomic absorption spectrometry
Solid phase extraction preconcentration of cobalt and nickel with 5,7-
dichloroquinone-8-ol embedded styrene–ethylene glycol dimethacrylate
polymer particles and determination by flame atomic absorption spectrometry
(FAAS)
Simultaneous preconcentration of cobalt, nickel and copper in water samples by
cloud point extraction method and their determination by flame atomic
absorption spectrometry
Simultaneous determination of nickel, cobalt and mercury ions in water samples
by solid phase extraction using multiwalled carbon nanotubes as adsorbent
after chelating with sodium diethyldithiocarbamate prior to high performance
liquid chromatography
Blitz-Raith, A. H., Paimin, R., Cattrall, R. W. and Kolev, S. D. (2007).
Talanta 71, 419–423
Citak, D., Tuzen, M. and Soylak, M. (2009). Food and Chemical
Toxicology 47, 2302–2307
Donati, G. L., Nascentes, C. C., Nogueira, A. R., Arruda, M. A. and
Nóbrega, J. S. (2006). Microchemical Journal 82, 189–195
Gil, R. A., Gásquez, J. A., Olsina, R., Martı́nez, L. D. and Cerutti, S.
(2008). Talanta 76, 669–673
Pacheco, P. H., Smichowski, P., Polla, G. and Martı́nez, L. D.
(2009). Talanta 79, 249–253
Pena-Pereira, F., Lavilla, I. and Bendicho, C. (2009). Spectrochimica
Acta Part B 64, 1–15
Pourreza, N., Zolgharnein, J., Kiasat, A. R. and Daystar, T. (2010).
Talanta 81, 773–777
Praveen, R. S., Daniel, S. and Prasada Rao, T. (2005). Talanta 66,
513–520
Xu, H., Zhang, W., Zhang, X., Wang, J. and Wang, J. (2013).
Procedia Environmental Sciences 18, 258–263
Zhou, Q. and Kuifu Zhao, A. X. (2014). Journal of Chromatography
A 1360, 76–81
168 Cobalt: Properties and Determination
precipitation. On the other hand, a lower flow rate of sample
produces a longer contact time with the solid phase, thus
increasing the percentage recovery. Finally, low sample vol-
umes result in a higher percentage recovery because the con-
tact time between the metal ions and the active sites of the
solid phase increases.
LLE is a preconcentration method in which the metal is
distributed between two immiscible liquid phases (usually an
aqueous phase and organic phase). Cobalt preconcentration is
achieved by adding a chelating agent to the aqueous phase,
followed by an extraction of the cobalt complexed into the
organic phase. For the analytic measurement of the extracted
cobalt ion, a back-extraction step to an aqueous medium,
usually acid, is recommended. Nevertheless, LLE has several
drawbacks, such as emulsion formation and the use of large
and contaminated solvent volumes, which make LLE expensive
and environmentally unfriendly. Searching for alternatives to the
conventional LLE using negligible volumes of extractant and a
minimum number of steps has led to the development of liquid-
phase microextraction techniques for cobalt preconcentration.
CPE is related to LLE but has emerged as an alternative due
to several reasons: (1) its excellent concentration factors, (2)
the smaller required sample size, (3) the use of a small volume
of organic solvent that is generally toxic, (4) the use of non-
toxic surfactants, (5) the reduction of laboratory residues, and
(6) being recognized as a safer, simpler, and cost-effective
procedure. CPE is based on the following phenomenon: in
much diluted solutions of nonionic surfactant, the monomers
are dispersed in the solvent. But above the critical micellar
concentration of the surfactant, these monomers associate
spontaneously, forming aggregates of colloidal dimensions
termed micelles, due to the diminished solubility of the sur-
factant in water. The surfactant solution becomes turbid
because it attains the cloud point. This clouding phenomenon
can be induced by changing the temperature, surfactant con-
centration, or the choice of a compatible pH buffer, resulting in
the separation of a single isotropic micellar phase into two
phases, a surfactant-rich phase of small volume and an aque-
ous phase. Choosing an appropriate chelating agent that is able
to form with the metal ion a hydrophobic complex, which is
entrapped by micellar structures, extraction and subsequent
preconcentration of cobalt in the surfactant-rich phase can be
achieved. Among the nonionic surfactants commonly used,
Triton X-114 (octylphenoxypolyethoxyethanol) is the most
feasible for preconcentration cloud point extraction. Regarding
chelating agents, 2-(5-bromo-2-pyridylazo)-5-(diethylamino)-
phenol (5-Br-PADAP), 1-(2-pyridylazo)-2-naphthol (PAN),
ammonium pyrrolidinedithiocarbamate (APDC), and 1-(2-
thiazolylazo)-2-naphthol (TAN) have been used for the deter-
mination of cobalt.
The separation of dissolved substances by precipitation may
occur in two ways. Direct precipitation is produced when a
reagent creates an insoluble and very selective precipitate.
Alternatively, indirect precipitation is formed when a precipi-
tate of a different compound is produced and the desired
analyte is retained by coprecipitation on the surface of the
formed precipitate. Further, centrifuging, filtering, or washing
separates this precipitate. The coprecipitation technique stands
out by its ease and the simultaneous preconcentration and
separation of the analytes from the food matrix. Hydroxides
of various metal ions as inorganic coprecipitants have been
used for the preconcentration of cobalt in food and environ-
mental samples such as magnesium(II), thulium(III),
zirconium(IV), tin(IV), and scandium(IV) hydroxide. Other
organic coprecipitants such as magnesium-8-quinolinate,
iron(III) hexamethylenedithiocarbamate, and bismuth(III)
diethyldithiocarbamate have been also employed in cobalt
coprecipitation studies, giving very high concentration factors.
Finally, another preconcentration technique is membrane
filtration in which cobalt ions are collected in the form of metal
chelates over a membrane filter from an aqueous solution by
filtration. The filter material has a strong affinity for hydropho-
bic species in water, which allow retention of these species
during filtering. Among the materials used as a filter to cobalt
ions are cellulose nitrate or cellulose acetate. APDC, poly(ethy-
leneimine) or carmine has been used as complexing agents.
Atomic Absorption Spectroscopy
In atomic absorption spectroscopy (AAS), electromagnetic
radiation at specific wavelength is passed through a cell con-
taining gaseous free atoms. Atoms are produced in two differ-
ent ways using AAS: flame atomic absorption spectrometry
(FAAS) and electrothermal atomic absorption spectrometry
(ETAAS) (graphite furnace and a L’vov platform). Thus, the
determination of cobalt using an atomic absorption spectro-
photometer requires a light source (wavelength l ¼ 240.7 nm)
and an atomization source. Standard working conditions for
analyzing cobalt by AAS are listed in Table 2.
FAAS uses an air–acetylene or nitrous oxide–acetylene as
the most common oxidant–fuel mixtures to produce atomiza-
tion. Preconcentration methods described in the preceding text
permit the use of FAAS for cobalt determination in foodstuffs
with detection limits of 0.30–2.4 ng ml1. However, when
conventional FAAS proves impractical because cobalt values
are below or near the detection limit of the instrument, it is
recommended to use ETAAS in which the atomization is pro-
duced by the passage of electrical current through a cylindrical
graphite tube (called graphite furnace). This process comprises
several steps: preheating, drying, pyrolysis, atomization, and
cleaning, which are achieved by a temperature–time interval
program (ramp heating). During the drying stage, liquid from
the sample is evaporated and driven off. In the pyrolysis stage,
some of the concomitants in the sample are removed. During
this process, the system is flushed with protective gas (usually
argon) to exclude air from the sample compartment and
to minimize oxidation of the atomizer material at high
Table 2 Standard conditions for cobalt analysis by AAS
Spectrometer settings Cobalt
Wavelength (nm) 240.7
Lamp current (mA) 5–7
Spectral resolution (nm) 0.1–0.2
Nebulizer Spoiler
Oxidant Air
Fuel C2H2
Flame condition Oxidizing
Deuterium lamp background correction

Table 3 Experimental conditions proposed for some authors for cobalt determination by ETAAS
Step Ramp(s) Hold(s)
Drying 1 1(10) 5 110(200)
Drying 2 5 15
Pyrolysis 10 30
Atomization 0 5
Cleaning 1 3
temperatures. Finally, the temperature is rapidly increased to
quickly vaporize and atomize the sample. Table 3 shows oper-
ation conditions used for cobalt determination by ETAAS.
ETAAS combined with preconcentration methods has been
used for cobalt determination. It can reach detection limits of
1.2–50 ng l1. In order to reduce matrix effects when ETAAS is
used, various matrix modifiers are added.
Inductively Coupled Plasma Optical Emission Spectrometry
(ICP-OES)
The earlier analytic techniques are sensitive enough to deter-
mine cobalt and other trace elements. However, as mono-
elemental techniques, they could become too slow and
tedious when trying to analyze a wide range of trace elements.
ICP-OES is a multielemental technique able to determine con-
centrations of multiple elements in a sample with a single
aspiration obtaining a high degree of variability and minimum
interferences. The technique is based upon the spontaneous
emission of light, with a characteristic wavelength, from atoms
and ions previously excited in plasma. The particular wave-
length leaving the monochromator is converted to an electrical
signal by a photodetector.
Plasma may be defined as a luminous volume of partially
ionized gas. It is generated from radiofrequency magnetic fields
induced by a copper coil wound around the top of a glass
torch. The sample solution is converted to an aerosol and
directed into the central channel of the plasma. The plasma
maintains a temperature of approximately 10 000
C in its core
where cobalt and other elements are released as free atoms and
excited. Therefore, argon plasma is needed to produce atomi-
zation and excitation. The nearly complete atomization of the
sample minimizes chemical interferences.
For cobalt determined by ICP-OES, a solution detection
limit of 5 mg l1 and a sample detection limit of 2.5 mg g1
can be achieved. Selectivity is important to minimize the spec-
tral overlap interferences resulting from rich line emission
spectra of cobalt and other elements such as tungsten,
niobium, and molybdenum. ICP-OES has been used for cobalt
determination in dried fruit, coffee, cocoa, and infant formula.
The emission spectral lines used for cobalt analysis are
228.616, 230.786, and 238.346 nm.
Inductively Coupled Plasma Mass Spectrometry (ICP-MS):
Cobalt Speciation
ICP can also be coupled with a mass spectrometer leading to
inductively coupled plasma mass spectrometry (ICP-MS). The
Cobalt: Properties and Determination 169
Furnace temperature (
C) Internal gas flow (l min1)
1.0
130(400) 1.0
700(800) 1.0
2200(2400) 0
2450(2700) 1.0
inductively coupled argon plasma is once again used as an
excitation source for the elements of interest. However, in
contrast to ICP-OES, the plasma in ICP-MS is used to generate
ions that are introduced in the mass analyzer through a sam-
pler cone, followed by a skimmer cone. Mass spectrometers are
instruments that separate ions according to their mass-to-
charge ratio (m/z) and then accurately quantify the resulting
ions once they pass through a detector.
There are five types of mass analyzers: quadrupoles, ion
traps, time-of-flight, magnetic sectors, and Fourier transform-
based ion cyclotrons. Quadrupole mass spectrometry has
become increasingly popular in recent years, being currently
the most used. Sample introduction can be static, through
direct insertion probes, or dynamic. The interconnection with
chromatographic equipment is implied in the latter. In fact, as
it will be discussed later, the use of high-performance liquid
chromatography with the mass spectrometry has recently
become a routine technique due to advances achieved in the
interconnecting interfaces.
ICP-MS is the method of choice for cobalt and other ultra-
trace element determinations due to its low detection limit,
multielement capabilities, fast routine, and wide linear calibra-
tion range. For cobalt, it has been widely applied in clinical
analysis, reaching quantification limits as low as 0.20 ng ml1
in oral mucosa cells, 0.01 ng ml1 in human serum, and
0.03 ng ml1 in whole blood.
Regarding interferences, spectroscopic interferences are
probably the largest ones in ICP-MS and are caused by atomic
or molecular ions that have the same m/z ratio as the analytes
of interest. In many cases, it can be avoided by using a different
isotope of the element of interest or by measuring another
isotope of the interfering element and subtracting from
the mass of interest on the basis of the known isotope ratio
of the interfering element. Current ICP-MS instrumental soft-
ware makes a correction for all known atomic ‘isobaric
interferences’; however, they do not provide corrections for
most of the polyatomic interferences. Such interferences are
caused by polyatomic ions that are formed from precursors
having numerous sources, such as the sample matrix, reagents
used for preparation, plasma gases, and entrained atmospheric
gases. Table 4 shows the main polyatomic interferences for
cobalt determination.
Currently, it is necessary to know not only the precise
quantity of trace element that is present in a food or biological
sample but also in what form it is present (speciation). Chem-
ical speciation comprises the identification and quantification
of different trace elements and the biomolecules to which they
are associated, their oxidation states, and the coordination
170 Cobalt: Properties and Determination
groups involved. In the last years, a novel scientific discipline
was arisen, known as metallomics or chemical speciation. To
this sense, cobalt can be present in the environment in differ-
ent forms. The main compounds of toxicological interest are
the metallic form (cobalt metal), the oxides (cobalt oxide and
tetroxide), and the salts (cobalt chloride, sulfide, and sulfate).
On the other hand, there are other forms of nutritional interest
such as a wide variety of cobalamins including cyanocobala-
min (CNCbl), hydroxocobalamin (OHCbl), methylcobalamin
(MeCbl), and 50 -deoxyadenosylcobalamin (AdoCbl).
To carry out these studies, several methods of high-
performance liquid chromatography (HPLC) and capillary
electrophoresis (CE) coupled with ICP-MS have been reported
for cobalt speciation analysis. The separation of cobalt species
is based on their retention times. The advantages of using
CE as a separation method, in comparison with other chro-
matographic techniques, are its low cost, applicability to small
sample volumes, and high resolving power and low reagent
consumption. It has been applied on pharmaceutical prepara-
tions of vitamin B12 achieving detection limits for the cobala-
mins ranging from 30 to 70 ng ml1. Another study using
EC-ICP-MS on nutritive supplements and chlorella foods
achieved lower detection limits of 0.3, 0.2, and 1.7 ng ml1
for CNCbl, OHCbl, and Co(II) ion, respectively.
In relation to HPLC-IPC-MS, the physical separation capa-
bilities of liquid chromatography with the analytic skill of mass
spectrometry are combined yielding high sensitivity and selec-
tivity. It has been used for the analysis of nutritive supplements
with detection limits of 0.008, 0.013, and 0.014 ng ml1 for
Table 4 Polyatomic interferences for the determination of cobalt in
ICP-MS
Isotope Abundance Interference
59 43
Co 100 Ca16Oþ, 42Ca16O1Hþ, 24Mg35Clþ, 40Ar23Naþ,
40 18 1 þ 40 19 þ
Ar O H , Ar F
Table 5 Cobalt speciation and analysis by ICP-MS
Investigation
Determination of cobalamins using capillary electrophoresis (CE) inductively
coupled plasma mass spectrometry
Simple and robust ICP-MS method for simultaneous determination of serum
Co and Cr in routine clinical practice
Determination of cobalamin in nutritive supplements and chlorella foods by
capillary electrophoresis-inductively coupled plasma mass spectrometry
Development and validation of an inductively coupled plasma mass
spectrometry (ICP-MS) method for the determination of cobalt,
chromium, copper and nickel in oral mucosa cells
Simultaneous analysis of 21 elements in foodstuffs by ICP-MS after closed-
vessel microwave digestion: method validation
An ORS-ICP-MS method for monitoring trace levels of cobalt and chromium
in whole blood samples from hip arthroplasty patients with metal-on-metal
prostheses
Cobalamin speciation using reversed-phase micro-high-performance liquid
chromatography interfaced to inductively coupled plasma mass
spectrometry
Combined use of HPLC-ICP-MS and microwave-assisted extraction for the
determination of cobalt compounds in nutritive supplements
Co(II), OHCbl, and CNCbl, respectively. The following table
(Table 5) shows some published studies on cobalt speciation
using these hyphenated techniques and on cobalt analysis by
ICP-MS.
Ion-Selective Electrodes
Ion-selective electrodes are one of the most frequently used
potentiometric sensors during laboratory analysis. The main
advantage over spectroscopic methods is that most of these
spectroscopic techniques are relatively expensive and involve
large infrastructure backup and support of expertise. In contrast,
ion sensors provide analytic procedures that overcome the
earlier drawbacks since they are fast and require minimal sample
treatment. Ionophore plays the main role in the sensitivity and
selectivity of ion-selective electrodes. A good ionophore shows
strong affinity for a particular ionic species than others.
A number of ion-selective electrodes for the determination
of cobalt concentration have been reported using materials
such as Schiff bases, calixarenes, isothiazoles, and mercapto
compounds. However, most of these sensors suffer from
disadvantages such as narrow working concentration range,
non-Nernstian potential response, high response time, high
detection limit, and poor reproducibility.
Recently, PVC membrane electrode (PME) and coated
graphite electrode (CGE) have been prepared by using 2-
((thiazol-2-ylimino)methyl)phenol as a good ionophore in
order to use it as cobalt-selective electrode. The electrodes
exhibit a Nernstian slope for cobalt ions with a limit detection
of 6.91  107 mol l1 for PME and 7.94  108 mol l1 for
CGE, which show a proper potentiometric response. Similarly,
azines, a condensation product of hydrazine with ketones or
aldehydes, can also be used as cobalt-selective electrodes. Thus,
palladium(II) dichloro acetylthiophene fenchone azine in a
plasticized PVC matrix reveals a good selectivity for cobalt(II)
with a detection limit of 8  107 mol l1. Finally, the
Reference
Baker, S. A. and Miller-Ihli, N. J. (2009). Spectrochimica Acta Part B 55,
1823–1832
Choi, H. J., Lim, S. J., Park, Y. S. and Lee, S. Y. (2015). Clinica Chimica
Acta 439, 91–96
Chen, J. H. and Jiang, S. J. (2008). Journal of Agricultural and Food
Chemistry 56, 1210–1215
Martı́n-Cameán, A., Jos, A., Calleja, A., Gil, F., Iglesias-Linares, A.,
Solano, E. and Cameán, A. M. (2014). Microchemical Journal 114,
73–79
Millour, S., Noël, L., Kadar, A., Chekri, R., Vastel, C. and Guérin, T.
(2011). Journal of Food Composition and Analysis 24, 111–120
Pei, K. L., Kinniburgh, D. W., Butlin, L., Faris, P., Lee, D., et al. (2012).
Clinical Biochemistry 45, 806–810
Yanes, E. G. and Miller-Ihli, N. (2004). Spectrochimica Acta Part B 59,
891–899
Yang, F. Y., Jiang, S. J. and Sahayam, A. C. (2014). Food Chemistry
147, 215–219

use of macrocyclic ligands (such as dipyridine–
hexaazacyclotetradecane derivatives) with PME and CGE has
also been developed with similar results (detection limits of
6.80  109 mol l1).
Colorimetric Methods
These types of techniques are less used for cobalt determina-
tion; however, they offer some advantages such as less tedious
sample pretreatments and more inexpensive instrumentation.
Colorimetric methods were introduced during the 1940s for
analyzing cobalt in biological materials and achieved detection
limits of 0.005 mg ml1 in blood samples. Recently, a simple
naked eye colorimetric method has been developed based on
controlling the oxidation level of methylene blue. This redox
dye could be tuned from blue to colorless, yellow, and green at
different oxidation states. The sensing system is just a simple
mixture of methylene blue, 2-aminothiophenol, and copper
nitrate, forming a complex that resulted in the reduction of
methylene blue from blue to colorless in 3 min. The addition
of cobalt ions to this system produces a color change from
colorless to brown at low cobalt concentrations and green at
high concentrations within 2 min. The sensing of cobalt ions
can be achieved by UV–visible spectra or by the naked eye. The
authors reported a linear range from 0.1 to 1.1 mM with a
correlation coefficient of 0.9982 and a detection limit of
0.04 mM.
Nondestructive Techniques
Neutron activation analysis is not a common technique for
cobalt analysis. However, it can be used as a multielement
technique when determining a large number of trace elements
without destroying the sample. Furthermore, it is a well-
established technique for multielement determination at ultra-
trace levels with high sensitivities (i.e., a detection limit of
0.6 ng g1 was achieved for cobalt in liquid milk samples).
The technique principle is that elements can be radioactive by
exposure to neutron irradiation. By monitoring the subsequent
decay of these radioisotopes, it is possible to identify and
accurately quantify the elements in the sample.
There are three main sources of neutrons for irradiation:
nuclear reactors, radioactive neutron sources, and electron–ion
accelerators. The former provide the highest neutron fluxes and
permit the highest sensitivities for detection and quantification
of various elements. Currently, this technique is being used to
Cobalt: Properties and Determination 171
authenticate the geographic origin of certain foods with a
quality mark (e.g., foodstuffs belonging to a protected
designation of origin).
x-Ray fluorescence (XRF) is another unusual nondestructive
technique for cobalt determination. It is based on bombarding
a small area of the sample with x-ray photons, which are
sufficiently energetic to cause ionization of inner electrons of
the atoms. Because of this unstable atomic configuration, an
electron from an outer orbital fills the vacancy of an inner
electron, which emits the difference of energy between the
two orbitals as a ‘secondary’ (or fluorescent) x-ray photon.
The main difference with ICP-OES is that in XRF, emitted
radiation from the ultraviolet–visible region of the spectrum
is covered. It has achieved a detection limit for cobalt of
1 mg g1 in fruits and of 0.3 mg g1 in dairy samples.
See also: Authenticity of Food; Cobalt: Toxicology; Infrared
Spectroscopy: Applications; Mass Spectrometry: Applications; Mass
Spectrometry: Principles and Instrumentation; Potassium: Properties
and Determination; Sodium: Properties and Determination;
Spectroscopy: Types; Zinc: Properties and Determination.
Further Reading
Broekaert JAC (2005) Analytical atomic spectrometry with flames and plasmas, 2nd ed.
Weinheim: Wiley.
Cornelis R, Caruso J, Crews H, and Heumann K (2005a) Handbook of element
speciation – techniques and methodology. Chichester, UK: Wiley.
Cornelis R, Caruso J, Crews H, and Heumann K (2005b) Handbook of element
speciation II – species in the environment, food, medicine and occupational health.
Chichester, UK: Wiley.
Davis JR and Davis & Associates (2000) ASM specialty handbook – nickel, cobalt and
their alloys. USA: ASM International.
Mester Z and Sturgeoin R (2003) Sample preparation for trace element analysis, 1st ed.
Amsterdam, The Netherlands: Elsevier.
Worsfold P, Townshend A, and Poole C (2005) Encyclopedia of analytical science, 2nd
ed. Amsterdam, The Netherlands: Elsevier.
Relevant Websites
http://www.thecdi.com/cdi/images/documents/facts/COBALT_FACTS-Metallurgical_%
20uses.pdf – Cobalt in Metallurgical Uses.
http://www.elementalanalysis.com/services/inductively-coupled-plasma-icp/ –
Elemental Analysis, Inc – Inductively Coupled Plasma (ICP).
http://www.s-ea.es/ – Sociedad de Espectroscopı́a Aplicada.
http://www.epa.gov/ttnatw01/hlthef/cobalt.html – United States Environmental
Protection Agency – Cobalt Compounds.
 Cobalt: Toxicology
F Cámara-Martos and R Moreno-Rojas, Universidad de Córdoba, Córdoba, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Cobalt Physiology
Cobalt is a trace element widespread in the environment and
human exposure is mainly attributed to air breathing and food
intake. Skin contact with soil or water containing cobalt may
also enhance exposure. This element has both beneficial and
harmful effects on human health. From a nutritional point of
view, cobalt is part of vitamin B12 in which this element is
complexed with four nucleic pyrroles joined in a ring called
corrin, similar to porphyrins. However, this element may be
toxic when human exposure occurs at levels higher than
recommended values. The respiratory system is the main target
organ as a result of inhalation of high doses of cobalt by
metallurgy workers, causing several diseases such as pneumo-
nia, nodular fibrosis, asthma, and lung cancer. It may also
cause thyroid damage, high blood pressure, heart effects,
vomiting, and diarrhea.
The human body contains an average of 1.1 mg of cobalt,
85% of it being in the form of vitamin B12. Usually, the main
source of cobalt in humans is through diet. Its absorption in
the digestive tract varies between 5% and 45% in different
individuals. This absorption is conditioned by both dietary
factors and physiological factors. Cobalt bioavailability is
determined by the cobalt intake from diet as well as on its
chemical form. Several studies show that cobalt species deter-
mine the kinetics of cobalt absorption both through diet and
by inhalation routes. Volunteer studies have found that chlo-
ride form of cobalt had higher absorption than the oxide form.
Moreover, early experiments showed that cobalt urinary excre-
tion is inversely proportional to the dose supply. On the other
hand, the digestive absorption of cobalt compounds is also
influenced by other concomitants present in the food. The
presence of albumin or lactose increases the absorption of
the element. Thus, administration of cobalt ions with cow
milk may increase the gastrointestinal absorption up to 40%.
On the other hand, the bioavailability in vivo of cobalt ions is
impaired in the presence of physiological concentration of
phosphates. Finally, a low iron status promotes cobalt absorp-
tion probably by active competition for the same transport
mechanism in enterocytes. In relation to physiological factors,
gastrointestinal uptake of cobalt differs according to sex, as
shown by a research study, where volunteers were supplied
with several amounts of soluble cobalt compounds showing
significantly higher urinary cobalt levels in women (median,
109.7 nmol mmol
1 creatinine) than in men (median, 38.4 -
nmol mmol
1 creatinine).
Once absorbed, cobalt is mainly present in the organism as
cobalt (II) ions and is distributed throughout the body, the
liver, kidneys, pancreas, and heart being the main organs
where cobalt is accumulated. Most of the cobalt present in
serum is bound to albumin, and the free fraction is estimated
between 5% and 12%. This latter fraction is in equilibrium
with free cobalt present in the interstitial fluid. It has also been
172 Encyclopedia of Food and Health
identified in serum a histidine-rich glycoprotein that binds
cobalt and other elements such as copper, nickel, and zinc.
There is not any standard biochemical marker of cobalt accu-
mulation in the human body with age.
Cobalt in the form of vitamin B12 is an established treat-
ment for pernicious anemia. It is proclaimed to increase eryth-
ropoiesis and physical performance, even in nonanemic
subjects. The molecular mechanism by which cobalt stimulates
erythropoietin production is not fully understood. It seems
that cobalt stabilizes the hypoxia-inducible factors (HIFs),
namely, HIF-a-prolyl hydroxylase and HIF-a-asparaginyl
hydroxylase, which increase the expression of the erythropoie-
tin gene. Eight non-corrin cobalt-containing enzymes have
been identified, which are shown in Table 1.
Cobalt may interact with other ion metals. Thus, in human
red cells, cobalt shares the same transport mechanism with
calcium. The uptake is practically irreversible since cobalt
bound in the cytosol is not itself extruded by the Ca pump.
Additionally, cobalt in vitro can replace iron in the heme group
of hemoglobin. Binding to hemoglobin is initially reversible;
however, a cobalt fraction starts later to bind strongly probably
due to oxidation in situ from cobalt (II) to cobalt (III). In
acute cyanide poisoning, the capacity of cobalt (II) ions to
form stable complexes with cyanide anion may be used to
neutralize this toxicant. On the other hand, cobalt can also
replace zinc in some enzymes such as alkaline phosphatase
and carboxypeptidase.
The main excretion route of cobalt is the urine followed by
feces. Studies of whole-body retention of inorganic cobalt in
human males after intravenous injection gave an excretion rate
after 8 days of 56% in urine and 11% in feces. This cobalt
excretion is lower in hemodialysis patients justifying the
importance of renal clearance. Urinary cobalt is used in
biological monitoring of occupational exposure to cobalt
compounds to discriminate occupationally exposed subjects
from nonexposed ones and to distinguish subgroups of
workers with different degrees of exposure.
Cobalt represents approximately a 4.3% of vitamin B12
weight. Considering that daily requirements of vitamin B12
are approximately 1 mg and that only 50% of vitamin B12 is
absorbed by the gastrointestinal tract, the Recommended Daily
Allowance (RDA) of vitamin B12 is 2.4 mg, which corresponds
to 0.10 mg of cobalt.
Exposure Sources
Toxic effects of cobalt may occur as a result of an exposure to
high concentrations or through chronic exposure for a long
time period at low concentrations (mainly occupational expo-
sure). Among the adverse effects of cobalt toxicity are skin
dermatitis and carcinogenic, mutagenic, cardiovascular, and
respiratory effects. Although the mechanism of cobalt toxicity
http://dx.doi.org/10.1016/B978-0-12-384947-2.00176-8

Table 1 Non-corrin cobalt-containing enzymes and its function in the
organisms
Enzyme Function
Aldehyde decarbonylase It is involved in the conversion of a fatty
aldehyde to a hydrocarbon. The only case
where the enzyme has been purified is
from a green alga and this decarbonylase
appeared to have cobalt and porphyrin
Bromoperoxidase Bromoperoxidase from Pseudomonas
putida catalyzes the formation of a
carbon–bromine bond in the presence of
peroxides. It is activated by incubation
with cobalt ions but not with other
transition metals such as iron, nickel, zinc,
and vanadate
Glucose isomerase It catalyzes the reversible isomerization of D-
glucose to D-fructose and is of outmost
commercial importance for the industrial
production of corn syrup
Lysine 2,3- A bacterial enzyme that also contains cobalt
aminomutase ions that catalyzes the interconversion of
L-lysine and L-beta lysine, the first step in
lysine degradation
Methionine It is a ubiquitous enzyme in both
aminopeptidase prokaryotes and eukaryotes that catalyzes
cotranslational removal of N-terminal
methionine from elongating polypeptide
chains during protein synthesis. It
contains two cobalt (II) ions per active
subunit that catalyze hydrolysis
Methylmalonyl-CoA It is a multienzyme complex from prokaryote
carboxyltransferase that couples two carboxylation reactions,
transferring a carboxyl group from (S)-
methylmalonyl-CoA to pyruvate to yield
propionyl-CoA and oxaloacetate
Nitrile hydratase It catalyzes the hydration of nitriles to their
corresponding amides. This enzyme is
also used as biocatalyst in acrylamide and
nicotinamide production, as well as in
environmental remediation for the removal
of nitriles from waste streams
Proline dipeptidase Also called prolidase. It is an enzyme that
catalyzed the cleavage of bonds between
aminoacyl and proline groups
remains unknown, it seems that cobalt ions in the presence of
hydrogen peroxide are able to produce hydroxyl radicals in a
manner possibly similar to Fenton reaction, causing oxidative
stress and oxidative damage to lipids, proteins, and DNA. This
would be favored by hyperoxic conditions with oxygen acting
synergically with cobalt to promote the formation of free rad-
icals. Cobalt also has a strong affinity for sulfhydryl groups
causing displacement of divalent cations in the ion center of
metal-activated enzymes and inhibition of vital enzymes such
as cytochrome P450, peroxidase, and tyrosine iodinase. This
effect has also been demonstrated for DNA repair enzymes
(incision and polymerization steps) in which cobalt ions inter-
act with zinc finger domains in the protein.
When the toxicity of cobalt compounds is investigated, it is
important to consider various factors. Speciation or chemical
form of the cobalt compound influences its solubility and
Cobalt: Toxicology 173
Table 2 Median lethal dose (LD50) of several cobalt compounds
in rats
Compound LD50 (mg kg
1)
Cobalt chloride 80
Cobalt oxide 202
Cobalt chloride hexahydrate 766
Cobalt sulfide >5000
Cobalt metal 6171
bioavailability. As a result, acute toxicity of these compounds
varies from each other (see Table 2). The route and the time of
exposure should also be considered for evaluating the risk
(single, repeated, short- and long-term exposure, oral, inhala-
tion, etc.). The organ or tissue whose damage gives rise to the
critical effect, called critical organ, must be selected. Aiming for a
quantitative assessment of risks related to toxicant exposure, risk
assessment is applied to environmental and occupational health.
Soil, Water, and Air
Cobalt is present in nature in a fairly widespread but dispersed
form, being detectable in trace quantities in many rocks, soils,
and manganese-rich marine nodules. Currently, the exploited
sources of cobalt are mainly those where it exists as a by-
product of more valuable metals, particularly copper, nickel,
zinc, lead, and platinum. Cobalt is also present in igneous
rocks that are rich in iron. The abundance of cobalt in the
Earth’s crust is 0.003%. In relation to sediments, cobalt con-
centration was determined in modern sediments in the Bristol
Channel (England), a region that receives a significant input of
industrial waste from coal mining, smelting, and other indus-
tries. A cobalt concentration of 22.8 mg kg
1 in suspended
particulates, 16.0 mg kg
1 in silts, and 6.4 mg kg
1 in sands
was found. Similarly, sediments from the Ems (Germany)
contained 40 mg kg
1 of cobalt and there was a significant
contribution from industrial discharges. Cobalt concentration
in soils is determined by several factors such as the amount of
organic matter, clay mineral content, oxidation–reduction
state, pH, and translocation to root and foliage plants. Usually,
cobalt concentrations in most soils ranged between 0.1 and
50 mg kg
1.
In seawater, the metal is present primarily as cobalt (II) ion
and its chloride, sulfate and carbonate complexes. Cobalt
values around 0.1–0.7 mg kg
1 are reported. An increase in
cobalt concentrations with depth in the oceans is related
to organic productivity. The low concentration of cobalt close
to the continents probably indicates removal through absorp-
tion, coprecipitation, and flocculation of solids. Thus, in shal-
low waters, up to 98% of the metal can be found in the
sediments and in suspended particle matter. Several studies
have found an average cobalt concentration in drinking water
over 2 mg l
1.
Cobalt concentration in the atmosphere depends on anthro-
pogenic activity and other natural sources that include
weathering and local geology, volcanic eruptions, and seawater
spray. The average concentration of cobalt in ambient air is
situated around 0.4 ng m
3. However, levels up to 0.61 mg m
3
174 Cobalt: Toxicology
have been detected in urban areas. The residence time of cobalt
in the atmosphere depends on the particle size and meteorolog-
ical factors. Rainwater solubilizes the cobalt species present in
the atmosphere. Studies have detected a cobalt concentration
in rainwater over 0.3 mg l
1 in rural areas against 1.7 mg l
1 in
highly industrial areas.
Dietary Sources
The average daily intake of cobalt from food is estimated to be
between 5 and 60 mg day
1 varying between countries. Several
studies have reported a mean dietary intake of cobalt of
4–29 mg day
1 in France, 11 mg day
1 in Canada and the
United Kingdom, and 60–65 mg day
1 in Turkey. Cobalt-rich
food sources include meat muscles, liver, fish, nuts, oats, and
green leafy vegetables such as broccoli and spinach. Several
studies have analyzed this element using ICP-OES or ICP-MS
obtaining an average concentration of 0.25–1.03 mg g
1 in
dried fruits, 0.06–0.18 mg g
1 in crucifers, 0.02–0.06 mg g
1
in cereals, and 0.17 mg g
1 in fish liver. As it will be seen
later, myocardial effects have been observed in heavy beer
consumers. This is because in the early 1960s, some breweries
added cobalt salts (chloride or sulfate) to stabilize beer foam,
resulting in elevated exposures to cobalt. Some people who
consumed excessive amounts of beer (8–25 pints per day)
suffered from severe heart problems, causing death.
Cobalt content in human milk depends on feeding mothers
and their lifestyles (dietary habits during pregnancy and
after the birth, taking medications, smoking, etc.). Cobalt
concentration in human milk from Central European women
has been found between 0.42 and 2.46 mg l
1. A transfer
factor from the mothers’ daily intake to milk was reported to
be equal to 8.4.
Anthropogenic Sources
Significant exposure to cobalt occurs predominantly at work
since environmental concentrations are usually low. Cobalt
compounds are used as a coloring agent (to enrich blue
color) for glass, enamels, and porcelains. It is also employed
to make heat-resistant superalloys. Some alloys of cobalt are
used in jet turbines and gas turbine generators, where high-
temperature strength is important. Cobalt compounds are also
important for nonmetallurgical applications, such as catalysts
for petroleum and chemical industries, as a drying agent, or as
part of lithium ion battery electrodes. Cobalt salts of the higher
carboxylic acids (cobalt soaps) are used to accelerate drying
in oil-based paints, varnishes, and inks. As a ferromagnetic
material, it forms permanent magnets, thus being used as an
electromagnet.
Cobalt levels present in the environment are reported to be
relatively high during cobalt powder production in grinding
processes, hard-metal industry, and ceramics industry. These
high levels of cobalt in the environment result in an increased
concentration in blood and urine of the exposed workers.
Cobalt exposure has also been detected, although to a lower
extent, among diamond polishers and dental technicians.
Spain has established a workplace exposure limit of
0.02 mg m
3 for cobalt.
58
Co and 60Co are radioactive isotopes produced from
routine operations of nuclear power plants. Although the
half-life of these isotopes is relatively short (t½ ¼ 71 days and
t½ ¼ 5.27 years), small amounts can be released into the envi-
ronment as cooling water pollutants or radioactive waste. Fur-
thermore, small amounts of cobalt could be released to the
atmosphere from incinerator plants that use carbon as fuel or
from the vehicle exhaust tubes.
As already mentioned, contaminated soils by highway and
airport traffic or other types of industrial pollution could con-
tain high cobalt concentrations. The mobility of cobalt present
in the soil depends on the acidity of the medium. Thus, cobalt
may be transferred from the plants’ edible parts, mainly fruits,
grains, and seeds, to the food industries. Although animals
eating these plants will accumulate this metal, this fact does
not seem to increase cobalt transmission and concentration
along the food chain.
Finally, over the last years, the use of cobalt alloys as bio-
material in orthopedic joint replacement has created a new
source of cobalt exposure. In dental usage, the term ‘steel’
became a synonym of cobalt–chromium alloys. On the other
hand, the use of metal bearings in hip joint arthroplasty may
cause a serious concern because of the wide applicability in
susceptible patients.
Allergic Effects
The existence of allergenic reactions to cobalt is well known
causing an erythematous and papular dermatitis. Cobalt ions
can act as hapten and bind to macromolecular components to
produce immunogenic products that can account for allergic
reactions. Cobalt allergy occurs as a result of exposure to sub-
stances containing this element such as detergents, hair dyes,
creams, and fertilizers. Also, the body adornments (jewels)
may contain cobalt. A sensitivity of 7.6% has been reported
by the North American Contact Dermatitis Group in individ-
uals patch-tested from 1998 to 2000. Similarly, a study carried
out in Denmark for 10 years (2003–12) has documented a
positive association between cobalt allergy and dermatitis
caused by nonoccupational exposure to leather goods.
Photosensibilization of cobalt in cement has been
described in bricklayers, itself being manifested by severe and
diffuse eczematous dermatitis in the body and limbs. A cross-
sectional study developed in the Swedish hard-metal industry
showed a prevalence of irritant reaction and hand eczema of
15% and 10%, respectively. Usually, skin cobalt allergy is
related to other allergic reactions caused by other metals such
as chromium and nickel (cross-reaction).
Dental technology students have an increased risk of develop-
ing contact dermatitis during their studies, owing to the use for
training purposes of alloys that release high amounts of cobalt
(0.0047–820 mg cm
2 week
1). Dental technicians and students
mostly have short and repeated contact with tools and
alloys containing this element, which may result in the deposi-
tion of large amounts of metal on their skin. A severe, generalized
pruritic eczematous rash in an 84-year-old woman with
cobalt–chromium prosthetic hip for a fractured femur neck has
also been diagnosed in Australia. The replacement of the cobalt–
chromium hip prosthesis with a titanium-on-polyethylene one

Table 3 Relevant researches on allergic and carcinogenic effects and genotoxicity of cobalt exposure
Investigation
Association between cobalt allergy and dermatitis caused by leather articles –
a questionnaire study
Cobalt, nickel, and chromium release from dental tools and alloys
Systemic allergic dermatitis caused by cobalt and cobalt toxicity from a
metal-on-metal hip replacement
Cobalt asthma – a case series from a cobalt plant
Lung cancer risk in hard-metal workers
Incidence of lung cancer among cobalt-exposed women
Lung cancer mortality in a site producing hard metals
Exploring the potential role of tungsten carbide–cobalt (WC–Co) nanoparticle
internalization in observed toxicity toward lung epithelial cells in vitro
The effect of nano- and micron-sized particles of cobalt–chromium alloy on
human fibroblasts in vitro
Absence of significant genotoxicity in lymphocytes and urine from workers
exposed to moderate levels of cobalt-containing dust: a cross-sectional
study
aided to overcome her dermatitis and the serum cobalt levels
decreased from 967 to 232 nmol l
1 after 3 months. Table 3
shows several investigations performed over allergic effects of
cobalt exposure.
Carcinogenic Effects
Studies on carcinogenic effect of cobalt are inconclusive.
Although inhalation of cobalt particles could be related to
the increased risk of lung cancer, this carcinogenic effect is
not so clear when is contacted by other means. In 1990, the
International Agency for Research on Cancer concluded that
the epidemiological evidence was inadequate in classifying
cobalt or cobalt compounds as human carcinogens and that
there was sufficient evidence for the carcinogenicity of
cobalt metal powder and cobalt oxide in experimental
animals.
Sarcomas in rats have been observed at the site of injection
of cobalt salts or cobalt metal powder. However, similar
injection studies have provided little evidence of cobalt-
induced cancer in mice, hamster, and guinea pigs. The
chronic exposure (0–3 mg m
3, 6 h day
1, 5 days week
1
during 104 weeks) to aerosols containing cobalt sulfate has
shown a major incidence of lung tumors in rats and mice of
both sexes. The study has also found a major prevalence of
liver hemangiosarcoma in male mice. However, in this case,
significant correlations could not be established due also to a
higher prevalence of infection by Helicobacter hepaticus. In
relation to the use of cobalt alloys as biomaterial, several
studies have evaluated the effects of implanted Co–Cr alloys
on the development of tumors in animals. Four out of six
studies reported tumors at the implant site, whereas two
studies reported no such increase.
Cobalt: Toxicology 175
Reference
Bregnbak, D., Thyssen, J. P., Zachariae, C., Menné, T. and Johansen, J.
D. (2014). Contact Dermatitis. doi:10.1111/cod.12319
Kettelarij, J. A., Lidén, C., Axén, E. and Julander, A. (2014). Contact
Dermatitis 70, 3–10
Wong, C. C. and Nixon, R. L. (2014). Contact Dermatitis 71, 108–128
Sauni, R., Linna, A., Oksa, P., et al. (2010). Occupational Medicine 60,
301–306
Moulin, J. J., Wild, P., Romazini, S., et al. (1998). American Journal of
Epidemiology 148(3), 241–248
Tiichsen, F., Jensen, M. V., Villadsen, E. and Lynge, E. (1996).
Scandinavian Journal of Work, Environment and Health 22, 444–450
Wild, P., Perdrix, A., Romazini, S., Moulin, J. and Pellet, F. (2000).
Occupational Environmental Medicine 57, 568–573
Armstead, A. L., Arena, C. B. and Li, B. (2014). Toxicology and Applied
Pharmacology 278, 1–8
Papageorgiou, I., Brown, C., Schins, R., et al. (2007). Biomaterials 28,
2946–2958
De Boeck, M., Lardau, S., Buchet, J. P., Kirsch-Volders, M. and Lison,
D. (2000). Environmental and Molecular Mutagenesis 36, 151–160
Two epidemiological studies were developed in France to
observe the incidence of lung cancer among hard-metal
workers. In this case, cobalt exposure is associated with tung-
sten carbide exposure in the hard metal-manufacturing indus-
try. It was found that these workers had an increased mortality
from lung cancer due to the simultaneous exposure to cobalt
and tungsten carbide. Although occupational risk was highest
among smoking workers, this excess mortality could not be
attributed to smoking alone. The toxicity of cobalt–tungsten
carbide has been also studied using lung epithelial cells in vitro.
The toxicity was higher in nanoparticles (80 nm) against
microparticles (4 mm), suggesting that internalization may
play a role in enhancing toxicity. Another research has esti-
mated the risk of cancer, especially lung cancer, among women
workers exposed to cobalt aluminate spinel used in the porce-
lain factories in Denmark. In that study, a woman group was
selected to be exposed to cobalt and further was compared with
a woman reference group from cobalt-free department in a
factory. The results showed a higher risk of lung cancer in
both groups if compared with the whole Danish women pop-
ulation, although the risk was only slightly higher for the
exposed group than for the reference group. Moreover, a higher
risk of cervical cancer was found in the exposed woman group
and of corpus uteri cancer in the reference group. Finally, an
increase in mortality from lung cancer was reported among
cobalt recovery workers in Soviet plants concerned with nickel
extraction. However, there was a simultaneous exposure to
nickel and arsenic that could explain this finding.
The use of Co–Cr alloys as hip implants has also been
studied in humans for long periods (3–7 years). Most of the
studies developed in patients showed that there is no evi-
dence that these metal-on-metal bearing surfaces are associ-
ated with an increased risk of any cancer diagnosis. Table 3
shows some relevant studies over carcinogenic effects of
cobalt exposure.
176 Cobalt: Toxicology
Cobalt Genotoxicity
Some in vitro studies, with human lymphocytes or mammalian
cells, indicate that cobalt (II) ions may affect DNA integrity
either directly through a Fenton-like mechanism or indirectly,
affecting the repair system of DNA damage. Another study,
comparing the effect of fine and nanoparticle alloys of
cobalt–chromium on human fibroblast cells, noted that the
nanoparticles generated free radicals, cell DNA damage, cyto-
toxicity and aneuploidy. This genotoxic effect has also been
shown with cobalt oxide nanoparticles in human hepatocarci-
noma cells through an increase in lipid hydroperoxide and
reactive oxygen species generation.
In vivo intraperitoneal administration of cobalt (II) ions
(50–100 mmol kg
1 as cobalt acetate) in rats produced oxida-
tive DNA damage in renal, hepatic, and pulmonary chromatin.
Similarly, chromosomal aberrations have also been found in
bone marrow cells of mice after a single oral administration of
high doses of cobalt chloride (20–80 mg kg
1). With all the
previously mentioned, there is considerable evidence to con-
sider that all soluble cobalt (II) salts have genotoxic effects in
animals and in vitro models. However, there is no evidence
available in humans. Thus, a human study realized by Belgian
researchers detected no significant increase of genotoxic effects
in workers exposed to cobalt-containing dust, at a mean level
corresponding to TLV-TWA (20 mg m
3), as compared to con-
trols. Although several epidemiological studies show that occu-
pational exposure to hard-metal particles is associated with an
increased risk of lung cancer and despite the increase of reactive
oxygen species tested with in vitro models, there is no evidence
of a genotoxic effect by cobalt compounds in humans. There-
fore, further investigation may be developed in this sense in the
future.
Respiratory Effects
Several compounds are thought to cause lung damage through
the generation of oxygen-derived free radicals and related reac-
tive compounds. The respiratory system is the most affected
organ resulting from cobalt exposure by inhalation. In addi-
tion to the possible increase of the incidence of lung cancer,
different types of toxic reactions related with respiratory system
have been reported, including the upper respiratory tract, the
bronchial tree, and the alveolar parenchyma.
Upper Respiratory Tract
Animal studies have shown degeneration of the olfactory epi-
thelium, squamous metaplasia of the respiratory epithelium,
and nonneoplastic lesions of the nose and larynx of rats and
mice exposed to aerosols containing cobalt sulfate hexahy-
drate. Furthermore, studies performed on humans showed
that inflammation of the nasopharynx seems to be frequent
in hard-metal workers. However, it is not clear whether the
occurrence of these symptoms is produced by particles con-
taining cobalt or from an immunologically mediated reaction
(allergic rhinitis). It has also been observed that subjects work-
ing in lignite mines have a high prevalence of atrophic rhinitis,
which may be related to an excessive pollution caused
by airborne contaminants such as chromium, nickel, cobalt,
and lead.
Bronchial Tree
Cobalt has been identified as an occupational agent, which is
involved in asthma diseases. Exposure to cobalt salts or cobalt
metal released from diamond-polishing activities may cause
typical bronchial asthma in a small proportion of workers
(generally <5%). Similarly, 7% of workers exposed to cobalt
dust at a hard-metal plant of Uppsala (Sweden) reported
asthma. Another study showed that chromium and cobalt
salts (in the form of cobalt chloride) can be responsible for
occupational asthma in workers exposed to higher metalwork-
ing fluid aerosols of an aerospace manufacturer of Birmingham
(the United Kingdom). A higher urinary excretion of chro-
mium and cobalt was observed in the occupational asthma
group than in the asymptomatic control group.
The pathophysiology of cobalt asthma may involve both
immunologic and nonimmunologic mechanisms. Japanese
researchers showed IgE antibodies to cobalt-conjugated
human serum albumin in some of the cobalt asthma patients.
For the remaining patients, the mechanism of cobalt-induced
asthmatic reaction remains unknown. In order to investigate
this mechanism, it could be noted that a positive lymphocyte
transformation test with cobalt (II) ions has also been reported
in hard-metal asthma, suggesting a role for cellular immunity.
On the contrary, a study conducted to analyze all the cases of
cobalt asthma encountered in a cobalt plant of Kokkola (Fin-
land) used skin prick tests for cobalt and common environ-
mental allergens. None of these patients had a positive reaction
against cobalt in skin prick test, indicating a nonimmunologic
mechanism.
Lung Parenchyma
In 1950, severe and fatal pulmonary edema and hemorrhage
were firstly described in rats treated intratracheally with cobalt
metal powder (500 mg per rat). Similarly, intratracheal instilla-
tion of 10–50 mg of cobalt metal produced acute pneumonia
with diffuse cellular infiltration and bronchiolitis obliterans.
Subchronic response assessed 8–12 months after the acute
dose was characterized by the presence of multinucleated
cells and a lack of cellular reaction within the alveolar walls.
Short-term exposure appears to have fewer adverse conse-
quences. Exposure of rats to aqueous suspension of ultrafine
metallic cobalt particles (20 nm) during 4 days at 5 h day
1
induced slight and reversible pulmonary injury including focal
hypertrophy or proliferation of the epithelium in the lower
airways.
Hard-metal pulmonary fibrosis has been known for over 70
years, in subjects exposed to dust produced during mixing,
shaping, and grinding of hard metals. Several studies have
shown a close relationship between cobalt exposure and lung
fibrosis. Thus, parenchymal reaction in some hard-metal
workers exposed to dust containing cobalt has been observed.
Histological changes varied from intense desquamative alveo-
litis with giant multinucleated cells to end-stage nonspecific
pulmonary fibrosis. A weak association of the disease with a

Table 4 Investigations performed on respiratory effects of cobalt exposure
Investigation
Inhalation toxicity and carcinogenicity studies of cobalt sulfate
The effect of environmental pollution on the respiratory system of
lignite miners: a diachronic study
Cobalt-induced bronchial asthma in diamond polishers
Lung function and respiratory symptoms in hard-metal workers
exposed to cobalt
An outbreak of occupational asthma due to chromium and cobalt
Cobalt asthma – a case series from a cobalt plant
Lung function and respiratory symptoms in hard-metal workers
exposed to cobalt
Hard-metal disease: eight workers with interstitial lung fibrosis due
to cobalt exposure
Reversible lung lesions in rats due to short-term exposure to
ultrafine cobalt particles
specific human lymphocyte antigen (HLA) polymorphism
(glutamate 69 in HLA-DP beta chain) has been reported.
According to the medical literature, bronchial asthma is
considered the main etiologic agent inducing hard-metal fibro-
sis. The essential factor to avoid the progression of the disease
is to separate the affected individuals from the work environ-
ment. However, in many cases, the disease remains despite the
cessation of exposure and not always is managed with steroid
treatment. To summarize, Table 4 shows several investigations
performed on respiratory effects of cobalt exposure.
Cobalt and Thyroid Function
Animal studies have confirmed that cobalt is a goitrogen
(a substance that suppresses the function of the thyroid gland
by interfering with iodine uptake). This effect may be related
with the capacity of cobalt ions to inhibit tyrosine iodinase
enzyme. In a cobalt production plant, a subclinical hypothy-
roid status has been observed in some workers. Similarly,
increased concentrations of total and free serum thyroid
hormone have been found in workers exposed to cobalt–zinc
sulfate.
Cardiovascular Diseases
Cobalt is considered responsible for a type of myocardiopathy
with a specific pathogenesis. As already mentioned, the first
cases reported of myocardiopathies associated with the intake
of high doses of cobalt were related to beer drinkers. In the
second half of the 1960s, in some areas of Canada, the United
States, and Belgium, a large number of subjects, all of them
great beer drinkers, showed a particular dilatative myocardio-
pathy associated with other symptoms such as cardiocircula-
tory insufficiency with dyspnea, hypotension, tachycardia,
cyanosis, enlarged heart with reduced cardiac output, and in
many cases a large pericardial effusion. The mortality rate of
Cobalt: Toxicology 177
Reference
Bucher, J. R., Hailey, J. R., Roycroft, J. R., et al. (1999). Toxicological Sciences
49, 56–67
Sichletidis, L., Tsiotsios, I., Chloros, D., et al. (2004). Medicina del Lavoro 95(6),
452–464
Geysens, B., Aurwerx, J., Van den Eeckhout, A. and Demedts, E. (1985). Chest 88
(5), 740–748
Rehfisch, P., Anderson, M., Berg, P., et al. (2012). Journal Occupational
Environmental Medicine 54(4), 409–413
Walters, G. I., Moore, V. C., Robertson, A. S., et al. (2012). Occupational
Medicine 62, 533–540
Sauni, R., Linna, A., Oksa, P., Nordman, H., et al. (2010). Occupational Medicine
60, 301–306
Rehfisch, P., Anderson, M., Berg, P., et al. (2012). Journal of Occupational and
Environmental Medicine 54(4), 409–413
Migliori, M., Mosconi, G., Michetti, G., Belotti, L., D´Adda, F. (1994). Science of
the Total Environment 150, 187–186
Kyono, H., Kusaka, Y., Kubota, H. and Endo-Ichikawa, Y. (1992). Industrial Health
30(2), 103–118
subjects was as high as 50% in some areas. It was observed that
these pathologies were attributed to cobalt added as a foaming
agent (1 ppm on average) in the form of sulfate or chloride.
This meant an estimated daily intake of 6–8 mg of cobalt.
Complications in the diagnosis of cobalt myocardiopathy
were also related to the possibility that alcohol would also be
able to induce such cardiopathy. Thus, cobalt myocardiopathy
could be an advanced state or a particular type of alcoholic
myocardiopathy. According to some authors, a fulminant form
of beriberi due to thiamine deficiency was caused by alcohol
and aggravated by cobalt. Finally, other studies claimed the
direct toxic role of cobalt.
Several cases of myocardial toxicity have been associated
with cobalt industrial exposure. Workers of a cobalt compound
plant showed an association between cumulative cobalt expo-
sure and left ventricular isovolumic relaxation time and decel-
eration time of the velocity of the early rapid filling wave,
indicating altered left ventricular diastole. Another study inves-
tigated cardiac function of a group of Italian hard-metal
workers with or without hard-metal fibrosis. The mean dura-
tion of exposure was 10.4 years, ranging from 1 to 40 mg m
3,
whereas that level of environmental exposure ranged between
0.009 and 13.6 mg m
3 cobalt. An abnormal left ventricular
ejection fraction during resting and at exercise was also found,
which could be explained by the formation of cobalt deposits
in the myocardium after industrial exposure. The subjects
also showed ventricular dysfunction and abnormality of the
right ventricular function, which could be a manifestation of
initial pulmonary artery hypertension or of early occult cor
pulmonale.
Several mechanisms about cobalt toxicity on the cardiovas-
cular system have been proposed:
(1) Impairment of antioxidant enzyme activities and myocar-
dial mitochondrial ATP production rate. An experimental
study showed a lower activity of manganese superoxide
dismutase in rats exposed to a cobalt-supplemented diet
for 24 weeks against control group. Activity in the
178 Cobalt: Toxicology
Table 5 Studies related with cobalt exposure and cardiovascular diseases
Investigation
Quebec beer-drinkers’ cardiomyopathy: 48 cases
Cobalt-beer cardiomyopathy: a clinical and pathological study
of 28 cases
Cardiac function study in hard-metal workers
Chronic cobalt exposure affects antioxidants and ATP
production in rat myocardium
The evolution of b-adrenergic dysfunction during the induction
of canine cobalt cardiomyopathy
Fatal myocardial disease associated with industrial exposure to
cobalt
respiratory chain enzymes, succinate–cytochrome c reduc-
tase, NADH–cytochrome c reductase, and cytochrome c
oxidase, was also reduced in the cobalt rats.
(2) Damage of the electromechanical matching of myocardial
tissues, probably connected with a decreased concentra-
tion of calcium ions in the cell due to damage of the
membrane transport pathway induced by cobalt.
(3) Inhibition of the sympathetic tone. A study on cobalt
cardiomyopathy in dogs showed changes in the b-
adrenergic system.
(4) Finally, an unspecific allergic mechanism was also
reported; however, the way that is induced remains
unknown.
Table 5 shows some studies related with cobalt exposure and
cardiovascular diseases.
See also: Cobalamin (Vitamin B12): Metabolism and Disorders;
Cobalt: Properties and Determination.
Reference
Morin, Y. L., Foley, A. R., Martineau, G. and Roussel, J. (1967). Canadian Medical
Association 97(15), 881–883
Alexander, C. S. (1972). American Journal of Medicine 53, 395–417
D´Ádda, F., Borleri, D., Migliori, M., et al. (1994). Science of the Total Environment
150, 179–186
Clyne, N., Hofman-Bang, Y., Haga, N., et al. (2001). Scandinavian Journal of Clinical
and Laboratory Investigation 61(8), 609–614
Unverferth, D. V., Fertel, R. H., Thomas, J., et al. (1984). Cardiovascular Research 18,
44–50
Kennedy, A., Dornan, J. D. and King, R. (1981). Lancet, 412–414
Further Reading
Cornelis R, Caruso J, Crews H, and Heumann K (2005) Handbook of element speciation
II – species in the environment, food, medicine and occupational health. Chichester:
Wiley.
Davis JRDavis & Associates (2000) ASM specialty handbook – nickel, cobalt and their
alloys. Materials Park, OH: ASM International.
Lison D, De Boeck M, Verougstraete V, and Kirsch–Volders M (2001) Update on the
genotoxicity and carcinogenicity of cobalt compounds. Occupational and
Environmental Medicine 58: 619–625.
Nordberg GF, Fowler BA, Nordberg M, and Friberg L (2007) Handbook on the
toxicology of metals, 3rd ed. Barlington: Elsevier.
Science of the Total Environment (1994) Cobalt and hard metal disease, vol. 150.
Amsterdam: Elsevier, Issues 1–3.
Relevant Websites
http://www.epa.gov/ttnatw01/hlthef/cobalt.html – United States Environmental
Protection Agency.
http://www.thecdi.com/cdi/images/documents/facts/COBALT_FACTS-Metallurgical_%
20uses.pdf – Cobalt in Metallurgical Uses.
 Cocoa: Composition and Health Effects
DD Mellor, University of Nottingham, Loughborough, UK
ã 2016 Elsevier Ltd. All rights reserved.
Description of Commodity
Cocoa is derived from the ‘bean’ from the cocoa or cacao tree
(Theobroma cacao), which is cultivated in the equatorial
regions, as it requires a hot climate with high levels of precip-
itation to grow and produce optimal yields. The cocoa ‘bean’
develops in large and often colorful pods that contain the bitter
seeds used in the manufacture of cocoa and, subsequently,
chocolate.
Sources and Production
Postharvest, the ‘bean’ requires a number of processing steps
that, ultimately, can influence many of the potential health
benefits and organoleptic properties of end products. Proces-
sing can not only degrade polyphenolic compounds, reducing
bitter flavors, which may be preferable to consumers, but also
reduce the availability of compounds that may be responsible
for the beneficial bioactive functions.
The initial step, following harvesting and splitting of the
pod to remove the ‘beans,’ is fermentation. The remaining pulp
around the ‘beans’ is removed, which allows the rich flavors
associated with cocoa to develop. The exact method varies
from country to country; it can be achieved by simply by
covering the piles of beans with leaves or with the use of
boxes designed especially for this purpose.
Following fermentation, which can take between 5 and
8 days depending on method and country, the ‘beans’ are
dried and winnowed where the ‘bean’ is cracked to separate
the shell from the nib (the part that is used to make cocoa and
chocolate). The nibs are roasted to further develop flavor.
These roasted nibs are ground before processing to produce
cocoa powder and cocoa butter. This has the effect of separat-
ing the fat-rich cocoa butter, which is used in chocolate pro-
duction along with many applications in the cosmetic
industry, and the cocoa powder, also often described as fat-
free cocoa mass. The distinction of fat-free cocoa mass from
cocoa solids is important, as a chocolate high in cocoa solids is
not necessarily high in fat-free cocoa mass, which is the cocoa
component rich in potentially beneficial polyphenolic com-
pounds. Chocolate, which typically lists cocoa solids as part of
the ingredient labeling information, is a composite of cocoa
butter and fat-free cocoa mass (cocoa powder).
The separation of the fat-free cocoa mass from cocoa butter
is important as it enhances the stability of final products, reduc-
ing any separation of fat that can result in unpleasant ‘bloom-
ing’ where a white fatty layer can be seen on the surface of
products, as was the case with eighteenth-century cocoa drinks
and poorly stored chocolate today. Separation is achieved using
methods such as the Dutch process that uses alkali to produce a
mellower-tasting product, which may be lower in polyphenols,
prior to hydrolytically pressing the powder from the fat or the
Broma process, which is a slower process using a combination
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00178-1
of sieving and temperature to produce what is known as natural
cocoa. Van Houten developed the former method, while the
latter was developed in the United States at the Ghirardelli
factory. These processes were accompanied by a number of
other technological advances in the nineteenth century that
helped change cocoa products from a bitter drink to the mod-
ern drinking cocoas and chocolates that are enjoyed by millions
today. Cocoa and chocolate are products often consumed for
pleasure owing largely to their hedonic qualities and luxury
image. Initially, this led to research aimed at understanding
potential harms, including tachycardia, but this has changed
dramatically over the last decade and a half with the investiga-
tion of potential health benefits.
The emergence of potential health benefits from com-
pounds found in cocoa is of great interest to both the scientific
community and the food industry. Arguably, a disproportion-
ate amount of work is being undertaken using cocoa and choc-
olate due to commercial interests, which may not exist to fund
work with other plant-based foods containing similar com-
pounds. Alongside potential financial interests, a key property
of cocoa and chocolate is that they are rich sources of polyphe-
nolic compounds, which are stable over time. Chocolate has
been shown to maintain its polyphenol content 2 years after
manufacture, with low levels of contaminants and bacteria
growth, characteristics not seen in fruit and vegetables. Whereas
other commodities, including teas, appear to be less stable, with
a shorter shelf life, they are yet to demonstrate similar levels of
bioavailability to the polyphenols found in cocoa or chocolate.
This, together with good consumer acceptability and desirabil-
ity, has resulted in over 2000 peer-reviewed papers describing
research on cocoa or chocolate in PubMed by August 2014.
A key challenge in the promotion of any potential health
benefits derived from the consumption of cocoa and chocolate
is the way it is considered as a food by society and the formu-
lations that are most popular. Many products based on cocoa
are energy dense, resulting from high levels of fat and sugar.
Chocolate as a formulation varies from country to country;
often, cocoa and cocoa butter can have added sugar and,
potentially, milk and/or other emulsifiers (typically soy leci-
thin), which in some countries are accompanied by added
vegetable fats; in Europe, only illipe, palm oil, sal, shea,
kokum gurgi, mango kernels, and copra oil can be used. But
this can mean that chocolate contains around 500–530 kcal
per 100 g (2050–2250 kJ), 27–32 g of fat of which 16–19 g is
saturated, and 45–64 g of added sugar (typically sucrose).
When sold as a powder, cocoa contains around 310 kcal per
100 g (1300 kJ), 22 g of fat of which 13 g is saturated, and only
a trace of sugar. However, cocoa is consumed rarely in this
form, and often, sugar and fat (often from added dairy fat) are
added, which can dramatically alter nutritional composition.
So, by definition, in some territories, chocolate cannot be
considered as a food with a health claim simply because of its
high fat and sugar content.
179
180 Cocoa: Composition and Health Effects
Availability, Absorption, and Metabolism
The mechanism(s) and exact bioactive compounds responsible
for the health benefits observed have also been an area of
debate. Epicatechin has been proposed as the key biologically
active polyphenol, in part not only due to its relatively low
molecular weight but also because it has been detected in
plasma following ingestion. However, it is equally likely that
many of the polyphenolic compounds found in cocoa and
chocolate are metabolized in the gastrointestinal tract,
degraded and transformed chemically both by stomach acid
and by the microflora of the colon prior to absorption. It has
been suggested that many of these metabolites could be
responsible for the beneficial effects of cocoa and chocolate
rather than the compounds native to cocoa after processing.
The complex nature of cocoa, as a source of polyphenols,
alongside other bioactive compounds such as theobromine, a
bioactive amine with similar effects to caffeine, has been
hypothesized to be responsible for many of the health benefits.
This is aside from its unique fatty acid and amino acid profiles
and the fact that cocoa is a good source of many micronutri-
ents, including magnesium and iron, which also may impact
human health. Potentially, this means that it is difficult to
identify if the benefits arise from one active compound or a
synergistic mix of compounds. In addition, the hedonistic and
socially ascribed qualities given to chocolate since the Aztecs
could infer benefits purely from its consumption in a
Hawthorne-type effect, where individuals improve or modify
an aspect of behavior in response to being observed.
Much of the initial work in vitro focussed on the antioxidant
properties of the phenolic rings in cocoa polyphenols. This is
demonstrated by the very high oxygen radical absorbance capac-
ity (ORAC) value for cocoa. However, the effect of these com-
pounds in vivo is less related to their antioxidant properties, as
their bioavailability is not sufficient to support this mechanism,
than the number of pathways targeted by cocoa polyphenols.
The most widely studied is perhaps inducible endothelial nitric
oxide synthase (eNOS), a key enzyme involved in how blood
vessels respond to the sheer stresses caused by changes in blood
flow and pressure. Cocoa polyphenols are associated with an
increase in nitric oxide, which improves the capacity of the blood
vessel to expand and reduces the risk of damage that might lead
to the development of atherosclerotic plaques and, ultimately,
cardiovascular disease. Other key enzymes acted on by cocoa
polyphenols include AMP-activated protein kinase, which can
influence insulin resistance and lipid metabolism in a positive
way, and the angiotensinogen-converting enzyme pathway,
which is key in determining blood pressure.
History of Consumption and Formulation Including
Some of the ‘Issues’
The consumption of cocoa can be traced back to the civilizations
of Central America, especially the Mayans and Aztecs, although it
appears to have originated in South America where a wide variety
of native cocoa trees can still be found. Cocoa has been cultivated
since about 1500 BC. Historically, a number of physiological
effects have been linked to cocoa consumption, with its use
initially linked to rituals; it was consumed as a bitter drink
mixed with chili or even blood by the ruling classes, priests,
and warriors. It was suggested by Montezuma II that a soldier
could march for a whole day on a single cup of cocoa without
getting fatigued. During the time of the conquistadors, Spanish
monks recorded in detail the potential health effects of cocoa,
including as a treatment for consumption (tuberculosis), diges-
tive problems, and general fatigue. Following these expeditions,
cocoa was brought to Europe, but dismissed in many royal
courts at the time as moldy almonds, although in Spain, a culture
of cocoa drinking did develop. This spread across Europe,
despite the fact that cocoa was bitter and the fat component
did not form a stable emulsion upon mixing with water. The
culture of cocoa drinking was a concern in France, where it was
considered a narcotic, most likely as a result of cocoa houses
becoming places where intellectuals would meet and, because
cocoa provided an alternative to the alcoholic beverages, began
to think more radically. However, it was not until the industrial
revolution when methods, such as the Dutch process and con-
ching, developed that modern cocoa and chocolate began to be
manufactured. Discovered by Rodolphe Lindt, conching physi-
cally redistributes fat in cocoa to improve the texture of the end
product, mixing cocoa powder for a long time using a paddle
that scrapes the surface of the bowl. Air and friction reduce the
amounts of volatile fatty acids and oxidize some of the bitter
compounds, thus mellowing the taste of the end product. How-
ever, this process and others while mellowing flavor have the
potential to reduce further levels of bitter bioactive compounds,
including epicatechin and theobromine. During this period,
Joseph Fry also discovered that, if cocoa butter was added back
to cocoa, the product could be molded allowing the first choco-
late bars to be produced.
Within much of northern Europe and North America, milk
chocolate is more popular than the bitter plain chocolate,
probably because of its mellower and sweeter flavor. Milk
chocolate was initially developed in Switzerland following
the invention of a process to dry milk. Until then, it was only
possible to mix milk with cocoa in a drink: Daniel Peter added
the dried milk developed by Henri Nestlé to cocoa liquor,
creating milk chocolate.
All these developments needed raw ingredients, namely,
cocoa and sugar, which came from plantations and colonies of
the Dutch, Spanish, and British empires and depended to a large
extent on the slave trade up until its abolition. The combination
of global trade and industrialization meant that cocoa and choc-
olate became not just a rare treat for the wealthy, but a product
consumed widely by the whole of society. Its high energy content,
including saturated fat and sugar, means that chocolate has been
implicated as a cause of a range of problems including obesity,
poor skin health, and dental caries. However, epidemiological
data do not support a link between chocolate consumption and
obesity; if anything, the data suggest the opposite. These findings
need to be taken with a note of caution, as such studies are prone
to a degree of selection and reporting bias, as they rely on partic-
ipants reporting what and how much chocolate they eat.
Health Effects
Early work studying the Kuna Indians, an indigenous popula-
tion inhabiting islands near Panama, provided modern insight

into the health effects of cocoa. This ethnic group has little age-
related increase in blood pressure or associated hypertension.
However, when members of this group moved to the urban
environment of Panama City, they display expected levels of
hypertension. This could be said to represent the classic ‘migra-
tion type’ study, suggesting a lack of a genetic effect, and the
increase in hypertension must be attributed solely to environ-
mental influences. The island-dwelling Kuna consume large
quantities of cocoa daily, both as drinks (up to five cups a
day) and as an ingredient in cooking. Those living in Panama
City, however, consume very little cocoa. Moreover, the island-
dwelling Kuna have a threefold higher level of nitric oxide
metabolites in their urine compared with those living in an
urban environment. These epidemiological data clearly sug-
gested an association between cocoa polyphenols (mainly fla-
vanols) and improved vascular function and support the view
that increased nitric oxide, via the induction of nitric oxide
synthase, could be the mode of action.
Epidemiological evidence of the effect of chocolate has
largely been derived from studies in which data collection
was initially undertaken as part of other studies, and the effect
of cocoa or chocolate consumption examined as post hoc
analysis. The majority of these data have been derived from
European prospective study data sets, which are supported by
cross-sectional data from the United States and Spain. The
cross-sectional natures of European cohort studies lack predic-
tive power because of their design. European studies, however,
reported hard end points, including death and myocardial
infarction, suggesting that chocolate consumption is associated
with a reduced rate of mortality.
Data from the Zutphen Elderly Study reported by Buijsse
et al. considered the effects of consuming foods containing
cocoa in elderly men over a period of 15 years. The findings
suggested no association between sugar confectionary con-
sumption and mortality but did demonstrate a significant
inverse association between habitual intake of cocoa-
containing foods and cardiovascular mortality rates (as well
as all-cause mortality rates). This was in agreement with the
cross-sectional data collected at the start of the study, suggest-
ing a significant inverse association between blood pressure
and habitual intake of foods containing cocoa. These data were
subsequently confirmed by analysis of the Potsdam cohort
from the European Prospective Investigation into Cancer and
Nutrition reported by Buijsse et al. In this group, chocolate
consumption and blood pressure were assessed at baseline and
found to be inversely associated in nearly 20 000 individuals
aged 35–65 years of age. When followed up, those who con-
sumed the greatest quantity of chocolate had a significantly
lower risk of myocardial infarction and stroke than those con-
suming the lowest amounts (RR ¼ 0.61 (95% confidence inter-
val 0.44–0.87)), with 12% of this effect (95% confidence
interval 3–36%) explained by the effect of chocolate on
blood pressure after an average follow-up period of 8 years.
Cocoa polyphenolic compounds have been reported to
have beneficial effects on cardiovascular risk, including the
lowering of blood pressure, in both animal models and clinical
trials with human participants. In rats, the consumption of
cocoa polyphenols demonstrated not only a reduction in
blood pressure but also an increase in the expression of a
number of genes associated with endothelial function, which
Cocoa: Composition and Health Effects 181
suggests the potential for an epigenetic effect of polyphenols.
Upregulated genes associated with reduced blood pressure
acted on endothelial function via increased nitric oxide and
prostaglandin production.
Many of the clinical studies demonstrating a reduction in
blood pressure attributed to the effect of polyphenols have
been credited to changes in endothelial function. However,
work in individuals with type 2 diabetes has shown an
improvement in endothelial function but no change in blood
pressure. The effects of the underlying condition and concom-
itant medication, which may reduce blood pressure but have
minimal effect on endothelial function, could explain this
apparent contradiction. It has been suggested that the improve-
ment in endothelial function derives from an increase in redox-
sensitive activation of the phosphatidylinositol 3-kinase/Akt
pathway, which results in the activation of eNOS. This is
thought to be via an increase in intracellular free calcium
concentration and activation of estrogen receptors. These
changes appear to go beyond just nitric oxide with cocoa poly-
phenols acting on other endothelium factors. The improve-
ments seen in endothelial function have been linked to
insulin signaling and, ultimately, the potential to reduce insu-
lin resistance. In individuals with type 2 diabetes, the results
are inconsistent. However, in studies with healthy individuals
and those with hyperinsulinemia consuming high doses of
chocolate (100 g day1) for 2 weeks, reductions in insulin
resistance have been reported consistently. Alongside the
potential for better endothelial function and insulin sensitivity,
it has been suggested that activation of the AMPK pathway in
both the skeletal muscle and liver could explain improvements
in insulin sensitivity.
Cocoa and chocolate polyphenols have also been associ-
ated with the beneficial effects on dyslipidemia across a range
of clinical and nonclinical groups, including individuals with
type 2 diabetes. This is in accordance with data that suggest
diets rich in plant-derived foods and polyphenols are associ-
ated with more favorable lipid and cholesterol profiles and
reduced cardiovascular risk. Data from mice models of diabe-
tes suggest that cocoa polyphenols might act via hepatocellular
AMP-activated protein kinase (AMPK) along with its down-
stream target, acetyl-CoA carboxylase. In 2006, Zang et al.
reported that the effects of these polyphenols were approxi-
mately 200 times more potent than metformin, the most com-
monly used pharmaceutical agent in type 2 diabetes, on lipid
metabolism. As in the case of blood pressure, there are a
number of alternate mechanisms by which cocoa polyphenols
might act to improve the lipid profile including the modula-
tion of the ATP-cassette binding protein. In data from human
studies, improvements in lipid profiles, specifically increased
high-density lipoprotein (HDL) cholesterol levels, have been
seen in populations with diabetes. Another research suggests
that polyphenols may reduce the overall amount and vulnera-
bility of low-density lipoprotein (LDL) cholesterol to oxidative
damage. Some of the data from clinical studies are challenging
to interpret, as often cardiovascular risk and LDL cholesterol
are calculated, rather than measured, creating a potential for
bias and/or confounding factors, meaning interpretation needs
to be undertaken with care.
Despite dismissing the potential of cocoa and chocolate as
having an antioxidant effect in vivo because the data from human
182 Cocoa: Composition and Health Effects
studies are largely equivocal, it is still important to consider what
is meant by oxidative stress and how it is measured. A number of
methods available can be insensitive to changes in response to
diet or can be directly influenced by their presence as compo-
nents of acute food intake, rather than showing any effect upon
endogenous redox biology. The relatively poor absorption of
cocoa polyphenols resulting in low plasma concentrations com-
pared with other antioxidant compounds, including uric and
ascorbic acids, suggests that any effects are likely to go beyond
just antioxidant capacity and act via multiple mechanisms. It is
logical that the beneficial effects of polyphenols on endothelium
function arise from increased synthesis of endothelium-derived
factors, such as nitric oxide and prostaglandins, which are also
free radical scavengers, resulting in an enhanced pool of poten-
tially beneficial compounds. Along with the effects of cocoa
polyphenols that have been demonstrated on superoxide dis-
mutase, this increase in free radical scavengers might explain the
reductions in oxidative stress markers including lipid peroxida-
tion and DNA damage.
Cocoa polyphenols may also have positive effects on coag-
ulation and clotting, having been demonstrated to inhibit
platelet aggregation, the potential mechanism being via
decreased production of superoxide anions and increased nitric
oxide synthesis, which in addition would reduce levels of oxi-
dative stress. A further potential effect of polyphenols on health
is as ‘an antinutrient’ or chelating agent. This could be consid-
ered as a ‘negative effect’ with increased risk of micronutrient
deficiencies and disease, such as iron-deficiency anemia. How-
ever, this chelating effect could potentially reduce chronic dis-
ease risk. Both in the gastrointestinal tract and, potentially, in
circulation, by binding metal ions that could otherwise be pro-
oxidants, oxidative stress and oxidation of LDL cholesterol
and/or DNA damage might be reduced. Currently, these effects
are little more than a hypothesis based on mechanistic work
in vitro. It is clear, however, that beneficial effects of cocoa are
likely to be derived from polyphenols and these compounds
appear to have multiple modes of action, with the mechanisms
described in this article often interacting and overlapping.
Cocoa and chocolate have been proposed to improve cog-
nition, based on mechanisms associated with nitric oxide
metabolism. This has been demonstrated in animals, but
work in humans has also reported the potential of improved
cognition. This concept has been hypothesized as perhaps
reducing progression in dementia, but consistent data are not
yet available. The effects of cocoa butter as a cosmetic product
have been observed for many years; the effect of chocolate on
the resistance of skin to ultraviolet light damage has also
shown limited promise in clinical studies.
Chocolate and cocoa drinks have been studied as a poten-
tial ergogenic aid in sport, for both professional athletes and
the general public. Initial studies have suggested that chocolate
might delay perception of fatigue and, thus, improve exercise
performance. This has been associated with a reduction in
glucose oxidation linked to the effects of polyphenols. Data
are yet to demonstrate this effect consistently. Other areas of
interest in exercise physiology are the potential of cocoa poly-
phenols to modulate oxidative stress and inflammation asso-
ciated with exercise; mechanistic work in vivo shows promise,
but the practical implications are yet to be demonstrated.
Endeavors to explore the health potential of chocolate are
less than 20 years old. From the initial work in vitro of
Waterhouse, Shirley, and Donovan in the mid-1990s to the
first human studies reported in 2000, attempts have been
made to see if cocoa has the potential to reverse or prevent
disease processing pertaining to cardiovascular disease and
cognitive decline. The vast majority of these studies have
been undertaken in healthy individuals, with a few undertaken
in populations with, or at increased risk of, cardiovascular
disease. Only five studies have been undertaken in individuals
with diabetes, for example, and two with heart failure or
following heart surgery. Some of these studies have combined
cocoa, typically in the form of chocolate, with other potentially
bioactive compounds, such as soy isoflavones and lycopene.
It is possible that favorable palatability and acceptability of
chocolate mean that it could be used as a carrier or vector for
other less palatable plant-derived bioactives in the future.
The key challenge when attempting to ascertain the poten-
tial health effects of cocoa and chocolate lies in the way many
studies are conducted. There is little consistency in the formu-
lations or even concentrations/doses of polyphenols being
consumed by participants. This reflects that cocoa and choco-
late manufacturers have, at least, supported many studies
financially and, therefore, are unlikely to share commercially
sensitive data with respect to recipes and composition. Addi-
tionally, many of the studies are limited in duration and often
sample size, with very few studies exceeding 100 participants or
lasting greater than 12 weeks. The reliability of many studies is
also brought into question as, although many studies state that
participants were blinded to the cocoa or chocolate they were
given, on closer examination, it is apparent that the control
product(s) was milk in studies of cocoa beverages or white
chocolate in studies of dark chocolate. Under such conditions,
it is highly plausible that informed study participants would be
fully aware of the arm to which they had been assigned. In a
review, Karen Cooper et al. summarized the first 10 years of
research on the health effects of chocolate and proposed an 11-
point checklist for planning future trials. These included the
design of the trials; need to consider the matrix or formulation
used; use of chocolate in preference to cocoa; transparency in
terms of both independency of the trial from industry and the
need to register human trials publically; careful selection of
biomarkers, especially those assessing antioxidant effects; and,
finally, publication of null or negative results.
Future and Health Claims
Increasingly, in many countries, claims associating food and
health are being regulated. In Europe, for example, the
European Food Safety Authority (EFSA) has a rigorous assess-
ment process for assessing health claims whether maintaining
health or preventing disease, which can be generic or proprie-
tary. The latter provides sole use for a claim for 5 years protect-
ing the investment in intellectual property. To be awarded a
health claim, foods need to have an identifiable, well-
characterized component with a defined mechanism(s) of
action and effect and clinical trial data to support the effect
in vivo. In 2012, the EFSA gave a positive opinion for a propri-
etary health claim by Barry Callebaut (BE) for a cocoa product
containing flavanols. This was granted under Article 13.5 and,
potentially, allows these cocoa and chocolate products to carry
the claim that Cocoa flavanols help maintain endothelium-

dependent vasodilation, which contributes to normal blood flow.
This claim acknowledges the growing body of data from
many sources for chocolate and cocoa that have been shown
to demonstrate this effect. However, it is perhaps less clear how
the general public would interpret this claim and if it would
influence purchasing decisions.
Not all health claims linked to cocoa and chocolate have
been as successful. In 2012, the USDA withdrew publication of
ORAC values from its Nutrient Data Laboratory website
(http://www.ars.usda.gov). This decision was based on mount-
ing evidence suggesting that there was no link between the
antioxidant capacity of foods and chronic disease risk. Using
the PASSCLAIM criteria, which are the basis for a positive
opinion for a health claim, it was found that reduction in
cardiovascular risk could not be attributed to the antioxidant
capacity of polyphenol-containing foods, such as cocoa. This
perhaps explains the EFSA judgment in 2010 that found a
causal relationship could not be demonstrated. The panel
also stated that any beneficial psychological effect (as with
physiological effects) could not be linked to the antioxidant
activity, content, or properties.
So, although one health claim has reached the EFSA thresh-
old, many have failed due to poor characterization of the
bioactive compounds, the absence of clear mechanism(s) of
action, or inadequate clinical trial data. This does not mean
that chocolate will eventually be labeled with health claims, as
nutritional composition and portion size are also important.
The Barry Callebaut claim was for 10 g of a specially formu-
lated chocolate, or 2.5 g of cocoa, containing 200 mg cocoa
flavanols, more than twice the more typical levels of polyphe-
nols. Otherwise, the energy content and added sugar would
make a health claim for chocolate incompatible with estab-
lished public health messages.
Many of the negative aspects of chocolate and cocoa con-
sumption have not stood up to rigorous examination, with no
apparent weight gain seen in studies or when observing popu-
lations. However, the shift in focus to consider the beneficial
effect of polyphenols and recommendations of higher intakes,
especially in an aging population, should be undertaken with
caution owing to the largely under studied potential of
food–nutrient–drug interactions. It has been suggested that
epicatechins and other flavanols can act on cytochrome
P450, including CYP3A4, which are responsible for the conju-
gation of many organic compounds, including a wide range of
drugs. Thus, any compounds altering the action of this and
similar enzyme systems could impact the circulating concen-
tration of pharmaceuticals and their half-life and, ultimately,
increase the risk of side effects or poor control of symptoms.
This could lead, in the future, to as yet undefined negative
health effects, which need to be studied before recommenda-
tions to increase intakes to above that achieved normally, for
example, levels seen among the Kuna of Panama.
Conclusion: Cocoa and Chocolate for Health
or Pleasure?
It is of note that the history of cocoa and chocolate consump-
tion has been linked with potential health benefits. However,
with industrialization, and the increased availability of sugar, a
sweeter product has been created, which is associated with
Cocoa: Composition and Health Effects 183
unproved negative health effects. This means that chocolate is
likely to remain a luxury, nonessential food. However, it should
not necessarily be considered as being inherently harmful and
has potential for a number of well-defined health benefits. This
does not mean that increasing consumption is wise, from both
the perspective of sustainability, as ecosystems would need to
be changed to grow more cocoa, and health, as eating more
chocolate is likely to lead to increased energy intake, weight
gain, and, potentially, long-term health consequences.
See also: Antibiotics and Drugs: Drug–Nutrient Interactions;
Antioxidants: Role on Health and Prevention; Cholesterol: Factors
Determining Blood Cholesterol Levels; Functional Foods; Phenolic
Compounds: Bioavailability and Health Effects.
Further Reading
Buijsse B, Feskens EJ, Kok FJ, and Kromhout D (2006) Cocoa intake, blood pressure,
and cardiovascular mortality: the Zutphen elderly study. Archives of Internal
Medicine 166(4): 411–417.
Cooper KA, Donovan JL, Waterhouse AL, and Williamson G (2008) Cocoa and health: a
decade of research. British Journal of Nutrition 99(1): 1–11.
EFSA (2010) Scientific opinion on the substantiation of health claims related to various
food(s)/food constituent(s) and protection of cells from premature aging,
antioxidant activity, antioxidant content and antioxidant properties, and protection of
DNA, proteins and lipids from oxidative damage pursuant to Article 13(1) of
Regulation (EC) No. 1924/20061 [Online]. EFSA Journal 8(2): 1489, Retrieved
from: doi:10.2903/j.efsa.2010.1489.
EFSA (2012) Scientific Opinion on the substantiation of a health claim related to cocoa
flavanols and maintenance of normal endothelium-dependent vasodilation
pursuant to Article 13(5) of Regulation (EC) No 1924/2006 [Online]. EFSA Journal
10(7): 21, Retrieved from: doi:10.2903/j.efsa.2012.2809.
European Union (2004) B directive 2000/36/EC of the European parliament and of the
council of 23 June 2000 relating to cocoa and chocolate products intended for
human consumption [Online]. October, (June 2000), 1–10. Retrieved from http://
eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri¼CELEX:32000L0036:EN:NOT
(accessed 30 December 2012).
Golomb B, Koperski S, and White H (2012) Association between more frequent
chocolate consumption and lower body mass index. Archives of Internal Medicine
172(6): 519–521.
Halliwell B, Rafter J, and Jenner A (2005) Health promotion by flavonoids, tocopherols,
tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not?
American Journal of Clinical Nutrition 81(1): 268S–276S.
Heuberger R (2012) Polypharmacy and food-drug interactions among older persons: a
review. Journal of Nutrition in Gerontology and Geriatrics 31(4): 325–403.
Hollenberg NK, Martinez G, McCullough M, et al. (1997) Aging, acculturation,
salt intake, and hypertension in the Kuna of Panama. Hypertension 29(1):
171–176.
Hooper L, Kay C, Abdelhamid A, Kroon PA, Cohn JS, Rimm EB, and Cassidy A (2012)
Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular health: a systematic
review and meta-analysis of randomized trials. American Journal of Clinical
Nutrition 95(3): 740–751.
Rein D, Lotito S, Holt RR, Keen CL, Schmitz HH, and Fraga CG (2000) Chocolate:
modern science investigates an ancient medicine epicatechin in human plasma:
in vivo determination and effect of chocolate consumption on plasma oxidation
status. Journal of Nutrition 130(8): 2109S–2114S.
Schroeter H, Heiss C, Balzer J, et al. (2006) ()-Epicatechin mediates beneficial effects
of flavanol-rich cocoa on vascular function in humans. Proceedings of the National
Academy of Sciences 103(4): 1024–1029.
Taubert D, Roesen R, Lehmann C, Jung N, and Schömig E (2007) Effects of low habitual
cocoa intake on blood pressure and bioactive nitric oxide: a randomized controlled
trial. Journal of the American Medical Association 298(1): 49–60.
USDA (2012). Oxygen radical absorbance capacity (ORAC) of selected foods, release 2
[Online]. Retrieved from http://www.ars.usda.gov/Services/docs.htm?
docid¼15866 (accessed 29 August 2012).
184 Cocoa: Composition and Health Effects

Waterhouse AL, Shirley JR, and Donovan JL (1996) Antioxidants in chocolate. Lancet Relevant Websites
348(9030): 834.
Zang M, Xu S, Maitland-Toolan KA, et al. (2006) Polyphenols stimulate AMP-activated http://www.icco.org/.
protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL http://www.thestoryofchocolate.com/.
receptor-deficient mice. Diabetes 55(8): 2180–2191. http://worldcocoafoundation.org/.
 Cocoa: Production, Chemistry, and Use
A Caligiani, A Marseglia, and G Palla, University of Parma, Parma, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Cocoa Tree and Beans
Cocoa beans represent the seeds of the fruits of cocoa tree
(Theobroma cacao L., order Sterculiacae), which is a native
species of tropical humid forests on the lower eastern equato-
rial slopes of the Andes in South America. Cocoa was domes-
ticated and consumed for the first time by the Mayas and
Aztecs. The growth of the cocoa tree requires warm-humid
climates with temperatures between 20 and 30
C and high
and constant humidity, conditions found in areas about 20

latitude north and south of the Equator. Abundant and well-
distributed rainfalls are essential because the cocoa tree toler-
ates a dry season of up to 3 months a year. The cocoa tree is a
rather delicate plant that does not tolerate direct strong winds
and direct sunlight. In fact, it is generally grown in the shade of
other tall trees (banana trees and coconut palms). Trees can
reach up to 20 m in height, but are usually kept below 5 m. The
fruit is 10–30 cm long and 7–10 cm wide and weighs
400–1000 g. It is an indehiscent drupe called a pod or
‘cabosside.’ It can be spherical, cylindrical, pointed or blunt,
smooth, or wrinkled. The pericarp has a thickness of
10–15 mm and can be of different colors depending on the
ripeness and variety, turning from green to yellow, from red to
orange. The optimum ripeness of the fruit varies according to
the variety and generally lasts from 4.5 to 7 months. The fruit is
harvested twice a year (in February/March and April/July). The
summer harvest usually produces fruit of better quality. The
seeds, in the number of 20–60 per fruit, are arranged in regular
rows and immersed in a mucilaginous acidic pulp containing
glucose and fructose and are called beans. Cocoa beans are the
only economically valuable part of cocoa tree because they
represent the raw material for the production of cocoa-based
products. Each cocoa bean consists of two cotyledons (nibs)
and a small embryo, all enclosed in a skin (shell). The cotyle-
dons contain two types of cells: storage or parenchyma cells,
containing fat globules, protein and starch granules, and pig-
mented cells, containing polyphenols and methylxanthines.
Cocoa Varieties and Geographical Diffusion
Two major botanical groups of cocoa are currently recognized:
Criollo and Forastero. Criollo (native) corresponds to the sub-
species ‘cacao’ and represents the original cocoa, previously
consumed by pre-Columbian people. Its diffusion resulted
from the dissemination through the Andes toward the low-
lands of Venezuela, Colombia, and Ecuador and northward to
Central America and Mexico, and to a large number of Carib-
bean Islands. Today the subspecies Criollo is found in Mexico,
Colombia, and Venezuela. It represents only 5% of the world’s
production due to its great susceptibility to disease; making it
difficult to cultivate. Criollo pods are green to red; the seeds are
pale, ferment easily, have a pleasant and penetrating aroma,
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00177-X
and have an optimum taste. Therefore they are considered an
excellent quality cocoa. The Criollo is rarely used alone; most
frequently it is used to fortify other mixtures having a weak and
not persistent aroma. It can be subdivided into Central
American Criollo and Venezuelan Criollo. The Forastero
cacao (foreigner) corresponds to the subspecies ‘sphaerocar-
pum’ and resulted from dissemination toward the Amazon
Valley in Northern Brazil and the Guyanas. In the nineteenth
century, Forastero cocoa seeds were taken from South America
to the islands of Sao Tomé of West Africa, then to Ghana. From
Ghana, Forastero spread to other African countries, the most
important of which are the Ivory Coast, Cameroon, and Nige-
ria. In these countries, there was an immediate increase in
cultivated area, and they are now the largest producers of
cocoa in the world. Forastero represents about 85% of the
world’s cocoa production because it is very strong, resistant to
disease, and easily cultivated, especially in Africa. The seeds
have a strong flavor, not very aromatic and of poor quality, and
are generally used in a mixture with other more valuable
varieties. Forastero cocoa is now a group very differentiated
in several subspecies and hybrids. The best-known Forasteros
are the ‘Amelonados’ with pods resembling melon, which were
the predominant types traditionally cultivated in West African
countries. The Amazonian subgroups show a wide genetic
variability. Some Forastero plants growing in particular areas
give cocoa of excellent quality (cru) such as Arriba, the national
cocoa of Ecuador.
Natural hybridization between Criollo and Forastero led
to the origin of a third variety of cocoa named ‘Trinitario,’
not found in the wild. It has been reported that the Criollo
population from Venezuela and the Amelonado-type Forastero
from Guyana could have been involved in hybridization lead-
ing to the production of Trinitario. It is difficult to specify
the characters of Trinitarios as they may have pod and bean
characters ranging from those typical of Criollos to those of
Forasteros.
Cocoa Beans Harvesting, Fermentation, and Drying
These steps of the process are carried out in the countries of
origin. Fresh cocoa seeds undergo fermentation and a drying
process to be ready and stable for transport to the countries in
which they will be processed to chocolate and related products.
Local or regional variations in cocoa plant materials, fermenta-
tion procedures, and drying processes lead to a traded good
typical of the country of origin; therefore, the composition of
the fermented cocoa beans, which is one of the most important
factors influencing taste and flavor of the cocoa products,
depends not only on the cocoa variety but also on the geographic
origin.
Cocoa bean fermentation can be considered the first key
stage in cocoa products and chocolate production because
185
186 Cocoa: Production, Chemistry, and Use
the series of biochemical reactions occurring in the beans is
necessary for inducing the pleasant characteristics of cocoa-
based products.
The fully ripe cocoa pods are carefully cut from the trees, are
gathered into heaps, and can be stored for 2–5 days. This
enhances prefermentation activity inside the pods. The har-
vested pods are broken by hitting against a hard surface, and
the seeds are extracted from the fruit together with the sur-
rounding sugary mucilaginous pulp (wet beans) and kept for
fermentation immediately. The fresh bean is bitter and is not
suitable for manufacture of cocoa products because it does not
have any flavor, aroma, or taste of cocoa products. All the
standard methods of fermentation essentially involve keeping
together in heaps, baskets, boxes, or perforated barrels a mass
of a reasonable quantity of wet beans for periods ranging from
4 to 6 days and mixing the mass of beans on alternate days. The
natural beans’ fermentations are generally carried out accord-
ing to a traditional process. Among the various methods
adopted for fermentation in the different cocoa-producing
countries, the heap, tray, and box methods are considered the
standard, widely adopted methods. The real substrate of the
fermentation is not the bean, but the surrounding pulp that
contains about 84.5% water, 10% glucose and fructose, 2.7%
pentosan, 0.7% sucrose, 0.6% protein, 0.7% acids, and 0.8%
inorganic acids and therefore it is a good substrate where a
wide range of microorganisms can develop. Wild yeasts
(Kloeckera and Saccharomyces spp.) and bacteria of genera
such as Lactobacillus, Bacillus, Pediococcus, Acetobacter, and Glu-
conobacter, are involved in cocoa fermentation, determining
alcoholic, lactic acid, and acetic acid fermentations.
The first consequence of fermentation is the loss of most of
the pulp around the beans because the pulp substrates are
broken down through microbial action; however, more impor-
tant are the biochemical changes inside the cocoa beans that
contribute to a reduction of their bitterness and astringency
and improve their color and flavor.
In the first stage of the fermentation, yeasts proliferate and
convert sugars to alcohol, then the development of lactic acid
bacteria occurs, which contributes to sugar breakdown produc-
ing lactic acid. After the pulp has run off, the conditions
become more aerobic, and the presence of oxygen allows acetic
acid bacteria to take over from the yeasts and convert alcohol
to acetic acid. Acids that are synthesized from pulp sugars move
into the beans and lower the internal pH. The acetic acid
diffusing through the beans causes a breakdown of the poly-
phenol and lipid membranes of the vacuoles of the cell, and
the cell contents become mixed, allowing various enzymatic
reactions to take place. One of the most important conse-
quences is the oxidation of polyphenols and their conversion
to insoluble forms due to the reactions with proteins; these
reactions are responsible not only for the removal of the bitter
taste from the beans but also for the strong reduction of total
polyphenol content, thus affecting the antioxidant and health
properties of cocoa. Oxidation of polyphenols is also respon-
sible for the change of bean color, from violet to brown.
During and after fermentation, another fundamental biochem-
ical change occurs: internal beans’ autolytic enzymes, in par-
ticular carboxypeptidase and aspartic endoprotease, are
activated by the low pH; the proteins in the cotyledons
undergo hydrolysis, giving rise to amino acids and
oligopeptides, essential precursors for the development of
cocoa aroma, arising from the reaction of amino acids with
sugars (Maillard reactions) during the subsequent step of
roasting.
The duration of fermentation ranges from 1.5 to 10 days,
depending on the cocoa variety, climate, volume of cocoa
mass, and the method adopted. Criollo ferments in a relatively
shorter period of 2–3 days, whereas Forastero takes 5–7 days.
For the manufacturer of chocolate or cocoa powders the
degree of fermentation of the beans is a major quality criterion.
Fully fermented cocoa beans have a brown color. It has been
shown that too high contents of nonfermented (slaty color) or
partly fermented (violet color) beans result in a lack of cocoa
flavor in the end product. The slaty beans cause a very acid and
astringent flavor profile, whereas the violet beans cause a bitter
and harsh flavor.
The fermented beans have a moisture content of about
55%, too high to permit the storage of the beans. The moisture
content has to be lowered to about 6% for safe storage and
transportation. Immediately after fermentation, cocoa beans
are sun-dried or dried utilizing hot air. Depending on climatic
conditions, the beans are exposed to the sun for about 12–20
days. This method generally gives good-quality beans in tradi-
tional areas of cocoa production where the weather is suffi-
ciently sunny, as in West Africa. In the areas where the climate
remains unsuitable for drying, artificial drying methods
became necessary, utilizing hot air with a maximum tempera-
ture of about 60
C. The humidity % after drying is 5–8%, with
a weight loss of two-thirds with respect to the fresh bean. The
dried beans are packed in jute bags of about 60 kg capacity and
traded to transformation countries, where all the following
steps (Figure 1) for production of chocolate and cocoa prod-
ucts will take place.
Processing of Fermented Cocoa Beans: Roasting
and Production of Cocoa Liquor
A key step of chocolate production is roasting. Roasting is
performed in the transformation countries, and flavor is
formed during this step from the precursors developed during
fermentation and drying of cocoa beans. The aroma precursors
in cocoa beans, which include free amino acids,
low-molecular-weight peptides, and reducing sugars, develop
into the cocoa specific aroma through Maillard reactions dur-
ing roasting.
Before roasting, cocoa beans are cleaned to remove stones,
metals, and other extraneous materials. Beans are cleaned by
passing through a series of screens and magnets. In some cases
a preroasting process is applied. The roasting can be performed
on whole cocoa beans or on cocoa nibs. In most cases the
traditional roasting is performed on whole cocoa beans by
hot air treatment, and cocoa shells are removed by aspiration
(winnowing). Temperatures of roasting are generally lower
that 150
C and for times of from 30 to 120 min. Roasted
cocoa beans are then lightly crushed to avoid dust formation,
obtaining cocoa nibs.
Roasted cocoa nibs are milled to break down the cell walls
and expose the cocoa butter. The resulting product is a homo-
geneous flowing cocoa paste called cocoa liquor containing
 Cocoa: Production, Chemistry, and Use 187

FERMENTED DRIED BEANS

Cleaning

Roasting

Winnowing COCOA SHELLS

Grinding

COCOA NIBS

Milling

COCOA LIQUOR
Alkalinization Mixing Sugar, milk etc.

Pressing Refining

COCOA CAKE COCOA BUTTER Mixing

Grinding Conching

COCOA POWDER Tempering

CHOCOLATE

Figure 1 An overview on cocoa bean processing.

about 55% fats. To produce cocoa liquor with improved
dispersibility, the cocoa nibs can be subjected to an
alkalinization process (Dutch cocoa process): roasted nibs are
treated with a diluted alkali solution at 75–100
C, then neu-
tralized and dried. This treatment causes starch swelling and
the formation of porous cell structure of cocoa mass that
prevents the formation of sediments in cocoa drinks.
Production of Cocoa Powder and Cocoa Butter
Cocoa powder is widely used in the manufacturing of cocoa-
based products as drinks, cake fillings, ice cream, and so on. To
convert cocoa liquor (generally alkalinized cocoa liquor) to
cocoa powder, a defatting process is performed by pressing
liquor in a mechanical or hydraulic press at 400–500 bar and
a temperature of 90–100
C. In this way part of the fat (cocoa
butter) is removed, and cocoa cake (compressed cocoa pow-
der) is produced. Cocoa powder is obtained by grinding cocoa
cake. Generally the fat content of cocoa powder is 10–24%.
Cocoa butter obtained from pressing is separated, filtered, and
reused as an ingredient for chocolate and many cocoa-derived
products.
Production of Chocolate
Chocolate is obtained from nonalkalinized cocoa liquor mixed
with sucrose, cocoa butter, emulsifying agents (lecithins),
flavoring compounds (vanillin), and eventually other ingredi-
ents (milk, hazelnuts, almonds, etc.). Ingredients are mixed to
obtain a homogeneous chocolate paste that is then refined to
obtain finer particles of < 30–40 mm. The refining step is
performed by single or multiple refining rollers. Further key
steps in chocolate production are conching and tempering.
Refined chocolate paste is powdery and has a harsh and sour
flavor, and conching is necessary to obtain fine chocolate of
optimal flavor, smoothness, and texture. Conching is per-
formed in round or oblong rotary conche pots in which the
chocolate mass is mixed, ground, and kneaded at temperatures
of 65–75
C. Conching times vary from a few hours to many
days, depending on the desired final chocolate quality. Gener-
ally during conching steps, flavors, emulsifier, and cocoa butter
are added. The effect of conching on chocolate paste is a
reduction of acidity due to the loss from evaporation of acetic
acid and other volatile compounds, loss of moisture, and a
more uniform distribution of fats that form a film around each
cocoa particle.
Tempering is one of the most critical steps to obtain a
product with a stable crystalline form of cocoa butter respon-
sible of the good melting properties and the glossy surface of
good-quality chocolate. Tempering is performed by cooling
under stirring the cocoa mass derived from conching (from
40–50 to 18–28
C). Cocoa is maintained at this low temper-
ature for about 10 min, and then is heated to 32
C. In this way
the polymorphic form V of cocoa butter is obtained, with a
melting point of 34
C, close to the human body temperature.
Different chocolate typologies are produced (Table 1) and
can be classified according to the different percentages of cocoa
liquor and cocoa butter in the final products or according to
the addition of ingredients different from cocoa. Today much
of the chocolate consumed is in the form of sweet chocolate,
with the addition of sugars (sucrose). Milk chocolate is sweet
chocolate that in addition contains milk powder. White choc-
olate contains cocoa butter, sugar, and milk, but no cocoa
liquor.
188 Cocoa: Production, Chemistry, and Use
Table 1 Examples of formulations for different kind of chocolate
(g/100 g)
Chocolate Chocolate Milk White
Ingredient (50% cocoa) (70% cocoa) chocolate chocolate
Cocoa liquor 35 70 12 – amino acid content recorded in fermented beans ranges from
Added cocoa 15 – 22 30
butter
Sugar 50 30 51 50
Whole milk – – 15 20
powder
Chemical Composition
Lipids
Cocoa fat (cocoa butter) represents about 50–58% of the cocoa
beans, and its triacylglycerols (97–98% of cocoa butter) mainly
consist of palmitic acid (25% of total fatty acids), stearic acid
(37%), and oleic acid (34%), with a low amount of linoleic
acid (3%). Oleic acid is primarily esterified at the 2-position of
glycerol, so the main triacylglycerols are 1,3-dipalmito-2-olein,
1-palmito-3-stearo-2-olein, and 1,3-distearo-2-olein. Cocoa
butter is solid at room temperature and melts at temperatures
between 30 and 40
C, depending on the polymorphic form.
The amount of cocoa butter in chocolate is 21–35%, depend-
ing on the addition of cocoa butter to the cocoa liquor.
Protein, Peptides, Amino Acids, and Amines
Proteins make up 10–15% of the dry weight of cocoa seeds, the
second most abundant constituent after cocoa fat. Proteins are
the cocoa fraction that undergoes the most intensive modifica-
tion during fermentation, where microbiological and enzy-
matic reactions lead to extensive breakdown of cocoa seed
proteins, yielding peptides and amino acids that are the impor-
tant flavor precursors.
Cocoa beans contain four main proteins, albumin, globu-
lin, prolamin, and glutelin. Albumin and globulin are the most
important both quantitatively and qualitatively. Globulins are
vicilin-like storage proteins consisting of three subunits with
molecular masses of 47, 31, and 15 kDa, which are derived
from a common 66-kDa precursor. The albumin fraction was
identified as a 21-kDa cocoa seed protein having trypsin inhib-
itory properties. During fermentation, peptide and free amino
acids increase and total protein concentration decreases. The
globulin protein fraction is the most degraded during fermen-
tation. Cocoa proteins are cleaved to hydrophilic and hydro-
phobic peptides as well as amino acids through autolysis by
two endogenous enzymes, aspartic endoprotease and carboxy-
peptidase. Fermentation of cocoa beans is fundamental for the
activation of these two enzymes by microbial metabolites
(such as acetic acid). Changes in the protein composition of
cocoa beans have been noted not only during fermentation but
also as a consequence of roasting. A decrease in total protein,
free amino acids, and albumin are seen during roasting.
Regarding amino acids composition, nonfermented beans
contain low levels of free amino acids with a 1:1 ratio between
hydrophobic/acidic amino acid, whereas in fermented cocoa
seeds this ratio significantly increases to 3:1. The increase in the
concentrations of hydrophobic amino acids, such as leucine,
alanine, and phenylalanine, is explained by the activity of
carboxypeptidase that releases single hydrophobic amino
acids and aspartic endoprotease, which hydrolyzes proteins
preferentially at the hydrophobic amino acids sites. The free
500 to 1800 mg/100 g, with a prevalence of the hydrophobic
amino acids responsible of the formation of Strecker aldehydes
and pyrazines, important compounds for cocoa aroma.
Among nonprotein amino acids, relevant contents of gamma-
aminobutyric acids, ranging from 30 to 100 mg/100 g, were
detected in fermented cocoa beans, therefore cocoa can be
considered an important natural source of this inhibitory neu-
rotransmitter amino acid.
Low amounts (20 mg kg1) of biogenic amines deriving
from microbial decarboxylation of amino acids occurring dur-
ing fermentation, as 2-phenylethylamine, tyramine and trypt-
amine have been found in cocoa. Biogenic amines can be also
originated by thermal decarboxylation of amino acids, so their
amount is higher in roasted products.
Carbohydrates and Organic Acids
The primary carbohydrates in fermented dried cocoa beans are
starch (6%) and cellulose (9%). Soluble carbohydrates include
glucose, fructose, sucrose (0.08–1.5%), raffinose, and sta-
chyose. Sucrose is partially hydrolyzed during fermentation,
providing reducing sugars precursors of aroma development
during roasting. Fiber fraction aside from cellulose contains
pentoses (1.5%), galactans, and polymers of galacturonic acid.
Fibers are concentrated in cocoa shells.
Organic acids (1.2–1.6%) are primarily formed during
cocoa bean fermentation, and the most represented are acetic
acid (0.2–0.7%), citric acid (0.4–0.7%), and oxalic acid
(0.3–0.5%). Acetic acid is partly lost during cocoa processing,
in particular during conching.
Methylxanthines
Cocoa, as coffee and tea, is generally considered a stimulating
food, due to the high levels of alkaloids. Theobromine and
caffeine in particular are the principal alkaloids found in cocoa.
Theobromine (3,7-dimethylxanthine) represents 1.2–2% of
cocoa beans, where it is partially bound to tannins in cotyledon
cells. During fermentation, the development of acetic acid per-
mits the release of theobromine that migrates from cotyledons to
shells. Cocoa shells in fact contain about 1.5% of theobromine,
and generally they are reused to extract this alkaloid.
Polyphenols
Cocoa is a rich source of polyphenols: the defatted unfermented
cocoa beans contain about 120–180 g kg1 of polyphenolic
compounds, representing one of the most concentrated natural
sources. The polyphenols in cocoa beans are stored in the
pigment cells of the cotyledons. Depending on the amount
of anthocyanins, pigment cells are white to deep purple. Three
groups of polyphenols can be distinguished: catechins or flavan-
3-ols (37%), anthocyanins ( 4%), and proanthocyanidins
( 58%). The main catechin is ()-epicatechin representing up

to 35% of polyphenol content. In smaller amounts, (þ)-catechin
as well as traces of (þ)-gallocatechin and ()-epigallocatechin
have been found. Procyanidins in cocoa consist of oligomers
and polymers of catechin and epicatechin. Anthocyanins iden-
tified in cocoa include cyanidin-3-galactoside and cyanidin-3-
arabinoside. Small quantities of quercetin, quercetin glycosides,
naringenin, luteolin, apigenin, clovamide, and phenolic acids
such as caffeic, ferulic, gallic, and p-coumaric acid have also been
found in cocoa products.
The high level of polyphenols in raw cocoa beans is progres-
sively reduced during cocoa processing. During fermentation
polyphenols diffuse with cell liquids from their storage cells
and undergo oxidation to condensed high-molecular mostly
insoluble tannins. These reactions are both nonenzymatic and
enzymatic catalyzed by polyphenol oxidase. It is reported that
epicatechin and catechin content, respectively, are reduced to
10–70% during fermentation. Roasting causes a dramatic
reduction of some phenolic substances, in particular clovamide,
together with an overall decrease of the antiradical and antiox-
idant properties of cocoa beans/nibs. High temperatures during
the cocoa bean roasting process and also the alkali treatments
on the cocoa powder induce the epimerization reaction of epi-
catechin to catechin, reducing its bioavailability. Moreover,
some recent works have achieved chiral separations of catechin
and epicatechin enantiomers, showing that the prevalent form
of catechin in roasted cocoa products is ()-catechin, a
nonnatural in cocoa beans with less bioavailability than
(þ)-catechin.
Flavor Compounds
Chocolate and cocoa flavors reside in their volatile fraction,
which is composed of a complex mixture of up to 500 com-
pounds. The aromatic profile of cocoa beans is very complex
and is also dependent on the method and duration of fermen-
tation and drying practices applied. Alcohols, aldehydes, and
ketones have been reported as the major groups of compounds
found in raw cocoa and at the beginning of the fermentation
process (1 or 2 days). Alcohols, esters, and acids (acetic acid
mainly) were developed in the middle of fermentation
(3–5 days), becoming the most important groups of volatile
compounds at the end of fermentation (6–8 days). Alcohols,
esters, and pyrazines contents increased during the sun-drying
process.
Cocoa fermentation is crucial not only to the formation of
significant volatile fractions but also for the development of
cocoa–chocolate flavor precursors as amino acids and reducing
sugars. Via Maillard reactions, cocoa roasting converts flavor
precursors formed during fermentation to two main classes of
odorant compounds: pyrazines and Strecker aldehydes, and
three of these had a strong chocolate flavor: 2-ethylpropanal,
2-methylbutanal, and 3-methylbutanal. Pyrazines were recog-
nized as cocoa/nutty notes: 2,3-dimethylpyrazine, trimethyl-
pyrazine, tetramethylpyrazine, 3(or 2),5-dimethyl-2(or
3)-ethylpyrazine, 3,5(or 6)-diethyl-2-methylpyrazine, and fur-
furylpyrrole. Conching has an effect on cocoa aroma, although
no new key odorant is synthesized during the heating process,
and levels of 2-phenyl-5-methyl-2-hexenal, furaneol, and
branched pyrazines are significantly increased, whereas most
Strecker aldehydes are lost by evaporation.
Cocoa: Production, Chemistry, and Use 189
World Production and Human Consumption
The cocoa-producing countries are all developing countries
and localized in Africa, Central America, South America, Asia,
and Oceania. World production of cocoa beans is constantly
growing: it has increased from 31 000 tons in 1880 to more
than 3 000 000 tons in 2002 and about 5 000 000 tons in 2012.
Africa is the continent with the highest production (67%). The
largest state producer is the Ivory Coast (about 30% of world
production). World cocoa production has risen at an average
annual growth rate of 3.3% during the period 2002–12.
Between 2002 and 2011/2012, primary cocoa processing
growth at an average rate of 2.9% per annum was registered.
Europe remained by far the largest cocoa-processing region,
followed by the United States. However, with an annual
growth rate of 5.6%, the largest regional processing increase
occurred in Asia and Oceania. Moreover, in the last 10 years
processing in the countries of origin has increased, supported,
in some countries, by government policies favoring the export
of value-added semifinished products rather than raw cocoa
beans.
European regions are also the largest cocoa consumers,
accounting for 48% of total world consumption of cocoa
followed by the Americas, at 33%. Growing consumption has
been observed in Asia and Africa. Consumption of chocolate
confectionery products increased by 10% between 2002 and
2010 in selected countries, including the major European
countries, the United States, Brazil, Japan, and Australia, corre-
sponding to an annual growth rate of 1.2%. World per capita
consumption of cocoa has also seen a similar pattern of growth
over the review period, rising from 0.54 kg in 2002 to 0.61 kg
in 2010.
Consumer preferences, especially of European and Ameri-
can consumers, has changed over the last 10 years: chocolate
aficionados are asking for single-origin premium chocolate
and ‘high cocoa content’ products, with their own distinctive
flavors. Cocoa industries are driven to use innovation to
appeal to consumers in saturated markets: new flavors,
new packaging, and new sizes but also chocolates with
health-promoting properties for health-conscious consumers.
Sustainable sourcing is also growing in importance for con-
sumers: demand for cocoa grown in a responsible manner
is rising, as a consequence of the companies’ response to
consumer preferences.
See also: Cocoa: Composition and Health Effects.
Further Reading
Afoakwa EO, Paterson A, Fowler M, and Ryan A (2008) Flavor formation and character
in cocoa and chocolate: a critical review. Critical Reviews in Food Science and
Nutrition 48(9): 840–857.
Kratzer U, Frank R, Kalbacher H, Biehl B, Wöstemeyer J, and Voigt J (2009) Subunit
structure of the vicilin-like globular storage protein of cocoa seeds and the origin of
cocoa- and chocolate-specific aroma precursors. Food Chemistry 113: 903–913.
Lima LJR, Almeida MH, Rob Nout MJ, and Zwietering MH (2011) Theobroma cacao L.,
“The food of the Gods”: quality determinants of commercial cocoa beans, with
particular reference to the impact of fermentation. Critical Reviews in Food Science
and Nutrition 51(8): 731–761.
190 Cocoa: Production, Chemistry, and Use
Prabhakaran Nair KP (2010) Cocoa (Theobroma cacao L.). In: The agronomy and
economy of important tree crops of the developing world, pp. 132–180. Boston,
MA: Elsevier.
Rohsius C, Matissek R, and Lieberei R (2006) Free amino acid amounts in raw
cocoas from different origins. European Food Research and Technology
222: 432–438.
Schwan RF and Wheals AE (2004) The microbiology of cocoa fermentation and its role
in chocolate quality. Critical Reviews in Food Science and Nutrition 44(4):
205–221.
Voigt J, Biehl B, Kamaruddin S, and Wazir S (1993) The major seed proteins of
Theobroma cacao L. Food Chemistry 47: 145–151.
Voigt J, Biehl B, Heinrichs H, Kamaruddin S, Gaim arsoner G, and Hugi A (1994)
In-vitro formation of cocoa-specific aroma precursors: aroma-related peptides
generated from cocoa seed protein by co-operation of an aspartic endoprotease and
a carboxypeptidase. Food Chemistry 49: 173–180.
Wollgast J and Anklam E (2000) Review on polyphenols in Theobroma cacao: changes
in composition during the manufacture of chocolate and methodology for
identification and quantification. Food Research International 33: 423–447.
Relevant Websites
http://faostat.fao.org/ – Food and Agriculture Organization of the United Nations.
http://www.icco.org/ – International Cocoa Organization.
http://worldcocoafoundation.org – World Cocoa Foundation.
 Codex Alimentarius
I Stankovic, University of Belgrade, Belgrade, Serbia
ã 2016 Elsevier Ltd. All rights reserved.
About Codex Alimentarius
The Codex Alimentarius, or the food code, is a collection of
internationally adopted food standards and related texts devel-
oped by the Codex Alimentarius Commission (CAC) and pre-
sented in a uniform manner. Codex standards, guidelines, and
codes of practice aim at protecting consumers’ health and
ensuring fair practices in the food trade. Since its beginning,
the Codex Alimentarius evolved in the most important inter-
national reference point in matters concerning food safety and
quality and international food trade.
Codex Alimentarius Commission
The CAC was established by the Food and Agriculture Organiza-
tion (FAO) of the United Nations and the World Health Organi-
zation (WHO), as an intergovernmental body to implement the
Joint FAO/WHO Food Standards Programme, which was estab-
lished by a FAO Conference resolution in 1961 and a World
Health Assembly resolution in 1963. Its principal objective is to
protect the health of consumers and to facilitate the trade of food
by setting international food standards and other texts that can be
recommended to governments for acceptance. The CAC also
coordinates all food standards work done by international gov-
ernmental and nongovernmental organizations.
The CAC is open to the governments of all member nations
or associate members of FAO and WHO. It currently has 185
members, and more than 200 intergovernmental and interna-
tional nongovernmental organizations are accredited as
observers. Codex members cover 99% of the world’s popula-
tion. More and more developing countries are taking an active
part in the Codex process, in many cases assisted by the Codex
Trust Fund, which strives to finance and train participants from
such countries to enable efficient participation. Being an active
member of the Codex helps countries to compete in sophisti-
cated world markets and to improve food safety for their own
population.
The body of Codex Alimentarius consists of CAC, Executive
Committee, General Subject Committees, Commodity Com-
mittees, ad hoc Intergovernmental Task Forces, and FAO/WHO
Coordinating Committees. All Codex Committees, their cur-
rent status, and host governments are presented in Table 1.
The Procedural Manual of the CAC describes the legal foun-
dations and practical functioning of the CAC and its subsidiary
bodies. It sets out the basic rules of procedures for the elabora-
tion of Codex standards and related texts, basic definitions, and
guidelines for the operation of Codex committees. It is intended
to help member governments participate effectively in the work
of the joint FAO/WHO Food Standards Programme.
Member countries communicate with the CAC through the
Codex Contact Points that act as the link between the Codex
Secretariat and member countries in order to coordinate all
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00180-X
relevant Codex activities within their own countries. In order to
provide better transparency and to facilitate more efficient
participation in Codex activities, member countries are encour-
aged to establish National Committees for Codex Alimentarius
that should include representatives of the relevant ministries,
technical experts, decision-makers from the industry, and
consumer organizations.
FAO/WHO Trust Fund for Participation in the Codex
FAO and WHO help developing countries to apply Codex
standards, to strengthen national food control systems, and
to take advantage of international food trade opportunities.
In 2002, FAO and WHO recognized that developing coun-
tries and countries with economies in transition were not
participating fully in the work of the CAC. In response, the
director-generals of WHO and FAO launched the FAO/WHO
Project and Fund for Enhanced Participation in Codex (Codex
Trust Fund) on 14 February 2003 with the objective of helping
developing and transition economy countries enhance their
level of effective participation in the Codex activities.
Codex Standards, Codes of Practice, Guidelines, and
Recommendations
The Codex Alimentarius includes standards for all the principal
foods, whether processed, semiprocessed, or raw, for distribu-
tion to the consumers, containing provisions with respect to
food hygiene, food additives, residues of pesticides and veter-
inary drugs, contaminants, labeling and presentation, methods
of analysis and sampling, and import and export inspection
and certification.
For food safety and nutrition matters, the CAC, as a risk
manager, establishes its standards using the principles of risk
analysis and bases its work on the scientific advice provided by
the independent international risk assessment bodies such as
the Joint FAO/WHO Expert Committee on Food Additives
(JECFA), Joint FAO/WHO Meeting on Pesticide Residues
(JMPR), Joint FAO/WHO Expert Meetings on Microbiological
Risk Assessment (JEMRA), or ad hoc consultations organized
by FAO and WHO. Codex standards also address issues related
to food quality to ensure fair practices in the food trade. Food
standards, guidelines, and recommendations established by
the CAC are recognized as reference points for food under the
relevant World Trade Organization (WTO) agreements. While
being recommendations for voluntary application by mem-
bers, Codex standards serve in many cases as a basis for
national legislation.
Public concerns about food safety issues are often placing
Codex at the center of global debates. Biotechnology, pesticides,
veterinary drug residues, food additives, and contaminants are
some of the issues discussed in Codex meetings. Creating
191
192 Codex Alimentarius

Table 1 List of all Codex Alimentarius bodies with their status and host countries

Acronym Name Status Host

CAC Codex Alimentarius Commission Active FAO (Rome) and WHO

(Geneva)
CCEXEC Executive Committee of the Codex Alimentarius Commission Active FAO (Rome) and WHO
(Geneva)
General subject committees
CCCF Codex Committee on Contaminants in Foods Active The Netherlands
CCFA Codex Committee on Food Additives Active China
CCFAC Codex Committee on Food Additives and Contaminants Renamed and —
reestablished
CCFH Codex Committee on Food Hygiene Active The United States
CCFICS Codex Committee on Food Import and Export Inspection and Certification Systems Active Australia
CCFL Codex Committee on Food Labelling Active Canada
CCGP Codex Committee on General Principles Active France
CCMAS Codex Committee on Methods of Analysis and Sampling Active Hungary
CCNFSDU Codex Committee on Nutrition and Foods for Special Dietary Uses Active Germany
CCPR Codex Committee on Pesticide Residues Active China
CCRVDF Codex Committee on Residues of Veterinary Drugs in Foods Active The United States
Commodity committees
CCCPC Codex Committee on Cocoa Products and Chocolate Adjourned sine die —
CCCPL Codex Committee on Cereals, Pulses and Legumes Adjourned sine die —
CCFFP Codex Committee on Fish and Fishery Products Active Norway
CCFFV Codex Committee on Fresh Fruits and Vegetables Active Mexico
CCFO Codex Committee on Fats and Oils Active Malaysia
CCIE Codex Committee on Edible Ices Abolished —
CCM Codex Committee on Meat Abolished —
CCMMP Codex Committee on Milk and Milk Products Adjourned sine die —
CCMPH Codex Committee on Meat Hygiene Adjourned sine die —
CCNMW Codex Committee on Natural Mineral Waters Adjourned sine die —
CCPFV Codex Committee on Processed Fruits and Vegetables Active The United States
CCPMPP Codex Committee on Processed Meat and Poultry Products Abolished —
CCS Codex Committee on Sugars Active Colombia
CCSB Codex Committee on Soups and Broths Abolished —
CCSCH Codex Committee on Spices and Culinary Herbs Active India
CCVP Codex Committee on Vegetable Proteins Adjourned sine die —
Ad hoc Intergovernmental task forces
CGECPMMP Joint FAO/WHO Committee of Government Experts on the Code of Principles Renamed and —
Concerning Milk and Milk Products reestablished
CXTO Joint CODEX/IOOC Meeting on the Standardization of Table Olives Abolished —
GEFJ Joint ECE/Codex Alimentarius groups of experts on standardization: Fruit Juices Abolished —
GEQFF Joint ECE/Codex Alimentarius groups of experts on standardization: Quick Frozen Abolished —
Foods
TFAF Ad Hoc Intergovernmental Task Force on Animal Feeding Dissolved —
TFAMR Ad hoc Codex Intergovernmental Task Force on Antimicrobial Resistance Dissolved —
TFFBT Ad Hoc Intergovernmental Task Force on Food Derived from Biotechnology Dissolved —
TFFJ Ad Hoc Intergovernmental Task Force on Fruit and Vegetable Juices Dissolved —
TFPHQFF Ad hoc Codex Intergovernmental Task Force on the Processing and Handling of Dissolved —
Quick Frozen Foods
FAO/WHO coordinating committees
CCAFRICA FAO/WHO Coordinating Committee for Africa Active Cameroon
CCASIA FAO/WHO Coordinating Committee for Asia Active Japan
CCEURO FAO/WHO Coordinating Committee for Europe Active The Netherlands
CCLAC FAO/WHO Coordinating Committee for Latin America and the Caribbean Active Costa Rica
CCNASWP FAO/WHO Coordinating Committee for North America and South West Pacific Active Papua New Guinea
CCNEA FAO/WHO Coordinating Committee for Near East Active Lebanon

standards that at the same time protect consumers and ensure
fair practices in the sale of food is a process that involves special-
ists in numerous food-related scientific disciplines, together with
consumer organizations, production and processing industries,
food control administrators, and traders. The finalization of
food standards and their compilation into a code that is credible
and authoritative require extensive consultation and the collec-
tion and evaluation of information, followed by confirmation of
final results and sometimes objective compromise to satisfy
differing sound, scientifically based views.
 Codex Alimentarius 193

There are two main groups of Codex standards: Codex gen-
eral standards such as the General Standard for Food Additives,
General Standard for Contaminants and Toxins in Food and
Feed, and General Standard for the Labelling of Prepackaged
Foods and specific standards of which the largest number is the
group called ‘commodity standards.’ Representatives of some of
the major commodity standards included in the Codex with a
year of last modification are presented in the Table 2.
Commodity standards tend to follow a fixed format set out
in the Procedural Manual of the CAC. The format consists of
the following categories of information:
– Scope includes the name of the food to which the standard
applies and, in most cases, the purpose for which the com-
modity will be used.
– Description includes a definition of the product or products
covered with an indication, where appropriate, of the raw
materials from which they are derived.
– Essential composition includes information on the composi-
tion and identity characteristics of the commodity, as well as
any compulsory and optional ingredients.
– Food additives includes the names of the additives and the
maximum amount permitted to be added to the food. Food
additives must be cleared by FAO and WHO for their safety,
and the use of food additives must be consistent with the
Codex General Standard for Food Additives.
– Contaminants contains the limits for contaminants that may
occur in the product(s) covered by the standard. These limits
are based on the scientific advice of FAO and WHO and must
be consistent with the Codex General Standard for Contami-
nants and Toxins in Food and Feed. Where appropriate, refer-
ence is also made to the Codex maximum limits for pesticide
residues and for residues of veterinary drugs in foods.
– Hygiene makes reference to relevant Codex Codes of
Hygienic Practice for the commodity concerned. In almost
all cases, it is required that the product shall be free from
pathogenic microorganisms or any toxins or other poison-
ous or deleterious substances in amounts that represent a
hazard to health.
– Weights and measures contains provisions such as fill of the
container and the drained weight of the commodity.
– Labelling includes provisions on the name of the food and
any special requirements to ensure that the consumer is not
deceived or misled about the nature of the food. These pro-
visions must be consistent with the Codex General Standard
for the Labelling of Prepackaged Foods. Requirements for
the listing of ingredients and date-marking are specified.
– Methods of analysis and sampling contains a list of the test
methods needed to ensure that the commodity conforms to
the requirements of the standard. References are made to
internationally recognized test methods that meet the CAS’s
criteria for accuracy, precision, etc.
The Codex Alimentarius contains more than 200 standards in
the prescribed format for individual foods or groups of foods.
Codex codes of practice define the production, processing,
manufacturing, transport, and storage practices for individual
foods or groups of foods that are considered essential to ensure
the safety and suitability of food for consumption. For food
hygiene, the basic text is the Codex General Principles of Food
Hygiene, which introduces the use of the Hazard Analysis and
Critical Control Point (HACCP) food safety management sys-
tem. A code of practice on the control of the use of veterinary
drugs provides general guidance in this area.
Codex guidelines fall into two categories:
– Principles that set out policy in certain key areas
– Guidelines for the interpretation of these principles or for
the interpretation of the provisions of the Codex general
standards
In the cases of food additives, contaminants, food hygiene, and
meat hygiene, the basic principles governing the regulation of

Table 2 Representatives of the Codex commodity standards and the year of last modification

Reference Title Last modified

CODEX STAN 199-1995 Standard for Wheat and Durum Wheat 1995
CODEX STAN 152-1985 Standard for Wheat Flour 1995
CODEX STAN 198-1995 Standard for Rice 1995
CODEX STAN 171-1989 Standard for Certain Pulses 1995
CODEX STAN 175-1989 Standard for Soy Protein Products 1989
CODEX STAN 211/1999 Standard for Named Animal Fats 2013
CODEX STAN 210/1999 Standard for Named Vegetable Oils 2013
CODEX STAN 33-1981 Standard for Olive Oils and Olive Pomace Oils 2013
CODEX STAN 311-2013 Standard for Smoked Fish, Smoke-Flavoured Fish and Smoke-Dried Fish 2013
CODEX STAN 3-1981 Standard for Canned Salmon 2011
CODEX STAN 219-1999 Standard for Grapefruits (Citrus paradisi) 2011
CODEX STAN 260-2007 Standard for Pickled Fruits and Vegetables 2007
CODEX STAN 247-2005 General Standard for Fruit Juices and Nectars 2005
CODEX STAN 89-1981 Standard for Luncheon Meat 1991
CODEX STAN 96-1981 Standard for Cooked Cured Ham 1991
CODEX STAN 207-1999 Standard for Milk Powders and Cream Powder 2014
CODEX STAN 275-1973 Standard for Cream Cheese 2010
CODEX STAN 212-1999 Standard for Sugars 2001
CODEX STAN 86-1981 Standard for Cocoa Butter 2001
CODEX STAN 87-1981 Standard for Chocolate 2003
194 Codex Alimentarius

these matters are built into the relevant standards and codes of
practice.
There are free-standing Codex principles covering the
– addition of essential nutrients to foods,
– food import and export inspection and certification,
– establishment and application of microbiological criteria for
foods,
– conduct of microbiological risk assessment (MRA),
– risk analysis of foods derived from modern biotechnology.
Interpretative Codex guidelines include those for food labe-
ling, especially the regulation of claims made on the label. This
group includes guidelines for nutrition and health claims;
conditions for production, marketing, and labeling of organic
foods; and foods claimed to be ‘halal.’ There are several guide-
lines that interpret the provisions of the Codex Principles for
Food Import and Export Inspection and Certification and
guidelines on the conduct of safety assessments of foods from
genetically modified plants and microorganisms.
The uniform Codex procedure for the Elaboration of Codex
Standards and Related Texts, according to the CAC Procedural
Manual, includes eight steps (Figure 1).
An alternative accelerated 5-step procedure for the elaboration
of Codex standards and related texts, as a subject of an accelerated

Step 1: The Commission decides, taking into account the outcome of the critical review
conducted by the Executive Committee, to elaborate a World-wide Codex Standard and
also decides which subsidiary body or other body should undertake the work.

Step 2: The Secretariat arranges for the preparation of a proposed draft standard.

Step 3: The proposed draft standard is sent to Members of the Commission and interested
international organizations for comment on all aspects.

Step 4: The comments received are sent by the Secretariat to the subsidiary body or other body
concerned which has the power to consider such comments and to amend the proposed
draft standard.

Step 5: The proposed draft standard is submitted through the Secretariat to the Executive
Committee for critical review and to the Commission with a view to its adoption as a
draft standard.

Step 6: The approved draft standard is sent again by the Secretariat to all Members and
interested international organizations for comment on all aspects, including possible
implications of the draft standard for their economic interests.

Step 7: The comments received are sent by the Secretariat to the subsidiary body or other body
concerned, which has the power to consider such comments and amend the draft
standard.

Step 8: The draft standard is submitted through the Secretariat to the Executive Committee for
critical review and to the Commission, together with any written proposals received from
Members and interested international organizations for amendments with a view to its
adoption as a Codex standard.

Figure 1 An 8-step procedure for the elaboration of Codex standards.


elaboration process, may also be performed if it is identified by
the CAC, or the relevant subsidiary body. The accelerated proce-
dure omits steps 5, 6, and 7 of the regular Codex procedure.
Codex Contact Points are responsible for ensuring that
papers are circulated to those concerned within their own
country and for ensuring that all necessary action is taken by
the date specified.
Risk Analysis
As defined by the CAC, the risk analysis should follow a struc-
tured approach comprising the three distinct but closely linked
components of risk analysis: risk assessment, risk management,
and risk communication, each component being integral to the
overall risk analysis. There should be a functional separation of
risk assessment and risk management, in order to ensure the
scientific integrity of the risk assessment, to avoid confusion
over the functions to be performed by risk assessors and risk
managers, and to reduce any conflict of interest. Effective com-
munication and consultation with all interested parties should
be ensured throughout the risk analysis.
Scientific Basis for Codex Work
Codex committees, when developing standards, apply risk
analysis and rely on the independent scientific advice provided
by expert bodies organized by FAO and WHO. These bodies
also give direct advice to member governments.
From the very beginning, Codex activity is based on the
sound science. Experts in a wide range of scientific disciplines
have contributed to every aspect of the code to ensure that its
standards withstand the most rigorous scientific scrutiny.
The main principles of developing scientific advice are as
follows:
– Excellence: Use of internationally recognized expertise, sup-
ported by the creation of a platform for global scientific
discussions based on best practices in elaborating guidance.
– Independence: Experts contribute in their own capacity and
not on behalf of a government or institution. They are
required to declare possible conflicts of interest.
– Transparency: Procedures and methods to ensure all inter-
ested parties understand the processes for the development
of scientific advice and have access to the reports, safety
assessments and evaluations, and other basic data.
– Universality: A broad base of scientific data is critical for the
elaboration of international standards-setting activities.
Therefore, institutions and all interested parties throughout
the world are invited to make data available.
Experts responsible for risk assessment should be selected in a
transparent manner on the basis of their expertise, experience,
and independence with regard to the interests involved.
Risk assessment should incorporate the four steps, hazard
identification, hazard characterization, exposure assessment,
and risk characterization, and should be based on all available
scientific data and on realistic exposure scenarios, with consid-
eration of different situations that include consideration of
susceptible and high-risk population groups.
FAO/WHO expert bodies responsible for the scientific
advice to CAC and their subsidiary committees are the JECFA,
Codex Alimentarius 195
the JMPR, the JEMRA, and other ad hock expert groups con-
ducted by FAO and WHO.
The JECFA is an international expert scientific committee
administered by FAO and WHO. It has been meeting since
1956, initially to evaluate the safety of food additives. Its
work now also includes the evaluation of contaminants, natu-
rally occurring toxicants, and residues of veterinary drugs in
food. JECFA has evaluated more than 2600 food additives,
approximately 50 contaminants and naturally occurring toxi-
cants, and residues of approximately 95 veterinary drugs. The
committee has also developed principles for safety assessment
of chemicals in foods that are consistent with current thinking
on risk assessment and take account of developments in toxi-
cology and other relevant sciences.
The JMPR is an expert ad hoc body administered jointly by
FAO and WHO in the purpose of harmonizing the require-
ment and the risk assessment on the pesticide residues. The
JMPR has met annually since 1963 to conduct scientific evalu-
ations of pesticide residues in food. It provides advice on the
acceptable levels of pesticide residues in food moving in inter-
national trade.
The JEMRA began in 2000 in response to requests from the
CAC and FAO and WHO member countries and the increasing
need for risk-based scientific advice on microbiological food
safety issues. JEMRA aims to develop and optimize the utility of
MRA as a tool to inform actions and decisions aimed at improv-
ing food safety and to make it equally available to both develop-
ing and developed countries. JEMRA focuses on the following
main areas of work: providing risk assessments for selected path-
ogens (pathogen–commodity combination risk assessments) to
the CAC and to member states, developing guidelines for risk
assessment of microbiological hazards in food and water, and
providing expert advice on risk management.
FAO and WHO provide expert scientific advice on many
aspects of food quality, safety, and nutrition relevant to the
work of the CAC. While not officially part of the CAC structure,
the FAO/WHO expert consultations provide independent sci-
entific expert advice to the commission and its specialist
committees and task forces. The issues being addressed include
biotechnology and nanotechnologies.
Codex and International Food Trade
During the last century, a quantity and variety of food traded
internationally has grown exponentially. According to FAO
statistics, international food trade is more than 500 billion
dollars per year market, with billions of tonnes of food pro-
duced, transported, and marketed. The increasing global mar-
ket requires uniform food standards and the Codex
international food standards, guidelines, and codes of practice
contribute to the safety, quality, and fairness of this interna-
tional food trade. Consumers can trust the safety and quality of
the food they buy and importers and distributers can trust that
the quality of food they ordered will be in accordance with
their specifications. Reference to the Codex Alimentarius
occurs in many bilateral and plurilateral trade agreements.
The WTO Agreement on the Application of Sanitary and
Phytosanitary Measures (SPS Agreement) and the Agreement
on Technical Barriers to Trade (TBT Agreement) both encour-
age the international harmonization of food standards, and
196 Codex Alimentarius
Codex standards have become the benchmarks against which
national food measures and regulations are evaluated within
the legal parameters of WTO agreements. WTO members that
wish to apply stricter food safety measures than those set by
Codex may be required to justify these measures scientifically.
See also: Codex Alimentarius Commission: Role in International Food
Standards Setting; Food and Agriculture Organization of the United
Nations; Food Classification and Description; Food Composition
Databases; Food Fraud; HACCP and ISO22000: Risk Assessment in
Conjunction with Other Food Safety Tools Such as FMEA, Ishikawa
Diagrams and Pareto; Heavy Metal Toxicology; Nutrition and Health
Claims for Food: Regulatory Controls, Consumer Perception, and
Nutrition Labeling.
Further Reading
Codex Alimentarius (2006) Understanding the Codex Alimentarius, 3rd ed. Rome:
Codex Secretariat FAO.
Codex Alimentarius (2007) Food labelling, 5th ed. Rome: FAO and WHO.
Codex Alimentarius (2007) Working principles for risk analysis for food safety for
application by governments, 1st ed. Rome: FAO and WHO.
Codex Alimentarius (2012) Prevention and reduction of food and feed contamination,
1st ed. Rome: World Health Organization and Food and Agriculture Organization of
the United Nations.
Food and Agriculture Organization of the United Nations and World Health Organization
(2007) FAO/WHO Framework for the provision of scientific advice on food safety
and nutrition, 1st ed. Rome: FAO Food Quality & Standards Service (AGNS).
Food and Agriculture Organization of The United Nations and World Health Organization
(2011) FAO/WHO Guide for application of risk analysis principles and procedures
during food safety emergencies, 1st ed. Rome: FAO Food Quality & Standards
Service (AGNS).
Joint FAO/WHO Food Standards Programme (2013) Codex Alimentarius Commission
procedural manual, 21st ed. Rome: FAO and WHO.
Relevant Websites
www.codexalimentarius.net – Codex Alimentarius.
www.fao.org – Food and Agriculture Organization of the United Nations (FAO).
http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/en/ – JECFA at FAO.
http://www.fao.org/agriculture/crops/core-themes/theme/pests/jmpr/en/ – JMPR at
FAO.
http://www.fao.org/food/food-safety-quality/scientific-advice/jemra/en/ – JEMRA at
FAO.
http://www.fao.org/food/food-safety-quality/scientific-advice/other-scientific-advice/
en/ – Other scientific advice at FAO.
ftp://ftp.fao.org/codex – Codex ftp link.
www.standardsfacility.org – Standards and Trade Development Facility.
www.who.int/foodsafety/codex/trustfund/en/ – Codex Trust Fund.
www.who.int – World Health Organization (WHO).
http://www.who.int/foodsafety/chem/jecfa/en/ – JECFA at WHO.
http://www.who.int/foodsafety/chem/jmpr/en/ – JMPR at WHO.
http://www.who.int/foodsafety/micro/jemra/en/ – JEMRA at WHO.
http://www.who.int/foodsafety/en/ – Other scientific advice at WHO.
www.wto.org – World Trade Organization (WTO).
 Codex Alimentarius Commission: Role in International Food Standards Setting
V Kotwal, Telecom Regulatory Authority of India, New Delhi, India
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Since ancient times, food standards have been laid down by
competent authorities or governments for the protection of
consumers and the facilitation of trade. Food laws are enacted
to protect the consumers against unsafe, adulterated, and mis-
branded food, and also to facilitate the movement of food
across borders. With technological advancements in storage
and transportation, the twentieth century has witnessed an
exponential increase in food trade. However, the independent
development of food standards in different countries led to
barriers in trade.
The establishment of the Food and Agriculture Organiza-
tion (FAO) in 1945 and the World Health Organization
(WHO) in 1948 led to an increased focus on food and health
and thus began a series of joint expert meetings. In 1950,
experts at the first meeting of the Joint FAO/WHO Expert
Committee on Nutrition stated, “Food regulations in different
countries are often conflicting and contradictory. Legislation
governing preservation, nomenclature, and acceptable food
standards often varies widely from country to country. New
legislations not based on scientific knowledge is often intro-
duced, and little account may be taken of nutritional principles
in formulation regulations.” The Committee noted that the
conflicting nature of regulations may be an obstacle to trade
and may therefore affect the distribution of nutritionally valu-
able food and suggested that FAO and WHO study these prob-
lems more closely.
The fourth session of the FAO/WHO Expert Committee on
Nutrition stated, “The increasing, and sometimes insufficiently
controlled, use of food additives, has become a matter of
public and administrative concern.” It also noted that the
means of solving problems related to food additives may differ
from country to country, and this fact “must in itself occasion
concern, since the existence of widely differing control mea-
sures may well form an undesirable deterrent to international
trade.” In the same year, 1955, the FAO and WHO convened
the first joint FAO/WHO Conference on Food Additives. The
Joint FAO/WHO Expert Committee on Food Additives (JECFA)
began work and, in its first meeting, articulated the general
principles for the use of food additives; a text that still forms
the framework for the consideration of food additive use.
At the same time, there were other developments taking
place at the international level involving food standards. In
1958, the United Nations Economic Commission for Europe
(UNECE) established the Geneva Protocol, in which a harmo-
nized layout for food commodity standards was proposed. The
layout still forms the basis of most food commodity standards
worldwide, including Codex standards. Similarly, the Interna-
tional Dairy Federation, founded in 1903, had worked on
standards and labeling requirements for milk and milk prod-
ucts. This work was taken over by the Joint FAO/WHO Expert
Committee of Government Experts on the Code of Principles
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00397-4
concerning Milk and Milk Products. The Committee developed
formal procedures for the elaboration of its standards, which
involved consultation with the governments between the
meetings of the Committee itself. After the Second World
War, there was a regional effort to harmonize the food stan-
dards in Latin America and Europe.
In February, 1961, the Director-General of the FAO, B.R.
Sen, actively entered into discussion with the WHO, the
UNECE, the Organisation for Economic Cooperation and
Development, and the Council of the Codex Alimentarius
Europaeus, with proposals that would lead to the establish-
ment of Joint FAO/WHO Food Standards Programme. In
November, 1961, the eleventh session of the conference of
the FAO passed the resolution by which the Codex Alimentar-
ius Commission (CAC) was established.
The Joint FAO/WHO Food Standards Conference, con-
vened in Geneva in 1962, established the framework for coop-
eration between the two agencies. The CAC was to be the body
responsible for implementing the Joint FAO/WHO Food Stan-
dards Programme, and it was also endorsed in May 1963 at the
sixteenth World Health Assembly.
The first session of the CAC was held in Rome in October
1963, and it was attended by delegates from 30 countries
and 16 international organizations. Currently, the CAC has
186 Codex Members, 185 member countries, and 1 member
organization (EU); 220 Codex Observers, 50 international
governmental organizations, 154 nongovernmental organiza-
tions (NGOs), and 16 UN agencies. The thirty-seventh session
of CAC, held in 2014 in Geneva, was attended by delegates
from 170 countries; one Member organization; and 28 inter-
national governmental, and nongovernmental organizations,
including UN agencies. The headquarters of Codex Alimentar-
ius is based in Rome and the Codex Secretariat is also located in
the FAO building in Rome. However, the meetings of CAC are
held in every alternate year in either Rome or Geneva.
Purpose
The Codex Alimentarius is a collection of internationally
adopted food standards and related texts presented in a uni-
form manner. It develops harmonized international food stan-
dards, guidelines, and codes of practice to protect the health of
consumers, and to ensure fair practices in the food trade. These
food standards and related texts aim at protecting consumers’
health and ensuring fair practices in the food trade. The pub-
lication of the Codex Alimentarius is intended to guide and to
promote the elaboration and establishment of definitions
and requirements for foods to assist in their harmonization,
and, in doing so, to facilitate international trade. The Commis-
sion also promotes the coordination of all food standards
work undertaken by international governmental and non-
governmental organizations.
197
198 Codex Alimentarius Commission: Role in International Food Standards Setting

Scope Structure of Codex Alimentarius Commission

The Codex Alimentarius includes standards for all the principle
foods, whether processed, semiprocessed, or raw, for distribu-
tion to the consumer. Materials for further processing into
foods should be included to the extent necessary to achieve
the purposes of the Codex Alimentarius, as defined. The Codex
Alimentarius includes standards/guidelines/codes of practices
for food hygiene, food additives, residues of pesticides and
veterinary drugs, contaminants, commodities (e.g., milk,
meat, fruits and vegetables, and processed food), labeling and
presentation, methods of analysis and sampling, and import
and export inspection and certification. Thus, it looks at both
horizontal and vertical standard setting so far as food is
concerned.
Codex standards and related texts are not a substitute for, or
an alternative to, national legislation. Every country’s laws and
administrative procedures contain provisions with which it is
essential to comply. Codex standards and related texts contain
requirements for food aimed at ensuring for the consumer a
safe, wholesome food product free from adulteration, correctly
labeled and presented. A Codex standard for any food or foods
should be drawn up in accordance with the Format for Codex
Commodity Standards and contain, as appropriate, the sec-
tions listed therein.
In 1995, the Codex standards, guidelines, and Codes of
practice became a reference for food safety in the World
Trade Organisation (WTO) Agreement on Sanitary and Phyto-
sanitary measures. The only other organizations mentioned are
the World Organization for Animal Health for animal health
issues and the International Plant Protection Convention for
plant health.
and Its Subsidiary Bodies
The CAC consists of the following main organizational ele-
ments, each of which has a role to play in the achieving the
mandate of the Commission (Figure 1):
(a) The Commission
(b) The Executive Committee
(c) The Codex Subsidiary bodies
(d) The Codex Secretariat
Codex Alimentarius Commission
While passing the resolutions at the eleventh session of the
FAO Conference in 1961 and the sixteenth World Health
Assembly in 1963, which led to the establishment of the
Codex Alimentarius Commission, the two bodies also adopted
the Statues and Rules of Procedure for the CAC, the main
decision making body of the Joint FAO/WHO Food Standards
Programme. The legal basis for the Commission’s work is
provided by the Statutes. The Membership to the Commission
is open to all Member Nations and Associate Members of the
FAO and/or WHO. The Commission meetings, which are held
annually (in July) in Rome or Geneva, are chaired by the
Chairperson, and he/she is assisted by the three Vice-
Chairpersons.
Executive Committee of the Codex Alimentarius Commission
The Executive Committee consists of the Chairperson and
three Vice-Chairpersons of the Commission, as well as the

Codex
Alimentarius
Commission

Executive
Secretariat
Committee

Ad hoc Inter- Joint FAO/WHO

General Subject Commodity Regonal
governmental
Committees Committees Coordinating
Task Force
Committees

Presently, there Currently, there 5 Ad hoc There are 6

are 10 active are 6 Commodity Intergovernment Regional
General Subject Commiitteees, for al Task Forces Coordinating
Committees, for e.g. Fresh Fruits have been Committees for
e.g. on food and Vegetables, established till e.g., Near East,
labelling, hygiene Spices & Culinary date & none is Asia, Europe,
etc. Herbs etc. active now. Latin America etc.

Figure 1 Organizational structure of the Codex Alimentarius Commission.

 Codex Alimentarius Commission: Role in International Food Standards Setting
Regional Coordinators, together with seven further Members
elected by the Commission at regular sessions from among the
Members of the Commission, one each coming from the follow-
ing geographic locations: Africa, Asia, Europe, Latin America and
the Caribbean, Near East, North America, and South-West
Pacific. The Executive Committee acts on behalf of the Commis-
sion as its executive organ between sessions of the Commission.
In particular, the Executive Committee may make proposals to
the Commission regarding general orientation, strategic plan-
ning, and programming of the work of the Commission; study
special problems; and assists in the management of the
Commission’s program of standards development, namely by
conducting a critical review of proposals to undertake work and
by monitoring the progress of standards development.
Subsidiary Bodies Under the CAC
There are two kinds of Subsidiary bodies, the Codex Commit-
tees, which prepare draft standards for submission to the Com-
mission and the Coordinating Committees, through which
regions or groups of countries coordinate food standards activ-
ities in the region. The Commission may also approve a third
type of subsidiary body called a Codex ad hoc Intergovernmen-
tal Task Force (TF), which is a Codex Committee with very
limited terms of reference (TOR), established for a fixed period
of time. Section V of the CAC Procedural Manual enlists the
table of Committees and their TORs.
The Codex Committees are further divided into the General
Subject Committees and the Commodity Committees.
General subject committees
These Committees are so called because their work has rele-
vance for all Commodity Committees, and this work applies
across all Commodity Committees. These are sometimes
referred to as ‘horizontal committees.’ Currently, the following
ten General Subject Committees are functioning:
• Codex Committee on Contaminants in Foods (CCCF)
• Codex Committee on Food Additives (CCFA)
• Codex Committee on Food Hygiene (CCFH)
• Codex Committee on Food Import and Export Inspection
and Certification Systems (CCFICS)
• Codex Committee on Food Labeling (CCFL)
• Codex Committee on General Principles (CCGP)
• Codex Committee on Methods of Analysis and Sampling
(CCMAS)
• Codex Committee on Nutrition and Foods for Special Die-
tary Uses (CCNFSDU)
• Codex Committee on Pesticide Residues (CCPR)
• Codex Committee on Residues of Veterinary Drugs in foods
(CCRVDF)
Commodity Committees
Commodity Committees are responsible for developing stan-
dards for specific foods or classes of foods based on the compo-
sition. They are often referred to as the ‘vertical standards.’ These
Committees convene as necessary and go into recess or are
abolished when their work is complete. There are currently six
active Commodity Committees, as follows:
• Codex Committee on Fish and Fishery Products (CCFFP)
199
• Codex Committee on Fresh Fruits and Vegetables (CCFFV)
• Codex Committee on Spices and Culinary Herbs (CCSCH)
• Codex Committee on Fats and Oils (CCFO)
• Codex Committee on Processed Fruits and Vegetables
(CCPFV)
• Codex Committee on Sugars (CCS)
The following Commodity Committees work through corre-
spondence or are in recess:
• Codex Committee on Cereals, Pulses, and Legumes
(CCCPL)
• Codex Committee on Cocoa Products and Chocolate
(CCCPC)
• Codex Committee on Meat Hygiene (CCMH)
• Codex Committee on Milk and Milk Products (CCMMP)
• Codex Committee on Natural Mineral Waters (CCNMW)
• Codex Committee on Sugars (CCS)
The following Commodity Committees that have been abol-
ished are as follows:
• Codex Committee on Edible Ices (CCIE)
• Codex Committee on Meat (CCM)
• Codex Committee on Meat and Poultry Products (CCMPP)
• Codex Committee on Soups and Broths (CCSB)
Joint FAO/WHO Regional Coordinating Committees
The Membership is open to all Member Nations and Associate
Members of the FAO and/or WHO, which are also members of
the CAC within the relevant geographic location. There are six
Regional Coordinating Committees, as given below:
• FAO/WHO Coordinating Committee for Africa
(CCAFRICA)
• FAO/WHO Coordinating Committee for Asia (CCASIA)
• FAO/WHO Coordinating Committee for Europe
(CCEURO)
• FAO/WHO Coordinating Committee for Latin America and
the Caribbean (CCLAC)
• FAO/WHO Coordinating Committee for North America
and South-West Pacific (CCNASWP)
• FAO/WHO Coordinating Committee for Near East
(CCNEA)
Each Committee meets once in a year or once every 18 months
or 2 years, depending upon its nature. The Codex Alimentarius
Commission has designated a member country of the Com-
mission, which has indicated its willingness to accept financial
and other responsibility, such as appointing a Chairperson of
the Committee. This country is referred to as the host country.
The host country is responsible for appointing the Chairperson
of the Committee from among its own nationals. Should this
person for any reason be unable to take the chair, the host
country shall designate another person to perform the func-
tions of the chairperson for as long as the chairperson is unable
to do so.
Ad hoc Intergovernmental Task Force
As per Statute 7 of the CAC, like other subsidiary bodies, the
Commission may establish an ad hoc Intergovernmental Task
Force (TF) for a specific purpose and specified period.
200 Codex Alimentarius Commission: Role in International Food Standards Setting
Presently, there are no TFs that are active. However, the follow-
ing TFs were established and then abolished on completion of
their assigned work as per their TORs:
• Task Force on Animal Feeding (TFAF): 1999–2004 and
2011–2013
• Task Force on Foods Derived from Biotechnology (TFFBT):
1999–2003 and 2004–2008
• Task Force on Fruit and Vegetable Juices (TFFJ): 1999–2005
• Task Force on the Processing and Handling of Quick Frozen
Foods (TFPHQFF): 2006–2008
• Task Force on Antimicrobial Resistance (TFAM):
2006–2011
The Codex Secretariat
The Codex Secretariat is located at FAO headquarters in Rome.
The Secretariat organizes the meetings of the Commission
and the Executive Committee, and it facilitates the work of
the Subsidiary bodies in close coordination with the secretariat
of the host country.
Scientific Basis for Codex Work
Within Codex, risk analysis is defined as “a process consisting
of risk assessment, risk management, and risk communica-
tion” (Figure 2). It is a structured, systematic approach that
examines the potential adverse health effect consequential to a
hazard, or conditions of food, and it develops options for
mitigating the risk. Risk analysis should be:
• applied consistently;
• open, transparent, and documented;
• conducted in accordance with both the Statements of Prin-
ciple Concerning the Role of Science in the Codex Decision-
making Process and the extent to which other factors are
Risk
Assessment
(Science
Based)
Risk Management
(Policy Based)
Risk
Communication
Figure 2 Process of risk analysis.
taken into account and the Statements of Principle Relating
to the Role of Food Safety Risk Assessment; and
• evaluated and reviewed as appropriate in the light of newly
generated scientific data.
Risk analysis is an integral part of the decision-making process
of the Codex, and the Commission has adopted the definition
of risk analysis, as well as the Working principles for risk analysis
for application in the framework of the Codex Alimentarius. Risk-
analysis principles to be applied by the Committees on Food
Additives; Contaminants in Food; Residues of Veterinary Drugs
in Foods; Pesticide Residues; Food Hygiene and the Nutrition
and Food for Special Dietary Uses have been adopted by the
Commission.
Risk Assessment
When developing standards, Codex Committees apply risk
analysis and rely on the independent scientific advice provided
by expert bodies organized by the FAO and WHO, as illustrated
in Figure 3. However, scientific advice may also be provided
through a series of ad hoc meetings on a given topic or ad hoc
expert consultations that are convened only once to address a
specific topic.
These bodies also give direct scientific advice to Member
Governments. There are four FAO/WHO expert committees,
namely: (1) Joint FAO/WHO Expert Committee on Food
Additives (JECFA); (2) Joint FAO/WHO Meeting on Pesticide
Residues (JMPR); (3) Joint FAO/WHO Expert Meetings on
Microbiological Risk Assessment (JEMRA); and (4) Joint
FAO/WHO Expert Meeting on Nutrition (JEMNU), with
other scientific advice provided by the FAO and WHO. The
funds for Codex Scientific Advice are primarily provided by the
FAO and WHO, and several countries also support the FAO
and WHO in this important work.
A four-step process for risk assessment (Figure 4) is fol-
lowed by these bodies.
Advice from the JECFA is considered by CCFA, CCCF, and
CCRVDF, as it is responsible for providing scientific advice on:
• Food additives
• Contaminants in foods
• Residues of veterinary drugs in foods
Similarly, the JMPR is responsible for evaluating exposure to
pesticides and recommending maximum residue levels (MRLs)
for the new and periodic review of compounds for dietary
intake purposes. There is well-established criteria for the prior-
itization of compounds for evaluation by the JMPR by the
CAC. It is thus responsible for performing the risk assessment
upon which the CCPR and, ultimately, the CAC base their risk
management decisions.
The JEMRA are commissioned by the CCFH through the
FAO and WHO for performing international risk assessments
upon which the CCFH and the CAC base microbiological risk
management options. The specific request to the FAO or WHO
for microbiological risk assessment by the CCFH will include
the risk profile document, a clear statement of purpose and the
scope of the work to be undertaken, any time constraints facing
the committee that could impact the work, and the specific risk
management questions to be addressed by the risk assessors.
 Codex Alimentarius Commission: Role in International Food Standards Setting 201

International Risk International Risk

Assessment Manager

JECFA
(food additives,
contaminants,veteri
nary drug residues)

JMPR Scientific
(pestcide residue in Advice
food)

Codex Alimentarius
Commission
JEMRA
(microbiological
hazards in food)

Request
for
Ad hoc expert scientific
consultations on advice
different areas

Figure 3 Roles of risk assessor and risk manager.

• Identification of biological, chemical and physical agents capable of causing

adverse health effects
Hazard
identification
• May be present in a paticular food or groups of food

• Qualitative and/or quantitative evaluation of the nature of the adverse health

efffect associated with biological, chemical and physical agents which may be
Hazard present in food
characterization

• Qualitative and/or quantitative evaluation of the likely intake of biological,

chemical and physical agents via food as well as exposures from other sources, if
Exposure relevant
Assessment

• Qualitaive and/or qiuantitative estimation based,including related uncertainities,

of the probability of occurrrence and severity of known or potential adverse health
Risk effects in a given pouplation based on hazrard indetification, characterization and
Characterization exposure assessment

Figure 4 A four-step risk assessment process.

202 Codex Alimentarius Commission: Role in International Food Standards Setting
Call for data
Codex Alimentarius
Commission
Calll for experts
Issues and priorties
FAO,WHO member
countries
Figure 5 Process for the provision of scientific advice by expert bodies.
So far as the nutritional risk assessment advice to the
CCNFSDU and CAC is concerned, the FAO and WHO are
acknowledged as the primary sources of this advice.
Procedure for the provision of scientific advice by expert
bodies
The steps involved in the provision of the scientific advice are
listed in Figure 5.
Identification and prioritization of issues referred by the
CAC or by member countries of the FAO and WHO.
• Call for data to facilitate the evaluation of the identified
issue: The data can be provided by the industry, wherever
applicable, or national data can be provided by the regula-
tory agencies or scientific institutes.
• The data submitted is examined by a group of experts who
are elected in their personal capacity and not as representa-
tives of their country or of the institute where they may be
employed. In appointing experts, the FAO and WHO con-
sider the scientific and technical excellence, diversity and
complementarity of scientific backgrounds and opinions,
and geographical as well as gender balance. The exact com-
position of the group will depend on the nature of the
expert advice required, but it may include representatives
of the natural sciences, including chemists, biologists, tox-
icologists, and public health specialists, as well as experts
from other fields.
• Based on the data that has been submitted and the expert
group consultations, the outcomes of the risk assessment
are published.
Risk Management: Elaboration of Standards by the CAC
When a Codex subsidiary body (i.e., a committee or a task
force) proposes to elaborate a standard, code of practice, or
related text within its items of reference, it should consider the
following:
• Priorities that were established by the Commission in the
Strategic Plan of Work
• Any specific relevant strategic project currently being under-
taken by the Commission
Statutes and guidelines
Meeting of the Expert
bodies
Results and
publications
Roster of experts
• Feasibility of completing the work within a reasonable
period of time
The CAC has well-established criteria, to be applied in deter-
mining priorities for the inclusion of tasks in the program of
work of committees and ad hoc task forces. These criteria are
generally addressed when a member country makes a submis-
sion to a committee for new work or for the review of an
existing, or adopted, Codex text. If the proposal falls outside
of the committee’s terms of reference, the proposal is referred
to another committee or reported to the Commission in
writing, together with proposals for amendments to the
committee’s terms of reference.
The initial proposal for a new or ammended Standard or
related text comes from a country or group of countries that
raises the issue at a Codex committee or an FAO/WHO coor-
dinating committee. A committee proceeds with work on a
new standard only once it has been approved by the
Commission.
When a committee or task force starts to elaborate a stan-
dard whose development has been approved by the
Commission, there is a step procedure to be followed. The
normal procedure has eight steps, although an accelerated
five-step procedure may be used if agreed to by at least two-
thirds of the Members of the Commission. Most of the Codex
documents are elaborated through this step process. Some
Codex documents are developed outside of the step process,
such as internal documents to guide the work of a specific
Committee.
Project documentation
When a committee or other subsidiary body of the Commis-
sion is considering elaborating a standard code of practice, or
related text, the committee will prepare project documentation
for submission to the Executive Committee and the
Commission. This documentation will provide the informa-
tion required by the Commission to determine whether or not
the work should be approved, and will be the basis for the
Executive Committee’s critical review and monitoring of the
progress of the work. This project documentation is not
required for individuals maximum residue limits for pesticides
 Codex Alimentarius Commission: Role in International Food Standards Setting
or veterinary drugs, or the maintenance of standards and texts
such as the General standard on food additives or international
numbering system.
Project documentation should consist of the following:
• Purpose of the proposed standard
• Indication of its relevance to the Codex strategic objectives
• Scope of the proposed standard
• Assessment of the proposed standard against the criteria for
the establishment of work priorities
• Proposed time line for completion of the work (including,
as a minimum, start date, proposed date for adoption at
step 5, and proposed date for final adoption by the
Commission)
• Identification of expert advice requirements
• Identification of any issues related to the needs of develop-
ing countries.
The preparation of this project documentation is the responsi-
bility of the Member proposing the new work. It should be
prepared in sufficient time for the committee to reach
consensus on whether or not to recommend the work and
subsequent consideration by the Executive Committee and
the Commission.
Specific criteria for determining priorities in the work
of committees and task forces
The specific criteria that are applied while evaluating a pro-
posal for the elaboration of a standard are different for general
and the vertical subject committees as given below:
Criteria applicable to general subject committees
• Contribution to the protection of consumers’ health and
prevention of fraudulent practices
• Diversification of national legislation and apparent resul-
tant or potential impediments to international trade
• Scope of the work undertaken and the establishment of
priorities between the various sections of the work
• Work already undertaken by other international organiza-
tions in this field will be considered
Criteria applicable to commodity committees
• Contribution to the protection of consumers’ health and
prevention of fraudulent practices
• Volume of production and consumption in individual
countries and volume and pattern of trade between
countries
• Diversification of national legislation and apparent resul-
tant or potential impediments to international trade
• International or regional market potential
• Amenability of the commodity to standardization
• Coverage of the main consumer protection and trade issues
by existing or proposed general standards
• Number of commodities that would need separate stan-
dards indicating whether raw, semiprocessed, or processed
products are to be included in the standard
• Work already undertaken by other international organiza-
tions in this field
203
Procedure for the elaboration of Codex standards
Part 3 and Part 4 of Section II of the Codex Procedural Manual,
which is updated as and when necessary, lays down the pro-
cedures for the elaboration of codex standards and related texts
through an eight- or five-step process.
The eight-step uniform procedure for the elaboration is as
follows:
Step 1: The Commission decides to elaborate a standard and
assigns the work to a committee, after taking into account
the outcome of the critical review conducted by the Execu-
tive Committee. A decision to elaborate a standard may also
be taken by a committee, but it is subject to endorsement by
the Commission. In the case of regional standards, the
Commission bases its decision on the proposal of the
majority of the members belonging to the region or group
of countries submitted at the CAC session.
Step 2: The Secretariat arranges the preparation of a proposed
draft standard. In the case of MRLs for residues of pesticides
or veterinary drugs, as well as MLs in case of the contami-
nants in food, the Secretariat distributes the recommenda-
tions of the JECFA and JMPR and any other information on
risk assessment work conducted by the FAO and WHO.
Step 3: The proposed draft standard is sent to members of the
commission and interested international organizations for
comment on all aspects.
Step 4: The comments received are forwarded by the Secretariat
to the concerned subsidiary body or other body concerned,
which, as per the TOR, has the mandate to consider the
comments and to propose amendments to the draft
standard.
Step 5: The proposed draft standard is submitted through the
Secretariat to the Executive Committee for critical review
and to the Commission for its adoption as a draft standard.
The Commission gives due consideration to the outcome of
the critical review and the comments that may be submitted
by any of its members.
Step 6: The draft standard is sent by the Secretariat to all
Members and interested international organizations for
comment on all aspects, including possible implications
of the draft standard for their economic interests.
Step 7: The comments received are sent by the Secretariat to the
concerned subsidiary body or the other body concerned,
which has the power to amend the draft standard.
Step 8: The drafts standard is submitted through the Secretariat
to the Executive Committee for critical review and to the
Commission for adoption as a Codex standard. At this step
again, the Commission gives due consideration to the out-
come of the critical review and comments submitted by the
members, and members may take part in the debate.
The five-step uniform, accelerated procedure for the elaboration
On the basis of a two-thirds majority of votes cast, and taking
into account the outcome of the critical review conducted by
the Executive Committee, the CAC shall identify those stan-
dards that shall be subject to an accelerated elaboration pro-
cess. While making a decision to use the accelerated
elaboration process, relevant consideration could be:
• Matters concerning new scientific information
• New technologies
204 Codex Alimentarius Commission: Role in International Food Standards Setting
• Urgent problems relating to trade or public health
• Revision or updating the existing standards
Once the Commission decides to elaborate a standard on the
basis of a two-thirds majority of votes cast using the accelerated
procedure and assigns the work to a subsidiary body or other
body, the Secretariat arranges the preparation of a proposed
draft standard, and the proposed draft standard is sent to
members of the Commission and the interested international
organizations for their comments. During step 2, the recom-
mendations of the risk assessment done by the JECFA, JMPR,
and other FAO/WHO consultations are made available. In step
3, the proposed draft standard is sent to the members of the
Commission and interested international organizations for
their comments, and when standards are subject to the accel-
erated procedure, members of the Commission and the inter-
ested international organizations are notified.
In step 4, the Secretariat forwards comments to the subsid-
iary body or other body for consideration and amendments to
the proposed draft standard, if any. In step 5, the proposed
draft standard subject to the accelerated elaborating procedure
is sent to the Executive Committee for critical review and then
to the Commission through the Secretariat, together with any
written proposals from Members and interested international
organizations, for adoption as a Codex standard.
Unlike in the eight-step procedure, the proposed draft stan-
dard or related text is submitted to the members of the Com-
mission and other international organizations and to the
Executive Committee only once.
The final adoption of the Codex standard, either through
the eight- or the five-step procedure, can be made only at the
plenary session of the CAC.
Measure to facilitate consensus while adopting standards
At the Twenty-sixth Session of the Commission in the year
2003, it was decided that every effort should be made to
reach agreement on the adoption or amendment of standards
by consensus. All efforts should be made to ensure that the
process of standard-setting is open, transparent, inclusive, and
science-based.
Risk Communication
After the adoption of the Codex standard by the CAC, it is
published and issued to all Member states and Associate Mem-
bers of the FAO and/or WHO, as well as to international
organizations. They are also available on the Codex Alimentar-
ius website (www.codexalimentarius.org).
Table 1 List of adopted Codex standards and related texts
Type of text
1. Codes of practice, both general and for specific production and handling processes
2. Guidelines
3. Individual food standards
4. Miscellaneous
The Codex Standards for Food Additives (GSFA, General Standards of Food Additives) list the food additives that have been adopted by the CAC (updated
up to 36th session).
Codex MRLs for pesticides (updated up to the 36th session of the CAC), veterinary drugs (updated up to the 35th session of CAC), and residues
Source: www.codexalimentarius.org.
The documents published by Codex can be classified as:
• Standards and MRLs, which contain mandatory require-
ments (are identified as CODEX STAN and CAC MRL/ML)
• Codes of practices, which are recommendations in general
or for specific manufacturing or handling process (are iden-
tified as CODEX RCP)
• Guidelines, directed at the Codex subsidiary bodies and
national governments, which are usually advisory texts
(are identified as CAC GL)
Methods of analysis are considered as part of the standard or
the MRL. Till date, the CAC has adopted the following Codex
standards and related texts (Table 1).
Role of Codex Standards in the Framework
of International Trade
Following the Marrakesh Agreement in 1994, which led to the
creation of the WTO in 1995, the Agreement on the Applica-
tion of Sanitary and Phytosanitary measures (the SPS agree-
ment) and the Agreement on Technical Barriers to Trade (TBT
Agreement) also went into effect. The SPS agreement has rec-
ognized and chosen the Codex standards, guidelines, and
codes of practices, as well as the recommendations for food
additives, veterinary drugs and pesticide residues, contami-
nants, methods of analysis, and sampling. Similarly, the
Codex standards have assumed considerable significance
under the Technical Regulations and standards provisions con-
tained in Article 2 of the TBT agreement.
The recognition of the scientifically based Codex standards
and the related texts within the framework of the SPS and TBT
agreements has given a big flip to the work of Codex and also
generated more interest in its Members. There has been
increased Member attendance at Codex meetings, particularly
from the developing countries.
Conclusion
At its Thirty-sixth Session in 2013, Codex Alimentarius cele-
brated its fiftieth year of establishment. It is a matter of great
satisfaction that the membership has grown from 30 in 1963 to
186. It reinforces the confidence in the organization’s work
and the extremely important responsibility of laying down
the global parameters for the quality and safety of food prod-
ucts for human consumption that the organization is shoul-
dering. However, to maintain relevance in the rapidly changing
Number
49
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4
 Codex Alimentarius Commission: Role in International Food Standards Setting
environment, where everyday new scientific developments are
taking place, new food safety concerns are emerging, consumer
preferences are changing rapidly, and there is increased global
trade, the CAC must be more nimble-footed in setting
standards. There is a need to make the process of standard-
setting more inclusive by making an extra effort to encourage
the developing countries to submit the necessary data.
The Codex Strategic Plan for the period 2014–19 that was
adopted at the Thirty-sixth Session would need to be followed
and monitored to ensure that the twin mandates of the Codex
Alimentarius work are followed in letter and spirit.
See also: Food and Agriculture Organization of the United Nations;
World Health Organization.
Further Reading
Codex Alimentarius Commission Procedural Manual, 22nd ed. (2014). Rome: WHO
and FAO of the United Nations.
Enhancing participation in Codex activities, an FAO/WHO training package (2005). FAO
and WHO.
Food Agriculture Organization/World Health Organization (1950) Joint FAO/WHO
Expert Committee on Nutrition: report of the first session. Geneva: World Health
Organization, Technical Report Series No. 16.
205
Food Agriculture Organization/World Health Organization (1955) Joint FAO/WHO
Expert Committee on Nutrition: report of the fourth session. Geneva: World Health
Organization, Technical Report Series No. 97.
Food Agriculture Organization/World Health Organization (1997) Uniform procedure for
the elaboration of Codex standards and related texts. In: Procedural Manual of the
Codex Alimentarius Commission, 10th ed. Rome: FAO.
General Agreement on Tariffs and Trade (1994) The results of the Uruguay Round of
Multilateral Trade Negotiations: the legal texts. Geneva: GATT Secretariat.
Randall AW (2000) International food standards: the work of codex. In: Rees N and
Watson D (eds.) International standards for food safety, pp. 3–10. Gaithersburg,
MD: Aspen Publication.
Stanton G (2000) Food safety and international trade: the role of WTO and the SPS
agreement. In: Rees N and Watson D (eds.) International standards for food safety,
pp. 11–25. Gaithersburg, MD: Aspen Publication.
van der Heide RF (1999) Structure, organization, and practical operation of
International/Intergovernmental Food Safety Regulation Bodies. In: van der
Heijden K and Younes M, et al. (eds.) International food safety handbook:
science, international regulation and control. New York/Basel: Marcel Dekker, Inc.,
p. 617.
Understanding the Codex Alimentarius, 3rd ed. Produced by Codex Secretariat,
FAO, Rome.
Relevant Websites
www.codexalimentarius.org – About Codex.
www.wto.org – The WTO and the FAO/WHO Codex Alimentarius.
 Coenzymes and Cofactors
RB Rucker, University of California, Davis, CA, USA
W Chowanadisai, Oklahoma State University, Stillwater, OK, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Metabolism represents an array of chemical reactions that
usually involve the transfer of specific functional groups
from which substances are produced, maintained, or
destroyed; and energy is made available to produce heat or
work. Such transfer reactions often depend on aid from
cofactors. Herein, cofactors are described that are involved
in (1) bond making and breaking steps, (2) redox reactions,
(3) the transfer of functional groups, and (4) changes in
cellular energy or chemical potentials. In general, cofactors
facilitate the coupling of the spontaneous processes of catab-
olism to the nonspontaneous processes of anabolism. As
biocatalysts, cofactors provide the possibility of differing
transition states that lower the activation energy of given
reactions. As such, cofactors may increase reaction rates or
enable reactions at lower temperatures. Cofactors can also
affect the reaction environment or bind to substrates to
change physical and chemical properties that make them
more susceptible to chemical alteration. When associated
with a corresponding apoenzyme, the presence of a cofactor
extends the catalytic capacity of the resulting holoenzyme or
functional enzyme (i.e., an apoenzyme plus cofactor). More-
over, when linked to specific metabolic processes, a cofactor
may provide additional regulatory functions by acting as
allosteric modulators.
For clarity and organization, the term cofactor will be
used throughout to describe both organic and inorganic
compounds that are involved as catalysts in enzymatic or
biologically relevant chemical reactions. In this regard, it is
also important to note that some terms have very specific
meanings. For example, ‘coenzyme’ will be used to define
organic cofactors that work in conjunction with apoen-
zymes. The term prosthetic group will be used when a
given cofactor is nondissociable with respect to its corre-
sponding enzyme; cosubstrate will be used when binding
is dissociable. When metals serve as cofactors, they usually
fall into two categories, that is, metals important to metal-
loenzymes or metals important to metal–enzyme complexes.
The stability of the interaction defines whether the term
metalloenzyme or metal–enzyme complex is used. Metal-
loenzymes have metal binding constants of 108–109 or
greater. Metal–protein complexes have constants of 105 or
less. To broaden the discussion, comments regarding biofac-
tors that are responsible for nonenzymatic chemical cataly-
sis, such as the tocopherols, are also included. Throughout
the article, a number of examples and descriptions of cofac-
tor function are described; however, our primary descriptive
goal is to portray the broad range of mechanistic approaches
that are involved in biological conversions important to
metabolism in various animal species.
206 Encyclopedia of Food and Health
Biochemical Functions
Role of Cofactors in Catalytic Processes
Cofactors aid in creating alternative routes for reactions and, as
a consequence, aid in reducing the amounts of energy required
to reach the reactions’ highest activation transition state.
Accordingly, more of a given substrate can proceed to the
formation of product. Cofactors improve enzyme–substrate
interactions by combinations of the following mechanisms:
(1) effecting conformation changes that improve interactions
and the binding of a substrate to an enzyme, (2) causing bond
strain to align a portion of a substrate so it is better positioned
to engage in a reaction, (3) altering the nucleophilic or elec-
trophilic properties of a substrate, (4) altering the environment
at the active site of an enzyme (e.g., water structure around the
active site), (5) forming transient covalent bonds with residues
within the active site to lessen the energy of substrate moiety
transfers, and (6) enhancing electron tunneling (increasing
movement of electrons to an active site). The active site of
enzymes is usually found in a 3-D groove or pocket. Cofactors
extend chemical options or improve on entropic consider-
ations by providing an extendable arm to place a substrate or
an intermediate closer to the active center of an enzymatic
process. Cofactors facilitate transition-state formation, a tran-
sition in which the targeted molecule is no longer a substrate,
but not yet a product.
As shown in Figure 1, one may describe chemical reactions
in terms of the ‘activation’ or ‘free energy’ needed to initiate a
chemical transition or reaction. In this regard, many reactions
proceed with multiple steps and reaction intermediate transi-
tion states. Note that the activation energy is lowered, but the
initial energy of the starting reactants or their final products is
not changed.
Listed in Table 1 are well-recognized cofactors derived from
water-soluble vitamins and vitamin K, as well as important
nonvitamin-derived cofactors. In addition, examples of essen-
tial dietary minerals utilized as cofactors are summarized in
Table 2, particularly those involved in redox reactions.
Role in Entasis (Facilitation of Energized States in Proteins
and Metalloproteins)
The term entasis comes from the Greek meaning ‘to stretch or
strain tight.’ When metal cofactors are involved, the state of
entasis (e.g., conformational strain) is relieved by a loss of an
electron from metals that are a part of a protein complex. The
electron transfer is more thermodynamically favorable. In
many enzymes for which elements, such as calcium, magne-
sium, zinc, sodium, and potassium, are required or influence
http://dx.doi.org/10.1016/B978-0-12-384947-2.00181-1
 Coenzymes and Cofactors 207

Activation energy without enzyme/catalyst
Activation energy with enzyme/catalyst
Free energy (G)
catalytic activity, their roles are often to facilitate entasis-like
processes.
In contrast, cofactors derived from organic compounds are
less capable of hydrolyzing or changing the chemical character
of biological compounds, because of their lower redox poten-
tials. The typically higher reduction potentials of metal-

containing complexes and/or the energy associated with entasis

Initial energy
of reactants
ΔG
Energy of final products
Reaction coordinates or progress
Figure 1 The relationship between reaction progress, activation
energy, and the function of enzymes as catalysts. The activation energy
(also known as the Gibbs free energy (G) or free enthalpy) is the
minimum energy required to start a reaction. With respect to enzyme-
catalyzed reactions, simply stated, an enzyme (often with the aid of a
cofactor) acts to lower the activation energy needed to start the reaction.
circumvent this problem. Metalloenzyme complexes are often
several orders of magnitude more proficient as redox catalysts
than organic-derived cofactors capable of redox. As it relates to
‘entasis,’ simple metal complexes tend to equilibrate quickly
with substrates and products and other components in the
reaction environment. However, when combined in a complex
protein structure (i.e., an enzyme), entatic strain caused by the
inclusion of metal cofactors makes the initial phases of the
reactions much more energetically favorable. The reaction cen-
ter is also less compromised by nonspecific interactions.
Accordingly, an active site of an enzyme can be thought of as
existing more in isolation than a component that is in equilib-
rium with cellular compounds and compounds not directly
related to the reaction.

Table 1 Coenzymes and cofactors

Vitamin-derived coenzymes and cofactors

Common abbreviations for Modifications leading to Chemical group(s) transferred

Cofactor name the cofactor Precursor cofactor formation or types of reactions catalyzed

Ascorbic acid Vitamin C The biosynthesis of ascorbic Ascorbic acid is a reducing

acid in many animals starts agent and antioxidant. It
with the formation of UDP often is utilized as a cofactor
glucuronic acid in a pathway for monooxygenase
important for D-glucuronic (hydroxylase)-catalyzed
acid synthesis. L- reactions
gulonolactone, a product of
the pathway, reacts with
oxygen, catalyzed by the
enzyme L-gulonolactone
oxidase, to produce
ascorbic acid. In humans,
L-gulonolactone oxidase is
missing; thus, a dietary
source of ascorbic acid is
required
Biotin; coenzyme R Biotin (vitamin H and Biotin as a cofactor is Biotin is essential for
vitamin B7) covalently linked. The carboxylation reactions
enzyme biotinidase involving the addition of
catalyzes the cleavage of CO2, important in the
biotin from biotinyl peptides synthesis of fatty acids,
(the proteolytic degradation isoleucine, and valine and in
products of holo-(biotin- gluconeogenesis
requiring) carboxylases,
which allows recycling and
reutilization of biotin)
Cobalamins: B12, OHCbl, or B12a Vitamin B12, In humans, the various forms Together with folic acid,
Hydroxocobalamin MeCbl or MeB12 cyanocobalamin are rapidly interconverted cobalamins are essential for
Methylcobalamin AdoCbl (commercial DNA synthesis.
Adenosylcobalamin preparation of B12 in Methylcobalamin serves as
which a CN moiety a source of transferable
‘CH3
’ groups.

(Continued)
208 Coenzymes and Cofactors

Table 1 (Continued)

Vitamin-derived coenzymes and cofactors

Common abbreviations for Modifications leading to Chemical group(s) transferred

Cofactor name the cofactor Precursor cofactor formation or types of reactions catalyzed

replaces the methyl Adenosylcobalamin serves

group) as a cofactor for
isomerization reaction,
rearrangements in which a
hydrogen atom is directly
transferred between two
adjacent atoms (e.g., the
conversion methylmalonyl-
CoA to succinyl-CoA by
methylmalonyl-CoA mutase)
Coenzyme A CoASH Pantothenic acid Coenzyme A is synthesized in CoASH aids in the transfer of
(vitamin B5) a multistep process that acetyl group and other acyl
requires four ATPs, groups (e.g., TCA-related
pantothenate, and cysteine oxidations and fatty acid
metabolism)
Flavin adenine FAD Riboflavin (vitamin B2) FAD consists of a riboflavin Catalyzes high-energy
dinucleotide moiety bound to the electron transfer reactions
phosphate group of an ADP important in oxidative
molecule phosphorylation, common
cofactor for enzymes with
reductase activity
Flavin mononucleotide FMN Riboflavin (vitamin B2) Produced from riboflavin by Functions as a prosthetic
or riboflavin-5- the enzyme riboflavin kinase group for various
phosphate oxidoreductases including
NADH dehydrogenase
Nicotinamide adenine NAD or DPN Niacin (vitamin B3) In animal cells, de novo As a cofactor, catabolic
dinucleotide or, in tryptophan degradation plus oxidative/reduction
older notation, ATP addition or, reactions; as a cosubstrate,
diphosphopyridine alternatively, ATP addition to polyribosylation reactions
nucleotide diet-derived niacin
Nicotinamide adenine NADP or TPN Niacin (vitamin B3) Conversion of NAD into NADP Most often anabolic oxidative/
dinucleotide by NAD kinase reduction reactions
phosphate or, in (requires ATP)
older notation,
triphosphopyridine
nucleotide
Pyridoxal-50 - PLP Pyridoxine Conversion of pyridoxine into Carries out transamination,
phosphate PLP by pyridoxal kinase decarboxylation, and
(requires ATP) racemization reactions (also
see Table 2)
Tetrahydrofolic acid THFA, H4FA, MTHF Folic acid (vitamin B9) The synthesis of THFA and Transfer of methyl, formyl,
derivatives (N5, methylene, and formimino
N10-methylene-THFA, groups and the so-called
N5-methyl-THFA, N5- one-carbon transfers,
formimino-THFA, N10- essential for amino acid,
formyl-THFA, N5, and N10- purine, and DNA synthesis
methenyl-THFA) starts with
dihydrofolate reductase
(essential to THFA formation
from H2FA). The reactions
that lead to derivatives are
FA ! H2FA ! THFA $
methylene-THFA ! methyl-
THFA
Thiamine TPP, TDP, ThDP Thiamine (vitamin B1) Thiamine is converted into Carries out two-carbon group
pyrophosphate (i.e., TPP by thiamine kinase transfer reactions, a-carbon
thiamine (requires ATP) cleavage (e.g., oxidative
diphosphate) decarboxylations),
transamination reactions
 Coenzymes and Cofactors 209

Table 1 (Continued)

Vitamin-derived coenzymes and cofactors

Common abbreviations for Modifications leading to Chemical group(s) transferred

Cofactor name the cofactor Precursor cofactor formation or types of reactions catalyzed

Thiamine triphosphate TPPP, TTP, ThTP
Vitamin K; 2-methyl-1, Vitamin K1 (phylloquinone),
4-naphthoquinone vitamin K2 (menaquinone,
derivatives: also MK-n, where M stands
phylloquinone, for menaquinone, K stands
menaquinone for vitamin K, and n
represents the number of
isoprenoid side chain
residues)
Thiamine (vitamin B1) ThTP is currently suggested to
be synthesized by apparent
chemiosmotic mechanisms,
analogous to ATP synthesis
Vitamin K Vitamin K1 is synthesized by
plants: the menaquinones
are synthesized in the
intestinal lumen by bacteria
Nerve cell energy metabolism,
sodium gating in nerve cells;
in E. coli, TTP is used in
metabolic processes
induced during amino acid
starvation
Vitamin K (in animals) is
involved in the carboxylation
of certain glutamate
residues in proteins to form
gamma-carboxyglutamate
(Gla) residues. Gla residues
facilitate subsequent
calcium binding that causes
activation of enzymes
important to blood
coagulation and regulation
of calcification in bone and
smooth muscle

Nonvitamin-derived coenzymes and cofactors

Common
abbreviations for Chemical group(s) transferred or types of
Cofactor name the cofactor Chemical modifications leading to cofactor formation reactions catalyzed

Adenosine triphosphate ATP ATP is formed by the action of ATP synthase. ATP
synthase is a mitochondrial membrane enzyme that
uses transmembrane electrochemical proton potential
differences facilitate the phosphorylation of adenosine
diphosphate (ADP) to ATP
Adenosine 3,5-cyclic cATP cAMP is derived from ATP, synthesized by adenylyl
monophosphate cyclase
Cytosine triphosphate CTP CTP synthetase catalyzes the last committed step in
pyrimidine nucleotide biosynthesis:
ATP þ UTP þ glutamine ! ADP þ Pi þ CTP þ glutamate
Coenzyme Q, CoQ, CoQ10 Biosynthesis occurs in 3 major steps: (1) creation of the
ubiquinone benzoquinone structure (using phenylalanine or
tyrosine), (2) addition of isoprene side chain units, and
(3) condensation of the isoprene chain with
benzoquinone
Glutathione (GSH) GSH GSH is a tripeptide (Gly-Cys-Glu) with a gamma peptide
linkage between the carboxyl group of the glutamate
side chain and the amine group of cysteine, which is
attached by peptide linkage to glycine. Glutathione
reduces disulfide bonds formed within cytoplasmic
proteins to cysteines by serving as an electron donor.
In the process, glutathione is converted to its oxidized
form, glutathione disulfide (GSSG). Once oxidized,
glutathione can be reduced back by glutathione
reductase, using NADPH as an electron donor
Guanosine-50 - GTP The synthesis and regulation of GTP are complex and
triphosphate involve enzymes, such as inosine-50 -monophosphate
dehydrogenase, GMP synthase, GMP kinase, and
nucleoside-diphosphate kinase
Adenosine triphosphate (ATP) is a
coenzyme involved in energy transfer
reactions including biosynthetic
reactions, motility, and cell division
cATP is important in cell signaling and cell
signaling transduction
CTP is a high-energy molecule similar to
ATP but is limited to a smaller subset of
metabolic reactions (e.g., synthesis of
glycerophospholipids and glycosylation
of proteins)
CoQ10 is found in the membranes of many
organelles; the highest concentration is
found on the inner membrane of the
mitochondria, where it facilitates
electron flow. CoQ also has antioxidant
potential
Major reducing agent in cells (e.g., is
maintained at mM levels). A
cosubstrate for glutathione peroxidases
and glutathione S-transferase. It is a key
component of the
glutathione–ascorbate cycle, a system
important to maintaining safe hydrogen
peroxide levels in cells
Analogous to ATP, GTP acts as a
substrate for the synthesis of RNA
during the transcription process or DNA
during DNA replication. It is used as a

(Continued)
210 Coenzymes and Cofactors

Table 1 (Continued)

Nonvitamin-derived coenzymes and cofactors

Common
abbreviations for Chemical group(s) transferred or types of
Cofactor name the cofactor Chemical modifications leading to cofactor formation reactions catalyzed

source of energy for specific steps in

protein synthesis and gluconeogenesis
Guanosine 3,5-cyclic cGMP cGMP is derived from GTP, synthesized by adenylyl cGMP is essential to signal transduction,
monophosphate cyclase in particular with G proteins, in second-
messenger mechanisms where cGMP
is converted to guanosine diphosphate
Heme (various types) Heme Heme consists of an Fe ion located at the center of Hemes are components of hemoglobin
heterocyclic organic ring, called porphyrin. Porphyrins and are at the active sites in numerous
consist of four pyrrolic groups joined together by enzymes and protein important to
methine bridges oxygen metabolism (e.g., myoglobin,
cytochromes, catalase, and nitric oxide
synthase)
a-Lipoic acid or thioctic ALA ALA is an organosulfur compound derived from octanoic Essential for the transfer of acetyl groups
acid acid. As a cofactor, ALA is covalently attached by an in pathways important to energy
amide bond to a terminal lysine residue in the lipoyl regulation and production
domain component of the active site of lipoic acid
requiring enzymes
Molybdopterin, Mo cofactor Molybdopterins are synthesized utilizing guanosine The molybdenum cofactor is used by
molybdenum cofactor triphosphate as a principle substrate sulfite oxidase, xanthine
oxidoreductase, and aldehyde oxidase.
The absence of molybdenum cofactor
leads to the accumulation of toxic levels
of sulfite
30 -Phosphoadenosine- PAPS PAPS is a derivative of adenosine monophosphate that is PAPS is a coenzyme in sulfotransferase
50 -phosphosulfate phosphorylated at the 30 -position with a sulfate group reactions
attached to the 50 phosphate
Pyrroloquinoline PQQ Tricyclic quinone derived from the annulation of PQQ is utilized as a dehydrogenase redox
quinone glutamic acid with tyrosine cofactor in many bacteria and recently
discovered to serve a similar role in
lower eukaryotes. Although it is not
currently viewed as a classical cofactor
important to enzymatic functions in
animals, it is ubiquitous in nature and is
present in all tissues. It appears to play
a role as a redox agent in cell signaling
by interaction with receptors important
to Janus kinase (JAK) and signal
transducer and activator of
transcription (STAT) pathways and
mitogen-activated protein kinase
(MAPK) pathways
S-Adenosylmethionine SAM-e, SAMe, SAM is made from ATP and methionine by methionine Serves as a cosubstrate in methyl group
SAM adenosyltransferase transfer reactions. Transmethylation,
transsulfuration, and aminopropylation
are the metabolic pathways that use
SAM as a cosubstrate
Tetrahydrobiopterin BH4, THB Tetrahydrobiopterin is synthesized by reactions THB serves as a cofactor for 3 aromatic
involving GTP as a substrate and GTP cyclohydrolase amino acid hydroxylase enzymes,
as an important enzyme associated with the synthetic which are important to the degradation
pathway of phenylalanine and synthesis of
serotonin, melatonin, norepinephrine,
and epinephrine. BHT is also a cofactor
for the production of nitric acid by nitric
oxide synthases
 Coenzymes and Cofactors 211

Table 2 Cofactors involved in redox reactions

Cofactor Description

Metals
Manganese Utilized in the mitochondrial antioxidant system (e.g., Mn-dependent superoxide dismutase) and enzymes, such as malic
enzyme and pyruvate dehydrogenase
Iron Utilized in numerous oxidases, mono- and dioxygenases, and reductases, as well as cytochrome P450 enzymes, important in
secondary metabolism and mitochondrial function. Iron is also found in heme proteins, such as hemoglobin and myoglobin.
Hemoglobin binds oxygen cooperatively via coordination with iron. Active sites containing iron often exist as iron–sulfur
clusters containing sulfide-linked di-, tri-, and tetrairon centers in variable oxidation states. For example, both mitochondrial
complex I and complex II have multiple Fe–S clusters
Zinc Utilized in various enzymes such as alkaline phosphatase, alcohol dehydrogenase, matrix metalloproteinase, carboxypeptidase
A, and carbonic anhydrase
Cobalt Utilized in corrin-containing proteins (e.g., as cobalamin) and enzymes, such as methionine aminopeptidase 2 (eukaryotes) and
nitrile hydratase (bacteria)
Nickel Utilized in plant urease, the NiFe hydrogenases in bacteria, and the nickel-tetrapyrrole coenzyme, cofactor F430, which is used by
methyl coenzyme M reductase, and minor forms of bacterial superoxide dismutase and glyoxalase
Copper Utilized in numerous oxidases: superoxide dismutase, mono- and dioxygenases, and reductases. Copper is also used in
hemocyanins that transport oxygen in some invertebrate, for example, crustaceans living in environments with low oxygen
pressure (e.g., hemocyanins bind oxygen noncooperatively and less efficiently than hemoglobin). Terrestrial arthropods (e.g.,
spiders and scorpions) also utilize hemocyanins
Molybdenum Utilized in aldehyde, sulfite, and xanthine oxidases
Nonmetals
Selenium Utilized in glutathione peroxidase (reaction: 2 GSH þ H2O2 ! GSSG þ 2H2O) and Se-dependent deiodinases that convert T4 to T3
(the active hormone) by removing an iodine atom from the outer tyrosine ring
Sulfur Utilized in redox (cysteinyl residues at the active site of proteins). Inorganic sulfur is a part of iron–sulfur clusters in proteins with
redox activity (cf. Fe and ferredoxins), which serve as electron shuttles in cells. In bacteria, nitrogenase enzymes contain
Fe–Mo–S clusters (important in nitrogen fixation)
H, C, N, O Utilized in all biologically important organic compounds. Distinguished by being the smallest of the elements that can form
stable multiple bonds
Vitamin-derived cofactors
Ascorbic Utilized in oxidation reactions catalyzed by monooxygenases (hydroxylases), dioxygenases, and oxidases. Such enzymes are
acid/vitamin C often iron- or copper-containing. Ascorbate acts as a reductant to maintain Fe and Cu in their reduced states
Niacin Utilized as a precursor of the coenzymes: nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide
phosphate (NADP). Reactions involve the removal of two hydrogen atoms from the reactant in the form of a hydride ion (H:)
and a proton (Hþ). The proton is released into solution, while the reductant RH2 is oxidized and NADþ is reduced to NADH by
transfer of the hydride to the nicotinamide ring of NAD or NADP. As a general observation, NAD is used mostly in catabolic
processes and NADP in synthetic processes
Pyridoxine Utilized in the hydrolysis of ether linkages in glycogen by glycogen phosphorylase. The phosphate group on the pyridoxal-50 -
(vitamin B6) phosphate donates a proton to an inorganic phosphate molecule, allowing the inorganic phosphate to in turn be deprotonated
by the oxygen forming the a-1,4 glycosidic linkages in glycogen
Riboflavin Utilized as a component of the cofactors FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) utilized by a variety
of oxidases. During the catalytic cycle, a reversible interconversion of the oxidized, semiquinone, and reduced forms of flavin
occurs. FMN is a stronger oxidizing agent than NAD and is particularly useful because it can take part in both one- and two-
electron transfers; importantly, because oxygen as a reactant prefers one-electron transfers.

Types of Cofactor-Catalyzed Reactions
Many cofactors, particularly those derived from vitamins, are
designed for highly specific chemical modifications and reac-
tions. Some of the more important types of reactions are out-
lined in this section.
Oxidation–reduction reactions
Redox chemical reactions occur with a transfer of electrons
(appreciating that some redox reactions occur with no appar-
ent electron net gain or loss in electrons, e.g., reactions involv-
ing covalent bonds). When redox reactions occur, the process
involves one of the following mechanisms: (1) direct electron
transfers, (2) hydrogen atom transfers, (3) hydride ion
transfers, and (4) direct combinations with oxygen. Cellular
energy release is controlled by means of redox reactions. In this
regard, redox proteins and metabolic steps in the regulation of
their associated cofactors are often linked or colocated, which
provides regulatory continuity, for example, the positioning of
mitochondria in cells and the interdependence of mitochon-
drial and nuclear genes in energy regulation.
Of the vitamin-derived cofactors, flavin adenine dinucleo-
tide (FAD) and riboflavin-50 -phosphate (flavin mononucleo-
tide (FMN)) derived from riboflavin and nicotinamide
adenine dinucleotide (NAD) and nicotinamide adenine dinu-
cleotide phosphate (NADP) derived from niacin are perhaps
the most common (cf. Tables 1 and 2). However, in animals,
more than a dozen metals, nonmetals, nonvitamin-derived
cofactors, and ascorbic acid and pyridoxal-50 -phosphate
(PLP) play important roles in redox.
212 Coenzymes and Cofactors

Generation of leaving groups
As noted, cofactors function in part by allowing the possibility
of differing transition states that lower the activation energy of
given reactions. With regard to the generation of leaving
groups, this usually implies enriching the nucleophilic or elec-
trophilic character of transition-state intermediates to better
engage in nucleophilic or electrophilic substitutions. The
vitamin-derived cofactors, thiamine pyrophosphate (TPP)
and PLP, serve as excellent examples of such functions.
Thiamine pyrophosphate (TPP) consists of aminopyrimi-
dine and thiazole rings linked by a methylene bridge. The
thiazole moiety acts as an ylid (a neutral dipolar molecule
containing a negatively charged atom attached to a heteroatom
with a formal positive charge). The carbanion character of
TPP’s thiazole ring is retained in transition-state intermediates.
Examples of enzymes in which TPP is used for this purpose are
pyruvate, a-ketoglutarate, or branched-chain amino acid dehy-
drogenase complexes and 2-hydroxyphytanoyl-CoA lyases (see
Figure 2). TPP catalyzes the reversible cleavage of a substrate at
the carbon–carbon bond connecting a carbonyl group to an
adjacent reactive group (usually a carboxylic acid or an alco-
hol). In the reverse reactions, the TPP–substrate bond is bro-
ken, reforming a TPP ylid and substrate carbonyl.
TPP is also a cofactor for transketolases, enzymes associated
with the pentose phosphate pathway in all organisms and the
Calvin cycle of photosynthesis. In transketolases, TPP accepts a
2-carbon fragment from a 5-carbon ketose (e.g., D-xylulose-5-
P) and then transfers the fragment to a 5-carbon aldose (e.g.,
D-ribose-5-P) to form a 7-carbon ketose (e.g., sedoheptulose-
7-P). The second reaction catalyzed by transketolase in the

TPP

H3C
N R1
N
R2 N+ CH3
H2N CH2
O=C [+] [–] + H+
R2 N+ CH
3 S
O=C[+] [H] CH2 R2
S O O O R1
R1 CH2CH2 P P
-O -
-O O
TPP
Thiamine pyrrolophoshate (TPP) R1
R2
N+ CH3
TPP
O=C
R1 R1 S
CH2 R2
[H]O [-] N CH3

R1 TPP
S
CH2 R2 R1
N CH3 + [R2]
-O

(a) R1 S CH2 R2

Pyruvate dehydrogenase
complex NADH
FAD
Pyruvate SH
Lip Dihydrolipoyl
TPP SH dehydrogenase
Pyruvate
Dihydrolipoamide Subunit III
dehydrogenase
acyltransferase FADH
Subunit I
Subunit II NAD+

S
CO2 Lip CoASH
+ S Acetyl CoA
Acetyl-TPP-PD Acyl-lipoate-DA
Transferase
(b) Acetyl-CoA
Figure 2 Thiamine. Thiamine (2-[3-[(4-amino-2-methyl-pyrimidin-5-yl) methyl]-4-methyl-thiazol-5-yl] ethanol) as the pyrophosphate derivative, TPP,
is exquisitely designed to facilitate the metabolism of carbohydrates. The thiazole moiety of thiamine has carbonium ion character, which is
retained in transition-state intermediates. As a consequence, decarboxylation of a-ketoacids derived from glucose may enter energy-generating cycles,
such as the tricarboxylic acid (TCA) cycle and citric acid cycle (a). As an example, thiamine pyrophosphate is an essential cofactor for pyruvate
dehydrogenase, a component of the pyruvate dehydrogenase complex (b).

pentose phosphate pathway involves the transfer of a 2-carbon
fragment from D-xylulose-5-P to erythrose-4-phosphate,
which results in fructose 6-phosphate and glyceraldehyde-3-
P. This reaction connects the pentose phosphate pathway to
glycolysis (Scheme 1).
PLP-dependent enzymes are particularly critical in the
metabolism of amino acids. The 4-formyl substituent of PLP
condenses with the a-amino group of an amino acid, to form
an azomethine (Schiff base) linkage. Next, the conjugated
double-bond system extends into PLP’s pyridinium nitrogen.
This weakens bonding around the a-carbon of the amino acid
substrate, which, depending on the enzyme, can lead to trans-
aminations, decarboxylations, or subsequent substitutions at
the carbon adjacent to the a-carbon. In PLP-dependent
enzymes, the moiety is often attached to a lysyl group within
the enzyme. This has the advantage of setting the stage for an
initial transimidation with an amino acid, which is a faster
reaction than an aldehyde with an amino acid, that is, a clas-
sical transamination. Further, the transimidation mechanism
allows PLP to be retained in its original state in association
with the enzyme at the conclusion of a catalytic cycle. Accord-
ingly, although the binding of PLP to transaminases can be
viewed as potentially dissociable, the net result is more cova-
lent in character (Scheme 2).
Acyl activation and transfer reactions
Cofactors involved in the transfer of acyl and acetyl groups
include coenzyme A (CoA), lipoic acid, and small molecular
weight proteins, acyl carrier proteins (ACPs), that are associ-
ated with fatty acid and polyketide synthesis complexes. A
phosphopantetheinyl moiety serves as the principle functional
component within the structure of CoA and ACP. The phos-
phopantetheinyl moiety provides a thiol group that reacts with
the carboxylic acid group associated with acetate or acyl func-
tions to form thioesters. This step is a prelude to numerous
acetyl and acyl group transfers and enolizations. The carbonyl
carbon of a thioester is more positively polarized than a corre-
sponding oxy ester, because oxy esters are more stabilized by
resonance than thioesters. Activation as a thioester sets the
stage for eventual acetyl and acyl transfers. The phosphopan-
tetheinyl moiety also provides a flexible arm to move sub-
strates from one site to the next in complexes, such as those
designed for fatty acid and polyketide synthesis (Scheme 3).
Ribulose-5-phosphate
Xylulose-5-phosphate Ribose-5-phosphate
Transketolase
Sedoheptylose-7-phosphate Glyceride-3-phosphate
Transketolase
Erythrose-4-phosphate Fructose-6-phosphate
Transketolase Xylulose-5-phosphate
Fructose-6-phosphate
+ boxylase active site, which acts as an electrophile that accepts
Glyceraldehyde-3-phosphate
Scheme 1 Transketolase reactions.
Coenzymes and Cofactors 213
In an analogous fashion, lipoic acid, a-lipoic (thioctic) acid,
provides a functional sulfhydryl group with an extended flex-
ible arm attached via a peptide bond to lysyl residues in sub-
units usually associated with dehydrogenase enzyme
complexes. For example, the role of lipoic acid in pyruvate
dehydrogenase is in part outlined in Figure 2. Given that lipoic
acid has two adjacent sulfhydryl groups, which are oxidized to
form a dithiolane ring, oxidized lipoic acid is sufficiently elec-
tronegative to catalyze the transfer of TPP intermediates with
carbanion character. The resulting thioester can then be trans-
ferred to form CoA intermediates.
Carboxylations
D-(þ)-Biotin is a cofactor designed for the transfer of CO2 in
carboxylase and transcarboxylase enzymes, such as acetyl-CoA
carboxylase-a, acetyl-CoA carboxylase-b, and methylcrotonyl-
CoA, propionyl-CoA, and pyruvate carboxylases. Biotin is cova-
lently attached to the epsilon-amino group of specific lysine
residues at the active site in such enzymes, which are essential to
fatty acid synthesis, branched-chain amino acid catabolism,
and gluconeogenesis.
The mechanism involves tautomerization of the ureido
nitrogen, a process that enhances its nucleophilicity. Phos-
phorylation of bicarbonate by ATP represents an independent
step to produce carbonyl phosphate, an electrophilic mixed-
acid anhydride. The carbonyl phosphate then reacts to generate
an active carboxylbiotinyl enzyme. These reactions change the
carbon in CO2 to one with carbanion character, which facili-
tates subsequent reactions as the conversion of acetate to
malonate or pyruvate to oxaloacetate (Scheme 4).
In addition to biotin, the active forms of vitamin K acts as a
cofactor that is essential for certain types of carboxylation
reactions, particularly those involved for posttranslational
modifications of proteins utilized in blood coagulation
cascades (e.g., thrombin) and pathways important to the reg-
ulation of extracellular matrix (ECM) calcification. Vitamin K-
dependent carboxylases catalyze the carboxylation of glutamyl
residues to g-carboxyglutamyl (GLA) residues. The presence of
GLA residues facilitates the binding of calcium ions, important
to the activation of GLA-containing enzymes (i.e., those
involved in blood coagulation) and GLA-containing ECM pro-
teins, such as osteocalcin, important to the control of ECM
mineralization. In contrast, in plants and bacteria, vitamin K
either functions as an electron acceptor in photosystem I or is
important to anaerobic respiration. Compounds with vitamin
K activity are derived from 1,4-naphthoquinone derivatives
that are combined with varying numbers of isoprenoid units
to form the so-called phytyl side chains. Vitamin K includes
two natural vitamers designated vitamin K1 (phylloquinone,
synthesized in plants) and vitamin K2 (menaquinone, present
in animal tissues) (Scheme 5).
Vitamin K-dependent carboxylases use vitamin K epoxida-
tion to drive the carboxylation reactions that lead to GLA
formation. GLA formation occurs in two independent steps:
(1) deprotonation of a targeted glutamic acid residue to form a
carbanion intermediate, followed by (2) the addition of CO2.
The mechanism is facilitated by a histidyl residue at the car-
the negative charge on the g-carbon of the modified glutamyl
residue carbanion (Scheme 6).
214 Coenzymes and Cofactors

Enzyme
Enzyme

(CH3)4 HB
(CH3)4 B−
NH2
H O HN+
C H
C

−O
CH2OPO3−3 −O
CH2OPO3−3

H3C + H +
N H 3C H
N
H H
H
:O H

H
Enzyme
H or R C COOH
(CH3)4 : NH2
NH2

Transaminations
Elimenation reactions
H
R group elimenations and additions H C COOH Decarboxylations
NH+
HC

−O CH2OPO3−3

H3C + H
N
H
Scheme 2 Pyridoxal-5’-phosphate reactions.

+
O H O
R C S R′ R C X + R′ SH

X Head activation O
R C S R′ O
H O O
−
R C O
R″ C C S R′ R″ CH C S R′
R″ CH C S R′
H B R′ SH
Tail activation
Scheme 3 Thiol-facilitated acyl transfer reactions.

Single-carbon transfer reactions compounds composed of 4-(pteridin-6-ylmethyl)-

A number of cofactors are involved in single-carbon transfer aminobenzoic acid that is conjugated with one or more
reactions, in higher organisms, namely, derivatives of folic L-glutamate units. The reactions that involve folic acid deriv-
acid, the cobalamins, and S-adenosylmethionine (SAM). atives include the generation and utilization of formaldehyde,
The cofactors derived from folic acid are heterocyclic formimino, and methyl groups. For these conversions to
 Coenzymes and Cofactors 215

+
X X
O O − O
O Pi
2’
rN H −
HN3’ HN N C O HN N CO2
−
OPO3H

S S S

−
Product CO2 Substrate

Scheme 4 Biotin activation.

CH3 CH3 CH3 CH3

CH3 CH3
OH OH

CH3 CH3
CH3 CH3 n = 4 or 5
OH OH
Vitamin K1 Vitamin K2

Scheme 5 Forms of vitamin K.

HN CO HN CO important to the interconversion and catabolism of amino

HC HC acids (Scheme 8).
Clotting proteins
CH2 or CH2 Further, the relative binding of THFA derivatives to given
HC bone-GLA protein HC enzymes appears related to the number of the glutamyl resi-
precursors dues attached to the 4-(pteridin-6-ylmethyl)-aminobenzoic
HOOC COOH HOOC COOH
acid moiety of folic acid. The number of glutamyl residues
Peptidyl-Glu O2 Peptidyl-γ- also dictates specificity and partitioning into given THFA
CO2 carboxy- apoenzymes.
glutamic acid The biochemical basis of the interrelationship between
(Gla) THFA and cobalamin is the maintenance of two functions:
(1) nucleic acid synthesis and (2) methylation reactions that
Vitamin K-dependent are involved in reactions that range from the silencing of genes
carboxylase
Vitamin K Vitamin K epoxide (DNA methylations) to the synthesis of lipotropic agents and
compounds involved as methyl donors, such as choline, beta-
ine, or methylated derivatives of glycine. The most important
linkage between folate and vitamin B12 occurs at the methio-
nine synthetase step, where 5-methyl-THFA serves as a sub-
Epoxide reductases strate with homocysteine to form methionine.
Scheme 6 Vitamin K-dependent carboxylase reaction. There are two forms of vitamin B12. Both forms of vitamin
B12 consist of a porphyrin-like structure of tetrapyrrole rings
containing cobalt at its center. A 50 ,60 -dimethylbenzimidazole
occur, folic acid must be reduced to tetrahydrofolic acid nucleotide may also linked to the tetrapyrrole rings via a phos-
(THFA) (Scheme 7). phate sugar linkage (see F, Rearrangements on vicinal
Reduction of the pteridine ring brings the two nitrogens at carbons). Methylated vitamin B12 (cobalamin) catalyzes the
positions five and ten closer together, which changes their conversion of homocysteine to methionine by transferring a
electrochemical properties to facilitate the formation of THFA methyl moiety derived from methyl-THFA to methionine. In
single-carbon derivatives. The potential derivatives of THFA are effect, vitamin B12 in this reaction acts somewhat analogous to
N5-methyl-THFA, N5-formimino-THFA, N10-formyl-THFA, a Grignard reagent (e.g., an organometallic complex involved
N5,10-methylene-THFA, N5,10-methenyl-THFA, and N5- in the transfer of an alkyl group) (Scheme 9).
methyl-THFA. The differing oxidation states of a carbon unit As noted, for the transfer of methyl groups, SAM also plays a
are needed for the range of reactions catalyzed by THFA. For predominate role. SAM is essential to the methylation of
example, the formyl, methanyl, and methylene forms of THFA phospholipids and production of methylated forms of various
are utilized for purine and thymidylate (i.e., DNA-related) amino acids and carbohydrates. SAM serves as the principle
synthesis. Methylated forms of THFA participate in reactions methyl source in DNA methylation. The methyl group attached
216 Coenzymes and Cofactors

Folate γ-Glu tail

Pterin pABA Glu

O O COOH
H 10
N CH2 N C N CH
HN 5
6 H H H
7 H (CH2)2 COOH
8
H2N N N H C N CH
H H
O (CH2)2 COOH

C N CH
H
O
n (CH2)2
COOH
Scheme 7 Folic acid.

NH

H HN CH3 HN CH HN
10 10 10
N N
5 5 5

N N N

H H H

Tetrahydrofolic Acid (THFA) N5-Methyl-THFA N5-Formimino-THFA

OHC
H N H2C N HC N
10 10 10
N N N
5 5 5

N N N

H H H

N10-Formyl-THFA N5-N10-Methylene-THFA N5-N10-Methenyl-THFA

Scheme 8 Derivatives of tetrahydrofolic acid.

to the methionine sulfur atom in SAM is chemically reactive.
This allows donation of this group to an acceptor substrate in
transmethylation reactions (Figure 3; Scheme 10).
Rearrangements on vicinal carbons
The rearrangement of vicinal carbon atoms (e.g., an ethenyl
group minus one hydrogen atom [
CH]CH2] or R
CH]
CH2 where R can be any other group of atoms) depends on the
50 , 60 -dimethylbenzimidazolyl nucleotide form of vitamin B12.
Examples include reactions catalyzed by methylmalonyl-CoA
mutase that allows for the oxidation of odd-chain fatty acids
and methylation of branched-chain fatty acids, which is impor-
tant to neural membrane assembly (Figure 4).
Control of reactive oxidants
Reactive oxidant species (ROS) are reactive oxygen ions and
peroxides that include superoxide anion, hydrogen peroxide,
hydroxyl radicals, and their derivatives (e.g., hypochlorous
acid, HOCl). ROS are by-products of the normal metabolism
of oxygen, particularly related to mitochondrial oxidations and
reactions catalyzed by cytochrome P450 oxidases. High levels
of ROS initiate reactions that may alter the structures of impor-
tant molecules, such as DNA, RNA, proteins, and lipids,
 Coenzymes and Cofactors 217

HO
HO N
O N
N
CONH2
N NH2
H CONH2
CH3 CH CONH2
CONH2
3
H CONH2
CONH2 CH2 CH3
H3C
N CH3
CONH2 CH3 N H3C
CONH2 N CH3
Co+3 N
CONH2 CH3
N CONH2
H N Co+3
CH3 N
O H N
H CH3 CH3
O
H3C N H CH3
NH CONH2 N
H3C
H3C N NH CONH2
CH3
HO H3C N
CH3
O O HO
O
P O OH O
OH O
O CH2OH P
O CH2OH
Methylcobalamin 5′-Deoxyadenosylcobalamin

Scheme 9 Derivatives of cobalamin.

Methionine

Tetrahydrofolic acid (THFA) S-Adenosyl-methionine

Methionine
N5-N10-Methylene-THFA synthetase
B-12
–CH3
N5
-N
TH 10-M
FA e N5-Methyl-THFA S-Adenosyl-homocysteine
red thyl
uc ene
tas - Homocysteine
e

Cystathionine synthrtase

Cystathionine
Figure 3 One-carbon metabolism pathway involves the interaction between folic acid, vitamin B12, and S-adenosylmethionine (SAM). Reduced
folic acid serves as donors of single carbons in any one of its three forms: 5-methyl-THFA, 5,10 methylene-THFA, and 10-formyl-THFA. The
single-carbon donor, 5-methyl-THFA, is used to convert homocysteine into methionine, which can then go to form S-adenosylmethionine, the key
substrate in methylation reactions. 5,10-Methylene-THFA is important for the conversion of deoxyuridylate into thymidylate (not shown),
and the donor 10-formyl-THFA is used de novo purine synthesis (not shown).

particularly lipids comprising cellular membranes. As a conse-
quence of the potentially destructive nature of ROS, a number
of systems and compounds have evolved to counteract poten-
tially destructive ROS reactions. A partial list of antioxidant
cofactors including some dietary biofactors is given in Table 3.
Of the vitamins and related derivatives, many are apolar
and reside mostly in the lipid membranes of cells or cellular
organelles. The best example of an essential nutrient that acts
as an antioxidant is vitamin E, a group of ten lipid-soluble
compounds that includes both tocopherols and tocotrienols.
Of the differing forms of vitamin E, g-tocopherol is the most
common (found in many plant oils), and a-tocopherol is
considered to be the most biologically active. Compounds
with antioxidant activity are often referred to as the first line
of defense with respect to cellular membrane protection. Of the
other small molecular weight compounds (vitamin-derived
cofactors and related metabolites), most appear to act as
reductants.
ADP-ribosylation reactions
About half the niacin present in cells as NAD or NADP is used
as a substrate for mono- and polyribosylation reactions, that is,
218 Coenzymes and Cofactors
−OOC
+H N
3
SAM
Methionine
SAM
Acceptor
Methionine
cycle
Methylated
Acceptor Homocysteine
SAH
Scheme 10 Relationship between the methionine and folate cycles.
multiple groups of ADP-ribose moieties are transferred
to proteins to form long branched chains, in a reaction
called poly(ADP-ribosylation). Mono- and polyribosylations
are important in the regulation of a broad range of enzymes.
In the nuclei of cells, polyribosylation of specific histones is an
essential part of DNA repair. ADP-ribosylation is the addition
of one or more ADP-ribose moieties to a protein (Scheme 11).
Cofactor Regulation and Generation
Transport and Transporters
The transport of organic cofactors, their intermediates, and
metals in and out of cells is an important regulatory mecha-
nism. Integral membrane proteins with specific permeability
and/or affinity for cofactors control cellular transport. With
regard to metals, given that metal ions are already at their
chemically basic level, intracellular metabolism is focused on
direct interactions between the metal and its transport path-
ways. Indeed, in most cases, simple diffusion plays a very
minor role. For example, it has been estimated that the ‘free’
cellular copper and zinc concentrations in cells are no more
than a few atoms per cell, because of binding to highly specific
intracellular transporters. In general, absorption of metals
from intestinal lumen cells into circulation or from circulation
into targeted cells occurs through association with protein
complexes, such as in the case for iron and transferrin through
the transferrin receptor and cobalamin and intrinsic factor
through the receptor cubilin, or through a transporter with
permeability for the cofactor, as in the case for the SLC39
family of zinc transporters or sodium-ascorbate cotransporters
SVCT1 and SVCT2.
For nonmetal-, vitamin-, or nonvitamin-derived cofactors,
the processes are also highly regulated and complex. For exam-
ple, for the vitamin-derived cofactors, disassembly is required
for intestinal absorption. Appreciate that vitamins in foods are
usually present as cofactors or in highly modified forms. Pan-
creatic and intestinal cell-derived enzymes (nucleosidases,
NH2
CH3 N
N
+
S
O N
N
HO OH
ATP Serine
THF
Glycine
Methionine Folate
synthetase Methylene-THF
B-12 cycle
Methyl-THF
phosphatases, and peptidases) are key factors in the initial
digestive process with the goal of creating smaller or more
soluble moieties designed to increase the statistical probability
of interacting with luminal receptors for transport out of
the intestinal compartment. In circulation, cofactors or their
precursors may be specifically bound to transport proteins
or loosely associated with proteins, such as albumin and
immunoglobulins.
In addition to dietary deficiencies of given precursors (e.g.,
essential vitamins and minerals), impairments in absorptive
mechanisms, through genetic disorders or other pathophysio-
logical conditions, can also lead to cofactor deficiencies. For
example, pernicious anemia in the elderly can result from
impaired cobalamin absorption in spite of adequate intake
due to insufficient gastric intrinsic factor secretion from gastric
parietal cells. Vitamin B12 absorption is directly dependent on
the interaction between intrinsic factor-bound cobalamin
and the cubilin receptor, which binds this nutrient–protein
complex.
Because various gene products handle the intracellular and
whole body metabolism of cofactors, mutations or polymor-
phisms in such genes can lead to disruptions in cofactor
metabolism with significant health consequences. For exam-
ple, mutations in genes required for intestinal absorption of
cofactors can result in a phenotype with close resemblance to
symptoms of the corresponding dietary deficiency. Elemental
iron absorption is mediated through DMT1, which is encoded
by the gene SLC11A2. Mutations in SLC11A2 in both rats and
mice lead to microcytic anemia, which is also present in iron
deficiency anemia. Likewise, at the cellular level, as would be
expected, transporters also play essential and important roles
in cofactor regulation. Some examples are listed in Table 4.
Further, mutations in the biosynthesis, recycling, or resto-
ration pathways can resemble the dietary deficiency. For
instance, L-gulono-gamma-lactone oxidase is a critical enzyme
in the L-ascorbic acid biosynthesis pathway, and mutation of
this gene in rats leaves it prone to scurvy. Interestingly, humans
and many other primates cannot synthesize their own vitamin
 Coenzymes and Cofactors 219

CoA O
H
S C C COO[H]
H C H
H
− −
O2C O2C
H H
HC C H HC C H

CoA-S H CoA-S C
C
O O
Ado - CH3
Ado - C
N N
N N
CO+2
N N
N N B-12
B-12

−
O2C
H
Methylmalonyl-CoA

HC C H
mutase

Transition CoA-S C
B12

state O−
intermediates
Ado - CH3

N N
CO+2
N N
B-12

−
O2C
−
H O2C
H
H C C H
HC C H
H C O
C O
S-CoA
S-CoA
Ado - CH3
Ado - CH
N N
N N
CO+2
CO+2
N N
N N
B-12
B-12
H
H C COO[H]
S C C H
H
CoA O
Figure 4 Mutase reactions begin with the homolytic cleavage of the bond between the 50 , 60 -dimethylbenzimidazolyl nucleotide form of B-12 and the
Co3þ. A free radical is formed; accordingly, coenzyme B-12 in the catalytic process acts as a free radical generator. A free radical is then formed on
the vicinal carbon of the substrate, which sets the stage for rearrangements, such as the isomerization of methylmalonyl-CoA to succinyl-CoA.

C because of mutations in the gulonolactone oxidase and as a
consequence are sensitive to dietary vitamin C deficiency.
Another example of a mutation in cofactor biosynthesis lead-
ing to a cofactor deficiency is X-linked sideroblastic anemia,
which is caused by a mutation in 5-aminolevulinate synthase
gene ALAS2. Mutations in ALAS2 affect the heme biosynthesis
pathway, and impairment in 5-aminolevulinate synthase leads
to impaired heme synthesis and microcytic anemia concomi-
tant with iron overload due to ineffective use of available iron.
It is also important to note that the phenotype because of
220 Coenzymes and Cofactors

Table 3 Cofactors, related enzymes, and selected biofactors with antioxidant activity

Essential compounds (vitamins) and related derivatives Function

a-, b-, g-, and d-Tocopherols (vitamin E) Lipid peroxide initiated chain reaction breakers
b-Carotenes Singlet oxygen quencher
Lycopene Singlet oxygen quencher
Ubiquinol (CoQ-10) Radical scavenger
Ascorbic acid Reductant
Glutathione Reductant
Urate Radical scavenger
Bilirubin Radical scavenger
NADH, NADPH, FADH2, FMNH2 Sources of reducing potential
Proteins and enzymes
Superoxide dismutase, depending on the organism, utilizes metals (Cu, Dismutation of superoxide anions:
Zn, Mn, and Ni) as cofactors [M(nþ1)þ]-SOD þ O2
! [Mnþ]-SOD þ O2
[Mnþ]-SOD þ O2
þ 2H þ ! [M(nþ1)þ]-SOD þ H2O2.
where M ¼ Cu (n ¼ 1); Mn (n ¼ 2); Fe (n ¼ 2); Ni (n ¼ 2)
Glutathione peroxidase (utilizes Se as a cofactor and glutathione as a Reduces lipid hydroperoxides to their corresponding alcohols and H2O2 to
cosubstrate) water: 2GSH þ H2O2 ! GSSG þ 2H2O
Myeloperoxidase (utilizes Fe as a cofactor) Controls hypochlorous acid production from hydrogen peroxide and
chloride anion
Hydrogen peroxidases (utilizes Fe as a cofactor) Converts hydrogen peroxide to water
Catalase (utilizes Fe as a cofactor) Catalyzes the decomposition of hydrogen peroxide to water and oxygen
a-1-Microglobulin (utilizes a critical cysteine residue at its active center to Degrades heme and is a radical scavenger as well as a reductase
facilitate reactions)
Metal binding proteins (e.g., metallothionein, chaperones, and Sequestration, transport, and regulation of transition metals capable of
ceruloplasmin) nonspecific redox when not protein bound
Diet-derived antioxidants/nutraceuticals
Polyphenol antioxidants (e.g., pyrroloquinoline quinone, hydroxytyrosol, Over 4000 distinct species, many have antioxidant activity; others can
and flavonoids like various flavones; flavanols, such as catechins or effect changes in cell-to-cell signaling, receptor sensitivity,
epicatechins; flavanones; isoflavone phytoestrogens, such as daidzein inflammatory enzyme activity, or gene regulation
and genistein; flavonols such as quercetin (like rutin); and stilbenoids
such as resveratrol)
Phenolic acids (e.g., cinnamic acid, caffeic acid, salicylic acid, and vanillin) Redox cycling, general antioxidant activity
Carotenoids (e.g., lutein, zeaxanthin, and carotenes) Over 600 known carotenoids in two classes xanthophylls (which contain
oxygen) and carotenes (which are hydrocarbons and contain no oxygen)
quench singlet oxygen

N CONH2
N

N N N NAD+
Ribose Ribose

P P
Nicotinamide
Protein
Poly(ADP-ribose) Adenosyl acceptor
polymerase
Adenosyl Ribose Ribose

Adenosyl Ribose Ribose P P

Ribose Ribose P P Poly(ADP-ribose)
glycohydrolase
P P
ADP-ribose
n
ADP-ribose and oligomers
Potential chain
branching
Scheme 11 Poly(ADP-ribose) polymerase reactions.
 Coenzymes and Cofactors 221

Table 4 Examples of transporters important to the regulation of vitamins and minerals

Solute
Transporter name/symbol carrier Genetic disease

Vitamin examples
Thiamine Organic cation transporter 1/OCT1 SLC22A1 Thiamine-responsive megaloblastic anemia
(vitamin B1) Thiamine transporter 1 SLC19A2 Lethal encephalopathy, basal ganglia disease
Thiamine transporter 2 SLC19A3
Riboflavin Riboflavin transporter 1/RFVT1 SLC52A1 Brown–Vialetto–Van Laere syndrome, motor neuron disease
(vitamin B2) Riboflavin transporter 1/RFVT2 SLC52A2
Riboflavin transporter 1/RFVT3 SLC52A3
Folate Reduced folate carrier/RFC SLC19A1 Hereditary folate malabsorption, cerebral folate transport
Proton-coupled folate Transporter/PCFT/ SLC46A1 deficiency
HCP1a
Folate receptor/FOLR1
Ascorbate Sodium-dependent vitamin C transporter SLC23A1
(vitamin C) 1/SVCT1 SLC23A2
Sodium-dependent vitamin C transporter
2/SVCT2
Mineral examples
Iron Divalent metal transporter 1/DMT1 SLC11A2 Microcytic anemia with hepatic iron overload, hereditary
Ferroportin SLC40A1 hemochromatosis
Zinc Zinc, IRT-like, protein 4/ZIP4 SLC39A4 Acrodermatitis enteropathica; Ehlers–Danlos syndrome
Zinc, IRT-like, protein 13/ZIP13 SLC39A13 (spondylocheiro dysplastic)
Copper Copper-transporting ATPase 1/ATP7A SLC31A1 Menkes disease
Copper-transporting ATPase 1/ATP7B Wilson’s disease
High-affinity copper uptake protein 1/CTR1
Heme Heme carrier protein 1/HCP1/PCFT* SLC46A1 Cerebral folate transport deficiency
a
PCFT/HCP1 can transport both folate and heme.

mutations in cofactor transporter genes can also be lethal. For
example, mutation of SLC23A1, which encodes the ascorbic
acid transporter SVCT2, in mice leads to perinatal lethality and
symptoms that do not resemble scurvy or vitamin C deficiency.
The differences (lethal vs. nonlethal) may be due to the devel-
opmental timing of the mutations. Mutations that occur very
early in development are often more likely to be lethal.
Another consideration is that the some minerals and vita-
mins may have other biological roles than strictly cofactor func-
tions, in which case the phenotype evident in the mutation of
transporter genes may reflect a combination of a lack of cofactor
activity and the noncofactor associated effects caused by the
reduced transport of the same nutrient. For example, zinc is a
cofactor for some enzymes such as carbonic anhydrase and
alcohol dehydrogenase, and zinc is also present in many DNA-
binding proteins as zinc fingers and can modulate cell signaling.
Cellular Synthesis Pathways and Chemical Modifications
Important to Cofactor Cellular Regulation or Modulation
of Activity
The regulation of given cofactors occurs at a number of levels.
In addition to regulation at the level of transport, many vita-
mins are subject to chemical modifications that transform
them into coenzymes. Thus, the amounts of available cofactors
may be controlled by chemical modifications and some cases
synthesis. The following sections focus on specific chemical
modifications important to organic cofactor activations to
highlight the range of cellular strategies used.
Phosphorylation. The addition of a phosphate (PO3
4 ) group
is often used as a major homeostatic control. In many proteins
and enzymes, phosphorylation serves as a type of ‘on/off’
switch, thereby altering function or activity. Enzymes desig-
nated as kinases usually catalyze phosphorylation reactions
using ATP as a cosubstrate. Phosphorylation can serve to (1)
alter hydrophilic charter and size so that the cofactor is less
likely to be transported out of a cell or cellular compartment,
(2) improve or alter binding of the cofactor to given enzymes,
(3) change a given chemical characteristic of the cofactor, or
(4) contribute to overall cellular control because of linkages to
the cell’s chemical potential, as defined by ADP/ATP concen-
trations. Some important examples include the following:
• Phosphorylation of ascorbic acid to ascorbate-2-phosphate,
which renders the molecule less likely to act as a redox
initiator by inhibiting keto–enol tautomerism (e.g.,
[
COH]COH–] $ [
CO–CO–])
• Phosphorylation of vitamin B6 to PLP, which allows PLP to
act as an acid catalyst in glycogen phosphorylase, that is, in
addition to its role in transaminases and other PLP-
requiring enzymes
• Phosphorylation of NAD to NADP, which results in a
cofactor that is more likely to function in a synthetic process
(NADP) than catabolic process (NAD)
• Phosphorylation of TPP to thiamine triphosphate, in which
neural tissue is involved in ion gating and transport
• Phosphorylation of pantotheine, which facilitates covalent
attachment of pantotheine-40 -phosphate to other moieties,
222 Coenzymes and Cofactors
such as intermediates in the coenzyme A synthesis pathway
or to ACP subunits
Adenylylations. Examples of adenylylation reactions include
conversion of niacin to NAD(P), pantothenic acid to CoA, and
vitamin B12 to adenosyl B-12 and also the formation of non-
vitamin-derived cofactors, such as SAM and 30 -phosphoadeno-
sine-50 -phosphosulfate (an activated form of sulfate). The
presence of an adenylyl function can improve binding to tar-
geted protein/enzyme binding domains. Analogous to certain
types of phosphorylations, adenylyl transfers represent another
way of linking cofactor generation to changes in a cell’s chem-
ical potential.
Methylations and Amidations. Examples of methylations
include the following:
• Methylation of ascorbic acid to 2-methyl-ascorbic acid. This
type of modification can be important in the regulating
ascorbic acid’s redox activity (inhibits keto–enol
tautomerism).
• Methylation of niacin to 1-methylnicotinamide, which
facilitates the removal of excess niacin from cells.
• Methylation of aromatic cofactors, which can change the
location of reactions on resonating ring structures. For
example, the presence of a methyl group can direct electro-
philes to attack aromatic molecules at ortho- and/or para-
positions relative to the location of the methyl group.
With regard to amidations, the conversion of nicotinic acid to
nicotinamide changes the –[COO-] moiety from an acid to a
weak base. The hydrogen bonding potential with water and
other protic solvents is altered. Similar to methylation, the
presence of a –[CONH2] group is ortho–para directing, that is,
incoming electrophiles are directed to ortho- or parapositions,
which results in the stereospecific transfer of hydride ions from
NAD or NADP.
Conjugations utilizing glutamate and isoprene units. As noted,
folic acid in cells is a pteroyl derivative containing polygluta-
myl residues in g-linkage. The number of g-linked glutamyl
residues in part determines the binding of folic acid (i.e.,
pteroylpolyglutamates) to given enzymes. For example, liver
thymidylate preferentially binds pteroylpolyglutamates with
four glutamyl residues, but derivatives with two to seven glu-
tamyl residues all bind at least 30-fold more tightly than pter-
oylmonoglutamate. Polyglutamylation can also influence folic
acid solubility. For transport in fluids and in and out of cells,
the principle form of folic acid is monoglutamylated, which is
the most soluble form. In the intestine and cells, conjugated
forms of folic acid in foods are converted to the monoglutamyl
folic acid by enzymes with g-linked peptidase activity in order
to increase solubility and eventual transport into the body and
into cells.
Isoprene conjugation is important to the synthesis of phytyl
side chains for numerous biological active biofactors and
cofactors (e.g., carotenoids, tocopherols, and phyllo- and
menaquinones). As an example of a dietary biofactor, preny-
lated flavonoids are produced in plants with varying degrees of
phytoestrogenic and antioxidant activity. Isoprene units (2-
methyl-1,3-butadiene, CH2]C(CH3)CH]CH2) are converted
to dimethylallyl diphosphate and isopentenyl diphosphate in
pathways designed for synthesis of compounds that have
highly divergent functions (e.g., from rubber to biologically
actives sterols). For the fat-soluble vitamins, a hydrophobic
(phytyl) side chain composed from isoprene units allows
such vitamins to partition into lipid membranes of the hydro-
phobic domains of targeted proteins.
Isomerism. The isomeric form of a cofactor also plays a role
in the regulation of given catalytic activities. As an example,
dehydrogenases and reductases often show preferences for one
or the other of the two isomeric forms of NAD[P]H (the A or
pro-R form or B or pro-S form). The two hydrogen atoms of
niacin at the 4-position are prochiral. The binding of NAD to
dehydrogenases or reductases is such that hydride transfer
involves only one side of the nicotinamide moiety. Binding
of the nonpreferred form is generally weaker, and the maxi-
mum velocity is slower. Examples of dehydrogenase and
reductases that prefer the A or pro-R form are alcohol, lactate,
and isocitrate dehydrogenases and dihydrofolate reductase,
whereas glyceraldehyde-3-phosphate, 3-phosphoglycerate,
glutamic acid, and glucose-6-phosphate dehydrogenases and
glutathione reductase prefer the B or pro-S form (Scheme 12).
Covalent Attachment and In Situ Cofactor Formation. Many
cofactors are covalently attached to the enzymes that they
serve. Of the vitamins and vitamin-like compounds, good
examples are biotin, lipoic acid, pantothenic acid (as pan-
totheine), and riboflavin-derived cofactors, FMN and FAD.
Indeed, more than 20 flavoenzymes have been described, for
which five novel FAD or FMN covalent linkages have been
identified (e.g., the covalent linkages to histidyl, tyrosyl, or
cysteinyl residues). PLP could also be included, because as
noted, imine bonds, although capable of dissociation, are
also covalent.
Covalent binding has obvious impact on cofactor metabo-
lism (e.g., metabolic turnover of the cofactor becomes more
dependent on protein catabolism before it can be released and
potentially reutilized). The thermodynamics constraints of
reactions can also be significantly altered (e.g., changes in
entropic considerations).
With regard to in situ cofactor formation, in the past two
decades, the best examples come from the discovery of four
novel redox cofactors derived from posttranslational modifica-
tion of specific amino acids within corresponding enzymes.
These include the topa quinone cofactor, found in copper
amine oxidases; lysine tyrosyl quinone, found in lysyl oxidase;
tryptophan tryptophylquinone, found in bacterial methyl-
amine dehydrogenases; and cysteine-cross-linked tyrosine,
HS Hg
Hg
N2H Hs
O
C C
NH2 O
N: N:
RO RO
O O
OH OH OH OH
H H
pro-R (anti-conformation) pro-S (syn-conformation)
Scheme 12 Isomeric forms of NADH.
 Coenzymes and Cofactors 223

O O

N CH C N CH C
H H
CH2 H2C
2
3 1 NH
H2C N H
H N
CH2 1′ 4
2′ 7 O
3′ 5 6
O
CH2
O
CH2 CH2 O
N CH C C CH N
H H
O O
Lysine tyrosylquinone Tryptophan tryptophylquinone
(LTQ) (TTQ)

NH
O
O CH2 CH
N CH C
H C O
CH2 Cu(II) S
O
1
2 6
3 5 CH2
4
O
N CH C
O− H
O
Topa-quinone Galactose oxidase cofactor
(TPQ)

Scheme 13 Quinone cofactors.

found in galactose oxidase. The topa quinone cofactor of cop-
per amine oxidase is generated by copper-assisted self-
processing of the precursor protein (Scheme 13).
Conclusion
Many of the aforementioned cofactors are found and used in
all forms of life. This suggests that they were present in our
earliest of ancestors. The recent assignment of quinone deriv-
atives as the main compound class composing ‘interstellar’
dust grains is important in this regard. For example, one of
the quinones that was identified, pyrroloquinoline quinone, is
an important redox cofactor used by methylotrophic bacteria
and present in all biological tissues examined to date. Further,
considerable evidence supports the notion that the formation
of RNA was a dominant process in the eventual evolution of
life. RNA is capable of self-replication and catalyzing specific
biochemical reactions. Accordingly, it is not difficult to envi-
sion that cofactors, such as ATP, evolved by a process similar to
the formation of RNA (and DNA) as major components of
reactions important to sustaining energy relationships.
With regard to the discovery and conceptual understanding
of cofactors as catalyst, most of the advances have come in this
century. NAD was the first organic cofactor to be identified
extending from the early work of Arthur Harden and William
Young. They observed that a boiled and filtered yeast extract
accelerated alcoholic fermentation in yeast capable of fermen-
tation. As methods important to the study of protein structure
and enzyme kinetics improved, the relationships that cofactors
play in influencing induced fit and the so-called ‘lock and key’
mechanisms, as well as entropic parameters, entasis, and quan-
tum tunneling, became more apparent.
Although detailed discussions of specific catalytic mecha-
nisms are beyond the scope of this article, our goal nevertheless
was to provide basic descriptions of cofactor functions. Many
of which represent the bases for biochemical lesions associated
with nutritional deficiency syndromes and conditions. For
more detailed discussions of mechanisms, the reader should
consult the references listed in section ‘Future Readings.’ For
additional physiological and nutritional descriptions, the
articles that are cross-referenced should be consulted, particu-
larly those related to vitamins and minerals that serve as
cofactors.
See also: Antioxidants: Role on Health and Prevention; Ascorbic Acid:
Physiology and Health Effects; Calcium: Physiology; Cobalamin
(Vitamin B12): Metabolism and Disorders; Copper: Physiology;
Enzymes: Functions and Characteristics; Folic acid and Folates:
Physiology and Health Effects; Hypovitaminosis A; Iodine: Physiology;
Iron: Biosynthesis and Significance of Heme; Iron: Physiology of Iron;
224 Coenzymes and Cofactors
Magnesium; Manganese; Retinol: Physiology; Riboflavin: Physiology;
Thiamin: Physiology; Tocopherols: Physiology and Health Effects;
Trace Minerals and Trace Elements; Vitamin K: Physiology; Vitamins:
Overview; Zinc: Physiology and Health Effects.
Further Readings
Ames BN, Elson-Schwab I, and Silver EA (2002) High-dose vitamin therapy stimulates
variant enzymes with decreased coenzyme binding affinity (increased Km): relevance
to genetic disease and polymorphisms. American Journal of Clinical Nutrition
75: 616–658.
Harris E (2014) Minerals in foods: bioactivity, metabolism, nutrition, 1st ed. Weimar,
TX: C.H.I.P.S.
Krueger FR, Werther W, Kissel J, and Schmid ER (2004) Assignment of quinone
derivatives as the main compound class composing ‘interstellar’ grains based on
both polarity ions detected by the ‘Cometary and Interstellar Dust Analyser’ (CIDA)
onboard the spacecraft STARDUST. Rapid Communications in Mass Spectrometry
18: 103–111.
McCormick DB (2007) Bioorganic mechanisms important to coenzyme functions.
In: Zempleni J, Rucker RB, McCormick DB, and Suttie J (eds.) Handbook of
vitamins, 4th ed., pp. 175–190. Boca Raton, FL: CRC Press.
Relevant Websites
http://www.javeriana.edu.co/Facultades/Ciencias/neurobioquimica/libros/
metabolismo/metabolismo_archivos/Coenzymes%20and%20Cofactors.pdf –
Coenzymes and Cofactors by Joan B Broderick - Article Pontificia Universidad
Javeriana Bogota.
http://www.rose-hulman.edu/brandt/Chem330/Vitamin.pdf – Vitamins and
Coenzymes PDF - Rose-Hulman Institute of Technology.
http://www.worthington-biochem.com/introbiochem/Enzymes.pdf – Introduction to
Enzymes PDF - Worthington Biochemical Corporation.
 Coffee: Analysis and Composition
MC Cid and M-P de Peña, University of Navarra, Pamplona, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Coffee is the most traded commodity in the world after oil.
Although native to Africa (Ethiopia (Abyssinia)), nowadays, it
is cultivated in regions between the Tropic of Cancer and the
Tropic of Capricorn.
The generic name Coffea covers approximately 70 species, but
only two of them are economically important: Coffea arabica,
commonly arabica coffee, which accounts for three-quarters of
world production, and Coffea canephora var. robusta, commonly
robusta coffee. Differences between these two species include
ideal growing climate, physical aspects, chemical composition,
and characteristics of the brew made with the ground roasted
seeds.
Coffee is one of the most consumed beverages in the world
and a rich source of antioxidants. The amounts of these anti-
oxidants are influenced by several technological factors.
Besides, the antioxidants identified in coffee (chlorogenic
acids (CGAs) and volatile and nonvolatile Maillard reaction
(MR) products) contribute in different proportions to the
overall antioxidant capacity. Several chronic diseases, such as
cancer, cardiovascular, inflammatory, and neurodegenerative
pathologies, are associated with oxidative stress. The antioxi-
dants present in coffee, together with caffeine, have been pro-
posed as those compounds that mainly contribute to its health
properties.
Green Coffee
Composition of Green Coffee
Arabica and robusta green coffees show both qualitative and
quantitative differences in their composition (Table 1).
Green coffee composition is dominated by carbohydrates
(60% dry matter), more abundant in arabica coffee than in
robusta. Most carbohydrates present in green coffee are insoluble
polysaccharides (cellulose and hemicellulose, arabinogalactan,
and galactomannan) accounting for 50%. The soluble fraction
(10%) includes disaccharides (sucrose) and monosaccharides
(glucose, galactose, arabinose, fructose, mannose, mannitol,
xylose, and ribose) in traces.
Proteins, peptides, and free amino acids account for
10–15% of the green coffee dry matter. The main amino
acids, both protein-bound and free, are asparagine, glutamic
acid, alanine, aspartic acid, and lysine. Several other
N-compounds are present in coffee, such as caffeine,
trigonelline, and nicotinic acid. The caffeine concentration in
robusta coffee is approximately double that in arabica.
Lipid contents in green coffee account for 8–18% of its dry
matter. The total content in arabica seeds is approximately
twofold than that in robusta. Fatty acids in coffee are found
primarily in combined forms; most are esterified with glycerol
in the triacylglycerol fraction (75%), 20% esterified with diter-
penes, and a small proportion esterified in sterol esters
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00185-9
(sitosterol (54%), stigmasterol, and campesterol). Most fatty
acids in coffee are unsaturated, being linoleic, oleic, and
linolenic acids the most abundant. Also, palmitic, stearic,
araquidic, lignoceric, and behenic acids and pentacyclic diter-
penes (methylcafestol, cafestol, and kahweol) are present.
Regarding minerals, potassium accounts for approximately
40% of the mineral content of ground coffee, and phosphorus,
for 4%. The remaining mineral content consists of approxi-
mately 30 different elements, including sodium, magnesium,
calcium, and sulfur. Trace minerals in coffee include zinc, sili-
con, manganese, iron, copper, and aluminum, among others.
Green coffee also contains a large amount and variety of
polyphenols. The main components of the phenolic fraction of
green coffee are CGAs (7–12% dry matter), which are more
abundant in robusta coffee. These phenolic acids are formed
by the esterification of one molecule of quinic acid and one to
three molecules of trans-hydroxycinnamic acids in different
positions. The main groups of CGA are caffeoylquinic acids
(CQAs), dicaffeoylquinic acids (diCQAs), feruloylquinic acids
(FQAs), p-coumaroylquinic acids (pCoQAs), and mixed dies-
ters of caffeoylferuloylquinic acids (CFQAs; Figure 1). CQAs
are the most abundant CGAS in coffee, being 5-O-CQA the
most abundant. More recently, numerous minor CGAs
were found in coffee including isomers of dimethoxycinna-
moylquinic acids, dimethoxy-hydroxycinnamoylquinic acids,
dihydroxy-methoxycinnamoylquinic acids, diferuloylquinic
acids, di-pCoQAs, and mixed diesters of the earlier described
hydroxycinnamic and methoxycinnamic acids. In addition,
at least three free cinnamic acids were also identified
in green coffee, namely, caffeic acid, ferulic acid, and
3,4-dimethoxycinnamic acid.
Phenolic compounds different from CGAs and related com-
pounds, such as cinnamoyl glycosides, cinnamoyl amino
acids, anthocyanidins, and lignans, have also been identified
in green coffee in minor amounts.
Roasting Coffee
Roasting Process
The characteristic properties of the coffee beverage, such as flavor
and aroma, are developed during the roasting of coffee. The
beans are heated to 180–250  C, from 2 to 25 min, depending
on roast technique and desired roasting degree (light, medium,
or dark). The initial changes occur at or above 50  C when the
protein in the tissue cells denatures. Three major phases are
drying, pyrolysis, and cooling. During the drying phase, most
of the free water evaporates keeping the bean temperature at
100  C. Temperature rise indicates the second phase. At about
150  C, there is a release of volatile products (water, CO, and
CO2), which results in an increase of 50–80% in bean volume. At
170–200  C, pyrolytic reactions drastically modify the chemical
composition of the bean by the formation of hundreds of sub-
stances that give coffee its characteristic aroma and taste. At the
225
226 Coffee: Analysis and Composition

Table 1 Chemical composition of green Coffea arabica and Coffea modified, resulting in the development of the characteristic
robusta seeds color, aroma, and flavor of coffee. These transformations
have been extensively studied, but the composition of roasted
Concentrationa (g/100 g)
coffee is still far from being elucidated. Table 2 provides a
Component Coffea arabica Coffea robusta rough overview of the main components of roasted arabica
and robusta coffee.
Soluble carbohydrates 9–12.5 6–11.5 Roasting leads to profound changes in the chemical com-
Sucrose 6.0–9.0 3–7 position of coffee with a simultaneous decrease in the naturally
Reducing sugars 0.1 0.4 occurring substances in green coffee and the generation of
Polysaccharides 3–4 3–4 many other compounds derived from the MRs, carbohydrate
Insoluble polysaccharides 46–53 34–44
caramelization, and pyrolysis of organic compounds.
Hemicelluloses 5–10 3–4
Cellulose, b-1,4-mannan 41–43 32–40
Although some compounds formed during roasting might be
Nitrogenous compounds responsible for the health properties of the coffee brew, poten-
Protein/peptides 10–11 11–15 tial carcinogenic compounds, such as acrylamide and furan,
Caffeine 0.8–1.4 1.7–4.0 can also be formed.
Trigonelline 0.6–1.2 0.3–0.9 Most of the carbohydrates present, such as cellulose and
Lipids polysaccharides of mannose, galactose, and arabinose, are
Triglycerides 15–17 8–12 insoluble. During roasting, some of them are degraded to
Diterpenes (free and esterified) 0.5–1.2 0.2–0.8 low-molecular-weight carbohydrates, which are soluble.
Minerals 3–4.2 4.4–4.5 Sucrose present in raw coffee is decomposed in roasted coffee
Chlorogenic acids 6.7–9.2 7.1–12.1
up to half.
5-O-Caffeoylquinic acid 3.0–5.6 4.4–6.6
Phenolic compounds suffer great changes during the heat-
Content varies according to cultivar, agricultural practices, climate, soil composition, ing processing of coffee due to their thermal instability. The
and methods of analysis. content of CGAs drops during roasting as shown in Table 3.
a
%Dry matter; water content of raw coffee 7–13%. During the drying phase, when there is still adequate water
content, isomerization of CGAs occurs, leading to an increase
end of roasting, the beans then burst due to the increase of in 4- and 3-CQAs in roasted coffee. Additionally, some of the
internal pressure, and the popping of the beans indicates that CGAs are hydrolyzed to cinnamic acids and quinic acid. The
roasting must be stopped by cooling (quenching). Despite cinnamates may be decarboxylated and transformed into low-
chemical changes, also, the physical parameters of coffee change molecular-weight compounds. Later in roasting, part of the
considerably. The bean color changes to cinnamon brown, an CGAs lactonize giving rise to several isomers of caffeoylquinic,
11–20% loss in weight occurs, and the specific gravity falls from feruloylquinic, and diCQA lactones (Figure 1). Decomposi-
1.2 to 0.6 due to volume increase. tion of CGAs has a great impact on the quality attributes of
Torrefacto is a roasting process in which sugar is added to roasted coffee and is directly related to flavor development.
coffee, usually robusta. This roasting technique is used in The MR involves the condensation of the carbonyl group of
several countries of southern Europe and South America the reducing sugars with the amino group of amino acids and
where some segments of the population prefer coffees with a proteins. It consists of a complex network of chemical reactions
dark-brown color, intense aroma, and a strong taste with a during which a myriad of products of different chemical com-
tendency to bitterness. This kind of roasting process was ini- positions are formed, commonly known as Maillard reaction
tially used to mask negative sensorial attributes in robusta products (MRPs). The polymeric brown-colored final products
coffees. Nowadays, torrefacto-roasted coffee is usually blended of the MR are called melanoidins and result from the
with conventional roasted coffee (robusta) for commercial use. cyclizations, dehydrations, retroaldolizations, rearrangements,
The degree of roast is reflected on the external color of the isomerizations, and condensations of other MRPs. Melanoi-
beans (from light to dark brown due to pyrolysis of organic dins are one of the major components of roasted coffee,
compounds). Despite visual evaluation, color analysis is tradi- accounting for up to 25% of dry matter. These compounds
tionally carried out on ground roasted coffee using techniques have different chemical compositions, which are still not fully
based on the reflectance of the light in the visible or in the elucidated, and consequently different properties: nutrient or
NIR range of the color spectrum. The use of a standardized contaminant binding, antioxidant or prooxidant, and muta-
tristimulus colorimeter provides lightness (L*) and chromaticity genic or antimutagenic. Because phenolic compounds also
(a* and b*) parameters that can also be applied to calculate participate in the MR, they are at least partly incorporated in
the hue angle (H ) and chroma (C*). The lower the lightness, coffee melanoidins through noncovalent or covalent bonds.
the darker the roast. Similarly for the Agtron-type spec- Besides color development, the MR plays a key role in aroma
trophotometer, which uses NIR illumination, other techniques generation. For example, pyrazines, resulting from the degra-
using lasers have been developed to be used directly on coffee dation of instable MRPs, are potent odorants in roasted coffee.
beans without grinding. Other main classes of aromatic compounds formed during the
MR are aldehydes, ketones, pyridines, oxazoles, thiazoles, and
thiophenes.
Influence of Roasting Process in the Composition of Coffee
Furthermore, the addition of sugar at the end of the torre-
The roasting process causes several changes to the composition facto roasting process might intensify the development of MRs
of the coffee beans, because some compounds are degraded or and, consequently, increase the antioxidant capacity of coffee.
 Coffee: Analysis and Composition 227

O HO O OH
OH HO O OH
HO O O
OH OH
OH OH
OH
O O
OH OH OH OH
OH OH O O
OH
5-Caffeoylquinic acid 4-Caffeoylquinic acid 3-Caffeoylquinic acid

O HO O OH
O HO O OH
HO O O CH3
OH OH
OH OH
CH3 OH
O O CH3
OH OH O O
OH OH O
OH O
5-Feruloylquinic acid 4-Feruloylquinic acid 3-Feruloylquinic acid

O HO O OH
HO O OH
HO O O
OH OH
OH OH
OH
O O
OH OH
OH OH OH O O
5-p-Coumaroylquinic acid 4-p-Coumaroylquinic acid 3-p-Coumaroylquinic acid

HO O OH O
O
OH OH HO O O OH
HO O O
O
OH OH OH OH
O OH OH OH
OH
OH OH
O OH O O OH
OH OH
O O O

3,4-Dicaffeoylquinic acid 3,5-Dicaffeoylquinic acid 4,5-Dicaffeoylquinic acid

HO O OH O O
OH O O
HO O O CH3 HO O O CH3
O CH3
OH O OH OH
O OH OH OH
OH
OH OH
O OH O OH O OH
OH
O O O

3-Caffeoyl-4-feruloylquinic acid 3-Caffeoyl-5-feruloylquinic acid 4-Caffeoyl-5-feruloylquinic acid

HO O OH O O
OH OH HO O O OH
HO O O
O OH
OH OH OH
O OH OH OH
OH OH
O CH3 OH O CH3
O CH3
O O OH O
O O O

3-Feruloyl-4-caffeoylquinic acid 3-Feruloyl-5-feruloylquinic acid 4-Feruloyl-5-feruloylquinic acid

O O
O O O O
OH OH OH
OH
OH
O OH O O CH3
OH OH OH O
OH OH
O O O
3-Caffeoylquinic acid lactone 4-Caffeoylquinic acid lactone 4-Feruloylquinic acid lactone
Figure 1 Main chlorogenic acids in coffee.

Protein is subjected to extensive changes when heated in the
presence of carbohydrates. There is a shift of the amino acid
compositions of coffee before and after bean roasting. The total
amino acid content of the hydrolysates drops by about 30%
because of considerable degradation. Arginine, aspartic acid,
cysteine, histidine, lysine, serine, threonine, and methionine,
being especially reactive amino acids, are somewhat decreased
in roasted coffee, while the stable amino acids, particularly
alanine, glutamic acid, and leucine, are relatively increased.
Free amino acids occur only in traces in roasted coffee because
they are involved in a series of reactions, for example, the MR,
giving rise to potent aroma compounds. Mercaptans, thio-
phenes, thiazoles, alkylpyrazines, pyrroles, pyridines, and pyr-
rolizines result from the degradation of sulfur amino acids,
hydroxy amino acids, and proline.
The caffeine content of raw arabica coffee is 0.8–1.4%,
whereas in the robusta variety, it is 1.7–4.0%. Caffeine is
quite thermostable and decreases only slightly during roasting.
228 Coffee: Analysis and Composition

Table 2 Chemical composition of roasted Coffea arabica and Coffea type of roasting machine. The formation of volatile com-
robusta pounds depends on the stability of their precursors and loca-
tion within the seed. More than 900 compounds with a wide
Concentrationa (g/100 g)
variety of functional groups have been identified until now
Component Coffea arabica Coffea robusta after roasting in different types of coffee.
The classes of volatile compounds found in roasted coffee
Carbohydrates/fiber are furans, pyrans, pyrazines, pyrroles, aldehydes, ketones,
Sucrose Tr – 4.2 Tr – 1.6 phenolics, hydrocarbons, alcohols, acids, esters, lactones, thio-
Reducing sugars 0.3 0.3 phenes, oxazoles, thiazoles, pyridines, amines, and various
Polysaccharides 31–33 37 sulfur and nitrogen compounds. Not all the volatiles in coffee
Nitrogenous compounds
are odorants, and their contribution to flavor is not usually
Protein/peptides 7.5–10 7.5–10
Caffeine 1.1–1.3 2.4–2.5
directly related to their abundance.
Lipids The complex aroma is formed during the roasting process
Triglycerides 17 11 by pyrolysis of the water-soluble components, such as sugars,
Diterpenes 0.9 0.2 amino acids, and trigonelline. A number of them may be
Melanoidins 25 25 produced by more than one route. The main pathways by
which the aroma precursors are degraded are the MR; Strecker
Content varies according to cultivar, agricultural practices, climate, soil composition,
degradation and formation of pyrazines and oxazoles; degra-
and methods of analysis.
a dation of trigonelline, phenolic acids, lipids, and sugars; break-
%Dry matter; water content of roasted coffee 1–5%.
down of sulfur and hydroxy amino acids; and proline and
hydroxyproline degradation. Generally, carbohydrates pro-
duce furans, aldehydes, ketones, and phenols; proteins,
Table 3 Chlorogenic acid content (%) as a function of the degree
peptides, and amino acids produce ketones, pyrroles, and pyr-
of roasting
azines; lipids are responsible for only small amounts of alde-
Raw/degree of roasting Arabica Robusta hydes and ketones given their resistance to changes during the
roasting process; CGAs produce phenolic volatile compounds
Raw 6.9 8.8 (e.g., catechols, pyrogallol, and phenol); trigonelline produces
Light 2.7 3.5 pyrroles, pyridines, and pyrazines. Almost all thiophenes,
Medium 2.2 2.1
oxazoles, and thiazoles are formed during roasting, since they
Dark 0.2 0.2
are not usually detected in green coffee.
Source: Belitz, H. D., Grosch, W. and Schieberle, P. (2009). Food chemistry. Germany: 2-Furfurylthiol is the most important contributor to the
Springer. aroma of coffee. Its precursors are polysaccharides containing
arabinose, for example, arabinogalactans, and cysteine in the
free and bound forms. Part of furfurylthiol and the other thiols
In contrast, trigonelline, which is present in green coffee up to are present in roasted coffee as disulfide bound to cysteine, SH-
1.2%, is 50–80% proportionally decomposed to roasting tem- peptides, and proteins.
perature. The main degradation products are nicotinic acid, Robusta coffees contain alkylpyrazines and phenols in sig-
pyridine, and several alkylpyridines and pyrroles. nificantly higher concentrations than arabica, being earthy and
The lipid fraction appears to be very stable and survives the smoky/phenolic notes in the aroma profile more intensive.
roasting process with only minor changes. Linoleic acid is Arabica coffees are usually richer in the odorants of the
the predominant fatty acid, followed by palmitic acid. The sweet/caramellike group. In torrefacto coffee, where coffee is
major compounds in roasted coffee beans (coffee oil) are roasted with sugar, pyrazines, pyridines, and furans are formed
triacylglycerols (78.8%) and diterpene esters (15%). The in greater quantity than in conventional roasted coffee due to
main diterpenes are cafestol, 16-O-methylcafestol, and kah- increases in the Maillard and caramelization reactions.
weol. Cafestol and kahweol are partially degraded by the roast- At least 28 volatile compounds are reported as key odorants
ing process. Sitosterol and stigmasterol are the major of ground and brewed coffees and contribute to the aroma.
compounds of the sterol fraction. Most of these odorants have been assigned to sweet/caramel,
The potassium is predominant in coffee ash (1.1%), fol- earthy, sulfurous/roasty, and smoky/phenolic notes. The
lowed by calcium (0.2%) and magnesium (0.2%). The pre- remaining odorants have a fruity or spicy odor. Several authors
dominant anions are phosphates (0.2%) and sulfates (0.1%). suggested that only those compounds which concentrations
Many other elements are present in trace amounts. surpass their odor thresholds are odor-active in food. However,
The aroma of roasted coffee is very complex, being com- the evaluation of the odor activity values for all of the volatiles
posed of a large number of volatiles with different odor qual- in coffee is very laborious because concentration and odor
ities, some pleasant, but many others probably below their threshold data must be determined by a large number of
detection threshold. The concentration, proportion, and syn- compounds (Table 4).
ergistic and antagonistic effects among volatile compounds One of the first practicable methods to convert the results of
influence the final aroma quality. instrumental analysis of the volatiles into sensory data was
The volatile composition of roasted coffee depends on many combined hedonic aromatic response measurement analysis
factors, including coffee species/variety, growing and harvest- and aroma extract dilution analysis. In both procedures, serial
ing conditions, storage prior to roasting, degree of roast, and dilutions of an extract containing the volatile fraction of a food

Table 4 Volatile compounds in coffee
Class of compound
Hydrocarbons
Alcohols
Aldehydes
Ketones
Carboxylic acids
Esters
Pyrazines
Pyrroles
Pyridines
Other bases (e.g., quinoxalines and indoles)
Sulfur compounds
Furanes
Phenols
Oxazoles
Others
Total
Source: Nijssen, L. M., Visscher, C. A., Maarse, H., Willemsens, L. C. and Boelens,
M.H. (1996). Volatile compounds. In: Food qualitative and quantitative data (7th ed.),
pp. 72. 1–72.23. Zeist, The Netherlands: TNO Nutrition and Food Research Institute.
are analyzed by gas chromatography–olfactometry (GCO). In
this technique, the separated compounds at the effluent from a
GC column are evaluated qualitatively one by one by human
assessors. An integrated approach involving the joint determi-
nation of the volatile compounds by gas chromatography–
mass spectrometry (GC–MS) and GCO provides useful infor-
mation about the most important active contributors to the
aroma of different coffees.
One of the key factors in the analysis of the profile of
volatile compounds is their extraction. Several methods have
been used to study the aroma fraction of roasted coffee and
coffee brews. One of the first was the preparation of coffee
extracts where volatile compounds were dissolved with organic
solvents, as in steam distillation extraction. This method
should be performed under mild conditions. The temperature,
in particular, is a critical point, due to the instability of, for
example, thiols and disulfides. To avoid the formation of art-
ifacts, the temperature during the isolation of the volatiles may
not exceed 50  C for a longer period.
As an alternative to the injection of an organic solvent
extract, the vapor phase surrounding the coffee sample (head-
space) can be directly analyzed. This method gives the most
accurate composition of volatiles. However, under the usual
operating conditions, the headspace gas sample is considerably
diluted by the carrier gas. This problem can be solved by the
injection of the headspace gas directly into the interior of the
capillary column (on-column injection), using a purge-and-
trap system with an adsorbent or using a static headspace (SH)
sampler. Headspace sampling has progressively replaced tech-
niques such as steam distillation or solvent extraction because
it is the most suitable for studying high volatile compounds
and because its composition represents better the aroma per-
ceived by the consumer. The direct and accurate analysis of
volatiles in coffee by SH (SH-GC) requires a careful standard-
ization of instrumental parameters such as sample size, equil-
ibration time and temperatures, and chromatographic
conditions required for the separation of volatile compounds.
Coffee: Analysis and Composition 229
As the injection procedure can be automated using a headspace
sampler, errors associated with manual handling are removed.
Number Solid-phase microextraction has been extensively applied to
80 the study of coffee aroma as a simple, rapid, solvent-free, and
24 inexpensive method. The selection of fibers and optimization
37 of both extraction and desorption parameters are necessary.
85 Polydimethylsiloxane (PDMS)/divinylbenzene (DVB) coating
28 had the highest overall sampling sensitivity, whereas carboxen
33 (CAR)/PDMS was the most effective for small molecules and
86 acids. In coffee, the DVB/CAR/PDMS fiber has been chosen
66 because its three-component composition gives high recoveries
20
for analytes with different structures and polarities.
52
The separation of volatile compounds is usually achieved
100
126 by GC. The components eluting from the GC can be detected
49 with an FID, although specific detectors, including the NPD
35 and FPD, are also widely used for coffee volatiles, but MS is
20 nowadays the most common detector used to identify and also
841 to quantify (by total ionic current) coffee volatiles.
The change in the flavor profile from the ground coffee to
the brew is caused by a change in the concentrations of these
compounds and not by the formation of new odorants. The
aroma of coffee is not stable; the fresh note is rapidly lost. Of
the highly volatile odorants, methanethiol evaporates the fast-
est, followed by acetaldehyde.
CGA contributes to body and astringency; sucrose contrib-
utes to the color, aroma, bitterness, and sourness; proteins
remain perfectly stable, but minor protein components, such
as free amino acids, are highly reactive. Trigonelline generates
pyridine and may consequently be responsible for some objec-
tionable flavors. Caffeine has no function other than a contri-
bution to the bitterness.
Coffee Brew
The brewing process is essential in brew composition, because
the contact of water with roasted coffee grounds is the crucial
step for the extraction of coffee compounds. Other factors,
such as origin or variety of coffee beans, blending, roasting
degree, and grinding, also play a key role in coffee brew
composition.
Among the several brewing techniques, filter coffee (drip
filter) is the most widely used coffee brew obtained by infusion
method, whereas espresso coffee (EC) is the most appreciated
coffee brew produced by pressure method. Moreover, in
southern European countries such as Italy and Spain, the use
of the mocha coffee maker is much extended at domestic level,
and the plunger coffee maker is being used more often for
coffee aroma lovers. In each case, the technical conditions
applied, such as the coffee/water ratio, water temperature,
and water pressure, also contribute to the different chemical
compositions of coffee brews.
In drip filtration methods, water at 92–96  C flows through
a hardly compressed ground coffee bed, and the extract drips
from the brewing chamber into the pot. Turbulence in the
brewing chamber prevents water from becoming saturated.
This method involves extracting water-soluble compounds,
but most of the lipophilic fraction remains in the filter with
the solid materials.
230 Coffee: Analysis and Composition
In pressure methods, water at approximately 9 bar and
88–92  C is forced to go through coffee grounds compacted
in a small brewing chamber (coffee cake). Rapid brewing time
and fine particle size are necessary. In this case, the composi-
tion and quality of the brew also depend on the water pressure.
Usually, the extraction of water-soluble components
including CGAs, caffeine, nicotinic acid, trigonelline, soluble
melanoidins, and hydrophilic volatile compounds is greater at
higher temperature and pressure. Galactomannans and arabi-
nogalactans are the predominant polysaccharides that pass
into the brew.
Caffeine is the single most frequently determined com-
pound in coffee products, and the methods applied to its
analysis have changed dramatically over the last 25 years.
Caffeine absorbs strongly in the ultraviolet region (272 nm in
water and 276 nm in chloroform), and this provides the basis
of countless spectrophotometric methods. At present, chro-
matographic methods, and mainly reversed-phase HPLC, pro-
vide the most reliable way to quantify caffeine in coffee
products. A simple aqueous extraction produces very complex
extracts, which cannot be directly used. A similar chloroform
extract is significantly cleaner but still contains interfering
compounds that absorb at 276 nm. The level of these interfer-
ing components may be reduced by simple clarification with
lead acetate and filtration to remove polymeric materials in
samples, solution cleanup with a C18 Sep-Pak cartridge, and
the column chromatography. In many cases, the chromato-
graphic conditions allow the simultaneous determination of
trigonelline and caffeine. A smaller number of papers have
described alternative liquid chromatographic techniques, for
example, ion-exchange and even gel-filtration.
The most abundant phenolic compounds of coffee are CGAs.
The main hydrophilic antioxidant compounds in coffee are
3-, 4-, and 5-monocaffeoylquinic acids and 3,4-, 3,5-, and
4,5-diCQAs. Also, little amounts of caffeic acid, ferulic acid,
p-coumaric acid, 4-hydroxybenzoic acid, and sinapic acid can
be found in coffee. Traditionally, the Folin–Ciocalteu colori-
metric method has been used to routinely measure the total
phenolic compounds. Although this is the most popular
method to evaluate the total phenolic compounds, the Folin–
Ciocalteu reagent can be reduced by many electron donors, not
only phenolic compounds. Furthermore, to identify and quan-
tify single phenolic compounds is necessary to apply HPLC
analysis. Detection of phenolic compounds is usually recorded
at 325 nm. Quantification of 5-caffeoylquinic acid (5-CQA) is
made by comparing the peak area with that of the standard.
Because of the lack of commercial standards, in many cases,
quantification of the other caffeoylquinic acids (and other
CGAs) is performed using the area of 5-CQA standard com-
bined with the molar extinction coefficients of the respective
CQA. Also, in recent years, MS has become the most suitable
method to identify and quantify phenolic acids.
The lipid fraction (not water-soluble) of coffee is partially
extracted due to the high temperature of the water and is
present in the brew as an emulsion. However, oil particles are
likely to be retained in filters made of paper or similar types of
materials. The high pressure used to make espresso and the
absence of a filter made of paper, or another lipophilic material
to retain the lipids, facilitate their extraction into the brew.
Thus, unfiltered coffee and EC brews contain higher amounts
of these compounds, including bioactive diterpenes and ste-
rols. The emulsified lipid fraction can be determined by
liquid–liquid solvent extraction with, for example, trichloro-
methane. Total lipids are quantified by weight after evapora-
tion of the solvent. Different amounts have been reported in
both espresso and non-EC beverages. For an EC prepared with
a 100% arabica coffee, 5% of the total solids may be consid-
ered as typical. Within the unsaponifiable fraction of coffee
lipids, two compounds belonging to the diterpene family –
cafestol and kahweol – are of relevance to blood cholesterol
level; present in roasted arabica beans at levels around 0.6%,
they are transferred to the espresso brew in small amounts.
Browned compounds are quantified by measuring the absor-
bance of the sample at 420 nm after exactly 1 min with a
spectrophotometer connected to a thermostatically controlled
chamber (25  C).
Caffeine and the quinic acid lactones are the main bitter
substances in the coffee brew. Also, acidity of coffee beverages
is an important organoleptic parameter. The presence of acetic,
formic, malic, citric, and lactic acids has been detected in the
brew, along with quinic acid and CGAs. The content of the last
ones is of course smaller when using dark roasted coffee, in
which they have largely disappeared. A mineral acid –
phosphoric acid – has also been found in brews, deriving
from the thermal degradation of phytic acid, the phosphoric
acid ester of inositol. Coffee brew acidity cannot be described
just by the pH. The measurement of this variable, obtained
using electrode-type instruments, reports values ranging from
4.8 to 5.8 (optimum 4.9–5.2), showing an increase with roast-
ing degree and depending on extraction time.
EC is an increasingly popular drink. By definition, EC is a
“polyphasic beverage prepared from roast and ground coffee
and water alone, constituted by a foam layer of small bubbles
with a particular tiger-tail pattern, on top of an emulsion of
microscopic oil droplets in an aqueous solution of sugars,
acids, protein-like material and caffeine, with dispersed gas
bubbles and solids.” These characteristics of EC are responsible
for their peculiar sensorial properties. A fine EC should have a
great amount of persistent, consistent, and hazelnut foam with
‘tiger-skin’ effect, a bitter/acid balance taste, and a strong body.
A good EC cup could be prepared from approximately 7.5 g of
finely ground roasted coffee for a volume of 40 ml of water at
9 atm of pressure and 96  C temperature.
The quality of a good cup of EC is initially evaluated by the
quality of its foam. The foam index is defined as the ratio, in
percentage, of EC foam and liquid volumes measured imme-
diately after the extraction of EC using a 100 ml graduated
cylinder. The persistence of foam is defined as the time (in
minutes) that the liquid phase below the cream layer took to
appear during cooling at room temperature. Foamability seems
to be influenced by a protein fraction, while foam persistency
depends more on a polysaccharide fraction.
Density is little influenced by coffee solids. Differences with
respect to pure water are limited to the second decimal digit;
this could be explained by the presence of coffee lipids dis-
persed as an emulsion, which are less dense than water and
decrease the overall density. The addition of sugar can raise
density to around 1.08 g ml 1. Viscosity is influenced by the
presence of dispersed phases, related to the amount of lipid
droplets in emulsion. Increased viscosity has been associated

with improved body. Surface tension is a property of interface,
due to the tendency of molecules to diffuse from one material
to another: pure water–air interfaces.
The brewing method may influence the hydrolytic degrada-
tion of insoluble carbohydrate polymers, adding to the soluble
content. Total solids content is the most important character-
istic in the chemical composition of EC, often perceived by
consumers as ‘strength’; a typical figure for soluble carbohy-
drates is 8 g l 1, corresponding to around 15% of total solids.
The total solids are determined by oven-drying 40 ml of EC to a
constant weight (14 h, 102  3  C) without filtering. Accord-
ingly, suspended solid materials, emulsified lipids, and solutes
are included. The extraction is defined as the percentage of total
solids with respect to ground roasted coffee dose (7.5 g). The
concentration is defined as the percentage of total solids with
respect to the EC volume (40 ml). The total solids on filtrate are
determined by oven-drying 40 ml of EC after filtering with
Whatman 1 to a constant weight (14 h, 102  3  C). The true
soluble fraction, typically 90% of the total solids, can be deter-
mined by oven-drying after filtering the liquid.
Antioxidant capacity. During very intense heat treatments,
such as the roasting of coffee, the loss of antioxidant activity
of natural antioxidants – mainly represented by polyphenols –
by progressive thermal degradation has been found to be min-
imized by the formation of MRPs. The antioxidant activity of
coffee can be measured using different methods. Colorimetric
reactions are developed to evaluate the ability of a food com-
pound or product to quench and/or reduce radicals or metals
using DPPH, or ABTS radicals, or the FRAP assay, respectively.
The antioxidant capacity is usually compared with that of a
very well-known antioxidant, such as vitamin E, using Trolox
(a water-soluble vitamin E analog). For this reason, these
methodologies are usually called Trolox equivalent antioxidant
capacity (TEAC) assays. These colorimetric methodologies are
currently the most widespread, but all of them are based on
similar electron transfer redox reactions and evaluate more
than phenolic compounds. Other methodologies like the elec-
tron spin resonance spectroscopy have been proposed as much
more specific techniques, capable of distinguishing the contri-
bution of phenolic and nonphenolic antioxidants in coffee by
means of different stabilized radicals such as Frémy’s salt and
TEMPO. The basis is the absorption of microwave energy
by unpaired electrons, such as those of free radicals, when
they are in a magnetic field. Mainly, phenolic compounds
can be detected when Frémy’s salt is used as the stabilized
radical, whereas TEMPO is mainly scavenged by MRPs, such
as melanoidins.
ABTS: 2,2´-Azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt. DPPH: Potassium persulfate,
2,2-diphenyl-1-picrylhydrazyl. Trolox: 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid. Frémy’s salt:
Dipotassium hydrogen phosphate, potassium dihydrogen
phosphate, sodium chloride, Frémy’s salt (potassium
Coffee: Analysis and Composition 231
nitrosodisulfonate). TEMPO: 2,2,2,2-Tetramethyl-1-piperidin-
e-1-oxyl.
See also: Antioxidants: Characterization and Analysis; Antioxidants:
Role on Health and Prevention; Caffeine: Characterization and
Properties; Caffeine: Consumption and Health Effects; Coffee:
Decaffeination; Coffee: Health Effects; Coffee: Types and Production;
Maillard Reaction; Phenolic Compounds: Bioavailability and Health
Effects.
Further Reading
Belitz HD, Grosch W, and Schieberle P (2009) Food chemistry. Germany: Springer.
Bravo J, Juániz I, Monente C, et al. (2012) Evaluation of Spent Coffee obtained from the
most common coffeemakers as a source of hydrolytic bioactive compounds. Journal
of Agricultural and Food Chemistry 60: 12565–12573.
Clarke RJ and Macrae R (eds.) (1989) Coffee. In: Chemistry, vol. 1. London/New York:
Elsevier Applied Science.
Clarke RJ and Vitzthum OG (eds.) (2001) Coffee. Recent developments. London:
Blackwell Science.
Esquivel P and Jiménez V (2012) Functional properties of coffee and coffee by-
products. Food Research International 46: 488–495.
Farah A (2012) Coffee constituents. In: Coffee: emerging heath effects and disease
prevention, pp. 21–59. Wiley-Blackwell.
Farah A, de Paulis T, Trugo L, and Martin P (2005) Effect of roasting on the formation of
chlorogenic acid lactones in coffee. Journal of Agricultural and Food Chemistry
53: 1505–1513.
López-Galilea I, Fournier N, Cid C, and Guichard E (2006) Changes in headspace
volatile concentrations of coffee brews caused by the roasting process and the
brewing procedure. Journal of Agricultural and Food Chemistry 54: 8560–8566.
López-Galilea I, de Peña MP, and Cid C (2008) Application of multivariate analysis to
investigate potential antioxidants in conventional and torrefacto roasted coffee.
European Food Research and Technology 227(1): 141–149.
Ludwig IA, Sánchez L, Caemmerer B, et al. (2012) Extraction of coffee antioxidants:
impact of brewing time and method. Food Research International 48: 57–64.
Ludwig IA, Clifford MN, and Crozier A (2014) Coffee. In: Handbook of functional
beverages and human health. Boca Raton, FL: CRC Taylor & Francis Group.
Ludwig IA, Clifford MN, Lean MEJ, Ashihara H, and Crozier A (2014) Coffee:
biochemistry and potential impact on health. Food and Function 5: 1695–1717.
Maeztu L, Andueza S, Ibáñez C, et al. (2001) Multivariate methods for characterization
and classification of espresso coffees from different botanical varieties and types of
roast by foam, taste and mouthfeel. Journal of Agricultural and Food Chemistry
49: 4743–4747.
Nijssen LM, Visscher CA, Maarse H, Willemsens LC, and Boelens MH (1996) Volatile
compounds. In: Food qualitative and quantitative data, 7th ed. Zeist, The
Netherlands: TNO Nutrition and Food Research Institute, pp. 72, 1–72.23.
Stalmach A, Clifford MN, Williamson G, and Crozier A (2011) Phytochemicals in coffee
and the bioavailability of chlorogenic acids. In: Teas, cocoa and coffee: plant
secondary metabolites and health, pp. 143–168. Oxford: Wiley-Blackwell.
Relevant Websites
http://www.asic-cafe.org/ – Association for Science and Information on Coffee (ASIC).
http://www.coffeeandhealth.org/ – Coffee and Health (Institute for Scientific Information
on Coffee).
http://coffeechemistry.com/ – Coffee Chemistry.
http://www.coffeeresearch.org/ – Coffee Research Institute.
http://www.ico.org/ – International Coffee Organization (ICO).
 Coffee: Decaffeination
AS Franca, DEMEC/Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The term coffee is mostly employed in association with the
consumable beverage obtained by extracting roasted coffee
with hot water, but it actually comprises a wide range of
intermediate products, starting from the freshly harvested
fruit (coffee cherries), then to green and roasted coffee beans,
to the final product of consumption, the beverage itself. Decaf-
feinated coffee is defined as ‘coffee from which caffeine has
been extracted’ and, as a commercially available product, cor-
responds to roasted and ground coffee. The decaffeination
process is usually performed on coffee beans prior to roasting
(green coffee beans).
Background
Caffeine (1,3,7-trimethylxanthine) is an alkaloid that occurs
naturally in coffee, cocoa beans, kola nuts, and tea leaves. It is
the most widely consumed and studied psychoactive substance
in history, but the general conclusions on its effects on human
health are still controversial. Increased alertness and learning
capacity and better exercise performance have been cited as
some of the positive effects of low to moderate caffeine intake.
While some studies have associated caffeine intake with high
blood cholesterol, coronary diseases, and cancer, others suggest
that its consumption may lower the incidence of suicide and
hepatic cirrhosis. Nonetheless, excessive caffeine intake has
been associated with a wide variety of health problems, includ-
ing aggravated heartburn and acid indigestion, anxiety, tachy-
cardia, insomnia, and accelerated osteoporosis. It has also been
reported to cause mutation, inhibition of DNA repairs, adrenal
stimulation, cardiac arrhythmias, and increased heart output.
In light of some deleterious effects and the general controversy
around this specific compound, a great deal of effort has been
devoted to providing caffeine-free beverages, especially coffee.
Roasted coffee beans contain  1–2% of caffeine by weight.
The decaffeination process removes up to 97% of the caffeine,
so that a cup (150 ml) of decaffeinated coffee contains from 1
to 5 mg of caffeine, as opposed to 60–180 mg for the same cup
with regular coffee.
It should be kept in mind that coffee is a complex mixture
of chemical compounds, and, although earlier studies on the
effects of coffee on health have been mostly associated with
caffeine consumption, it should be emphasized that this com-
pound is just one of the several bioactive substances that are
present in coffee. Unfiltered coffee is a significant source of
cafestol and kahweol, and such diterpenes have been impli-
cated in the cholesterol-raising effects of coffee. Some results of
epidemiological research indicate that coffee consumption
may help prevent several chronic diseases, including type 2
diabetes, Parkinson’s disease, and liver disease. Overall, there
are little evidence of health risks and some evidence of health
232 Encyclopedia of Food and Health
benefits for adults consuming moderate amounts of coffee
(3–4 cups per day).
Decaffeination Procedures
In the production of coffee, green beans are roasted to generate
the coffee soluble solids and volatiles that impart flavor to
the brewed beverage. In order to avoid coextraction of aroma
components in the decaffeination process, green beans are
generally extracted. The conventional methods employed for
coffee decaffeination are organic solvent extraction, water
decaffeination, and supercritical carbon dioxide extraction. A
schematic overview of these procedures is displayed in Figure 1,
and a more detailed discussion on each specific procedure is
presented as follows.
Solvent Decaffeination
The original processes employed for coffee decaffeination
were based on solvent extraction from the green coffee beans.
Solvent extraction relies on the solubility of caffeine in various
organic solvents including acetone, benzene, ethyl acetate,
ethyl alcohol, ethyl ether, and methylene chloride. It can be
accomplished either by direct solvent extraction of the beans or
indirectly, employing water extraction of the beans, followed
by solvent extraction of the caffeine from the water extract.
The first commercial process was developed in Germany
(1905), established by the firm Kaffee HAG. Chlorinated com-
pounds were the first caffeine extractants used in this process.
Trichloroethylene was commonly employed as the solvent of
choice until it became the subject of US Food and Drug Admin-
istration investigations and was eliminated in 1977, because of
its suspected carcinogenicity, and then replaced by methylene
chloride. A wide variety of solvents have been tested over the
years, with dichloromethane (DCM) and ethyl acetate being
the most employed up to date. DCM has also been recently
tested as a caffeine extractor for coffee bean waste, that is, spent
coffee grounds. DCM is not selective as other solvents, includ-
ing methanol and water, although in this specific application,
the coffee was roasted as opposed to the green beans used in
the traditional processes. Also, caffeine levels will be much
lower in this type of waste because most of the caffeine will
be transferred to the beverage, given its high solubility in water
at elevated temperatures (>90  C).
The conventional decaffeination process can be divided
into four major steps: (1) steaming the beans, (2) extracting
caffeine with an organic solvent, (3) driving off the solvent by
steam to a tolerable level, and (4) drying the coffee beans. It
was early determined that direct organic solvent extraction was
either not very effective or rather slow, regardless of the high
solubility of pure caffeine in the specific solvent employed. In
order to improve extraction and expedite caffeine removal,
http://dx.doi.org/10.1016/B978-0-12-384947-2.00183-5
 Coffee: Decaffeination 233

Green coffee beans

WATER EXTRACTION
SOLVENT
CO2
EXTRACTION
EXTRACTION

Moisturizer Moisturizer

Heater

Extractor Extractor
CO2 + caffeine

Extractor
Extractor Extractor

CO2
Adsorption

Extractor Extractor

solvent

Stripper Green
Caffeine-loaded
extract
adsorbent
steam
Dryer

water

Decaffeinated coffee beans

Caffeine-rich green
extract

Caffeine rich-solvent
Caffeine recovery
adsorbent

Caffeine
Figure 1 Schematic overview of conventional decaffeination processes.

coffee beans are thus steamed. This will promote swelling of
the beans and improve solvent permeability and also break
down caffeine complexes. A process patented in 1939 sug-
gested that the coffee beans should be first contacted with
water (20  C) for approximately 1 h prior to steaming. This
immersion of the beans is intended to wet the outside surface
of the beans and also provides a so-called surface absorption of
water, since the water content is raised from 10% to 22%,
approximately. Other processes suggested a wetting step after
steaming in order to raise the moisture content of the beans
up to 40%.
The extraction itself is carried out in a battery of several
fixed bed columns with the solvent, at temperatures ranging
from 25 to 120  C, being contacted countercurrently with the
bed of beans, for a period of up to 10 h. The column that
contains the coffee beans with the lowest caffeine content is
separated and solvent-drained. It is then steam-stripped in
order to remove all the residual solvent. The decaffeinated
coffee beans are then discharged and dried prior to storage.
The caffeine-rich solvent that exits the first column of the batch
is sent to a caffeine recovery stage, and the resulting caffeine-
free solvent is returned to the process. See Figure 1 for a
schematic view of the process.
There are some studies introducing modifications over the
basic solvent extraction procedure. The extraction time can be
reduced down to 3 h by employing a solvent in turbulent flow.
234 Coffee: Decaffeination
The step of moisturizing the coffee beans can be skipped when
dimethyl sulfoxide is employed as a solvent. However, other
compounds including sugars are also dissolved, and thus,
their concentration must be kept near their saturation point.
Some experiments indicate that mixing a suspension of
caffeic acid crystals in water with methylene chloride or ethyl
acetate provides a significant increase in extraction capacity. A
caffeine–caffeic acid complex is rapidly crystallized at room
temperature, and the extent of decaffeination is much higher
in comparison with the same solvents mixed with water.
A patent from 1988 (General Foods Corporation, the United
States) proposed that contacting any type of caffeine-containing
extract solution with caffeic acid should result in an insoluble
caffeine–caffeic acid complex. Several types of extracts/solvents
(oil, water, and organic) were tested, employing both green and
roasted coffees.
Water Decaffeination
In 1941, a process that aimed at the removal of caffeine from
coffee employing the universal ‘green’ solvent was devised. The
so-called water decaffeination (Swiss Water process) consists
basically of replacing the commonly employed organic solvents
by water, taking advantage of the temperature-dependent solu-
bility of caffeine in such nontoxic solvent. However, there are
other water-soluble constituents that should not be extracted in
order to maintain flavor qualities. Therefore, in order to prevent
their extraction, the extracting water must contain equilibrium
quantities of these solubles, with little or no caffeine. Claimed
advantages of this process over solvent decaffeination include
increased extraction rates, elimination of the solvent stripping
system, purer extracted caffeine, and elimination of undesirable
compounds extracted by the organic solvents. Also, the pre-
steaming and pre-wetting steps are eliminated. The extraction
process itself is similar to the one employed for solvent decaf-
feination, with a battery of columns containing the green beans
being contacted countercurrently with the water extract (con-
taining up to 15% green coffee water solubles), for a period
of up to 8 h (see Figure 1). The decaffeinated beans are
washed with water and air-dried. The wash water is mixed
with the caffeine-free water extract in order to maintain the
required equilibrium composition of noncaffeine solubles.
The caffeine-rich extract is also recycled after caffeine removal.
Caffeine recovery is performed by either solvent extraction or
adsorption by activated carbon.
CO2 Decaffeination
The use of supercritical CO2 extraction for coffee decaffeina-
tion started in Germany in 1970. The major advantage of this
method is the use of an inert gas as solvent, while the disad-
vantages are the costly high-pressure equipment and batch
processing. The process is usually carried out at pressures rang-
ing from 180 to 300 bars and temperatures of 40–90  C.
Initially, the coffee beans are brought to a moisture content
of up to 50% (by wetting, steaming, or both) as in the case of
solvent extraction. The beans are then loaded into the extrac-
tion vessel, while a solid adsorbent (activated carbon) is
loaded into the adsorption vessel. As the CO2 is circulated
through the vessels, caffeine is extracted from the beans and
retained by the activated carbon (see Figure 1). The extracted
caffeine may also be removed from the supercritical fluid by
reducing pressure, thereby decreasing its solubility, or by pass-
ing the CO2 into a washing tower, where the caffeine is
absorbed by the water. An alternate of the method represented
in Figure 1 consists in placing both the green coffee beans and
activated carbon into a pressure vessel, being contacted with
CO2 at 220 bar and 90  C. The coffee beans are separated from
the granular activated carbon by a vibrating sieve. Decaffein-
ated beans are then dried down to 10% moisture as in the other
types of decaffeination procedures.
There are several patents introducing a few modifications
and variations over these methods. If a decaffeination rate of
over 97% is required, the caffeine/CO2 mixture can be passed
first through a washing tower and then through a column
containing the activated carbon. The use of supercritical
nitrous oxide has been proposed as a replacement for CO2,
given its higher solvent power under similar pressure and
temperature conditions. The use of a CO2-saturated caffeine-
free green coffee extract has also been proposed. After contact-
ing the beans for several hours at high pressures (up to
300 bar) and temperatures (up to 100  C), there is a rapid
drop in pressure, and the beans are washed with the liquid
for up to 2 h. The CO2-saturated caffeine-containing green
coffee extract is then decaffeinated with supercritical CO2 (the
caffeine is separated from the CO2 in a water absorption
tower). Other proposed modifications include (i) elimination
of the pre-wetting step by employing a varying temperature
profile during extraction, (ii) acidification of the beans during
the pre-wetting step in order to compensate for acidity losses,
and (iii) replacement of the activated carbon by ion exchangers
or zeolites. A recent study proposed simultaneous extraction of
caffeine and chlorogenic acids from coffee beans by employing
both water and supercritical CO2 flowing countercurrently
through the extractor filled with the beans. The nonpolar
recovery section height was varied (increased) by adding
glass beads to the bottom of the extractor. It was observed
that caffeine recovery in the supercritical CO2 increased with
increasing height of the nonpolar recovery section, because
it allowed caffeine already dissolved in the water phase to be
transferred to the supercritical CO2 phase.
Although the majority of CO2 decaffeination processes
employ supercritical conditions in order to obtain satisfactory
extraction, there is a patent from 1989 (Hermstein & Still,
Germany) that proposes the use of liquid CO2. Moisturized
green beans (45–55% moisture) are contacted with water-
saturated CO2 at relatively lower pressures (65–70 bar) in
comparison with supercritical extraction. Extraction time is
quite high, 60 h, but it is claimed that the flavor quality of
the decaffeinated coffee is very good. Caffeine is recovered by
decompression of the CO2–caffeine mixture.
Roasted Coffee Decaffeination
The major drawback of applying the aforementioned methods
directly to roasted coffee is the simultaneous extraction of
volatiles and aroma components. Nonetheless, a few proce-
dures have been proposed. A patent from 1984 (from Kaffee
HAG) described the application of supercritical CO2 decaffei-
nation to roasted coffee. Extraction takes place in two steps, the

first employing dry CO2 to extract aroma components and then
employing CO2 to remove the caffeine itself from wetted
roasted coffee. Afterward, the water-soluble components are
extracted from the decaffeinated coffee and mixed with the
aroma-containing CO2. The resulting extract can then be
freeze-dried to produce decaffeinated instant coffee. A few
years later, the same company proposed a procedure employing
liquid CO2 to remove caffeine from roasted and ground coffee
with minimal extraction of aroma volatiles. Moistened roasted
coffee is placed in a pressure vessel connected in a closed cycle
with another pressure vessel filled with a strong acid ion
exchanger that is selective for adsorption of caffeine. Extraction
takes about 2–3 h in a closed system and the CO2 is kept at low
temperatures (15–30  C). The small amount of roasted coffee
components that are absorbed by the CO2 can be separated by
CO2 evaporation and returned to the roasted coffee. It is
claimed that the flavor of the decaffeinated roasted coffee
obtained by this procedure is similar to regular roasted coffee.
Other Developments
Water-immiscible oils or fatty materials have also been pro-
posed as extractants for decaffeination processes. Examples
include sunflower, soybean, corn, peanut, and coffee oils.
The procedure consists basically of the conventional multistage
countercurrent extraction, with the coffee beans previously
moistened (40–60% water content). Caffeine extraction is car-
ried out at temperatures ranging from 90 to 120  C. The oil is
separated from the green beans by steaming and regenerated
by liquid–liquid water extraction. This type of caffeine extrac-
tant can also be employed for caffeine removal from the
caffeine-rich green extract obtained during water extraction.
Other ‘green’ solvents have been recently evaluated as alter-
natives for caffeine removal. A recent study showed the poten-
tial of ethyl lactate (ethyl 2-hydroxypropanoate) for such
purpose. Accelerated solvent extraction was employed at tem-
peratures ranging from 100 to 150  C and 10 min extraction
time. The recovery of caffeine was in the range of 50–90%.
Further studies are mentioned aiming at the use of supercritical
CO2 with ethyl lactate as a cosolvent.
Caffeine Recovery
Large amounts of caffeine are required for addition to bever-
ages and for pharmaceutical uses. Market needs are met either
by recovering and refining caffeine from the decaffeination
of coffee or via synthetic route. Caffeine removed from coffee
decaffeination processes is usually obtained from a caffeine-
rich solvent mixture, either an organic solvent or water, or from
a caffeine-loaded adsorbent. Caffeine mixed with a volatile
solvent can be recovered by solvent evaporation or distillation.
If the solvent presents a high boiling point, a countercurrent
water liquid–liquid extraction system can be employed.
Removal of caffeine from the water–caffeine mixture coming
from either this type of process or water decaffeination can be
accomplished by (a) liquid–liquid extraction with a volatile
solvent (that will be later evaporated) or (b) adsorption by an
activated carbon. As previously mentioned, caffeine recovery
from processes employing supercritical CO2 is also based on
adsorption.
Coffee: Decaffeination 235
Recovery of caffeine from the saturated adsorbent can be
performed in several ways. The most common procedure is
contacting the caffeine-loaded adsorbent with a chemical
agent that will provide desorption, usually an acidic solution.
Employed acids include acetic, formic, benzoic, citric, dichlor-
oacetic, and lactic, either pure or combined. Other desorption
agents include organic solvents such as methyl ethyl ketone,
ethyl acetate, and dichloromethane. Caffeine can then be recov-
ered from the resulting solution by employing several methods
such as solvent evaporation or distillation, membrane separa-
tion, and crystallization. Both water and CO2-based decaffeina-
tion procedures claim the major advantage of employing ‘green’
solvents. Naturally, such advantage can no longer be claimed
if the previously mentioned solvent-based caffeine recovery
systems are also employed. Thus, some solvent-free processes
have also been devised. In a process developed by Kaffee HAG,
in 1985, desorption is accomplished by treating the caffeine-
loaded activated carbon with hot pressurized water (300  C and
90–200 bar) for 1–3 h. The activated carbon is then ready to be
reused in the decaffeination process without further treatment.
As the adsorption capacity decreases slightly after each cycle of
use, the activated carbon must be eventually reactivated at
high temperatures. The caffeine is recovered from the aqueous
solution by membrane separation or evaporation.
The so-called ‘direct caffeine desorption’ process, based on
two patents from HAG (1986 and 1994), also avoids the use of
solvents in caffeine recovery. The caffeine-loaded adsorbent is
processed in a three-stage fluidized bed reactor. In the first
(upper) stage, caffeine desorption takes place by contacting
the adsorbent with an inert gas from combustion (oxygen-
depleted) at 360  C. In the second (middle) stage, non-
desorbed caffeine and inorganic impurities are decomposed
at a higher temperature. In the third (lower) stage, the activated
carbon is regenerated by the gas entering the reactor at 800  C.
The adsorbent is then water-quenched and returned to the
decaffeination plant. The caffeine-saturated gas stream is trea-
ted in a scrubber system, in which the caffeine is absorbed by
water and later crystallized.
Future Trends
There are a few available reports on attempts to produce genet-
ically decaffeinated coffee beans. Transgenic coffee plants with
suppressed caffeine synthesis using RNA interference (RNAi)
technology have been obtained. Overall, it was concluded that
RNAi seemed to confer a global effect on expression of several
relevant genes, indicating that reduction of caffeine levels was
not due to the suppression of a single enzyme but instead attrib-
uted to a disturbance of the whole biosynthetic pathway. It was
also concluded that theobromine is the major intermediate in
caffeine biosynthesis. Both theobromine and caffeine contents
of the plants were reduced by up to 70%, indicating that it
should be feasible to produce coffee beans that are intrinsically
deficient in caffeine. However, given that caffeine offers protec-
tion to young leaves and fruits from predators like larvae and
also avoids competition by preventing the growth of neighbor-
ing plant species, producing decaffeinated plants through
genetic manipulation still presents a few challenges.
236 Coffee: Decaffeination
Recent innovations in food technologies have led to the use
of traditional technologies, such as fermentation and enzyme
technology, to produce new health food ingredients, reduce or
remove undesirable food components, add specific nutrients
or functional ingredients, modify food compositions, mask
undesirable flavors, or stabilize ingredients. With that in
mind, understanding the mechanisms involved in the degra-
dation of caffeine by either microorganisms or enzymes can
help in the development of a bio-based process for caffeine
removal.
Bacterial strains found capable of degrading caffeine
belonged to Pseudomonas and Serratia genera, and there is an
indication that they can be used for reduction of the caffeine
content in bearing plants. A method has been proposed for
producing tea leaves with less caffeine content by growing
caffeine-degrading bacteria on the surface of the leaf, but no
studies on coffee have been reported. Many of the available
studies on caffeine degradation by biological action means
have focussed on lowering the caffeine content of coffee pulp
and husks (residues generated during the wet and dry proces-
sing of coffee, respectively) so they can be employed as a
supplement for animal feed. A few have focussed on the use
of these residues as fermentation substrates for the production
of value-added chemicals, and caffeine degradation was merely
a consequence of microorganisms’ growth. Anaerobic fermen-
tation of coffee pulp resulted in about 13–63% reduction of
caffeine in 100 days, whereas aerobic fermentation resulted in
100% degradation of caffeine in 14 days. Bioremediation of
coffee pulp and husks to reduce the caffeine content has been
mostly studied in fungal systems. Among the microbial
communities present in coffee pulp, only a few species like
Aspergillus, Penicillium, and Rhizopus were found capable of
degrading caffeine. Aspergillus and Penicillium species degraded
caffeine almost with 100% efficiency at 25  C, whereas the
efficiency of degradation decreased to 30% at 30  C. An effec-
tive method has been reported using coffee pulp and husks as
substrates for the growth of molds. Caffeine was degraded
during the growth of Lentinus edodes, whereas caffeine was
accumulated in the fruiting bodies of Pleurotus sp. Thus, Pleur-
otus sp. could be used to recover caffeine from coffee.
Though the search for a caffeine-degrading microorganism
began over 40 years ago, studies in this research area are still
needed. The degradation pathway in fungi is still not clearly
known. Even in microbial systems, studies have to be per-
formed to ensure that toxic metabolites are not formed by the
strain during its growth. More microorganisms that could
degrade caffeine need to be isolated. Also, studies employing
coffee beans or plants as growth substrates have yet to be con-
ducted. Biological detoxification of coffee pulp by solid-state
fermentation using fungi seems promising. However, the initial
caffeine concentration and external nitrogen concentration are
very crucial for caffeine degradation to be effective. Though the
enzymes involved in the degradation of caffeine are known,
in vitro enzymatic studies for caffeine degradation are not yet
available. Also, such enzymes are not very stable, and thus,
more studies on enzyme stability and biochemical characteri-
zation are required. Such enzymes could be purified and immo-
bilized in a bioreactor, so their caffeine-degrading ability could
be studied and optimized. In time, such studies can lead to the
development of a biological process for caffeine degradation.
See also: Caffeine: Characterization and Properties; Caffeine:
Consumption and Health Effects; Coffee: Health Effects.
Further Reading
Bichsel B (1979) Diffusion phenomena during the decaffeination of coffee beans. Food
Chemistry 4: 53–62.
Clarke RJ (2003) Decaffeination. In: Trugo LC and Finglas PM (eds.) Encyclopedia of
food sciences and nutrition, 2nd ed., pp. 1506–1511. Waltham, MA: Academic
Press.
Gokulakrishnan S, Chandraraj K, and Gummadi SN (2005) Microbial and enzymatic
methods for the removal of caffeine. Enzyme and Microbial Technology
37: 225–232.
Heilmann W (2008) Decaffeination of coffee. In: Clarke RJ and Vitzthum OG (eds.)
Coffee recent developments, pp. 108–124. Oxford: Blackwell Science.
Katz SN (1987) Decaffeination of coffee. In: Clarke RJ and Macrae R (eds.) Coffee –
volume 2: technology, pp. 59–71. London: Elsevier Applied Science.
Relevant Websites
http://www.coffeeandhealth.org/all-about-coffee/decaffeination/ – Coffee & Health.
http://www.ico.org/decaffeination.asp – International Coffee Association.
http://www.swisswater.com/trade/the-swiss-water-experience/science-of-decaffeination –
Swiss Water Process.
 Coffee: Health Effects
R Tofalo, University of Teramo, Mosciano Sant’Angelo (TE), Italy
G Renda and R De Caterina, G. d’Annunzio University of Chieti, Chieti, Italy
G Suzzi, University of Teramo, Mosciano Sant’Angelo (TE), Italy
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Beverages are an important component of our diet. Coffee
occupies an important place among beverages, since it holds
the second position in consumption after water. On average,
people consume 500 billion cups of coffee annually, with a
global production of 8 million tons per year. The major con-
sumers are the United States, Brazil, Europe, and Japan.
Coffee (Coffea L.) is considered as a valuable and enjoyable
beverage, and often, it is consumed due to its stimulatory
effects, mainly related to the presence of caffeine (1,3,7-
trimethylxanthine): generally, 240 ml of instant coffee con-
tains approximately 100 mg of caffeine. Caffeine content can
be, however, influenced by the different technologies used. For
instance, green bean dewaxing and wet processing can reduce
its content, which remains in the range of 0.65–2.30%.
In addition to caffeine, coffee contains more than a thousand
different chemicals, including carbohydrates, lipids, nitroge-
nous compounds, vitamins, minerals, alkaloids, and phenolic
compounds. Some of them, such as trigonelline, nicotinic acid,
chlorogenic acid (5-O-caffeoylquinic acid, CQA), melanoidins,
and diterpenes (cafestol and kahweol), together with caffeine
feature relevant nutritional or functional properties.
The coffee fruit (also called berry or cherry) is an oval drupe
of about 10 mm in size. Coffee beans present an outer skin
called pericarp, which is green in unripe and red-violet, deep
red, yellow, or orange, depending on the cultivar, in ripe fruits.
The pericarp covers the mesocarp (the soft yellowish, fibrous,
and sweet pulp), a pectin adhesive layer, which is a highly
hydrated layer of mucilage, and the endocarp, called the parch-
ment (Figure 1). Finally, a silverskin covers each hemisphere of
the coffee bean (endosperm).
Many factors influence coffee quality, among which the
action of microorganisms. Microbial metabolites produced
during coffee fermentation can diffuse into the grains and
influence the beverage’s final quality. There are different
kinds of coffee beverages, characterized by different nuances
in terms of body, aroma, acidity, and astringency.
Pectin Layer
Seed (Coffee Bean)
Endocarp
Silverskin
Outer Mesocarp
Pericarp
Figure 1 Coffee fruit structure and botanical classification of coffee plant.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00182-3
Coffee is a complex array of biologically active components
that can influence many metabolic processes. The main com-
ponents are alkaloids (caffeine and trigonelline), phenolic
compounds (chlorogenic acids), and diterpenes (cafestol and
kahweol) (Table 1).
The main mechanism of action of caffeine is to antagonize
adenosine receptors (with its A1 and A2A subtypes); a second-
ary effect is the inhibition of phosphodiesterases, with the
subsequent accumulation of cyclic adenosine monophosphate
and an intensification of the effects of catecholamines. Coffee
is metabolized in the liver by enzymes known as cytochrome
P450 1A2 (CYP1A2). Caffeine effects translate, in most people,
in a psychoactive response, which includes increased alertness
and attention, through the stimulation of the central nervous
system, and in a complex cardiovascular response, mainly
consisting of an acute increase in blood pressure. Some bene-
fits related to its consumption include mood enhancement,
better exercise performance, and shorter reaction times.
Caffeine also produces negative effects, such as sleeplessness,
anxiety, restlessness, tension, nervousness, palpitations,
tremulousness, and psychomotor agitation. Moreover, the
risk of rheumatoid arthritis and osteoporosis increases when
coffee is consumed on a regular basis. Some other negative
effects can arise strictly related to some special risk groups: for
such subjects, a reduced consumption of coffee is recom-
mended (Table 2).
Cafestol and kahweol are also present in coffee. They can
increase blood cholesterol and also feature some chemopre-
ventive activity. The way of action of these compounds on
lipoprotein metabolism is not completely understood. The
only scientific evidence available is that they are absorbed
intestinally. The positive association between coffee consump-
tion and serum cholesterol has been observed mainly in Scan-
dinavia and in the United States, where boiled coffee and
filtered coffee are very popular. Therefore, the brewing method
is probably critical to the cholesterol-raising effect of coffee.
Also, French press (cafetière) coffee contains relatively high
levels of cafestol and kahweol (6–12 mg/cup).
Kingdom Plantae
Division Magnoliophyta
Class magnoliopsida
Order Gentianales
Family Rubiaceae
Genus Coffea
Species Arabica; Canephora
237
238 Coffee: Health Effects

Table 1 Main physiological actions of coffee components

Compound Molecular formula Amount (mg/100 ml) Physiological action

Trigonelline O 0.4–1.2 nicotinic acid Antioxidant and antiinflammatory activities

−
O N+
CH3

Kahweol OH 0–0.1 (filtered coffee) Increased low density lipoprotein cholesterol and
H3C OH 0.7–10 (boiled coffee) total cholesterol
Free radical scavenging

O CH3
Caffeine N 30–54 Central nervous system stimulation
Elevation of blood pressure
H3C N N Increased alertness
Increased metabolic rate
N
O Decreased DNA degradation and reduced hydroxyl
H3C radicals
O
Antioxidant properties
Chlorogenic acid HO OH 35–175 CGA Antiinflammatory and antiangiogenic activities
(CGA) 17–87 caffeic acid Antioxidant properties
O
HO
O OH
OH
HO

OH
Cafestol H3C OH 0–0.1 (filtered coffee) Increased low density lipoprotein cholesterol and
0.5–8 (boiled coffee) total cholesterol
Free radical scavenging

Chlorogenic acids and caffeic acids are other important
healthy components present in coffee. Chlorogenic acids are
a family of esters formed between quinic and trans-cinnamic
acids, which are an important group of dietary phenols.
Caffeoylquinic acid (CQA) is the main compound among
chlorogenic acids. The content of chlorogenic acids has
been reported to range from 70 to 350 mg, which would
provide about 35–175 mg of caffeic acid. These compounds
show antioxidant activity in vitro. Probably, their action
in vivo is reduced, because the metabolites deriving from
their catabolism have lower antioxidant activity than the
parent compounds.
Finally, coffee contains some micronutrients, such as mag-
nesium, potassium, niacin, and vitamin E, which may contrib-
ute to the health effects of coffee consumption.
Antioxidant and Antibacterial Properties of Coffee
Lifestyle factors play a key role in human health. Traditional
diets rich in bioactive components are associated with a lower
risk of different illnesses; for example, the consumption of
antioxidants or antioxidant-rich foods induces a lower risk of
cancer (colorectal, hepatic, renal, ovarian, pancreatic, esopha-
geal, endometrial, and pharyngeal cancers), as demonstrated
by several meta-analyses.
Coffee is known as a dietary source of antioxidants and free
radical scavengers, such as caffeine, hydroxycinnamic acids (caf-
feic, chlorogenic, coumaric, ferulic, and sinapic acids), Maillard
reaction products, and melanoidins. Compared to other bever-
ages, coffee stands out for its antioxidant potential; some authors
reported levels of phenolic compounds in several beverages,
 Coffee: Health Effects 239

Table 2 Main groups subjected to caffeine’s adverse effects and safe limits

Groups Risks Limit (mg day 1)

Women of Conception: High intakes of coffee or caffeine ranging from 400 to 800 mg day 1 are 300
childbearing associated with delays in conception
age Pregnancy complications: Levels of at least 300 mg day 1 of caffeine increase the risk of
spontaneous abortion. This association could be explained by the relationship between
nausea and fetal viability
Fetal growth: Caffeine intakes ranging from 200 to 400 mg day 1 induce a decrease in
mean birth weight of about 100 g. Mothers of small for gestational age (SGA) infants had
higher caffeine intakes in the third trimester of pregnancy than mothers of non-SGA
infants
Lactation: High maternal caffeine intakes cause irritability and poor sleeping patterns in
infants
Children Caffeine doses higher than 3 mg kg 1 of body weight can result in some behavioral effects 45 for 4–6 years old, 62.5 for 7–9
(nervousness, anxiety, and sleep disturbances) years old, and 85 for 10–12 years
old
Older adults Higher plasma caffeine concentrations could increase the risk of drug interactions and 400
fracture risk, particularly in the presence of calcium and vitamin D insufficiency

drinks, and infusions in concentrations ranging from 0.07 to
4.16 mg l 1 in the following order: black tea > instant
coffee > coke > red wine > violet carrot juice > apricot nectar >
Turkish coffee > white wine. Coffee components exhibit some
degree of antioxidant activity, so that coffee has the potential of
reducing inflammation by decreasing oxidative stress.
Coffee antioxidant potential is associated with the presence
of both natural compounds and substances developed during
roasting. The temperature and time of the roasting process can
impact on total antioxidant activity. Melanoidins, which are
high-molecular-weight nitrogenous and brown-colored com-
pounds, are formed during the roasting process. They show
antioxidant, antimicrobial, anticariogenic, anti-inflammatory,
antihypertensive, and antiglycative activities. Melanoidins
from coffee showed higher antioxidant activity than those
isolated from other sources, such as beer. All types of coffee
preparations (filter, espresso, and Italian style) show antioxi-
dant capacity, since all are effective free radical scavengers.
Decaffeinated coffee has lower antioxidant capacity than regu-
lar coffee. The antioxidant capacity is about 6–7% after each
200 ml of coffee consumption, resulting in a reduction of
inflammation through the reduction of free radicals and
other reactive oxygen species.
Roasted coffee extract also features activity against some
microorganisms, such as Staphylococcus aureus and Streptococcus
mutans and several species/strains of Enterobacteriaceae (Serra-
tia marcescens, Enterobacter cloacae, and Salmonella enterica).
This activity is related to some coffee characteristic compo-
nents, such as caffeic acid, trigonelline, caffeine, chlorogenic
acid, and protocatechuic acid, all melanoidins with well-
known natural antimicrobial activity.
Cardiovascular Effects of Coffee
The effects of coffee on the cardiovascular system are largely
debated because the underlying mechanisms are complex and
because of the considerable variability in individual responses.
Many effects of coffee on the cardiovascular system have been
attributed to caffeine, although coffee contains hundreds of
chemical substances, many of which, such as polyphenols, are
pharmacologically active.
Several epidemiological studies have examined the associa-
tion between coffee consumption and the risk of coronary
heart disease, but the findings have been equivocal. Experi-
mental data from short-term and animal studies have sug-
gested detrimental effects of caffeine on blood pressure,
insulin resistance, and arrhythmia and have implicated coffee
as a potential cardiovascular risk factor. Moreover, cross-
sectional studies have shown an association between coffee
and plasma cholesterol concentrations. Additionally, evidence
from case–control studies has suggested that coffee consump-
tion is associated with a higher risk of cardiovascular disease.
On the other hand, prospective cohort studies generally have
not supported the existence of an association between coffee
consumption and a higher risk of cardiovascular diseases, indi-
cating that for most healthy people, moderate coffee consump-
tion is unlikely to adversely affect cardiovascular health.
Furthermore, several studies have suggested that also for patients
with established coronary heart disease, it is safe to continue
their habitual coffee consumption. A randomized clinical trial
involving patients with acute ST segment elevation myocardial
infarction (MI) has evaluated the effects of an acute ingestion of
coffee on the autonomic function and cardiovascular health.
Coffee ingestion was associated with an increase in parasympa-
thetic tone, and coffee did not increase cardiac arrhythmia. The
authors concluded that coffee ingestion is safe and not associated
with adverse cardiovascular outcomes in postmyocardial infarc-
tion patients.
The relation between coffee consumption and the risk of
arrhythmias has also been investigated. Although early animal
studies indicated that coffee appeared to cause arrhythmias in a
canine model, more recent studies have suggested that coffee
does not increase arrhythmias. Actually, long-term coffee
drinking might reduce the risk of abnormal cardiac rhythms,
including atrial fibrillation.
Controlled interventional studies have shown that in nor-
mal adults, even acute high-dose caffeine did not affect cardiac
240 Coffee: Health Effects
rhythm and rate and did not cause clinically significant ven-
tricular or supraventricular arrhythmias. Moreover, prospective
cohort studies did not find significant association between
coffee consumption and the risk of atrial fibrillation. Mecha-
nisms involved in this potential protection against arrhythmias
are still largely unknown, but it has been hypothesized that
caffeine attenuates negative effects of endogenous adenosine
on cardiac electrical conduction.
The cardiovascular risk factor more extensively studied in
relation to coffee is blood pressure: an elevated blood pressure
is a risk factor for coronary heart disease, congestive heart failure,
stroke, kidney disease, and all-cause mortality, and the relation
between blood pressure and subsequent outcomes is direct and
progressive throughout the usual range of blood pressure,
including the nonhypertensive range. Therefore, even a small
change in average blood pressure levels may have a major public
health effect. Evidence that caffeine is an antagonist of adenosine
receptors suggested the biological plausibility that coffee induces
vasoconstriction and elevates systolic and diastolic blood pres-
sure acutely. However, whether coffee consumption has chronic
effects on blood pressure and cardiovascular disease is far from
obvious and remains controversial. Dietary and other lifestyle
factors play an important role in hypertension and blood pres-
sure control: excess intake of salt or alcohol, suboptimal dietary
pattern, physical inactivity, and excess body weight are here the
most important factors. Coffee drinking might add as another
dietary factor causing hypertension, but whether coffee intake
per se is associated with detrimental long-term blood pressure
changes or increases the risk of hypertension remains debated.
A recent meta-analysis of 20 randomized, controlled trials and
five cohort studies has shown no clinically important effects of
long-term coffee consumption on blood pressure or the risk
of hypertension in coffee consumers.
Various confounding factors may have influenced the
equivocal results of different studies, including differences in
the study design, populations examined (age, sex, usual fre-
quency of coffee drinking, and smoking), types of coffee blend,
and types of preparations used. Particularly, the design of
epidemiological studies may affect findings about the relation
between coffee and blood pressure or cardiovascular risk.
Observational (cohort) studies usually have a large sample
size and can provide insight into the long-term effects of dif-
ferent doses of coffee on blood pressure and possible changes
in the effects by gender, age, race, cardiovascular risk profile,
and other characteristics. However, observational evidence
does not demonstrate causality, because coffee drinking is
part of an individual’s lifestyle and is related to many other
factors, such as alcohol intake, mental stress, and dietary
habits, which may also influence blood pressure.
Randomized clinical trials may provide insight into causal-
ity, because both the intervention (e.g., the use of coffee or
caffeine in tablets) and the control treatment (e.g., decaffein-
ated coffee or placebo) are randomly assigned to participating
subjects, and any confounder that could distort the relation
between coffee and blood pressure should be equally distrib-
uted in both groups. The limitation of currently available
randomized clinical trials on this subject is however that only
fixed doses of coffee or caffeine have been studied, and for a
relatively short period of time, the number of participating
subjects has been limited; and there have often been problems
of poor adherence to the assigned treatments. Furthermore,
trials may have suffered from unsuccessful randomization or
lack of blinding of the participants and/or investigators, which
could introduce bias. Therefore, any conclusion on the cardio-
vascular effects of coffee has to rely on the overall evidence
deriving from multiple sources of information.
The variability observed in the results of coffee studies and
meta-analyses may be in part explained by the development of
tolerance. For example, tolerance to caffeine-induced pressor
effects quickly develops in habitual coffee drinkers, and blood
pressure may be increased only temporarily in occasional cof-
fee drinkers, while it is usually not elevated in heavy drinkers. It
is possible that the complex set of counterregulatory hormones
maintaining blood pressure causes tolerance to the humoral
and hemodynamic effects of caffeine. In addition, above a
certain level of consumption, caffeine’s pressor effect might
be counterbalanced by other coffee ingredients, including
chlorogenic acid, flavonoids, melanoidins, quinide, magne-
sium, cafestol, and kahweol. Also, potassium, contained in
coffee, may lower blood pressure. This may help explain the
observed inverse ‘J-shape’ relation between habitual coffee
drinking and the risk of hypertension, which increased up to
three cups per day and then slightly decreased at higher
amounts of consumption.
Another reason underlying the heterogeneity of responses
to coffee drinking is the interaction between coffee compo-
nents and the individual genetic constitution (nutrigenetics).
For example, the enzyme accounting for approximately 95% of
caffeine metabolism (the CYP1A2 isoform) has a wide inter-
individual variability in activity, resulting in rapid or slow
metabolism of caffeine. It has been observed that subjects
with slow metabolism have an increased risk of MI. Also,
blood pressure responses are variable between subjects, with
most subjects experiencing an increase in blood pressure,
others showing no change, and some even showing blood
pressure reductions. An association between gene variants in
the adenosine and adrenergic receptors and blood pressure
responses to caffeine has been observed. In principle, the expo-
sure to higher blood pressure values as the result of such
nutrigenetic interactions in some genetically predisposed indi-
viduals may expose such individuals to a higher coffee-related
cardiovascular event. Moreover, it has been observed that
genetic variants of adenosine receptors may also explain the
interindividual variability in the susceptibility to anxiety and
sleep changes induced by caffeine.
Besides blood pressure, other cardiovascular risk factors
may be affected by coffee consumption. Some coffee com-
pounds other than caffeine also have insulin-sensitizing and
anti-inflammatory effects, and recent evidence from prospec-
tive cohort studies has suggested an inverse association between
coffee and the risk of type 2 diabetes mellitus. This relationship
is consistent across age, obesity, and study location (the United
States and Europe), independent of potentially confounding
dietary and lifestyle factors. Some proposed mechanisms able
to explain this association include effects on insulin sensitivity
and/or insulin secretion modulated by different minerals, anti-
oxidants, and phytochemical compounds found in coffee. The
role of caffeine in increasing or decreasing the risk of type 2
diabetes mellitus is still poorly understood. Most findings indi-
cated a dose–response relation, with diabetes risk reduction for

higher levels of coffee consumption. Generally, four or more
cups of coffee per day have generally been associated with a
lower risk of type 2 diabetes mellitus, whereas for lower levels of
consumption, results are controversial. Cross-sectional studies
have highlighted that high coffee consumption is associated
with lower postload plasma glucose concentrations among
persons without diabetes and a lower incidence of impaired
glucose tolerance and type 2 diabetes mellitus. In short-term
metabolic studies, caffeine intake acutely reduces insulin sensi-
tivity, exaggerating the blood glucose response to glucose loads.
This action is probably related to the ability of caffeine to exert
adenosine receptor antagonism or increase catecholamine
levels. Also, decaffeinated coffee consumption has been associ-
ated with a reduction of risk of type 2 diabetes mellitus. Caf-
feinated and decaffeinated coffee consumption has a beneficial
effect on insulin sensitivity because both reduce plasma
C-peptide concentrations. However, the beneficial effect of
decaffeinated coffee on glucose metabolisms requires further
study. Chlorogenic acid and quinides are also probably
involved in the reduction of risk of type 2 diabetes mellitus
because they act on glucose homeostasis in animal models of
diabetes. In humans, the components of coffee other than
caffeine may exert beneficial effects on glucose metabolism.
However, long-term studies on glycemic control are needed.
The previously considered harmful effects of coffee on the
lipid profile depend on how the beverage is prepared. In fact,
boiled coffee has higher concentrations of cholesterol-
increasing compounds, classified as diterpenes, such as cafestol
and kahweol. They are extracted from coffee beans by pro-
longed contact with hot water, while brewed/filtered coffee
has a much lower concentration of cafestol and kahweol
because of the much shorter contact with hot water and the
retention of diterpenes by filter paper. Accordingly, it has
been observed that the consumption of boiled coffee dose-
dependently increase serum total and low density lipoprotein
(LDL) cholesterol concentrations, whereas the consumption of
filtered coffee results in very little change in serum cholesterol.
Regarding the association between coffee consumption and
the risk of stroke, various findings have led to inconsistent
results. Two recent meta-analyses have concluded that moderate
coffee consumption has a preventive effect on stroke, and this is
probably due to the healthy effect of coffee on the main risk
factors for stroke, including hypertension, cardiovascular dis-
eases, and diabetes mellitus. Regarding heart failure, a meta-
analysis has indicated that there is a modest inverse association
between moderate coffee consumption and the incidence of
heart failure. Although potential modifiers of the coffee–heart
failure relationship are not known, it is possible that the benefi-
cial effects on blood pressure and other cardiovascular risk fac-
tors contribute to positively affect the risk of heart failure. In light
of these findings, the current heart failure prevention guidelines
have suggested a revision of the warning about coffee consump-
tion, due to the evidence showing that coffee may provide a
moderate protection against the incidence of heart failure.
Effects on Cancer
The relationship between coffee and cancer raises great interest,
and in fact, more than 500 papers in the last decades have
Coffee: Health Effects 241
estimated the association between coffee consumption and the
occurrence of cancer at 11 organ sites. Some studies found out
that coffee consumption is associated with a reduced risk of
hepatocellular, kidney, and, to a lesser extent, premenopausal
breast and colorectal cancers, while it is unrelated to prostate,
pancreas, and ovarian cancers. Since the results obtained are
often controversial, in 2007, the World Cancer Research Fund
(WCRF) conducted a comprehensive analysis of diet and can-
cer, using a standardized approach to review the overall evi-
dence. This report showed an inverse relationship between
coffee and the risk of pancreatic cancer and kidney cancer.
This influence on cancer development appears to be related
to the chemical composition of coffee and probably due to
compounds able to influence the risk of cancer. For instance,
caffeine can both stimulate and suppress tumors, depending
on the species and the phase of administration. The consump-
tion of caffeine is inversely related to hepatocellular injury.
A population-based case–control study in the United States
highlighted that caffeine was associated with a lower preva-
lence of abnormal alanine aminotransferase activity. Some
other compounds, such as cafestol and kahweol, show anti-
carcinogenic properties, including the induction of phase II
enzymes involved in carcinogen detoxification and the reduc-
tion of bile acid secretion associated with colon carcinogenesis.
Polyphenols and chlorogenic acid are characterized by antiox-
idant effects. In particular, chlorogenic acid reduces blood
glucose levels in rats and increases insulin sensitivity. Chronic
hyperinsulinemia and insulin resistance are confirmed markers
of high risk for some cancer sites. Moreover, caffeic acid
inhibits DNA methylation in human cancer cells: this epige-
netic regulation represents an important mechanism, because
tumor cells are hypermethylated. In fact, DNA hypermethyla-
tion is often associated with the inactivation of genes and
pathways involved in the tumorigenic process, including cell
cycle regulation, inflammatory and stress response, and apo-
ptosis. In addition, coffee-mediated stimulation of the Nrf2/
ARE signaling pathway induces an increase in defense mecha-
nisms against chemical stresses.
The influence of coffee consumption on the main types of
cancer is reported in Table 3.
Effects on Neurodegenerative Diseases
Coffee has been shown to reduce the risk of Alzheimer’s
dementia and other diseases of the central nervous system,
including Parkinson’s disease.
A trend towards a protective effect of caffeine on
Alzheimer’s dementia has been frequently reported. Coffee
may be the best source of caffeine to protect against
Alzheimer’s dementia due to a component in coffee that syner-
gizes with caffeine to selectively enhance plasma cytokines.
A quantitative review of four studies, despite heterogeneous
methodologies and results, indicated that coffee consumption
is inversely associated with the risk of Alzheimer’s dementia,
compared to nonconsumers.
Coffee and caffeine intake have also been associated with
a reduced risk of Parkinson’s disease, especially in men, in a
number of prospective and case–control studies. Caffeine
reduces the loss of dopaminergic neurons in animal models,
242 Coffee: Health Effects
Table 3 Coffee influence on cancer development
Type of
cancers Coffee effect
Breast cancer Coffee consumption has an inverse association with
breast cancer, in particular among premenopausal
women. Premenopausal women with a breast cancer
1, early onset (BRCA1) or breast cancer 2, early onset
(BRCA2) gene mutation who habitually drank six or
more cups of coffee per day experienced a 70%
statistically significant reduction in breast cancer risk
Colorectal Coffee is a protective factor against colorectal cancer.
cancer This action is due to the presence of cafestol and
kahweol that regulate the excretion of bile acids and
neutral sterols into the colon
Prostate No association between prostate cancer risk and
cancer cumulative lifetime daily coffee consumption,
duration of daily drinking, and age when daily
drinking was started has been established
Ovarian No association between coffee and ovarian cancer has
cancer been found. However, some studies showed a
modest inverse relationship between caffeine intake
and ovarian cancer. This effect is probably related to
caffeine modulation of estrogen level circulation
Pancreatic No association between coffee and pancreatic cancer
cancer has been found. A reduced risk was apparent among
men who drank at least three cups of coffee per day
Liver cancer Coffee consumption appears to reduce the risk of liver
cancer. Probably, coffee polyphenols may maintain a
relatively lower iron status and therefore reduce the
risk of liver injury
Kidney Coffee consumption is associated, but not significantly,
cancer with a lower risk of kidney cancer. This action is
probably due to the fact that caffeine has a diuretic
effect by blocking antidiuretic hormone and
antioxidant compounds reduce oxidative damage to
DNA, proteins, and other molecules. Moreover, coffee
consumption may reduce the risk of kidney cancer by
improving insulin sensitivity
and its neuroprotective function is attributed to the antago-
nism on adenosine 2A (A2A) receptors in the brain, which are
being increasingly targeted by anti-Parkinson therapies in clin-
ical trials.
Other Effects
Coffee consumption is also associated with various other
health effects. For instance, coffee may reduce the risk of
depression. Additionally, coffee may improve asthmatic symp-
toms, probably through caffeine, which is a methylxanthine
bronchodilator. Coffee may also enhance physical perfor-
mance in sustained high-intensity exercise. Coffee may also
prevent symptomatic gallstones and its consumption is associ-
ated with protection against some infectious and malignant
diseases, particularly of the liver.
High levels of caffeine may increase urine output and uri-
nary calcium and magnesium excretion, which have implica-
tions for bone health. Caffeinated coffee increases the risk of
bone loss and fractures.
Effects on Mortality
Some studies have also investigated the association between
coffee consumption and major causes of death, including heart
diseases, respiratory diseases, stroke, injuries and accidents,
diabetes, and infections. The results of these studies have
been variable, and data are lacking to clarify the association
between coffee drinking and mortality, to determine whether
there is a dose–response relationship, and to assess whether
associations are consistent across various subgroups. In a large,
prospective cohort study, a dose-dependent inverse association
between coffee drinking and total mortality was observed, after
adjusting for potential confounders (smoking status in partic-
ular). Although the observational nature of the study does not
prove a cause/effect relationship, the authors speculated that a
plausible mechanism by which coffee consumption might
have health benefits is the presence of antioxidant compounds,
including polyphenols. The significant inverse associations of
coffee consumption with death from all causes provide reas-
surance with respect to the concern that coffee drinking might
adversely affect health.
Conclusions
The currently available overall evidence on cardiovascular
effects related to habitual coffee consumption is largely reas-
suring. Moreover, coffee has a protective effect on cancer and
neurodegenerative disease. Therefore, coffee can be included as
part of a healthy diet. Many of coffee benefits probably derive
from its caffeine content, but other substances may have an
important role in health protection, particularly for their anti-
oxidant effect.
It is very difficult to establish the factors responsible for
coffee’s beneficial or harmful effects. In order to better
understand the function of these compounds, they should be
isolated and utilized in a controlled experimental situation,
using a well-established chemical balance of coffee components
and at doses nutritionally achievable. Finally, it is also possible
that coffee drinkers differ in other important dietary and socio-
logical aspects from nonconsumers, and coffee use may be a
surrogate marker of some other dietary or lifestyle risk factor.
Clinical trials need to verify the relationship between coffee
and diseases, controlling coffee type, the original coffee beans,
the roasting process, serving sizes, brewing process, and dura-
tion over a long period of time, with specific quantitative
information (in mg kg 1 day 1) on caffeine intake.
See also: Coffee: Analysis and Composition; Coffee: Decaffeination;
Coffee: Types and Production; Fermented Foods: Fermented
Vegetables and Other Products; Fermented Foods: Use of Starter
Cultures; Functional Foods.
Further Reading
Butt MS and Sultan MT (2011) Coffee and its consumption: benefits and risks. Critical
Reviews in Food Science and Nutrition 51: 363–373.

Costa J, Lunet N, Santos C, Santos J, and Vaz-Carneiro A (2010) Caffeine exposure and
the risk of Parkinson’s disease: a systematic review and meta-analysis of
observational studies. Journal of Alzheimer’s Disease 20: 221–238.
Daglia M, Papetti A, Grisoli P, Aceti C, Spini V, Dacarro C, and Gazzani G (2007)
Isolation, identification, and quantification of roasted coffee antibacterial
compounds. Journal of Agricultural and Food Chemistry 55: 10208–10213.
Dorea JG and da Costa THM (2005) Is coffee a functional food? British Journal of
Nutrition 93: 773–782.
Frost-Meyer NJ and Logomarsino JV (2012) Impact of coffee components on
inflammatory markers: a review. Journal of Functional Foods 4: 819–830.
Jee SH, He J, Appel LJ, Whelton PK, Suh I, and Klag MJ (2001) Coffee consumption
and serum lipids: a meta-analysis of randomized controlled clinical trials. American
Journal of Epidemiology 153: 353–362.
Mostofsky E, Rice MS, Levitan EB, and Mittleman MA (2012) Habitual coffee
consumption and risk of heart failure: a dose–response meta-analysis. Circulation:
Heart Failure 5: 401–405.
O’Keefe JH, Bhatti SK, Patil HR, Di Nicolantonio JJ, Lucan SC, and Lavie CJ (2013)
Effects of habitual coffee consumption on cardiometabolic disease, cardiovascular
health, and all-cause mortality. Journal of the American College of Cardiology
62: 1043–1051.
Coffee: Health Effects 243
Panagiotakos DB, Pitsavos C, Chrysohoou C, Kokkinos P, Toutouzas P, and
Stefanadis C (2003) The J-shaped effect of coffee consumption on the risk of
developing acute coronary syndromes: the CARDIO2000 case–control study.
Journal of Nutrition 133: 3228–3232.
Rebello SA and van Dam RM (2013) Coffee consumption and cardiovascular health:
getting to the heart of the matter. Current Cardiology Reports 15: 403.
Renda G, Zimarino M, Antonucci I, et al. (2012) Genetic determinants of blood
pressure responses to caffeine drinking. American Journal of Clinical Nutrition
95: 241–248.
Santos C, Costa J, Santos J, Vaz-Carneiro A, and Lunet N (2010) Caffeine intake and
dementia: systematic review and meta-analysis. Journal of Alzheimer’s Disease
20: 187–204.
Sofi F, Conti AA, Gori AM, Eliana Luisi ML, Casini A, Abbate R, and Gensini GF (2007)
Coffee consumption and risk of coronary heart disease: a meta-analysis. Nutrition,
Metabolism, and Cardiovascular Diseases 17: 209–223.
Steffen M, Kuhle C, Hensrud D, Erwin PJ, and Murad MH (2012) The effect of coffee
consumption on blood pressure and the development of hypertension: a systematic
review and meta-analysis. Journal of Hypertension 30: 2245–2254.
 Coffee: Types and Production
LR Batista, Federal University of Lavras, Lavras, Brazil
SM Chalfoun de Souza, Agricultural and Livestock Minas Gerais State Research Institution – EPAMIG, Lavras, Brazil
CF Silva e Batista and RF Schwan, Federal University of Lavras, Lavras, Brazil
ã 2016 Elsevier Ltd. All rights reserved.
Coffee Types
Coffee cultivation is spread all over the world, starting in Arabia
(one species is called Coffea arabica, and a variety is ‘Moka’,
named after an Arab village), passing through many countries,
such as Ceylon (Sri Lanka), Java, India, the Philippines, Hawaii,
and Vietnam, among others, some of which are important
producers to this day. Coffee was introduced in America from
plants previously adapted to the climate in Amsterdam and Paris
and planted in Martinique, Suriname, and French Guiana, from
where it was brought to Brazil. Nowadays, coffee is the second
beverage widely consumed in the world, being overcome only by
water, and Brazil is the largest producer in the world.
After oil, coffee is the most valuable traded commodity
worldwide, with global retail sales estimated to be US$ 90
billion. Coffee is the major export product of some countries
such as Brazil, Uganda, Burundi, Rwanda, and Ethiopia. About
70% of the world crop is grown on smallholdings smaller than
10 ha, and hence, it is often a family business that provides
maintenance for over 25 million people worldwide. On a
broader scale, the international coffee trade involves about
500 million people in its management, from cultivation to
the final product for consumption.
The coffee tree belongs to the tribe Coffeeae in the family
Rubiaceae. One hundred species are associated with the genus
Coffea, but only two species are agroeconomically important,
Coffea arabica and C. canephora. C. dewevrei, C. congensis,
C. eugenioides, C. kapakata, C. salvatrix, C. stenophylla, C. liberica,
C. racemosa, and others are primarily used in genetic crosses.
Presently, arabica coffee accounts for about 63% of coffee
produced, and robusta coffee 37%.
Coffea arabica (Arabica Coffee)
Coffea arabica was first described by Linnaeus in 1753. The best
known varieties are ‘Typica’ and ‘Bourbon,’ but from these,
many different cultivars have been developed, such as
‘Caturra’ (Brazil and Colombia), ‘Mundo Novo’ (Brazil),
‘Tico’ (Central America), the dwarf ‘San Ramon’, and the
‘Jamaican Blue Mountain’. Arabica plant is a large bush with
dark-green oval leaves. It is genetically different from other
coffee species, having four sets of chromosomes rather than
two. The fruits are oval and mature in 7–9 months; they usually
contain two flat seeds (the coffee beans) – when only one bean
develops, it is called a peaberry. Compared with robusta,
arabica trees are generally less vigorous and productive with a
higher cost of production and produce beans that contain
about half the amount of caffeine. Arabica coffee produces a
beverage with a typical sweet aromatic taste that can be con-
sumed alone or blended with C. canephora.
‘Catuai’ and ‘Mundo Novo’ are the most traditionally
cropped cultivars of C. arabica, but many others are also
244
economically important worldwide. Since arabica coffee is
often susceptible to attack by pests and diseases, resistance
is a major goal of plant-breeding programs. Arabica coffee is
grown throughout Latin America, in Central and East Africa, in
India, and to some extent in Indonesia.
Coffea canephora (Robusta Coffee)
The term ‘robusta’ is actually the name of a widely grown
variety of this species. It is a robust shrub or small tree growing
up to 10 m height, but with a shallow root system. The fruits
are round and take up to 11 months to mature; the seeds are
oval in shape and smaller than those of C. arabica. Robusta
coffee is harvested in West and Central Africa, throughout
Southeast Asia, and to some extent in Brazil, where it is
known as conilon. Robusta coffee constitutes a relatively new
commercial crop, so there is a great potential for genetic
improvement. ‘Robusta’ is the most widely cultivated variety
of C. canephora in the world, so that the name of this variety is
used to designate the common name of the species. Neverthe-
less, in Brazil, ‘Conilon’ (also known as ‘Kouillou’) is practi-
cally the sole cultivated variety of C. canephora.
Coffea canephora, produces a beverage that qualifies it to be
used for soluble coffee production and to be blended with arabica
coffee. Current research shows that in spite of giving beverage
with different sensory characteristics of the arabica, robusta coffee
produced and processed properly can be a source of desirable
characteristics in the composition of blends with arabica.
Coffea liberica (Liberian Coffee)
Liberian coffee grows as a large strong tree, up to 18 m in
height, with large leathery leaves. The fruits and seeds (beans)
are also large. Liberian coffee is grown in Malaysia and in West
Africa, but only very small quantities are traded.
Early planning of a coffee plantation has an important
meaning and can ensure the success of the activity to be started.
Because it is a permanent culture, mistakes in implementation
may be impossible to fix, seriously compromising the venture.
Thus, important aspects should be considered, such as avail-
able resources, proper choice of the area and cultivar, and the
planting system and management.
Coffee cultivation and treatment involve the following
steps: tree abatement; soil preparation; planting (small plants
are usually grown in nurseries in the same or in external
properties); treatment (soil correction, fertilizing, pest control,
and terrain cleaning manually or with herbicides); fruit picking
(ripe fruit is usually red and therefore called a berry; Figure 1);
sieving to get rid of impurities; transportation; washing to
remove pulp and membranes; sun drying, revolving grains
with a rake, or mechanical drying through hot air blasting;
hand separation of grains; and storing in silos and bagging.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00184-7

Skin
Bean Mucilage
Parchment
Pulp
Figure 1 Green coffee (top) and longitudinal cut of the mature fruit
coffee (cherry) (bottom) showing all the parts present in the coffee fruit.
Planting
One of the most important factors in coffee planting is the
production of well-developed healthy plants of good quality.
Seedlings must have a well-developed root system, have good
root/shoot ratio, be acclimated to the sun, since coffee is grown
in full sun, and be free of pest problems and nutritional defi-
ciencies. However, the formation of good quality planting
material depends primarily on the quality of the seeds.
Coffee Plant Propagation
Propagation by Seeds
Ripe red cherries of arabica coffees are collected and pulped,
and the mucilage is removed by fermentation. The freshly
picked coffee seeds (typically referred to as beans) can be either
planted immediately or dried for later use. Coffee drying takes
place on wire mesh trays in the shade. Coffee seeds with
approximately 10–15% of humidity can be used up to a year
or more if properly stored. Storing coffee beans correctly is
essential for a longer seed life.
Growing Coffee Seeds in Poly Bags
Poly bags, made of black diothene (200 gauge), are commonly
used and filled with a mixture of topsoil, well-rotted cattle
manure, coarse sand, gravel, coffee pulp, and coffee husks. A
ratio of three parts topsoil to one part coarse sand and one part
Coffee: Types and Production 245
cattle manure is often used. A top dressing of nitrogen is
applied by incorporating 20 g urea in 5.0 l of water per meter
of bed. The seedlings of arabica coffee can still be grafted franc
or foot type. A variety of C. canephora is most commonly used
as rootstock considering its resistance to nematodes. Tubes are
conical containers, made of hard plastic (polypropylene). The
production of seedlings in plastic pots in a greenhouse facili-
tates isolation of the nursery in relation to insects, reduces pest,
preserves the integrity of the root system, and increases the ease
of handling in producing grafted seedlings.
Coffee Cultivation
The basis of the success of the coffee activity relies on the
adoption of good agricultural and processing practices, which
can be considered as production processes based on recom-
mendations predetermined aimed at satisfying performance of
coffee farms, in an efficient way generating satisfaction and
safety for the whole production chain.
Good agricultural practices for cultivation are based on
three principles: food security; social and environmental
responsibilities, necessary for the development of a solid strat-
egy for the integration of production; and farm management
capable of monitoring and controlling over the production
process and when necessary making adjustments in the system
(inputs, safety procedures, and management training).
On the other hand, consumers are concerned with aspects
beyond the sensory quality of the product such as the preser-
vation of the environment, improvement of income of the
growers, and rural workers’ safety. Aiming to attest if these
requirements are met, several certifiers are responsible for
guiding, inspecting, and certifying the compliance of these
coffee requirements. The certification movement is growing
by promoting improvements in the entire coffee production
chain. Examples of accreditation in the culture of coffee are
Fairtrade, Rainforest Alliance, Organic, and others.
The trend towards a global economy, a growing
competition, and the search for technologies with higher pro-
ductivity also has effects upon coffee cultivation. Mechaniza-
tion is being disseminated and updated. Moreover, new
methods of cultivation are introduced, among them being
high-density cultivation, in which the distance between plants
is being reduced. This modern method increases the number of
coffee trees from 3000 or 4000 to 100 000 plants per hectare,
with an increase in productivity of around 50% over the tradi-
tional method. This procedure is important for workers’
health, since lower risks are involved and less herbicide is
applied, especially after the third year. On the other hand,
there is an increase in the frequency of tree cutting and higher
demand for control of fungus disease in the plants.
Coffee Harvesting
Depending on the variety, it takes approximately 3–4 years for
newly planted coffee trees to begin to bear fruit. The fruit
(coffee cherry) turns a bright, deep red or yellow when it is
ripe and ready to be harvested.
246 Coffee: Types and Production
Harvesting is one of the most important steps influencing
the coffee quality. Coffee processed from ripe cherries is natu-
rally sweet and shimmering with floral and fruit notes. Coffee
processed from unripe cherries may taste grassy, green, thin, or
astringent and finally processed from overripe, shriveled
cherries (sometimes called raisins) runs the risk of tasting
fermented, musty, or moldy.
Harvesting coffee is particularly challenging because coffee
fruit typically does not ripen uniformly; presenting more than
one bloom, the same branch may simultaneously display ripe
red cherries, unripe green cherries, and dry, overripe black
cherries. Studies with coffee fruits in various stages of matura-
tion showed that the highest quality of cherry fruit occurs in
stage, ideal harvest. The coffee harvested early with a high
percentage of green causes damage in the type of the coffee
(quality) and drink and can also achieve a rate of 20% of losses
on the final yield, in addition to affecting the appearance,
roasting, type, and drink. On the other hand, the longer it
stays in the coffee tree, the greater incidence of black rot grain
or loss of dry mass and quality.
In regions where labor is inexpensive, where families pick
their own small plots, or in mountainous areas, trees may be
picked repeatedly, and only ripe fruits are harvested during each
pass. In parts of the world where labor is scarce or expensive,
coffee may be stripped from the trees in a single picking. Ripe,
unripe, and overripe cherries are all gathered together, along
with some leaves and twigs. Although sophisticated sorting
methods can compensate to some degree for mass picking, no
expedient is quite as effective as repeated, skillful handpicking.
Machines have been developed that selectively pick ripe
cherries by vibrating the tree just vigorous enough to knock
loose the ripe fruit while leaving the unripe fruit still attached
to the tree. Such machines do not approach the selectivity of a
good handpicker and are used only in regions of the world –
Brazil, Australia, and parts of Hawaii – where labor is too costly
to support handpicking. The main exception is Brazil, where
the relatively flat landscape and immense size of the coffee
fields have permitted mechanization of the process. Almost
all fine coffee is still picked selectively by hand.
Harvesting Methods
Strip picked – the entire crop is harvested at one time. This can
be done either by machine or by hand. In either case, all of the
cherries are stripped off of the branch at one time.
Selectively picked – only the ripe cherries are harvested and
they are picked individually by hand. Pickers rotate among the
trees every 8–10 days, choosing only the cherries that are at the
peak of ripeness. Since this kind of harvest is labor-intensive,
and thus more costly, it is used primarily to harvest the finer
arabica beans. At the end of the day, each worker’s harvest is
carefully weighed and each picker is paid on the merit of his or
her work. The day’s harvest is then combined and transported
to the processing plant.
Coffee Processing
Coffee beans are the seeds of fruits that resemble cherries,
with a red skin (the exocarp) when ripe. Beneath the pulp
(the mesocarp), surrounded by a parchment-like covering
(the endocarp), lies two beans, flat sides together. When the
fruit is ripe, a thin, slimy layer of mucilage surrounds
the parchment. Underneath the parchment, the beans are
covered in another thinner membrane, the silver skin (the
seed coat). Each cherry generally contains two coffee beans; if
there is only one, it assumes a rounder shape and is known as a
peaberry. Coffee beans must be removed from the fruit and
dried before they can be roasted; this can be done in three
ways, known as the dry, wet, and semidry fermentation
methods. When the process is complete, the unroasted coffee
beans are known as green coffee (Figure 1).
Dry Method
The dry method (also called the natural method) is the oldest
and simplest method and requires little machinery. The
method involves drying the whole cherry and the process is
environmentally friendly because it produces low amounts of
solid and liquid wastes, with no production of effluents with
high organic matter content (Figure 2). The beverage quality
depends of the proper processing. This coffee has their own
market because the beverage is full-bodied and less acidic than
coffee from wet process.
The method involves drying the whole cherry and, most
often, is used after nonselective harvesting of different matura-
tion stages of the coffee fruit. There are variations on how
the process may be carried out, depending on the size of the
plantation and the facilities available that interfere in the final
quality obtained. The three basic steps, cleaning, drying, and
hulling, are described later in the text.
Firstly, the harvested cherries are usually sorted and
cleaned, to separate the unripe, overripe, and damaged cherries
and to remove dirt, soil, twigs, and leaves. This can be done by
winnowing, which is commonly done by hand, using a large
sieve. Any unwanted cherries or other materials not winnowed
away can be picked out from the top of the sieve. Ripe cherries
can also be separated by flotation (by different density) in
washing channels close to the drying areas.
The coffee cherries are spread out in the sun, either on large
concrete or brick patios or on matting raised to waist height on
trestles. As the cherries dry, they are raked or turned by hand to
ensure homogenous drying. It may take up to 4 weeks before
the cherries are dried to the 12.5% maximum moisture con-
tent, depending on the weather conditions. On larger planta-
tions, machine-drying is sometimes used to speed up the
process after the coffee has been predried in the sun for a
few days.
The drying operation is the critical stage of the process,
since it affects the final quality of the green coffee. Coffee that
has been overdried will become brittle and produce too many
broken beans during hulling (broken beans are considered
defective beans). Coffee that has not been dried sufficiently
will be too moist and prone to rapid deterioration caused by
the attack of fungi and bacteria during the storage time.
The dried cherries are stored in bulk in special silos until
they are sent to the mill where hulling, sorting, grading, and
bagging take place. All the outer layers of the dried cherry are
removed in one step by the hulling machine.
 Coffee: Types and Production 247

Fruit of coffee

Mechanical harvest Harvest derriça

Separation of leaves and twigs

Washing fruits for separations

of differents fractions

Ground for drying

Hulling

Storage of green beans

Figure 2 Main steps involved in dry or natural coffee fermentation. Reproduced from Schwan, R. F., Silva, C. F. and Batista, L. R. (2012). Coffee
fermentation. In: Hui, Y. H. (ed.) Handbook of plant-based fermented food and beverage technology. Boca, Raton, FL: CRC Press, Chapter 42.

The dry method is used for about 90% of the arabica coffee
produced in Brazil; Ethiopia, Haiti, and Paraguay; and India
and Ecuador. Almost all robusta coffees are processed by this
method. It is not practical in very rainy regions, where the
humidity is too high or where it rains frequently during
harvesting.
Wet Method
The wet method (also called the washed method) requires the
use of specific equipment and substantial quantities of water
(Figure 3). When properly done, it ensures that the intrinsic
qualities of the coffee beans are better preserved, producing a
green coffee, which is homogeneous and has few defective
beans. Hence, the coffee produced by this method is usually
regarded as being of better quality and commands higher
prices. This method is used for all arabica coffee, except in
Brazil, Ethiopia, and Yemen arabica. Just a few percentage of
robusta coffee is processed in this way.
Even after careful harvesting, a certain number of partially
dried and unripe cherries, as well as some stones and dirt, will
be present among the ripe cherries. As in the dry method,
preliminary sorting and cleaning of the cherries are usually
necessary and should be done as soon as possible after harvest-
ing. This operation can be done by washing the cherries in
tanks filled with flowing water. Screens may also be used to
improve the separation between the ripe and unripe, large and
small, cherries.
After sorting and cleaning, the pulp is removed from the
cherry. This operation is the key difference between the dry and
the wet methods, since in the latter, the pulp of the fruit is
separated from the beans before the drying stage. The pulping
is done by a machine, which squeezes the cherries between
fixed and moving surfaces. The flesh and the skin of the fruit
are left on one side and the beans, enclosed in their mucilag-
inous parchment covering, on the other. The clearance
between the surfaces is adjusted to avoid damage to the
beans. Pulping operation should also be done as soon as
248 Coffee: Types and Production
Fruits of coffee
Selective manual harvest
Mechanical pulping
Waste skin and pulp
Dry fermentacion Wet fermentacion
Ground for drying
Hulling
Storage of green beans
Figure 3 Main steps involved in wet coffee fermentation. Reproduced
from Schwan, R. F., Silva, C. F. and Batista, L. R. (2012). Coffee
fermentation. In: Hui, Y. H. (ed.) Handbook of plant-based fermented
food and beverage technology. Boca, Raton, FL: CRC Press, Chapter 42.
possible after harvesting to avoid any fruit deterioration, which
might affect the bean quality.
The pulped beans go on to vibrating sieves that separate
them from any unpulped or imperfectly pulped cherries, as
well as from any large pieces of pulp that might remain. From
the screens, the separated pulped beans then pass through
water-washing channels where a further flotation separation
takes place before they are sent to the next stage.
Because the pulping is done by mechanical means, it nor-
mally leaves some residual flesh as well as the sticky mucilage
adhering to the parchment surrounding the beans. The parch-
ment has to be completely removed from the coffee beans to
avoid contamination of products resulting from the mucilage
degradation. The newly pulped beans are placed in large fer-
mentation tanks in which the mucilage is broken down by
natural and microbial enzymes until it is dispersible, when it
can be washed away. Unless the fermentation is carefully mon-
itored, the coffee can acquire undesirable, sour flavors. For
most coffees, mucilage removal takes place between 24 and
36 h, depending on the temperature, thickness of the mucilage
layer, and concentration of the enzymes; the end of the fer-
mentation is assessed when the parchment surrounding the
beans loses its slimy texture and acquires a rougher ‘pebbly’
feel. When the fermentation is complete, the coffee is thor-
oughly washed with clean water in tanks or in special washing
machines.
Another version of this method is when the mucilage is
mechanically removed obtaining the coffee called demucilated
without the fermentation stage consequently with less final
flavor. An intermediary process consists in obtaining the
pulped beans with part of the mucilage and directly drying
them (dry fermentation), obtaining the coffee called dehulled.
The wet parchment coffee at this stage has of approximately
57% moisture. To reduce the moisture to a maximum 12.5%,
the parchment coffee is dried either in the sun, in a mechanical
dryer, or by a combination of the two. Sun drying is done on
extensive flat concrete or brick areas, known as patios, or on
drying tables made of fine mesh wire netting. The beans are
laid out in a layer of 2–10 cm and turned frequently to ensure
even drying. Sun drying should take from 8 to 10 days, depend-
ing upon ambient temperature and humidity. Coffee dries
more quickly if raised on tables because of the upward draft
of warm air. The use of hot air drying machines becomes
necessary to speed up the process in large plantations where,
at the peak of the harvesting period, there might be much more
coffee than can be effectively dried on the terraces. However,
the process must be carefully controlled to achieve satisfactory
and economical drying without any quality damage.
After drying, the wet-processed coffee, or parchment coffee
as it is commonly known, is stored and remains in this form
until shortly before export.
The final stages of preparation of the coffee, known as
‘curing,’ usually take place at a special plant just before the
coffee is sold for export. The coffee is hulled, to remove the
parchment, and then passes through a number of cleaning,
screening, sorting, and grading operations, which are common
to both wet- and dry-processed coffee. Electronic sorting
machines may be used to remove defective beans that cannot
be distinguished by eye.
The wet method is generally used for arabica coffees, with
the exception of those produced in Brazil and the arabica-
producing countries mentioned earlier in the text as users of
the dry method. This method is rarely used for robusta.
Semidry Method
The semidry process (or pulped natural) is a variation of wet
processing. It is an intermediate process between dry and wet
processing and results in what are called pulped natural cof-
fees. It combines a wet mechanical process to remove the pulp
and the spreading out of depulped beans as a thin layer on
cement patios for a period to allow further aerobic degradation
of mucilage (dry process; Figure 4). Some studies have focused
on the chemical properties of coffee beans under semidry
processing and the correlation between those properties and
beverage quality. No study has yet presented conclusive evi-
dence of the level of influence of this process on the final
quality of the beverage. However, it is possible to say that the
qualities of coffee beverages made from pulped natural coffee
lie somewhere between those obtained using dry and wet
processing. The green beans produced via semidry processing
are usually used in espresso blends.

Fruits of coffee
Selective manual harvest
Mechanical pulping
Grond for drying (fermentation)
Hulling
Storage of green beans
Figure 4 Main steps involved in semidry coffee fermentation.
Reproduced from Schwan, R. F., Silva, C. F. and Batista, L. R. (2012).
Coffee fermentation. In: Hui, Y. H. (ed.) Handbook of plant-based
fermented food and beverage technology. Boca, Raton, FL: CRC Press,
Chapter 42.
Microorganisms: Quality and Safety in Coffee
The method of coffee processing impacts on the native
microbiota and, therefore, with the groups of microorganisms
involved during fermentation. In some regions or in certain
producing years, because of the environmental conditions of
coffee, the quality can be inferior because the microbiota
changes to deteriorative microorganisms (like butyric and pro-
pionic acid bacteria). Nevertheless, in all processing methods
(dry, wet, and semidry), the objectives of fermentation, involv-
ing bacteria and yeasts, are to remove the mucilaginous layer
rich in polysaccharides (pectins) and to reduce the water
content present in the fruits. Coffee seeds have all the precur-
sors needed to generate typical flavor and aroma during the
roasting operation but the natural microbiota during the
fermentation/drying step confer special flavor in the coffee
beverage. The microorganisms naturally present in the produc-
tion environment use sugars in the pulp and mucilage and
excrete organic acids and other metabolites that may affect
Coffee: Types and Production 249
the final sensory characteristics of the beverage. In addition,
coffee fermentation and drying must be managed in order
to control the growth of filamentous fungi that can give off-
flavors and produce mycotoxins.
The three different methods for processing the fruit show
quantitative and qualitative differences on the microbiota. The
dry process has more microbial species than the other two
processes (wet and semidry) because the whole fruits are pro-
cessed. On the other hand, in the wet process, the skin and
pulp are removed, and consequently, the epiphytic microor-
ganisms are mechanically removed. The microbiota of the
semidry processing is still not well known but bacteria, yeast,
and filamentous fungi are detected in Brazil and India. The
review for this topic has been published by Silva.
Microorganisms Present in Dry Process
The presence of organic acid from fermentation (acetic, lactic,
butyric, and propionic acids) confirms the microbial action in
the fermentation process for natural coffees. In the beginning
of the process, the bacteria species predominate over yeast and
filamentous fungi, but this changes when the water activity and
physical–chemical conditions alter in the media and final
phase of the fermentation. Some species identified were Tatu-
mella ptyseos, Pseudomonas putrefaciens, P. mirabilis, Enterobacter
aerogenes, Acinetobacter, B. subtilis, and Paenibacillus amylolyticus.
The bacteria present on the surface of the coffee cherries
synthesize the acid that can migrate to the pulp and mucilage
and can interfere with the organoleptic quality of the beans.
The negative influence of bacteria on the quality of the coffee
beverage is linked to the species present and not to population
density. In natural coffee fruit rated very soft (i.e., as having
optimal sensory quality), the bacterial population can repre-
sent up to 80% of the total microbiota during this process.
Yeasts become predominant as the water activity declines
during the fermentation and drying periods, which can last up
to 20 days. Twenty-one species of yeasts have been identified,
including seven pectinolytic species (Debaryomyces hansenii, D.
polymorphus, Pichia anomala, P. holstii, P. burtonii, P. guilliermondii,
and Arxula adeninivorans). The presence of yeasts during the
whole process contributes to the availability of the substrate
and action of pectinases over the pulp and mucilage. The role
of yeast may be related not only to the fermentation process but
also to the control of filamentous fungi growth. Isolates belong-
ing to the genera Debaryomyces and Pichia have demonstrated the
ability to inhibit the growth of toxigenic fungi and may therefore
have the potential for biological control.
Studies of natural coffee microbial populations always
emphasize filamentous fungi isolation and identification, but
the microorganisms present during the fermentation and dry-
ing periods are predominantly bacteria and yeasts. Therefore,
the presence of filamentous fungi is not involved in the fer-
mentation process, and in general, their presence decreases the
sensorial and sanitary quality during the mycotoxin produc-
tion, especially ochratoxin A (OTA).
Microbiota Present in Wet and Semidry Processing
The microbial diversity of coffee beans in wet processing is
smaller compared with that in dry processing, because the
250 Coffee: Types and Production
fermentation time is shorter (up to 48 h) and there is a more
rapid pH decline from 6 to 4.3. This microbiota comprises few
bacterial and yeast species. There is no report of filamentous
fungi involved in wet fermentation. The mucilage adhered to
beans is the substrate used by bacteria and yeast during fer-
mentation. Higher microbial diversity in washed coffee fruits
could be observed using molecular identification techniques
that allow the detection of cultivable and uncultivable
microorganisms.
The molecular identification of microorganisms associated
with coffee showed that, in semidry process, the microbial
succession occurred: bacteria dominated at the beginning of
the process, but after 72 h, bacteria and yeast populations
reach similar values (average 106 CFU g
1) and after 192 h of
fermentation, the yeast population predominates.
Currently, there is still doubt regarding the apparent bacte-
rial role on coffee fruit fermentation. Recently, it was observed,
in wet process, that the predominant population was lactic acid
bacteria (LAB), especially Weissella, Lactobacillus, and Leuconos-
toc (W. cibaria and W. soli, Lb. plantarum and Lb. brevis,
L. mesenteroides, L. pseudomesenteroides, and L. citreum). Thus,
it is possible that the LAB in the wet processing are responsible
for the degradation of the mucilage; however, this group of
bacteria does not participate in the coffee fermentation in dry
fermentation. It is possible that the anaerobic or low oxygen
conditions present in wet fermentation favor the LAB develop-
ment. The action of LAB allows the pH to fall, preventing the
proliferation of other bacteria and promoting yeast growth.
About the role of yeast in the fermentation of the depulped
beans, some researchers do not believe the yeast action on the
mucilage degradation. However, pectinolytic yeasts have been
isolated during the wet processing of coffee in both robusta
and arabica coffees. The fermentation also occurs due to the
action of yeast, namely, Kluyveromyces sp., Saccharomyces cere-
visiae, Candida parapsilosis, C. tropicalis, C. pelliculosa, Torulopsis
famata, Pichia anomala, P. kluyveri, and Hanseniaspora uvarum.
Some species like Pichia kluyveri, P. anomala, and H. uvarum
showed an inhibitory effect on the growth of Aspergillus ochra-
ceus, a fungus that is potentially toxigenic and is often isolated
in coffee fruits and beans. Therefore, it is possible that yeasts
play a dual role during the processing of coffee fruits: they
facilitate mucilage degradation during fermentation and afford
biological control.
The microbiota of the semidry processing is still not well
known. A great diversity of prokaryotes and eukaryotes was
detected using polymerase chain reaction–denaturing gradient
gel electrophoresis (PCR-DGGE) in addition to conventional
techniques. PCR-DGGE allowed the detection of species not
isolated from conventional techniques, such as Enterobacter cow-
anii, Lactobacillus plantarum, Pantoea agglomerans, Bacillus macer-
ans, and an endophytic bacterium that could not be cultivated
and the yeasts S. cerevisiae and H. uvarum. The presence of
filamentous fungi was detected with populations below
103 CFU g
1 and only in recently harvested fruits, washed fruits,
and washed fruit fractions and in the skin þ pulp portion. The
filamentous fungi species found were Aspergillus sp., A. chevalieri,
A. foetidus, A. niger, A. ochraceus, A. tubingensis, A. versicolor, Cla-
dosporium sp., C. cladosporioides, C. macrocarpum, Cylindrocarpon
sp., Eurotium chevalieri, Fusariella sp., Fusarium sp., F. chlamydos-
porum, F. lateritium, F. nivale, F. solani, F. sporotrichioides,
Geotrichum sp., Mucor hiemalis, Penicillium sp., P. brevicompactum,
P. commune, P. decumbens, P. fellutanum, P. implicatum,
P. roqueforti, Phoma sp., and Ulocladium sp.
Ochratoxin in Roasted Coffee
In agricultural products, such as coffee, interactions between
different fungal species represent a natural phenomenon that
affects the development of these microorganisms and their
subsequent production of mycotoxins. Several studies have
been undertaken to assess the growth of mycotoxigenic fungi
and mycotoxin degradation. These interactions are influenced
by environmental conditions, microorganism diversity, agri-
cultural products, the type of processing, and the cultivation
system used. Interactions between mycotoxigenic and nontoxic
species can lead to the appearance of diseases and the produc-
tion of different types of mycotoxins and their degradation.
The results of these interactions are influenced by environmen-
tal conditions. OTA has been detected in various agricultural
products, including coffee. Coffee drinks significantly contrib-
ute to the ingestion of OTA in the human diet. OTA is primarily
produced by Aspergillus section Circumdati (A. westerdijkiae and
A. ochraceus) and section Nigri (A. carbonarius and A. niger)
species.
Coffee bean contamination with OTA may occur through-
out the entire production chain and is directly related to the
care and quality of the crop management, harvest, postharvest
storage, and type of roasting.
The toxic effects of OTA appear to be related to its ability to
inhibit protein synthesis by competing with phenylalanine in
the reaction catalyzed by phenylalanyl-tRNA synthetase and
other systems that require this amino acid. OTA also increases
lipid peroxidation, leading to greater mitochondrial and cell
damage. Furthermore, this mycotoxin is also considered neph-
rotoxic, cytotoxic, carcinogenic, teratogenic, and immunosup-
pressive and was classified by the International Agency for
Research on Cancer (IARC) as a member of group 2B, that is,
a possible human carcinogen.
Several studies have shown that the process of roasting is
effective in reducing the OTA concentration, but there is still a
lack of more conclusive studies about the effects of the vari-
ous stages of roasting and grinding and methods of preparing
the beverage on toxin stability. The type of roasting and
particle size interfere with the residual content of OTA in
the beverage. Therefore, the study helps to establish quality
requisites for roasted and ground coffee production within
national and international standards of quality, enabling
value to be added to a traditional product of Brazilian
agriculture.
See also: Caffeine: Characterization and Properties; Caffeine:
Consumption and Health Effects; Coffee: Analysis and Composition;
Coffee: Decaffeination; Coffee: Health Effects; Drying: Effect on
Nutrients, Composition and Health; Drying: Physical and Structural
Changes; Drying: Principles and Types; Mycotoxins: Occurrence and
Determination; Oxidation of Food Components.

Further Reading
Batista LR and Chalfoun SM (2014) Quality of coffee beans. In: Schwan RF and
Fleet G (eds.) Cocoa and coffee fermentation. Boca Raton, FL: CRC Press,
Chapter 13.
Batista LR, Chalfoun SM, Prado G, Schwan RF, and Wheals AE (2003) Toxigenic fungi
associated with processed green coffee beans (Coffea arabica L.). International
Journal of Food Microbiology 85: 293–300.
Blanc M, Pittet A, MunozBox R, and Viani R (1998) Behavior of ochratoxin A during
green coffee roasting and soluble coffee manufacture. Journal of Agricultural and
Food Chemistry 46: 673–675.
Chalfoun SM and Rebelles PR (2010) História da cafeicultura no Brasil. In: Reis PR and
Cunha RL (eds.) Café arábica do plantio a colheita, pp. 19–65. Brası́lia: Embrapa.
Clarke RJ and Macrae R (eds.) (1987) Coffee: technology. London: Elsevier Applied
Science Publishers.
Davies AP, Govaerts R, Bridson DM, and Stoffelen P (2006) An annotated taxonomic
conspectus of genus Coffea (Rubiaceae). Botanical Journal of the Linnean Society
152: 465–512.
Evangelista SR, Silva CF, Miguel MGPC, Cordeiro CS, Pinheiro ACM, Duarte WF, and
Schwan RF (2013) Improvement of coffee beverage quality by using selected yeasts
strains during the fermentation in dry process. Food Research International
61: 183–195.
Heilmann W, Rehfeldt AG, and Rotzoll F (1999) Behaviour and reduction of ochratoxin A
in green coffee beans in response to various processing methods. European Food
Research and Technology 209: 297–300.
Coffee: Types and Production 251
Mitchell HW (1988) Cultivation and harvesting of the arabica coffee tree. In: Clarke RJ
(ed.) Coffee: agronomy, pp. 43–90. New York: Elsevier Applied Science.
Oliveira G, da Silva DM, Pereira RGFA, Paiva LC, Prado G, and Batista LR (2013) Effect
of different roasting levels and particle sizes on ochratoxin A concentration in coffee
beans. Food Control 34(2): 651–656.
Schwan RF, Silva CF, and Batista LR (2012) Coffee fermentation. In: Hui YH (ed.)
Handbook of plant-based fermented food and beverage technology. Boca Raton, FL:
CRC Press, Chapter 42.
Silva CF (2014) Microbial activity during coffee fermentation. In: Schwan RF and Fleet G
(eds.) Cocoa and coffee fermentation. Boca Raton, FL: CRC Press, Chapter 11.
Taniwaki MH, Pitt JI, Teixeira AA, and Iamanaka B (2003) The source of ochratoxin A in
Brazilian coffee and its formation in relation to processing methods. International
Journal of Food Microbiology 82(2): 173–179.
Velmourougane K, Bhat R, Gopinandhan TN, and Panneerselvam P (2011) Impact of
delay in processing on mold development, ochratoxin-A and cup quality in arabica
and robusta coffee. World Journal of Microbiology and Biotechnology
27: 1809–1816.
Relevant Websites
https://www.embrapa.br/cafe – Brazilian Agricultural Research Corporation/Coffee.
http://www.fao.org/food/food-safety-quality/a-z-index/coffee/en/ – Food & Agriculture
Organization of United Nations. Good Hygiene Practices along the coffee chain.
http://www.ico.org/ – International Coffee Organization (ICO).
 Colon: Diseases and Disorders
R Arbizu and S Nurko, Harvard Medical School, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Functional Disorders Affecting the Colon
Constipation
Constipation is the most common gastrointestinal complaint
in the general population. It is particularly prevalent in women,
children, and older adults. The definition of constipation var-
ies, but based on epidemiological studies, it is commonly
defined as having a bowel movement frequency of 3 or less
per week. However, reports of bowel movement frequency are
usually inaccurate and often do not correlate with complaints
of constipation. This observation led to a more extensive defi-
nition or the Rome III criteria (Rome III, last revised in 2006)
for constipation that includes the presence of the following for
a period of at least 6 months:
1. Must include two of the following:
– Straining during at least 25% of defecations
– Lumpy or hard stools in at least 25% of defecations
– Sensation of incomplete evacuation for at least 25% of
defecations
– Sensation of anorectal obstruction/blockage for at least
25% of defecations
– Manual maneuvers to facilitate at least 25% of
defecations
– Fewer than three defecations per week
2. Loose stools are rarely present without the use of laxatives.
3. There are insufficient criteria for irritable bowel syndrome.
The prevalence of constipation varies according to the criteria
utilized for estimation. Overall, the prevalence of constipation
in North America varies between 2% and 27%. In children, the
worldwide prevalence ranges from 0.7% to 29.6%. In adults,
the prevalence rises dramatically, especially after 65 years of
age. In general, constipation can be regarded as disordered
movement of stool through the colon or anorectum. However,
secondary causes include neurological, metabolic, and endo-
crine disorders. Risk factors associated with constipation
include advanced age, female gender, lower level of education,
low socioeconomic status, nonwhite ethnicity, and use of cer-
tain medications. Cross-sectional studies, however, have failed
to link low intake fiber and constipation.
The initial evaluation of the patient with constipation
includes a careful history and physical examination. Laboratory
evaluation, endoscopic evaluation, and radiological studies
should be performed only in selected individuals if indicated.
Treatment of chronic constipation begins with lifestyle mod-
ifications, if indicated, and therapy with fiber. Osmotic and
stimulant laxatives, stool softeners, emollients, and enemas
sometimes are required to treat refractory intractable constipa-
tion. It is well recognized that fiber is important for normal
defecation. This is due primarily to the ability of fiber to increase
stool weight. The increased weight is secondary to the physical
presence of the fiber, water held by the fiber, and increased
bacterial mass from fermentation. Larger and softer stools
252 Encyclopedia of Food and Health
increase the ease of defecation and reduce transit time through
the intestinal tract, which may help to prevent or relieve consti-
pation. In general, cereal fibers are the most effective at increas-
ing stool weight. Wheat bran is considered the gold standard
when it comes to fecal bulking. Overall, the recommended
amount of dietary fiber is 20–35 g day 1 coming from meals
and supplements. The effect of fiber and low-digestible carbohy-
drates on gastrointestinal tolerance is a concern due to the devel-
opment of symptoms like bloating and distention, leading to
low compliance.
Irritable Bowel Syndrome
Irritable bowel syndrome (IBS) is a functional gastrointestinal
disorder characterized by chronic abdominal pain and dis-
turbed defecation without evidence of an organic etiology that
explains the symptoms. IBS can be associated with significant
emotional distress, impaired health-related quality of life, dis-
ability, and high health care costs. Psychosocial factors have an
important role in modulating the illness experience and its
clinical outcome.
The estimated prevalence of IBS in North America is approx-
imately between 10% and 15%. Although it can affect both men
and women of different age ranges, women and younger
patients are more likely to be diagnosed. The clinical diagnosis
of IBS is based on the Rome III criteria, which includes recurrent
abdominal pain or discomfort for at least 3 days per month in
the last 3 months associated with two of the following:
1. Improvement with defecation
2. Onset associated with a change in frequency of stool
3. Onset associated with a change in form (appearance) of
stool
The clinical presentation of IBS comprises a history of chronic
abdominal pain with variable intensity, locations, and periodic
exacerbations; altered bowel habits ranging from diarrhea and
constipation; and other gastrointestinal symptoms like bloat-
ing and increased gas production. Based on the symptoms, IBS
is classified in four different types:
• IBS with constipation: hard or lumpy stools  25%/loose or
watery stools <25% of bowel movements
• IBS with diarrhea: loose or watery stools 25% of bowel
movements/hard or lumpy stools <5% of bowel
movements
• Mixed IBS: hard or lumpy stools 25%/loose or watery
stools 25% of bowel movements
• Unsubtyped IBS: insufficient abnormality of stool consis-
tency to meet the previously mentioned subtypes
If performed, with the purpose of ruling out other conditions
with similar symptomatology, routine laboratory workup is
usually normal in IBS. Further workup should be done in the
http://dx.doi.org/10.1016/B978-0-12-384947-2.00188-4

presence of alarming symptoms like unexplained weight loss,
rectal bleeding, nocturnal symptoms, and abnormal laboratory
results or family history of inflammatory bowel disease, celiac
disease, or colorectal cancer.
The treatment strategy is based on the nature and severity of
symptoms, their correlation with food intake and/or defecation,
the degree of functional impairment, and the presence of psycho-
social difficulties and psychiatric comorbidity affecting the course
of the illness. If symptoms are mild and intermittent with low
impact on the quality of life, treatment is usually aimed towards
lifestyle and dietary modifications. However, if symptoms are
moderate to severe in intensity or have a significant impact on
the patient’s quality of life, then adjunctive pharmacological
therapy should be considered. Dietary modifications include
the exclusion of gas-producing foods or foods that increase
flatulence; the avoidance of lactose in patients with lactose intol-
erance; a diet low in fermentable oligo-, di-, and monosaccha-
rides and polyols in IBS patients with bloating; and a gluten-free
2-week trial in patients with diarrhea-predominant IBS. Although
the benefit of a high-fiber diet in IBS is controversial, its use
should be considered in patients with constipation-predominant
IBS. Lifestyle modification includes an increase in daily physical
activity. If adjunctive pharmacological therapy is indicated, then
it should be tailored according to the predominant IBS symptom.
Treatment options in constipation-predominant IBS include
osmotic laxatives (polyethylene glycol) and agents that enhance
intestinal fluid secretion and transit, like intestinal chloride
channel agonist (lubiprostone) and guanylate cyclase C agonist
(linaclotide). In the case of diarrhea-predominant IBS, antidiar-
rheal medications, like loperamide or bile acid sequestrants,
should be considered. As-needed basis antispasmodic agents are
indicated for short-term relief of abdominal pain in patients with
IBS. Patients with unremitting symptoms with associated psychi-
atric impairment may benefit from behavior modification tech-
niques in conjunction with antidepressants.
Colonic Diverticulosis and Diverticular Disease
A diverticulum is a saclike protrusion or a herniation of the
mucosa and submucosa through a defect of the muscularis.
Location is over the mesenteric border of the colonic wall and
its origin corresponds to the four sites of penetration of the
bowel wall by the major branches of the vasa recta. Strictly
speaking, it is truly a pseudodiverticula since it does not contain
all four layers of the colonic wall, only the mucosa and submu-
cosa. Diverticulosis is the term used to define the presence of
colonic wall diverticulum. Diverticular disease is a broader term
used to define symptomatic diverticulosis.
The exact incidence and prevalence of diverticulosis are not
exactly known because the majority of patients are asymptom-
atic. It is known, however, that the prevalence of diverticulosis
is age-dependent, < 20% at age 40 and increasing to >60% by
age 60. In addition, the distribution of diverticulosis also varies
by geography. Around 95% of patients in Western countries
have sigmoid diverticula. In Asian countries, the prevalence of
diverticulosis is predominantly right-sided.
Environment and lifestyle are important factors associated
with the development of diverticulosis. Increasing age, obesity,
diet rich in meats and low in fiber, living in Western countries,
Colon: Diseases and Disorders 253
and connective tissue diseases are factors that increase the risk
of developing diverticulosis. On the other hand, high-fiber
intake and living in predominantly rural areas decrease the
risk. Equivocal or factor that do not affect the risk include
gender, alcohol and coffee consumption, colorectal cancer,
and polycystic kidney disease. Smoking has been associated
with increased risk of perforated diverticulitis when compared
with nonsmoking.
As mentioned earlier, diverticula develop at well-defined
points of weakness in the colonic wall that correspond to
where the vasa recta penetrate the muscularis. Although still
unclear, abnormal colonic motility seems to be a predisposing
factor in the development of diverticula. Diverticula can vary in
number from one to literally hundreds. The typical size of a
diverticulum is 3–10 mm in diameter, but they can exceed
2 cm. Gross descriptions of the diverticula show a thickened
inner circular wall and shortening of the taeniae. Electron
microscopy studies have demonstrated a normal size and num-
ber of muscle cells, but demonstrated an increase in elastin and
collagen deposition between the muscle cells in the taeniae.
Clinically, most patients with diverticulosis remain asymp-
tomatic throughout their lifetime, and many cases go undetected.
On the other hand, diverticular disease is defined as symptomatic
diverticulosis due to diverticular bleeding, diverticulitis, segmen-
tal colitis associated with diverticula, or symptomatic uncompli-
cated diverticular disease. Diverticular bleeding is characterized
by painless hematochezia due to segmental weakness of the vasa
recta associated with a diverticulum. Diverticulitis is defined as
inflammation of a diverticulum that may be acute or chronic and
uncomplicated or complicated by a diverticular abscess, fistula,
bowel obstruction, or free perforation. Segmental colitis associ-
ated with diverticula or diverticular colitis is characterized
by inflammation in the mucosa without involvement of the
diverticular orifices. Symptomatic uncomplicated diverticular dis-
ease is characterized by persistent abdominal pain attributed to
diverticula in the absence of macroscopically overt colitis or
diverticulitis.
The diagnosis is based on clinical history and physical
examination and aided by the use of laboratory workup,
imaging, and endoscopic tools. Acute diverticulitis should be
suspected in patients with lower abdominal pain and abdom-
inal tenderness on physical examination, keeping in mind
other causes like appendicitis, ischemic or infectious colitis,
inflammatory bowel disease, and neoplasia.
Patients with acute diverticulitis can present with uncompli-
cated or complicated disease. The management of uncompli-
cated diverticulitis includes outpatient treatment with an oral
antibiotic aiming at Gram-negative rods and anaerobe coverage,
dietary modifications including a clear diet for 2–3 days, high-
fiber intake and avoiding nuts and seeds, and if possible the use
of topical anti-inflammatory medications. Generally, the elderly,
immunosuppressed, patients with significant comorbidities,
and patients with high fever or significant leukocytosis should
be hospitalized. The treatment of acute complicated diverticulitis
is based upon the specific complication: bleeding, perforation,
obstruction, peritonitis, abscess, or fistula formation. Manage-
ment involves hospitalization, bowel rest, and intravenous
hydration and empiric antibiotics. Failed medical management
and recurrent episodes of acute diverticulitis are indications for
operative management.
254 Colon: Diseases and Disorders

Colonic Inflammation (Colitis)
Inflammatory bowel disease is discussed in a separate article.
Infectious Colitis
Infectious colitis or infectious diarrhea is a lethal but prevent-
able problem that poses a significant health and economic
burden. The majority of deaths occur in developing countries
where sources of clean water and adequate sanitation amenities
are not readily available. In addition, these are regions where
the prevalence of malnourishment is high. In developing
countries, most of the cases occur in travelers or secondary
to outbreaks from food and water contamination. In these
circumstances, mortality is rare and generally occurs in immuno-
compromised patients. Clinically, inflammation of the infected
colon results in frequent, small-volume bowel movements,
which may contain mucous and blood. Other presenting symp-
toms include fever, dyschezia, and tenesmus. Onset is often times
acute, but can have a chronic duration. Treatment is frequently
guided according to the identified pathogen. A description of the
most common microorganisms responsible for infectious colitis
is shown in Table 1. A detailed depiction of specific pathogens is
described elsewhere.

Table 1 Common pathogens associated with infectious colitis

Risk factor Symptoms Diagnosis Treatment Complications

Bacteria
Campylobacter Foreign travel Fever; malaise; Stool culture Ciprofloxacin Guillain–Barré;
jejuni abdominal pain; cystitis;
diarrhea; pancreatitis;
hematochezia reactive arthritis;
toxic megacolon;
postinfectious IBS
Clostridium Antibiotic use; Diarrhea; fulminant Selective anaerobic stool First-line outpatient: Ileus; toxic
difficile hospitalization colitis; asymptomatic culture; toxin metronidazole. megacolon;
carrier immunoassay; PCR; cell First-line postinfectious IBS;
culture cytotoxicity assay hospitalized: protein-losing
metronidazole, enteropathy
vancomycin
Escherichia coli Contaminated food Hematochezia; Toxin assay Supportive care HUS; ischemic
O157:H7 abdominal pain; fever colitis; TTP; toxic
megacolon
Salmonella Contaminated food; Fever; abdominal pain; Stool, blood, and urine Ciprofloxacin; HUS; toxic
typhimurium foreign travel diarrhea; weight loss; culture penicillin; megacolon
hematochezia quinolones;
supportive care
Shigella spp. Travel to endemic area Fever; abdominal pain; Stool culture; PCR; DNA TMP/SMX; third- HUS; appendicitis;
diarrhea; tenesmus; probe; serology generation erythema
hematochezia cephalosporins, nodosum;
fluoroquinolones; meningitis;
nalidixic acid; postinfectious IBS;
supportive care toxic megacolon;
protein-losing
enteropathy; TTP;
perforation
Yersinia spp. Cold climate; winter Abdominal pain; Stool culture; serology Aminoglycosides; Erythema nodosum;
months; hematochezia TMP/SMX; exudative
contaminated food/ supportive care pharyngitis;
water; undercooked glomerulonephritis;
pork carditis;
polyarthritis;
reactive arthritis;
sepsis; perforation;
appendicitis; toxic
megacolon
Virus
Adenovirus AIDS; Diarrhea Viral culture; Supportive
immunosuppression immunohistochemistry
Cytomegalovirus IBD; Nonspecific Urine culture; Ganciclovir;
immunosuppression immunohistochemistry; valganciclovir
PCR

(Continued)
 Colon: Diseases and Disorders 255

Table 1 (Continued)

Risk factor Symptoms Diagnosis Treatment Complications

Herpes simplex Anal intercourse; Anorectal discharge; Intranuclear inclusion Acyclovir;

virus immunosuppression nonspecific bodies on biopsy; PCR; valacyclovir;
immunohistochemistry; famciclovir
antibody in formalin-
fixed tissue
Protozoa
Balantidium coli Food/water Nonspecific Identification of stool cyst, Tetracycline; Perforation;
contamination with precyst, or trophozoites; iodoquinol peritonitis
pig excrement trophozoites in muscle
biopsy
Cryptosporidium Public swimming Chronic diarrhea Microscopic stool analysis Paromomycin;
spp. pools; HIV; metronidazole;
malnourishment; nitazoxanide
age <2 years
Entamoeba Foreign travel; Diarrhea Identification of stool Metronidazole and Perforation;
histolytica pregnancy; trophozoites iodoquinol; appendicitis;
malnourishment; paromomycin; strictures;
immunosuppression nitazoxanide abscesses
Parasite
Strongyloides Living in endemic Hematochezia Microscopic stool analysis; Thiabendazole Perforation;
stercoralis areas serology; biopsy emphysematous
gastritis;
appendicitis
Trichuris Living in endemic Diarrhea; abdominal Identification of eggs in Mebendazole Rectal prolapse;
trichiura areas (tropic/ pain; tenesmus; stool neurotoxicity
subtropics) weight loss

AIDS, acquired immunodeficiency syndrome; DNA, deoxyribonucleic acid; HIV, human immunodeficiency virus; HUS, hemolytic uremic syndrome; IBD, inflammatory bowel disease;
IBS, irritable bowel syndrome; PCR, polymerase chain reaction; SMX, sulfamethoxazole; TMP, trimethoprim; TTP, thrombotic thrombocytopenic purpura.

Drug-Induced Colitis text). Dihydroergotamine is also associated with ischemic coli-

Certain medications are often associated with gastrointestinal
adverse effects ranging from nausea to severe colitis. Many
medications are known to cause colitis and only a few will be
discussed in this section.
Chemotherapy and radiation for underlying neoplastic
conditions are well-known causes of neutropenia. In this set-
ting, neutropenic colitis, necrotizing enteropathy, or typhlitis
can occur. This is a serious condition clinically manifested as
severe right lower quadrant abdominal pain and bloody diar-
rhea. Fever is indicative of sepsis, intestinal perforation, and
peritonitis. The pathology of the affected colonic segment fea-
tures transmural edema, mucosal inflammation, ulcerations,
denuded epithelium, hemorrhage, and necrosis. Treatment
includes supportive care in the intensive care unit, intravenous
fluids and antibiotics, and surgical resection if indicated.
Drug-induced hypomotility is associated with the use of opi-
oids, tricyclic antidepressants, and anticholinergic medications.
The use of vincristine is associated with paralytic ileus. Intestinal
hypomotility leads to luminal stasis and bacterial overgrowth.
Nonsteroidal anti-inflammatory drug-induced colitis has a
clinical presentation similar to inflammatory bowel disease,
presenting with generalized abdominal pain and diarrhea.
The use of these drugs is also associated with the development
of strictures and ulcerations, particularly in the right colon.
Mucosal healing and symptom relief is usually seen after dis-
continuing the offending drug.
Alosetron, a serotonin receptor antagonist, is associated
with the development of ischemic colitis (see the succeeding
tis secondary to prolonged vasoconstriction.
Chemical Colitis
Several agents are well-known causes of colonic inflammation.
The type of damage is thought to occur from a hypertonic or
direct toxic effect over the mucosa. The severity of damage is
related to the concentration of the substance, the duration of
exposure, and the presence of an underlying colonic disease.
The classic example is the use of detergents or soaps for enemas.
Other implicated substances include acetic acid, lye, hydrogen
peroxide, water-soluble contrast agents, formalin, alcohol,
vinegar, and glutaraldehyde. Soaps are alkaline agents that
produce liquefactive necrosis, inflammation that ranges from
mild to severe, as well as saponification of the colon. Damage
from water-soluble contrast agents, mainly in the proximal
colon, is thought to be secondary to its hypertonicity. Acute
inflammation can be self-resolving without permanent dam-
age. However, scarring, fibrosis, transmural inflammation, and
perforation have been reported. Treatment is mainly supportive
with intravenous fluids and antibiotics. Surgery is indicated in
severe cases of extensive necrosis.
Ischemic Colitis
Intestinal ischemia is caused by a reduction in blood flow,
which can be related to acute arterial embolic/thrombotic
occlusion, venous thrombosis, or hypoperfusion of the
256 Colon: Diseases and Disorders
mesenteric vasculature causing nonocclusive ischemia. Colon-
ic ischemia is a common disorder of the large bowel in the
elderly and is the most common form of intestinal ischemic
injury. The reason why the colon is more vulnerable to injury is
because it receives less blood flow compared to the rest of the
gastrointestinal tract. Perfusion to the colon can be compro-
mised by changes in the systemic circulation or by anatomic
or functional changes in the local mesenteric vasculature. The
three main mechanisms responsible for colonic ischemia are as
follows:
1. Nonocclusive ischemia: this is the most common cause
comprising  95% of the cases. This type of ischemia
tends to affect the ‘watershed’ areas of the colon that
include the splenic flexure and rectosigmoid colon.
2. Embolic and thrombotic ischemia: this is either from spon-
taneous emboli from proximal sources or from aortic
instrumentation.
3. Mesenteric vein thrombosis: this is a rare cause of colonic
ischemia, but when involved, it tends to affect the proximal
colon.
The injury to the colon following an ischemic event is due to
hypoxia and damage from reperfusion. The hypoxic compo-
nent causes rapid injury over the colonic mucosal surface.
Irreversible damage results from transmural necrosis and com-
plete injury to the tissue. Reperfusion injury usually takes place
in the setting of partial ischemia and it is secondary to the
release of free oxygen radicals that promote further injury.
Patients with acute colonic ischemia present with a history
of abdominal pain and tenderness of rapid onset, usually over
the left lower quadrant, as well as increased urgency to defe-
cate. Hematochezia is usually present within 24 h after the
onset of pain. Laboratory work results suggestive of ischemia
are leukocytosis, decreasing hemoglobin levels, and metabolic
acidosis. Plain abdominal films are usually nonspecific, but the
‘thumbprint’ sign can be seen. Abdominal computed tomog-
raphy is commonly the first test ordered. The typical finding of
thickening of the bowel wall in a segmental pattern is sugges-
tive but not specific for ischemia. The finding of hepatic portal
venous gas, although rare, is associated with extensive bowel
necrosis and predicts a high risk of mortality. Colonoscopy is
another test modality that should be performed early and with
minimal insufflation. Colonoscopy findings in the acute set-
ting frequently include pale mucosa with petechial bleeding.
Bluish hemorrhagic nodules may be seen representing submu-
cosal bleeding; these are the equivalent to the ‘thumbprint’
sign detected on radiological studies. Ulcerations and cyanotic
mucosa are indicative of chronicity. Arteriography is rarely
helpful in the diagnosis of colonic ischemia. It is not always
readily available, and patients with nonocclusive colonic ische-
mia are often markedly dehydrated and acidotic; therefore, if
used, it should be after careful fluid resuscitation.
Supportive care including bowel rest, intravenous fluids,
and continuous monitoring is the initial therapy in patients
with colonic ischemia. Although there is no strong evidence for
the use of antibiotics, they are recommended in cases of mod-
erate to severe ischemia. Anticoagulant therapy is indicated
for patients who develop ischemia due to mesenteric venous
thrombosis or due to cardiac embolization. Urgent abdominal
surgical exploration is required in patients with colonic
infarction and necrosis followed by postoperative hemody-
namic support and observation in the intensive care unit.
Microscopic Colitis
Microscopic colitis is an uncommon chronic inflammatory dis-
ease of the colon clinically manifested as chronic watery diar-
rhea, histological changes of chronic mucosal inflammation,
and normal colonoscopy findings. Microscopic colitis is classi-
fied into two subtypes according to their microscopic findings:
1. Lymphocytic colitis: intraepithelial lymphocyte count >20
per high-power field
2. Collagenous colitis: colonic subepithelial collagen band
>10 mm in thickness
The two subtypes are more common in patients aged 50–70,
but cases have been reported in children and adolescents. Both
also have a female predilection. Associated conditions include
celiac disease, arthritis, and other autoimmune disorders like
autoimmune thyroiditis and type 1 diabetes mellitus. Medica-
tions implicated as causative agents or triggers include non-
steroidal anti-inflammatory drugs. Smoking may play a role
in the development of microscopic colitis due to its effect on
colonic blood flow. The pathogenesis of microscopic colitis is
unclear; however, it is likely to be multifactorial, involving
mucosal immune responses to luminal factors in a genetically
predisposed individual. Although both subtypes have a similar
inflammatory cell response, it is uncertain whether they are
related. Abnormal collagen metabolism leading to increased
deposition and a defect in epithelial barrier function predispos-
ing to increased intestinal permeability have been proposed
as possible pathogenic explanations for collagenous and lym-
phocytic colitis, respectively.
Clinically, patients with microscopic colitis present with a
history of chronic, watery, nonbloody diarrhea. Onset is often
insidious. Bowel movement volume and frequency tend to
decrease with fasting. Other symptoms include abdominal
pain, urgency, incontinence, weight loss, and dehydration.
Extraintestinal manifestations, like uveitis and arthralgia, can
be encountered. Physical examination is usually unremarkable
and routine laboratory studies are also normal. Colonoscopy
examination is usually normal. Nonspecific abnormalities
including patchy edema, erythema, friability, and an abnormal
vascular pattern have been reported, whereas mucosal ulcera-
tions in the ascending and transverse colons have been reported
in a few patients with collagenous colitis.
Treatment is largely empiric. Evaluation of therapy is diffi-
cult, because both disorders usually exhibit a relapsing and
remitting course over many years. No single agent works in all
cases. Antidiarrheal agents like loperamide or bulking agents
like psyllium and methylcellulose are associated with clinical
improvement, but not histological remission. Other medica-
tions like 5-aminosalicylate, glucocorticoids, and bile acid
resins, alone or in combination, improve diarrhea and inflam-
mation in some patients. Trial with a 6–8 week course of
budesonide has shown good results in patients with collage-
nous colitis. Ileostomy should be considered only as a last
resort, but it appears to be effective in patients with disabling
and refractory symptoms.

Diversion Colitis
Diversion colitis is an inflammatory process that occurs in the
diverted segment of the colon and rectum after surgical diver-
sion of the fecal stream. Although it has been reported to occur
more commonly in patients with inflammatory bowel disease,
diversion colitis has been found in patients who have under-
gone surgical diversion for many indications. The prevalence of
diversion colitis is unknown and probably underestimated
since a proportion of patients with histological changes of
the diverted segment are asymptomatic.
The pathogenesis of diversion colitis is related largely to
lumen nutrient deficiency in the form of short-chain fatty
acids. Other luminal elements besides short-chain fatty acid
deficiency are likely to play a role, but the nature of such factors
is unknown.
The diagnosis is based on clinical history and endoscopic
findings of erythema, friability, nodularity, edema, aphthous
ulcerations, exudates, and frank bleeding. Histological findings
range from lymphoid follicular hyperplasia and mixed mono-
nuclear and neutrophilic infiltration to severe inflammation
with crypt abscesses, mucin granulomas, and Paneth cell meta-
plasia. Crypt architecture tends to be preserved. Caution must
be taken in patients with recurrent inflammatory bowel disease
and Clostridium difficile infection should be ruled out.
The treatment of choice is restoration of colonic continuity
that reverses symptoms and histological changes. If reanasto-
mosis is not feasible and symptoms are moderate to severe,
short-chain fatty acid enema with propionate, acetate, and
butyrate mixed in a saline solution has been described as a
treatment option.
Colonic Neuropathies: Hirschsprung’s Disease
Hirschsprung’s disease (HD) is an intestinal motor disorder
that results from the congenital absence of ganglion cells in the
rectum and distal colon. As a consequence, the affected agan-
glionic segment lacks receptive relaxation resulting in a func-
tional obstruction clinically manifested as constipation.
The incidence of HD is  1 in 5000 live births. The overall
male to female ratio is of 4 to 1, although it can be nearly 1 to 1
when the entire colon is involved. In  85% of the cases, the
aganglionic segment is limited to the rectum and distal colon;
in other cases, longer segments can be affected, and in 5%, the
entire colon is affected, including the distal ileum.
HD results from a defect in the craniocaudal migration of
neural crest cells during the first 12 weeks of migration. In
addition, defective differentiation of neuroblasts into ganglion
cells and accelerated ganglion cell destruction may also play a
part. The etiology of HD also has a genetic basis. Hereditary
patterns are not straightforward and at least eight genetic muta-
tions have been identified. The predominantly affected gene is
the RET proto-oncogene, accounting for nearly 50% of familial
and 20% of sporadic cases with long-segment disease. Other
involved genes include Down’s syndrome cell adhesion mole-
cule, endothelin 3, endothelin receptor type B, endothelin-
converting enzyme, SOX10, PHOX2B, and the neuregulin genes.
Up to one-third of patients with HD suffer other syndromes
or congenital anomalies. Down’s syndrome affects up to 2–3%
Colon: Diseases and Disorders 257
of patients with HD. Other monogenic syndromes include mul-
tiple endocrine neoplasia type 2, congenital central hypoventila-
tion syndrome, cartilage–hair hypoplasia, Mowat–Wilson
syndrome, Waardenburg syndrome type 4, Smith–Lemli–Opitz
syndrome, and Bardet–Biedl syndrome. Approximately 20–25%
of patients with HD have associated congenital anomalies that
involve the heart, kidney, or urinary tract.
HD is usually diagnosed during the neonatal period in
patients with symptoms of distal intestinal obstruction, but
can present at any age. Delayed passage of meconium passed
the first 48 h of life should raise the suspicion of HD. Abdom-
inal distention, feeding intolerance, bilious emesis, and explo-
sive expulsion of gas and stool after digital rectal exam are other
clinical presentations in infants with HD. Some infants with
HD may have normal stooling initially, especially if they are
breast-fed, and then develop severe constipation when other
foods are introduced into the diet. A life-threatening presenta-
tion is HD-associated enterocolitis, a severe diarrheal illness
that requires a high index of suspicion and prompt manage-
ment with aggressive fluid resuscitation, intravenous antibiotic
anaerobic coverage, and rectal irrigations. In extreme cases, a
diverting colostomy may be indicated. Older children with
less severe disease may present with a chronic history of intrac-
table constipation, abdominal distention, or failure to thrive.
Interestingly, there appears to be no correlation between the
severity of symptoms and the length of aganglionic segment.
The diagnostic steps depend on the level of suspicion for
HD. Abdominal radiographs may show signs of distal intesti-
nal obstruction. A contrast enema with barium may reveal a
more dilated colon proximal to a narrower distal aganglionic
colon. This radiological finding is called a ‘transition zone’ and
it may not be evident in patients with long-segment HD. If not
clearly identified, a postevacuation film 24 h later may reveal
residual barium in the colon. Anorectal manometry is a useful
screening test if performed by experienced personnel. The pres-
ence of internal anal sphincter relaxation, or rectoanal inhibi-
tory reflex, with balloon distention excludes the diagnosis of
HD. To confirm the diagnosis, a rectal biopsy should be done,
safely using a mucosal suction technique. If tissue sample is
inadequate, then a repeat full-thickness biopsy under anesthe-
sia is warranted. The diagnosis is confirmed if submucosal
ganglion cells are absent on light microscopy. Hypertrophied
nerve trunks, positive acetylcholinesterase, and negative calre-
tinin immunostains are supportive histopathologic findings.
Treatment is aimed at resecting the affected aganglionic
segment, the creation of a neorectum in close proximity with
the anus, and preserve sphincter function. Several surgical
techniques have been developed over time and are performed
in different stages. The decision depends on experience and
clinical characteristics of the patient. Currently, the majority of
cases are done over one stage with an operation known as the
Soave endorectal pull-through.
The majority of patients usually do well after surgical cor-
rection having normal to near normal anorectal function and
have a good quality of life. However, some bowel function
abnormalities are commonly encountered. Constipation or
intermittent obstructive symptoms can be caused by mechan-
ical obstruction, residual aganglionosis, or increased internal
anal sphincter pressure. Fecal incontinence is usually encoun-
tered after surgery. However, continence tends to improve with
258 Colon: Diseases and Disorders
age. Finally, despite surgical repair, patients are still at risk for
developing HD-associated enterocolitis. Risk factors include
the presence of a mechanical obstruction like an anastomotic
stricture and patients with long-segment disease, suggesting
that intestinal stasis may be a predisposing factor.
See also: Colon: Structure and Function; Diarrheal Diseases.
Further Reading
Brandt LJ and Boley SJ (2000) AGA technical review on intestinal ischemia. American
Gastrointestinal Association. Gastroenterology 118: 954–968.
Cappell MS (2004) Colonic toxicity of administered drugs and chemicals. American
Journal of Gastroenterology 99: 1175–1190.
Davila ML (2007) Neutropenic enterocolitis: current issues in diagnosis and
management. Current Infectious Disease Reports 9: 116–120.
Drossman DA, Camilleri M, Mayer EA, and Whitehead WE (2002) AGA technical review
on irritable bowel syndrome. American Gastrointestinal Association.
Gastroenterology 123: 2108–2131.
Eggenberger JC and Farid A (2001) Diversion colitis. Current Treatment Options in
Gastroenterology 4: 255–259.
Engum SA and Grosfeld JL (2004) Long-term results of treatment of Hirschsprung’s
disease. Seminars in Pediatric Surgery 13: 273–285.
Higgins PD and Johanson JF (2004) Epidemiology of constipation in North America: a
systematic review. American Journal of Gastroenterology 99: 750–759.
Sheth AA, Longo W, and Floch MH (2008) Diverticular disease and diverticulitis.
American Journal of Gastroenterology 103: 1550–1556.
Slavin J (2013) Fiber and prebiotics: mechanism and health benefits. Nutrients
5: 1417–1435.
Stewart DR and von Allmen D (2003) The genetics of Hirschsprung disease.
Gastroenterology Clinics of North America 32: 819–837.
Tabbers MM, DiLorenzo C, Berger MY, et al. (2014) Evaluation and treatment of
functional constipation in infants and children: evidence-based recommendations
from ESPGHAN and NASPGHAN. Journal of Pediatric Gastroenterology and
Nutrition 58: 258–274.
Veress B, Löfberg R, and Bergman L (1995) Microscopic colitis syndrome. Gut
36: 880–886.
 Colon: Structure and Function
R Arbizu and S Nurko, Harvard Medical School, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Structure
Embryology
During embryogenesis, the mature intestine develops from all
three germ layers: the endoderm that gives rise to the simple
columnar epithelial cell lining of the surface of the small and
large intestines, the mesoderm from which cells of the lamina
propria and muscularis layers originate, and the ectoderm that
gives rise to the enteric neurons and other components of the
enteric nervous system.
The primitive gut develops around the fourth week of ges-
tation from the primitive yolk sac, which is further divided
into three parts: foregut, midgut, and hindgut. The embryo-
logical origin of the colon is dual. The midgut gives rise to the
appendix, cecum, ascending colon, and proximal two-thirds of
the transverse colon. The distal third of the transverse colon,
the descending colon and sigmoid colon, the rectum, and the
upper portion of the anal canal originate from the hindgut. The
distal hindgut further divides into a ventral component that
becomes the urogenital sinus and a dorsal component that
forms the lower portion of the anal canal.
During the fifth week of gestation, the midgut rapidly elon-
gates with the formation of the primary intestinal loop. The
cecum originates as a small dilation of the caudal limb of the
primary intestinal loop by the sixth week of gestation. It ini-
tially lies in the right upper quadrant and then descends to the
right iliac fossa, placing the ascending colon and hepatic flex-
ure in the right side of the abdominal cavity. The appendix
originates from the distal end of the cecal bud, and its final
position is frequently retrocecal or retrocolonic. When the
caudal limb of the primitive intestine moves to the right side
of the abdominal cavity, the dorsal mesentery twists around
the origin of the superior mesenteric artery. After the ascending
and the descending portions of the colon reach their final
destinations, their mesenteries fuse with the peritoneum of
the posterior abdominal wall, and they become retroperitoneal
organs. The appendix, cecum, and descending colon retain
their free mesentery. The transverse colon fuses with the pos-
terior wall of the greater omentum.
Anatomy
Macroscopic features
The colon is a tubular structure that is continuous with the
small intestine proximally at the ileocecal valve and ends dis-
tally at the anal verge. In the full-term newborn, the colon is
30–40 cm in length, increasing approximately to 150 cm in
adults. The caliber is greatest in the cecum and gradually
diminishes as it reaches the rectum. The appearance of the
colon differs from the small intestine in several ways. The
outer longitudinal muscle fibers of the colon coalesce into
three distinct bands termed taeniae coli that extend continu-
ously from the base of the appendix to the rectum. The haustra
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00187-2
are outpouchings of the colon that give it a characteristic
sacculated appearance. The plicae semilunares are intermittent
creases that are visualized as irregular folds over the luminal
surface of the colon. In addition, small sacs of peritoneum
filled with adipose tissue, the appendices epiploicae, are
found on certain areas of the external surface of the colon.
The colon, as the small intestine, is enclosed by the mesen-
tery. However, most of the colon is fixated in a retroperitoneal
fashion, and only a small portion is suspended by mesentery.
The transverse colon and sigmoid colon are suspended by
mesentery. A small portion of mesentery termed mesocecum
fixes the mobility of the cecum. The ascending colon, descend-
ing colon, and rectum are partially covered by mesentery on
their anterior surfaces.
The cecum is the most proximal part of the colon. It lies in
the right iliac fossa and projects downward as a blind pouch
below the entrance of the ileum and projects superiorly to the
ascending colon. It is a saclike structure 6 cm long and 8 cm
wide in adults. The vermiform appendix is a blind, narrow,
elongated outpouching of the cecum, and its lumen is found
2.5 cm inferior to the ileocecal valve. Its length varies from 2
to 20 cm, being longest during childhood.
The ascending colon extends from the cecum in a distal
manner toward the posterior lobe of the liver where it makes a
sharp turn and forms the hepatic flexure. It is covered in
peritoneum; therefore, it is a retroperitoneal organ. The diam-
eter of the ascending colon is narrower than the cecum, and its
length reaches 20 cm in adults.
At the hepatic flexure, the colon emerges medially and
anteriorly into the peritoneum as the transverse colon. Distally,
it curves acutely and inferior to the lower pole of the spleen,
forming the splenic flexure. The transverse colon is the longest
segment of the colon with measured lengths from 40 to 50 cm.
It lies anteriorly to the stomach and small intestine. It is
completely covered in mesentery termed the transverse meso-
colon. It is the most mobile segment of the colon; in fact, it can
lie at the level of the pelvis when a person assumes an upright
position.
The descending colon, about 25–45 cm in length, arises
distal to the splenic flexure. It travels downward and posteri-
orly and assumes a retroperitoneal position ending at the level
of the left iliac crest.
Distally, the descending colon emerges at the pelvic brim
from a retroperitoneal position as the sigmoid colon. This is an
S-shaped segment of the colon that varies in length and is very
tortuous and mobile. It is suspended by mesentery termed
sigmoid mesocolon that shortens and disappears as it reaches
the rectosigmoid junction.
The rectum extends distal to the sigmoid colon at the level
of the third sacral vertebrae toward the anal canal, 2–3 cm in
front and below the coccyx. In adults, the rectum measures
10–12 cm in length. It lies below the peritoneum and in close
proximity to the pelvic structures.
259
260 Colon: Structure and Function
The anal canal is  5 cm in length in the adult and has a
discrete upper and lower demarcation. The anorectal ring is
located proximally and is composed of the upper portion of the
internal anal sphincter, the longitudinal muscle of the rectum,
the deep portion of the external sphincter, and the puborectalis
portion of the levator ani muscle. Distally, the anal verge
represents the transition of anoderm to true skin. The mucosa
of the distal 3 cm of the rectum and the anal canal contains
6–12 redundant longitudinal folds called the columns of Mor-
gagni. These columns are joined together by mucosal folds
called the anal valves, which are situated at the dentate line.
The muscularis mucosae disappears in the anorectal canal,
and the inner circular coat of muscularis propria thickens to
form the internal anal sphincter. The external anal sphincter
surrounds the anal canal, and its fibers blend with those of
the levator ani muscle to attach posteriorly to the coccyx and
anteriorly to the perineal body.
Vascular supply
The superior mesenteric artery, a direct branch of the abdom-
inal aorta, supplies oxygenated blood to the cecum, ascending
colon, and proximal two-thirds of the transverse colon. The
inferior mesenteric artery, also a branch of the abdominal
aorta, supplies the distal third of the transverse colon, the
descending colon and sigmoid colon, and the superior portion
of the rectum as the superior hemorrhoidal artery. The middle
and inferior hemorrhoidal arteries, which are branches of the
hypogastric and internal pudendal arteries, respectively, supply
the rest of the rectum and anal canal.
The superior and inferior mesenteric veins drain the same
regions of the large intestine supplied by the corresponding
arteries. The venous system of the anal region drains into the
systemic and portal systems. The internal hemorrhoidal plexus
drains the proximal anal region into the superior rectal veins
and then into the inferior mesenteric vein, which then,
together with the superior mesenteric vein, joins the splenic
vein to form the portal vein. The external hemorrhoidal plexus
drains the distal anal region through the middle rectal and
pudendal veins into the internal iliac vein.
Lymphatic drainage
The lymphatic drainage of the large intestine follows their
respective blood supply, first draining to the pericolic nodes,
intermediate nodes, and ultimately preaortic nodes that sur-
round the superior and inferior mesenteric arteries. The rectum
and anal canal drain into the perirectal nodes, which then
further drain into the iliac and inferior mesenteric nodes.
Innervation
The innervation of the large intestine, as well as the rest of the
gastrointestinal tract, comes from the intrinsic nervous system
and the extrinsic nervous system. The enteric nervous system
constitutes the intrinsic nervous system of the gastrointestinal
tract. It is derived from the neural crest cells and consists
essentially of an equal to the 100 million neurons in the spinal
cord. The nerve cell bodies of the enteric nervous system are
located in different plexuses of the gastrointestinal wall, mainly
in the submucosa and muscularis externa. The sympathetic
and parasympathetic nerves constitute the extrinsic nerve sup-
ply. The proximal colon receives its sympathetic neuronal
innervation from the celiac and superior mesenteric ganglia.
The parasympathetic innervation comes from the vagus nerve.
In the distal colon, the sympathetic innervation comes from the
branches of the lumbar segment of the sympathetic trunk,
whereas the parasympathetic nerves originate from the pelvic
splanchnic nerves. The sources of innervation of the internal
and external sphincters are different. In the anal canal, the
internal anal sphincter receives inhibitory innervation from
the intrinsic nervous system and from extrinsic input from the
lumbar sympathetic and sacral parasympathetic nerves. The
pudendal nerve innervates the external anal sphincter and
other pelvic floor muscles.
Microscopic features
In general, the intestinal wall of the gastrointestinal tract is
composed of the following four layers:
1. Mucosa
2. Submucosa
3. Muscularis externa
4. Serosa
Mucosa
The mucosa is the innermost layer of the colon. It is further
subdivided into three distinct sublayers: the glandular epithe-
lium, lamina propria, and muscularis mucosae, the outermost
sublayer. Compared with the small intestine, the mucosa of the
colon lacks villi. However, the glandular epithelium does con-
tain crypts of Lieberkühn that extend down through the lamina
propria to the muscularis mucosae. The glandular epithelium
is composed of the following several cell types:
• Stem cells are pluripotential cells located at the base of the
intestinal crypts. These cells give rise to all types of mature
intestinal epithelial cells and additional stem cells to main-
tain their numbers. Cell differentiation is regulated by dif-
ferent signaling pathways (Wingless, Notch, bone
morphogenetic protein, and Hedgehog) between epithelial
cells and cells in the lamina propria. In the colon, terminal
differentiation occurs in the upper one-third of the crypt,
and most cells migrate onto the mucosal surface. On the
surface of the colon, older cells are extruded to the intestinal
lumen by a process termed anoikis and expelled out of the
body by defecation.
• Absorptive cells, generally called enterocytes, are high
columnar cells located at the base near the lamina propria.
They have oval basal nuclei, an eosinophilic cytoplasm, and
a periodic acid–Schiff-positive free surface. Enterocytes are
composed of superficial microvilli that are more numerous
on the small intestine compared with the colon and contain
membrane-bound digestive proteins, transport proteins,
and other cellular elements necessary for nutrient absorp-
tion. In the colon, absorptive cells are termed principal
cells.
• Goblet cells are mucin-producing cells located in the crypts
characterized by a narrow base and oval apical surface,
flattened basal nucleus, and basophilic cytoplasm with
mucin-containing granules. They are much larger in num-
ber in the colon compared with the small intestine. The
ample amount of mucin produced by goblet cells in the

colon provides lubrication for adequate stool passage. Gob-
let cells also provide a protective barrier against shear stress.
This same barrier is thought to bind surface antigens and
inhibit their attachment to the epithelial surfaces.
• Paneth cells are pyramid-shaped cells located exclusively at
the base of the crypts. They have a basal nucleus and a
cytoplasm rich in eosinophilic granules. Their primary
function is to secrete antimicrobial products conferring
protection from enteric infection and contributing to the
maintenance of the gastrointestinal antimicrobial barrier.
In response to luminal bacteria and bacterial antigens, they
secrete different substances like phospholipase A2, lyso-
zyme, and a-defensins, the latter being the principal anti-
microbial molecule secreted by Paneth cells.
• Enteroendocrine cells, also known as gut endocrine or neu-
roendocrine cells, are specialized, tall, columnar cells
located in the crypts of the colon. They have a large nucleus
and prominent cytoplasmic secretory granules, distributed
mainly in the basal region of the cell. The enteroendocrine
lineage consists of at least 15 different cell types that are
categorized based on their morphology, specific regional
distribution, and peptide hormone expression.
The lamina propria is a thin layer of connective tissue that
provides support to the mucosal epithelial cell basement mem-
brane. In the colon, the lamina propria contains solitary lym-
phoid follicles that extend to the submucosa, being more
developed in the rectum. Furthermore, the lamina propria is
rich in arterioles, veins, lacteals, and nerve fibrils and various
cell types, including fibroblasts, lymphocytes, macrophages,
eosinophils, mast cells, and neutrophils.
The muscularis mucosae is the outermost layer of the
mucosa. It lies next to the submucosa and consists of an
inner circular layer and outer longitudinal layer of smooth
muscle cells ( 3–10 cells thick). The colonic muscularis
mucosae is thicker when compared with the small intestine,
and the thickness increases progressively from the cecum to the
anal canal.
Submucosa
The submucosa, located between the outermost layer of the
mucosa and the muscularis externa, is made of connective
tissue and several different cell types that include fibroblasts,
lymphocytes, eosinophils, macrophages, plasma cells, and
mast cells. The submucosa has a rich vascular supply and
lymphatic drainage, and, because of its close proximity to the
mucosa, it supports its electrolyte, fluid, and nutrient absorp-
tion. The enteric nervous system nerve cell bodies are located in
the submucosal plexus, which is divided into two networks,
Meissner’s plexus, which lies closer to the mucosa, and
Schabadasch’s plexus, which lies adjacent to the inner circular
muscle.
Muscularis externa
The muscularis externa is composed of smooth muscle cells
arranged in two distinct layers: the outer longitudinal layer and
the inner circular layer. As mentioned earlier, the outer longi-
tudinal layer fibers form the taeniae coli, which run in parallel
to the long axis of the colon throughout its entire length.
Within the two smooth muscle layers, there is a nerve fiber
Colon: Structure and Function 261
plexus of ganglionated cells, termed Auerbach’s plexus, that
also forms part of the enteric nervous system.
It is important to mention a specific group of cells, the
interstitial cells of Cajal, that are involved in the coordinated
activity between the enteric nervous system, autonomic ner-
vous system, and smooth muscle cells that direct gastrointesti-
nal motility. The interstitial cells of Cajal are modified smooth
muscle cells and are regarded as the pacemaker cells of the
gastrointestinal tract. These cells form networks that are widely
distributed within the submucosal, intramuscular, and inter-
muscular layers. Contractions of gastrointestinal smooth
muscle are initiated by these specialized pacemaker cells. Prop-
agation of contractions occurs as waves of membrane depolar-
ization spread via gap junctions between adjacent smooth
muscle cells.
Serosa
The serosa, an extension of the visceral peritoneum, is the
outermost layer of the colon. It is formed by a layer of meso-
thelial cells supported by connective tissue or adventitia. Occa-
sionally, scattered different cell types are found within the
serosa like macrophages, fibroblasts, and mast cells.
Functions
Water and Electrolyte Transport
The colon plays an integral role in intestinal homeostasis by
regulating the balance between water and electrolyte absorp-
tion and secretion. In general, colonic epithelial cells are pri-
marily responsible for absorption, and the crypt cells are
involved in both absorption and secretion.
The colon is a very efficient water-preserving organ. Nor-
mally, the colon receives between 1000 and 1500 ml of water
daily. Approximately 90% of this volume is reabsorbed by the
colon, and 100–150 ml is excreted in the feces. Passive diffu-
sion along an osmotic sodium luminal gradient is the main
mechanism in which water is transported along the colon via
paracellular pathways. However, the transmembrane protein
channels, termed aquaporins, also play a role in the transcel-
lular movement of water. Of the colonic segments, the ascend-
ing colon has the greatest absorptive capability because of its
longer exposure time to the large volume of small intestinal
fluid.
Under normal conditions, the colon reabsorbs sodium and
chloride and secretes potassium and bicarbonate. The recovery
of sodium is one of the main functions of the colon. Approx-
imately 130–140 mmol l 1 of sodium is delivered daily to the
colon, and only 40 mmol l 1 is excreted in the stool. Under
certain circumstances, the colon is able to salvage up to
800 mmol l 1 day 1, preventing hyponatremia. Throughout
the colon, the mechanism by which sodium is reabsorbed
varies. The proximal colon exhibits sodium/hydrogen
exchanger channels located in the apical surface of the epithe-
lial cells. These channels are coupled with a chloride/bicarbon-
ate exchanger channel. The distal colon and rectum have
electrogenic sodium-specific channels. Both types of transport
channels allow the passive diffusion of sodium along an elec-
trochemical gradient. Once inside the cell, sodium is then
exchanged for potassium via an energy-dependent pump on
262 Colon: Structure and Function
the basolateral membrane of the epithelial cell. Aldosterone,
short-chain fatty acids, a2 adrenergic agents, and somatostatin
further enhance the absorption of sodium.
Chloride is also recovered from the colon. As mentioned
earlier, the proximal colon has chloride/bicarbonate exchange
channels that absorb chloride and secrete bicarbonate. The
acidic environment of the colon enhances the absorption of
chloride, and the secretion of bicarbonate is responsible for the
alkaline pH of the feces.
Potassium is passively transported along a sodium gradient.
However, potassium is actively secreted in the proximal colon
via a transport channel and absorbed in the distal colon via an
energy-dependent hydrogen/potassium exchanger. The final
concentration of stool potassium is  50–90 mmol l 1.
Urea entering the colon from the small intestine is metab-
olized by colonic bacteria and converted to ammonia, which is
passively absorbed by the epithelial cells. Other sources of
ammonia that is further absorbed come from dietary nitrogen,
sloughed cells, and bacteria. Absorbed ammonia reaches the
enterohepatic circulation and is delivered to the liver where it is
reconverted to urea.
Colon Flora, Nutrient Absorption, and Fermentation
The human colon is one of the most diversely colonized and
metabolically active organs in the human body. Up to 1000
different species of bacteria reside in the colon with microbial
populations comprising 1011–1012 colony-forming units per
gram of content. The colon provides a favorable environment
for bacterial growth due to its slower transit time, readily
available nutrients, and favorable pH. Generally, bacteria hav-
ing an almost exclusive saccharolytic metabolism can be con-
sidered potentially beneficial. Such a metabolic profile is
typical for lactobacilli and bifidobacteria.
The colonic flora salvages energy via fermentation of undi-
gested carbohydrates that reach the colon. These substances
include resistant starches, nonstarch polysaccharides, non-
digestible oligosaccharides, and sugar alcohols. The end prod-
ucts of bacterial fermentation – mainly of soluble plant
residues – are the short-chain fatty acids butyrate, propionate,
and acetate and the formation of odorless gases, including
hydrogen, methane, and carbon dioxide. Approximately 90%
of these short-chain fatty acids are absorbed by the mucosa in
the proximal colon via short-chain fatty acid/bicarbonate
transport channels and other electrolyte-coupled transporters.
An average of 400 mmol day 1 of short-chain fatty acids is
produced in the colon, providing 5–15% of the total caloric
needs. Although butyrate is the least abundant of the short-
chain fatty acids, it acts as the primary energy source for colonic
cell health, supplying up to 90% of its energy needs. In addi-
tion, butyrate serves as an antidiarrheal agent as it promotes
the absorption of water, sodium, and chloride. Propionate
and acetate reach the liver via the bloodstream and participate
in gluconeogenesis and long-chain fatty acid synthesis,
respectively.
The colon is also involved in the metabolism and absorption
of other nutrients. Proteins, via fermentation, are converted in
the distal colon to short-chain fatty acids, branched-chained fatty
acids, amines, ammonia, phenols, indoles, and sulfurs. The
human colon cannot absorb long-chain triglycerides. In turn,
they bind to luminal calcium making oxalate readily available
for absorption. In patients with short bowel syndrome, medium-
chain fatty acids from supplements can be absorbed in the
remaining colon providing caloric energy. Absorption of calcium
is highest in the cecum. Calcium requires solubilization of
calcium salts enhanced by the acidic environment of carbohy-
drate fermentation. In addition, propionate and acetate also
enhance the direct absorption of calcium in the colon. Vitamin
K, in its K2 form, comprises a group of bacterial products termed
the multiprenyl menaquinones. Vitamin K2 is passively absor-
bed in the colon.
Motility of the Colon
The other important function of the colon is the transport of
unused material into the rectal area where it can be expelled in
a socially acceptable manner during defecation. Although
undigested food takes <2 h to reach the colon, it may take as
long as 2–5 days for it to be expelled as stool. The propelling
function of the colon is accomplished by haustrations (seg-
mental contractions) and mass movement (high-amplitude
peristaltic contractions (HAPCs)).
HAPCs are defined as contractions of at least 80–100 mmHg,
lasting 10 s and propagating for at least 30 cm (see Figure 1).
HAPCs can reach amplitudes > 200 mmHg. HAPCs originate in
the proximal colon and migrate distally > 95% of the time,
usually stopping in the distal sigmoid colon.
Food ingestion has an important influence on colonic
motility. The response lasts 2–3 h and is mostly composed of
segmental contractions, accompanied by an increase in colonic
smooth muscle tone and HAPCs, which have been described in
pediatric patients. The colonic response to eating is influenced
by the caloric content and meal composition. Fat and carbo-
hydrate represent an important stimulant, whereas protein
may have an inhibitory effect (Figure 2).
Defecation
Normal defecation is a complex process that involves a
sequence of events initiated by the entrance of stool in the
rectum. When the rectal wall becomes stretched by stool or
gas, the internal anal sphincter relaxes (also known as the
rectoanal inhibitory reflex), permitting a small amount of
stool to enter the anal canal. An enteric descending inhibitory
reflex mediates this process. At the same time, there is a brief
contraction of the external anal sphincter, maintaining conti-
nence. Extrinsic cholinergic parasympathetic pathways in the
pelvic nerves mediate this process. Sensory receptors in the
proximal anal canal provide a distinction between solid or
liquid stools and gas. The internal anal sphincter has a relaxa-
tion of longer duration when the rectal wall is distended by a
larger volume, producing a conscious voluntary contraction of
the external anal sphincter until the individual decides when it
is socially appropriate to evacuate the intraluminal contents.
Suppression of the defecation urge and rectal receptive accom-
modation allows for temporary storage of stool or gas in the
rectum or retrograde transport back to the sigmoid colon.
When socially appropriate, an individual may voluntarily
relax the external anal sphincter to allow defecation to proceed.
Intra-abdominal pressure is elevated to aid in the expulsion
 mmHg 200

P9 10

cecum
0
mmHg 200

P10 3

transverse
0
mmHg 200

HAPC
P11 1

descending 0
mmHg 200

P12 0

sigmoid 0
mmHg 200

P13 15

rectum 0
Time 13:52:26.20 40:00 13:42:00 13:44:00 13:46:00 13:48:00

Figure 1 Normal colonic motility. Colonic motility in a child with functional constipation. Note the high-amplitude peristaltic contractions (HAPCs)
that resulted after bisacodyl administration. Adapted and modified from Rodriguez, L. and Nurko, S. (2011). Gastrointestinal motility procedures.
In: Wyllie, R., et al. (eds.) Pediatric gastrointestinal and liver disease (4th ed.), pp. 686–698. Philadelphia, PA: Elsevier.

100 Caecum 1 2
Meal start Meal end
6

0
100 Ascending colon

−6

0
100 Tranverse colon

−1

0
100 Tranverse colon

−4 Postprandial increase in motility index

0
100 Splenic flexure

−5

0
100 Descending colon

−2

0
100 Sigmoid colon

−6

0
100 Rectum

0
35.20 7:00:00 8:00:00 9:00:00 10:00:00

Figure 2 Normal postprandial colonic motility. This colonic motility shows the normal postprandial increase in the motility index. Adapted and
modified from Rodriguez, L. and Nurko, S. (2011). Gastrointestinal motility procedures. In: Wyllie, R., et al. (eds.) Pediatric gastrointestinal and liver
disease (4th ed.), pp. 686–698. Philadelphia, PA: Elsevier.
264 Colon: Structure and Function
of feces. Evacuation is usually preceded by a deep breath,
which moves the diaphragm downward. The glottis then
closes, and contractions of the respiratory muscles on full
lungs and abdominal muscles elevate both intrathoracic
pressure and intra-abdominal pressure. Both activities serve
to increase the anorectal angle, thereby reducing resistance to
outflow. At the same time, the puborectalis muscles relax,
further increasing the angle, the levator ani muscles contract,
the perineum descends further, and stool is expulsed.
See also: Colon: Diseases and Disorders; Diarrheal Diseases.
Further Reading
Kahn E and Daum F (2010) Anatomy, histology, embryology and developmental
anomalies of the small and large intestine. In: Sleisenger and Fordtran’s
gastrointestinal and liver disease, 9th ed., pp. 1615–1639. Philadelphia, PA:
Elsevier.
Rodriguez L and Nurko S (2011) Gastrointestinal motility procedures. In: Wyllie R, et al.
(ed.) Pediatric gastrointestinal and liver disease, 4th ed., pp. 686–698. Philadelphia,
PA: Elsevier.
Sadler TW (2006) Digestive system. In: Langman’s medical embryology, 10th ed.,
pp. 203–225. Philadelphia, PA: Lippincott Williams & Wilkins.
Scoville DH, Sato T, He XC, and Li L (2008) Current view: intestinal stem cells and
signaling. Gastroenterology 134: 849–864.
Shroyer NF and Kocoshis SA (2011) Anatomy and physiology of the small and large
intestines. In: Wyllie R, Hyams JS, and Kay M (eds.) Pediatric gastrointestinal and
liver disease, 4th ed., pp. 324–336. Philadelphia, PA: Elsevier.
Slavin J (2013) Fiber and prebiotics: mechanism and health benefits. Nutrients
5: 1417–1435.
Szmulowicz UM and Hull TL (2014) Colon physiology. In: Beck DE, Wexner SD, and
Hull TL, et al. (eds.) The ASCRS textbook of colon and rectal surgery, 2nd ed.,
pp. 27–48. New York: Springer-Verlag.
Topping DL and Clifton PM (2001) Short-chain fatty acids and human colonic function:
roles of resistant starch and nonstarch polysaccharides. Physiological Reviews
81: 1031–1064.
Venkatasubramanian J, Rao MC, and Sellin JH (2010) Intestinal electrolyte absorption
and secretion. In: Sleisenger and Fordtran’s gastrointestinal and liver disease, 9th
ed., pp. 1676–1693. Philadelphia, PA: Elsevier.
 Colors: Health Effects
D Villaño and C Garcı́a-Viguera, CEBAS-CSIC, Murcia, Spain
P Mena, University of Parma, Parma, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
It is well known that color is one of the first criteria for iden-
tification and perception of food quality. This color is provided
by pigments, natural in fresh foods and natural or synthetic in
manufactured products. Even more, consumers associate food
color with safety, quality, and good food processing, a reason
why food industries spend high proportion of the product’s
cost to preserving or adding color.
Food colors or colorants are food additives added in order
to make foodstuffs more visually appealing; to restore their
original appearance if the visual acceptability has been dam-
aged as a consequence of processing, storage, packaging, and
distribution; and/or to add color to those foodstuffs otherwise
colorless or colored differently.
Nevertheless, even if color is the obvious property for pig-
ments, it is important to note that these molecules may have
other biological properties. Due to this, natural pigments from
plants are of growing interest as substitutes for synthetic dyes in
the food and pharmaceutical industry, and they increase their
added value if they possess positive effects on health. In most
countries, the use of food additives (including colorants) is
governed by strict regulation. The legislation specifies which
colorant may be used, the source, the purity, and to which food
and at what level they may be added.
This article reviews the main aspects of natural pigments
and food colorants dealing with human health.
Food Natural Pigments
Different food natural pigments can be identified:
• Anthocyanins
• Betalains
• Carotenoids
• Chlorophylls
• Curcumin
• Myoglobin and hemoglobin
Pigments naturally formed during food processing have also
been included in this section, as the melanoidins and the
Monascus pigments (Figure 1).
Anthocyanins
The anthocyanins are part of a widespread group of plant
constituents, known as flavonoids. The structure is based on
a C15 skeleton consisting of a chromane ring bearing a second
aromatic ring B in position two (anthocyanidin). The structure
is complemented by one or more sugar molecules bonded at
different hydroxylated positions (anthocyanins).
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00190-2
Six anthocyanidins (aglycones) are the most common
in nature and consequently used as natural colorants (viz.,
pelargonidin, peonidin, petunidin, malvidin, cyanidin, and
delphinidin).
Regarding their chemical stability, it is important to men-
tion that the color of these pigments is highly affected by pH
and four structures exist in equilibrium: the flavylium cation
(colored) at low pH, displaced toward the uncolored forms
(carbinol pseudobase and chalcone) or the quinoidal form
(red/blue) as the pH value increases (Figure 2).
Anthocyanins provide the red to purple or blue color to
pome fruits (apples, pears, chokeberries, etc.); stone fruits
(apricots, cherries, peaches, and plums); small fruits (currants,
strawberries, and all other berries); some tropical fruits (acer-
olas, lychees, mangos, etc.); other fruits such as grapes, pome-
granates, and red oranges; cereals (rice, barley, sorghum, etc.);
legumes (e.g., beans); roots, tubers, and bulbs (onions,
potatoes, etc.); brassica crops (broccoli, red radish, etc.); and
other crops (eggplant, perilla, rhubarb, tamarillo, etc.).
Due to the aforementioned chemical properties, anthocya-
nins, as food colorants, are used for red fruit drinks, sorbets,
water ices, jellies, jams, gums, etc., at pH values below 4.
In recent years, several studies have shown that anthocya-
nins display a wide range of biological activities, including
antioxidant, anti-inflammatory, and antimicrobial, and their
consumption is associated with a diminished risk for different
types of cancer owing to their antiproliferative, proapoptotic,
and antiangiogenic effects. Also, they play an important role in
blood vessels, platelets, and lipoprotein, able to reduce the risk
of coronary heart diseases. Nevertheless, the absorption and
metabolic fate of these pigments are still under study.
Their influence on prevention in visual performance and
ocular diseases, their role in skin aging and UV-induced skin
damage, and their neuronal and cognitive brain functions have
also been reported, being of special interest as they are one of
the fewer secondary metabolites able to cross the blood–brain
barrier.
Betalains
Betalains are water-soluble nitrogen-containing pigments,
which provide red-violet (betacyanins) and the yellow (betax-
anthins) colors to some fruits and vegetables. They are ammo-
nium conjugates of betalamic acid with cyclo-DOPA and
amino acids or amines, respectively.
With a focus on food, the scarce attention toward betalains
may be due to the fact that red beet has long been considered
the only edible betalainic source. However, other sources of
betalains are species of the genus Amaranthus; other Cactaceae
such as cactus fruits from Opuntia sp., Hylocereus polyrhizus, or
Myrtillocactus geometrizans; and Swiss chard.
265
266 Colors: Health Effects

The most interesting applicable feature of betalains is the Preliminary experiments on the biological and health-
stability in the pH range from 3 to 7, when processed at low promoting properties of betalains have been focussed on
pasteurization, in the presence of ascorbic acid, and stored at their antioxidant properties due to their chemical and in vitro
low temperature, which makes these pigments suitable for antiradical properties. However, their real physiological rele-
their application in a wide range of low-acid and neutral foods. vance has not been fully unraveled in intervention or clinical
From a nutritional point of view, betalains represent a group trials in humans. New studies conducted at the right physio-
of phytochemicals with a restricted occurrence in the diet. Only logical concentrations about the action of betalains and their
red beet, Swiss chard, Amaranthus, cactus pear, pitahaya, and circulating metabolites may open new ways of biological
some tubers, and their derived products provide betalains to actions of these phytochemicals against human disorders.
our diet. However, the use of betanin as food colorant and the
plant betalain-enriched extracts in functional foods increases
the consumption of this type of phytochemicals. Carotenoids
Betalains are bioavailable molecules, present as intact forms Carotenoids are the most widespread group of pigments in
at systemic level, but new studies should be carried out in order nature, responsible of the red, orange, or yellow colors of
to identify the complete pool of circulating compounds many vegetables and fruits. These molecules are isoprenoids
derived from betalain biometabolism once they are consumed. that consist of eight isoprene units in a long polyene chain,
which may extend from 3 to 15 conjugated double bonds.
b-Carotene is the most prominent member of the group of
R1 carotenoids. It belongs to the class of carotenoids named car-
3⬘
OH otenes, together with lycopene. The group of xanthophylls
B is formed from the hydrocarbon carotenes by the introduction
HO O+ of oxygen functions, as lutein, a- and b-cryptoxanthin, and
5⬘
R2
A C zeaxanthin.
Around 60 different carotenoids have been identified in
OH
fruits and vegetables.
OH At least 85% of our dietary lycopene comes from tomato
Anthocyanidin R1 R2 fruit and tomato-based products. Major sources of b-carotene
Pelargonidin H H include green leafy vegetables, carrots, red peppers, oranges,
potatoes, and broccoli. In a lesser extent, carotenoids are also
Cyanidin OH H present in eggs, poultry, and fish, especially when included in
Peonidin OCH3 H the animal feed. Lutein and zeaxanthin are highly present in
Delphinidin OH OH spinach and kale, broccoli, peas, and Brussels sprouts, and they
are the major carotenoids of corn and egg yolk; astaxanthin is
Petunidin OCH3 OH
the red pigment of crustaceans, salmon, and trout. Capsanthin
Malvidin OCH3 OCH3 and capsorubin are present in paprika flavoring. Bixin and
norbixin are apocarotenoids present in annatto, a natural
Figure 1 Structure of the main dietary anthocyanins.
food coloring.

R3⬘ R3⬘
OH OH

O O HO O+
R5⬘ R5⬘
⫺H+
OGlu OGlu
OH OH
Blue quinoidal base Red flavylium cation

H2O/⫺H+

R3⬘ R3⬘
OH OH
OH OH
HO O HO O
R5⬘ R5⬘

OGlu OGlu
OH OH
Yellow chalcone Carbinol pseudobase
Figure 2 Structural transformations of anthocyanins depending on pH changes.

Bioaccessibility of carotenoids is limited and conditioned
by different factors, as food processing and the composition
and structure of the food matrix. Pressure cooking and stir-
frying increase their bioaccessibility, whereas the presence of
dietary fiber has a negative effect. Because of their lipophilic
nature, the absorption of carotenoids is enhanced from a fatty
diet.
Carotenoids have a broad range of positive effects on
human health. The best established function is the provitamin
A activity (vitamin A is important for many functions in the
human body, being essential for normal growth and develop-
ment, immune function, and vision), restricted to those mol-
ecules with b-ring end groups, as b-carotene, zeaxanthin, and
b-cryptoxanthin.
Lutein and zeaxanthin are present in eye macula and might
protect from light-induced damage and scavenge free radicals
formed. Besides, UV protective effects have been reported in
the skin after supplementation of b-carotene, due to the pro-
tection against photooxidation. Consumption of tomato prod-
ucts, rich in lycopene, is associated with reduced low-density
lipoproteins (LDLs) oxidation implicated in vascular lesions,
and low serum levels of lycopene have been related to
increased risk of atherosclerotic events in middle-aged men.
In relation to cancer, carotenoids have shown to inhibit cell
proliferation and increase gap junctional communication. Epi-
demiological studies demonstrate that an increased consump-
tion of a diet rich in carotenoids is associated with a
diminished risk for different types of cancer, as it occurs with
the high lycopene consumption and prostate cancer and lung
and stomach cancer. However, the CARET (Beta-Carotene and
Retinol Efficacy Trial) study showed that lung cancer was
alleviated with b-carotene supplementation. It seems that the
dietary consumption of a combination of carotenoids and
other phytochemicals in fruit and vegetables is more effective
in the protection against cancer than the intake of single sup-
plements of any carotenoid.
Chlorophylls
Chlorophyll is the green pigment found in cyanobacteria and
chloroplasts of algae and plants and is critical in photosynthe-
sis as it allows plants to absorb energy from light.
The basic skeleton is composed of a large planar structure of
four pyrrole rings joined together with a central metal atom of
magnesium and a phytol hydrophobic group.
At least six chlorophyll molecules are known: chlorophylls
a, b (higher plants), c, d, and e (algae) and bacteriochloro-
phylls (photosynthetic bacteria). The most widely distributed
in plants is chlorophyll a.
Chlorophyllins are molecular analogs of chlorophyll more
stable and also available as food colorants. They are hydro-
philic, due to hydrolysis of the phytol tail, and the magnesium
atom is replaced with other metal complexes as copper.
Chlorophylls are abundant in leafy fresh vegetables, as
spinach and kale, lettuce, and basil. They are highly susceptible
to degradation during processing. The most common reactions
involve the loss of magnesium by heat or acidic conditions,
forming pheophytin, and the green color turns to an olive
green. Further decarbomethoxylation leads to the conversion
of pheophytin into pyropheophytin. These changes are
Colors: Health Effects 267
perceived by the consumer as a loss of quality. Storing the
product at low temperatures and the immediate processing
help to preserve the green color.
Native chlorophylls are poorly absorbed as such and are
easily converted into Mg-free pheophytins during the gastric
phase of digestion. Pheophytins are transferred to micellar frac-
tions for their optimal uptake by human intestinal cells and
likely represent the biologically relevant forms of these pigments.
Natural chlorophylls and chlorophyllins have been
reported to display antioxidant activity, in models of lipid
peroxidation, and antimutagenic and chemopreventive activi-
ties. Their porphyrin cycle can either scavenge free radicals or
form a complex with carcinogens. Human studies on popula-
tions at high risk of developing carcinoma, because of toxin
ingestion, have shown a reduction of urinary excretion of
damaged DNA catabolites, without reported toxicity effects,
possibly due to the formation of adducts of chlorophylls and
mutagen, interfering in the bioavailability of the latter.
Curcumin
Curcumin is a natural colorant extracted from the rhizomes of
Curcuma longa plant (turmeric). Three compounds have been
isolated: curcumin, demethoxycurcumin, and bisdemethoxy-
curcumin that impart the yellow pigmentation to the plant and
the products added, for example, curry.
The main pattern of consumption is in powder form, con-
taining a mixture of the three curcuminoids, used as spice in
Indian cuisine, to improve palatability and preservation of
foods and to impart color to condiments and dressings.
Curcumin exhibits poor bioavailability, a major limit for its
therapeutic efficacy and use in clinic. The major factors are its
poor absorption due to insolubility in water, fast metabolism,
extensive enterohepatic recirculation, and rapid systemic elim-
ination in the bile and urine. The 40% of orally administered
curcumin is excreted unchanged in the feces.
Curcumin is believed to have a wide range of biological
effects. It has been shown to directly interact with numerous
signaling molecules and activate/inhibit multiple regulatory
proteins, including transcription factors, enzymes, and cyto-
kines. In this way, curcumin may reverse conditions as insulin
resistance, hyperglycemia, hyperlipidemia, and other inflam-
matory symptoms associated with obesity and metabolic
diseases.
Because of its multitargeting properties, curcumin has
exhibited activities against some types of cancer as colorectal
and breast cancer. Histological improvement of precancerous
lesions has been observed in bladder cancer, oral leukoplakia,
and intestinal metaplasia of stomach, and a decrease in the
number and size of polyps in skin lesions has been observed in
phase I and phase II human clinical trials. In proinflammatory
diseases as ulcerative colitis, Crohn’s disease, pancreatitis, or
arthritis, curcumin has shown promising effects, with high
tolerability and scarce adverse effects. Efforts must be done to
improve its poor bioavailability.
Melanoidins
Melanoidins are brown-colored compounds, with high-
molecular-weight heterogeneous polymeric structure. These
268 Colors: Health Effects
products are formed through the Maillard reaction of sugars
and amino acids at high temperatures and low water activity.
Information about the structure of melanoidins is scarce
due to the high complexity and diversity of structures and the
difficulty of isolation of pure compounds.
Melanoidins are present in a large range of products, such as
barley malts, bread crust, bakery products, balsamic vinegar,
tomato sauce, and coffee.
Little is known about the metabolic fate of melanoidins.
They are largely resistant to digestion in the human gastroin-
testinal tract. The microbial gut degradation occurs for the less
complex melanoidin structures that are absorbed and can be
detected in urine in quantities up to 30% of intake.
Human studies have reported antioxidant activity of coffee
or beer melanoidins after ingestion. Melanoidins protect
against radical stress in the colon and behave as dietary fiber
by promoting the growth of gut bifidobacteria. Besides, inhib-
itory effects of melanoidins on matrix metalloproteases impli-
cated on tumor growth and development have been reported.
They also have antibacterial activity against Staphylococcus
aureus, Escherichia coli, and Salmonella enterica and could pre-
vent the adhesion of Streptococcus mutans to the tooth surface
playing a role inhibiting the development of dental caries.
Better knowledge of melanoidin structures would benefit
the understanding of their biological properties.
Monascus
Monascus-fermented rice, also referred as red yeast rice (RYR), is
a food product that is produced by inoculating fungi of the
genus Monascus into steamed rice. Monascus fungi produce
different main pigments: red colorants (rubropunctamine
and monascorubramine), orange colorants (rubropunctatin
and monascorubrin), and yellowish colorants (monascin and
ankaflavin).
RYR (known as beni-koji in Japan) has been used for cen-
turies in Eastern countries as China and Japan as natural food
colorant and flavor as well as food preservative of meat and
fish. Food examples are red rice vinegar, some types of tofu,
Peking duck, Chinese pastries and rice wines as Japanese sake,
imparting reddish color. The RYR is most often powdered and
sold as such but the pigments may also be extracted.
Healthy effects have been reported for RYR as cholesterol
lowering, antidiabetic activity, anti-inflammation, and preven-
tion of osteoporosis, and some of these activities have been
related to the pigments contained.
The red and orange pigments possess antibiotic activity
against bacteria, yeast, and filamentous fungi. Both orange
(rubropunctatin and monascorubrin) and yellow pigments
such as monascin and ankaflavin have shown immunomodu-
latory and anti-inflammatory properties. In addition, Monascus
pigments could prevent obesity development. Monascin and
ankaflavin might reduce triglyceride accumulation and prolif-
eration of preadipocytes. Cancer-chemopreventive activity has
been reported for all pigments, specially monascin and anka-
flavin, by inhibition of cancer cell progression and cytotoxicity
of tumoral cells.
However, the RYR can be contaminated by citrinin, a
hepatotoxic metabolite of Aspergillus and Penicillium species
that has led to the prohibition of Monascus pigments use as
food additive colorants in EU and United States, while in Asian
countries, it is accepted under monitoring and the establish-
ment of maximum limits for citrinin in foods.
Myoglobin and Hemoglobin
Meat color is due to the concentration of the pigments myo-
globin and hemoglobin. Myoglobin is a metalloprotein com-
posed of a polypeptide globin and a heme residue with iron
ion.
Color changes in meat occur because of the susceptibility of
myoglobin to alterations. Under low oxygen tension, iron in
myoglobin exists in Fe2þ state and meat is visually purple-red.
When it is exposed to oxygen for a short time, myoglobin binds
oxygen producing oxymyoglobin, which is bright pink or red.
Oxidization of iron to Fe3þ state produces metmyoglobin,
visually brown or gray-brown. Further degradation of metmyo-
globin gives green and dark brown pigments.
Biological function of myoglobin is related to storage of the
oxygen brought by the hemoglobin for the requirements of
living tissues. It binds reversibly O2 and facilitates its transport
to mitochondria. Myoglobin also scavenges reactive O2 species
and facilitates oxygen diffusion. An additional role of this
protein is to bind nitric oxide in skeletal muscle as nitric
oxide inhibits cytochrome c oxidase and thus impairs mito-
chondrial respiration.
Colorants Used as Additives in Food Industry
Fruit and vegetable juices can be added to foodstuffs but are
considered ingredients rather than additives. Because of this,
they do not carry an E number in the European Union (EU)
market, turning the foodstuff into a more natural product.
Accepted food colors and quantities allowed to be added to
each foodstuff are regulated by national laws (except for the EU
countries that share a common legislation).
According to the US Food and Drug Administration (FDA),
all color additives shall be declared. Food colorings exempt
from certification are those having generally a mineral, plant,
or animal origin and are declared as ‘artificial color,’ ‘artificial
color added,’ or ‘color added.’ Colors subject to certification
are usually identified by the terms ‘FDþC No.’ All the ‘FDþC’
color additives have to be declared, and in order to save label-
ing space, they can appear with their names without the prefix
‘FDþC No.’
This section will focus on natural pigments that are not a
food in itself and are not normally used as a characteristic
ingredient of foods, such as riboflavins, cochineal, vegetable
carbon, and some minerals. Artificial food colors, which are
produced by chemical synthesis, will also be reviewed.
Riboflavins
Riboflavin, also known as vitamin B2, occurs as yellow to
orange-yellow crystals. It is designated by the E number E 101
and can be found both in its free form (E 101i) and as
riboflavin-50 -phosphate (E 101ii). Riboflavin or vitamin B2 is
present naturally in animal products (meat, milk, and eggs),
vegetal ones (malted barley, nuts, and almonds), and yeast.

It is also used as food color in order to confer yellow shades to
some foodstuffs such as dairy products and confectionary. It
may be produced by chemical synthesis, biosynthesis from
Eremothecium ashbyii (aka Ashbya gossypii), Saccharomyces cerevi-
siae, and Bacillus subtilis, among other microorganisms, or iso-
lated from natural sources. Riboflavin-50 -phosphate is
prepared.
The health effects of riboflavin are mainly determined by its
role as a vitamin. Its physiological functions are described in
other articles of this Encyclopedia.
Cochineal, Carmine, and Carminic Acid
Carminic acid is a red-crimson anthraquinone coloring matter
that occurs naturally in some insects like cochineal (Coccus
cacti L.). The food color cochineal or cochineal extract is
obtained after hydroalcoholic extraction of bodies of insects.
Carmine is a more concentrated solution containing, at least,
50% of carminic acid.
Cochineal-derived dyes are mainly used to confer red
shades in alcoholic beverages, yogurts, juices, ice creams, and
confectionary, although it can be also found in jams and some
processed meat products.
Concerning its health properties, carminic acid may cause
allergic reactions and even anaphylactic shock to a sensitive
subset of populations. Nevertheless, it is not a significant haz-
ard to the general population.
Vegetable Carbon
Vegetable carbon or carbon black is produced by steam activa-
tion of carbonized plant material. It is mainly used in confec-
tionary and desserts, including dairy products.
No ADI has been established for vegetable carbon owing to
its scarce toxicological data, despite evidence on its safety has
been confirmed by the European Food Safety Authority
(EFSA).
Food Colors from Mineral Origin
There is a group of food colors that are obtained mainly from
mineral sources by diverse extraction processes. These food
colors are titanium dioxide that provides white color and
opacity; iron oxides, conferring yellow, red, or black shades;
aluminum; silver; and gold. These colors are mainly used as
surface coatings (e.g., iron oxides are present in sausage coat-
ings). Gold and silver can also be included in expensive eccen-
tric beverages. Calcium carbonate can also act as coating
colorant providing white color, although its principal role as
additive is to serve as anticaking agent, stabilizer, and acidity
regulator. Although they are accepted in the EU, most of these
cannot be used within the American market as they are banned
by the FDA. On the contrary, some color additives as ferrous
gluconate and ferrous lactate are accepted in the United States,
but not at the EU, for the coloring of ripe olives.
Information on the dietary exposure to these minerals
when used as food additives is very scarce (Table 1). Nonethe-
less, their contribution to the daily consumption of minerals
may be considered as negligible attending to their restricted
use. The absorption, distribution, metabolism, excretion, and
Colors: Health Effects 269
safety of these mineral food colors follow the same patterns as
when they are contained naturally in foods or they are present
as contaminant due to food processing operations.
Azo Food Colors
Most of the synthetic additives used as food colors share a
common chemical structure characterized by the presence of
an azo functional group. In addition, many of these colors are
sodium salts of sulfanilic acid. Tartrazine, Sunset Yellow FCF,
Azo Rubine, amaranth, Ponceau 4R, Allura Red AC, Brilliant
Black BN, Brown HT, Lithol Rubine BK, Orange B, and Citrus
Red No. 2 are part of this group.
Tartrazine is the trisodium salt of 5-hydroxy-1-(4-sulfona-
tophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole-
3-carboxylate. It provides orange-yellow color to some des-
serts and sweets, potato and corn chips, muesli, corn flakes,
noodles, tartar sauce, mustard, bouillon cubes, and energy and
sport drinks. Tartrazine is also available for consumer, as it is
one of the few food additives that can be purchased at grocery
stores. Sunset Yellow FCF, Orange Yellow S, or FDþC Yellow
No. 6 is an orange-yellow azo dye present not only in soft
drinks and desserts such as jellies but also in confectionary,
jams, chips, biscuits, ice creams, instant noodles, and food
decorations and coatings. Azo Rubine or carmoisine, a red
dye, is present in nonalcoholic flavored drinks, sugar
confectionary, and yogurts. Amaranth provides dark, reddish
brown color to alcoholic beverages like aperitif wines just as to
candied fruit, other confectionary products, yogurts, and ice
creams. Ponceau 4R, Cochineal Red A, or New Coccine is a red
color used mainly in soft drinks, confectionary, coatings, bis-
cuits, ice creams, and fine bakery wares. Allura Red AC can be
found in crisps, biscuits, nonalcoholic flavored drinks, drinks,
desserts and yogurts, sweets, ice creams, and jams. Brilliant
Black BN or Black PN and Brown HT are bisazo colors used to
confer dark shades in a broad array of foodstuffs that are
banned in many other countries outside the EU. Lithol Rubine
BK is a red dye used exclusively in cheeses as surface coating.
Orange B, banned in the EU market but allowed in the United
States, is only used in sausage coatings. Citrus Red No. 2 is only
for use in the United States to color orange skins.
Dietary exposure to azo food colors vary notably among
these additives because of their differences regarding quantities
used and pattern of consumption of foodstuffs containing
them (Table 1).
Azo food colors are extensively metabolized by the gut
microbiota to sulfanilic acid and aminopyrazolone after oral
administration. On the basis of this fact, urine excretion of
intact azo colors is negligible or very low (e.g., <5% for
tartrazine), while their metabolites, mainly free and conju-
gated sulfanilic acid, are absorbed to a greater extent.
Regarding health effects of azo colors, controversial litera-
ture has been reported. Some cell/animal studies pointed out
how tartrazine, Ponceau 4R, Allura Red AC, Brilliant Black BN,
and Brown HT might affect neurodevelopment or exert carci-
nogenic properties. Nevertheless, adverse effects of these food
colors on neurobehavioral development or cell proliferation
should not be expected to occur in humans, according to EFSA.
In general, there are no indications of any genotoxic/carcino-
genic potential of (bis)azo dyes or their metabolites. On the
270 Colors: Health Effects

Table 1 Characteristics, acceptable intake, and exposure for food colors accepted in United States and EU

EU regulationa US regulationc

E ADI (mg kg body Mean dietary exposure (mg kg body

Name number MLUb Name Certification?d weight1 day1)e weight1 day1)f

Curcumin 100 R Turmeric No 3 European children: 0.5–3.8

UK adults: 0.9
Riboflavins 101 q.s. Riboflavin No 0.5* Men: 2
Women: 1.5
Tartrazine 102 R FDþC Yellow No. 5 Yes 7.5 European children: 0.8–3.4
Hong Kong children: 0.35
Quinoline Yellow 104 R DþC Yellow No. 10 Banned 0.5 Irish children: 2.7
Hong Kong children: 0.0
Sunset Yellow 110 R FDþC Yellow No. 6 Yes 1 European children: 0.02–0.4
FCF/Orange Yellow Hong Kong children: 1.1
S
Cochineal, carminic 120 R Cochineal extract No 5*
acid, carmines or carmine
Azo Rubine, 122 R Banned 4 European children: 0.3–2.5
carmoisine Hong Kong children: 0.0
UK adults: 0.5
Amaranth 123 R Banned 0.15 European children/adolescents:
1.6–4.0  103, adults/elderly:
0.008–0.009  103
Hong Kong children: 0.11
Ponceau 4R, 124 R Banned 0.7 European children: 0.3–2.5
Cochineal Red A Hong Kong children: 0.06
UK adults: 0.5
Erythrosine 127 R FDþC Red No. 3 Yes 0.1 Europe: Negligible
Hong Kong children: 0.03
Allura Red AC 129 R FDþC Red No. 40 Yes 7 European children: 0.8–3.4
Hong Kong children: 0.15
UK adults: 0.9
Patent Blue V 131 R Banned 5 Toddlers: 1–4.5, children: 1.1–3.6
adolescents: 0.5–1.8, adults:
0.3–1.4, elderly: 0.2–0.6
Indigotine, Indigo 132 R FDþC Blue No. 2 Yes 5* Hong Kong children: 0.0
carmine
Brilliant Blue FCF 133 R FDþC Blue No. 1 Yes 6 European children: 0.5–3.4
Hong Kong children: 0.22
UK adults: 0.9
Chlorophylls, 140 q.s. Banned Not limited*
chlorophyllins
Copper complexes of 141 q.s. Sodium copper No 15
chlorophylls, chlorophyllin
chlorophyllins
Green S 142 R Banned 5 European children: 0.5–3.5
Hong Kong children: 0.0
UK adults: 0.9
Plain caramel 150a q.s. Caramel No Combined ADI: 300, Toddlers: 19–105, children: 31–83,
Caustic sulfite 150b q.s. Caramel No with an ADI of 100 adolescents: 12–56, adults: 15–57,
caramel to E 150c elderly: 9–35
Ammonia caramel 150c q.s. Caramel No
Sulfite ammonia 150d q.s. Caramel No
caramel
Brilliant Black BN, 151 R Banned 5 Below 5
Black PN
Vegetable carbon 153 q.s. Banned No established European children: 3–30
UK adults: 4
Brown HT 155 R Banned 1.5 European children: 0.3–2.2
UK adults: 0.5
Carotenes 160a q.s. b-carotene No 5* European children: 0.03–0.22
UK adults: 0.06

(Continued)
 Colors: Health Effects 271

Table 1 (Continued)

EU regulationa US regulationc

E ADI (mg kg body Mean dietary exposure (mg kg body

Name number MLUb Name Certification?d weight1 day1)e weight1 day1)f

Annatto, bixin, 160b R Annatto extract No 12*

norbixin
Paprika extract, 160c q.s. Paprika, paprika No
capsanthin, oleoresin
capsorubin
Lycopene 160d R Tomato lycopene No 0.5*
extract/
concentrate
Beta-apo-80 - 160e R Beta-apo-80 - No 0.05 European children: 0.5–3.4
carotenal carotenal UK adults: 0.9
Lutein 161b R Tagetes (Aztec No 1 0.6–2.2
marigold) meal
and extract
Beetroot red, betanin 162 q.s. Dehydrated beets No No established*
(beet powder)
Anthocyanins 163 q.s. Grape color No No established Toddlers: 1.5–4, children: 1.5–4.7,
extract/skin adolescents: 1–2.5, adults:
extract 0.7–1.9, elderly: 0.5–1.1
(enocianina)
Calcium carbonate 170 q.s. Calcium carbonate Banned No established
Titanium dioxide 171 q.s. Titanium dioxide No No established
Iron oxides/ 172 q.s. Synthetic iron No 0.5*
hydroxides oxide
Aluminum 173 q.s. Alumina Banned
Silver 174 q.s. Banned
Gold 175 q.s. Banned
Lithol Rubine BK 180 q.s. D&C Red No. 6 Banned 1.5 European children: 0.02–0.06
UK adults: 0.01–0.03
Fast Green FCF Banned – FDþC Green No. 3 Yes 25*
Banned – Ferrous gluconate/ No 0.8*
lactate
Banned – Orange B Yes
Banned – Citrus Red No. 2 Yes
a
Data according to the Commission Regulation (EU) No 1129/2011 of 11 November.
b
MLU, maximum level of use by EU laws; R, regulated on the basis of the foodstuff considered; q.s., ‘quantum satis,’ which means that no maximum level is specified and substances
shall be used in accordance with good manufacturing practice.
c
Data according to the Code of Federal Regulations Title 21 (Parts 73 and 74).
d
If the color does not require certification, it follows the rules established at the Part 73 of the Code of Federal Regulations Title 21; if it requires, it follows Part 74 of Title 21.
e
Acceptable daily intake (ADI) based on recent EFSA reevaluations of selected colors as food additives. Data with asterisk refer to values based on the Joint FAO/WHO Expert
Committee on Food Additives (JECFA).
f
Data based on the mean dietary exposure considering the maximum permitted levels (Tier 2) established by EFSA Panels after reevaluations of selected colors as food additives and on
Lok et al.

other hand, the effects of Sunset Yellow FCF on the testis,
sperm morphology, and sperm mobility remain unclear now-
adays, which motived the reduction of the ADI for this azo dye.
Tartrazine has been identified to cause migraine, asthma,
and certain cutaneous conditions (i.e., urticaria) in a very small
fraction of the population due to intolerance reactions. Sensi-
tive reactions after Sunset Yellow FCF, Azo Rubine, amaranth,
Ponceau 4R, or Allura Red AC consumption may also take
place in some individuals, overall when it is taken within
blends of other synthetic food colors. Finally, behavior
changes (hyperactivity, restlessness, and sleep disturbance)
might appear in children following exposure to some azo
colors in combination with other food additives like sodium
benzoate. This fact would be restricted to specific sensitive
individuals, and its clinical significance remains unclear.
Quinoline Yellow
It is a greenish yellow food dye, produced by chemical synthe-
sis that can be found in sport and energy drinks, ices, and
confectionary, among other products.
It has a limited absorption in mammal animal models, and
the bulk of the dose is excreted unchanged via the feces. None-
theless, this color additive is to some extent bioavailable, con-
cerning some systemic effects found in long-term toxicity
studies. Adverse sensitivity reactions (urticaria and rhinitis)
272 Colors: Health Effects
after intake of Quinoline Yellow have been reported although
no conclusive results on the induction of sensitivity by this
food color have been achieved. It may also have an adverse
effect on activity and attention in children, similarly to some
azo dyes.
Caramels
Caramel is a complex mixture of compounds resulting in a
dark brown liquid or solid material formed by the controlled
heat treatment of sugars. Caramelization is produced in the
presence of ammonia, sulfite, alkalis, or combinations thereof,
and caramels are classified, according to their processing, into
plain caramel, caustic sulfite caramel, ammonia caramel, and
sulfite ammonia caramel. Caramels are present in a plethora of
foodstuffs such as beer, biscuits, chocolate, confectionary,
cookies, crisps, ice creams, sauces, soups, vinegar, wood-aged
spirits, and, overall, colas.
The main contributors to mean exposures (Table 1) to
caramels in children through European countries are flavored
drinks, bakery, and desserts, including dairy products. Non-
alcoholic beverages, beer, cider, and confectionary account for
the higher amounts of the four caramel colors in adults. Some
population groups having high exposure to caramel color
could exceed the ADI of this color.
Caramels are complex mixtures of poorly characterized
compounds, and their bioavailability remains unclear.
Caramel food colors present low toxicity/carcinogenicity
both in short-term tests and in chronic studies. However,
certain low-molecular-weight compounds being part of these
additives, such as furan and 5-hydroxymethyl-2-furfural
(5-HMF), might have chemotoxic potential though their con-
tribution to the toxicity of caramel colors has not been fully
elucidated. There are no data concerning the long-term toxicity
or carcinogenicity of caramel colors so far. In contrast to other
synthetic additives, there are no reported cases of sensitivity
reactions.
Triarylmethane Colors
Brilliant Blue FCF, Erioglaucine disodium, Green S or
Lissamine Green B, and Fast Green FCF are synthetic triaryl-
methane dyes. They can be found mainly in ice creams and
other dairy products, peas, and drinks. According to the higher
level of intake of these dyes (Table 1), some adults may be
above the ADI of Brilliant Blue FCF and Green S. Anyway,
triarylmethane colors are poorly absorbed and mainly excreted
unchanged in feces. Brilliant Blue FCF lacks significant geno-
toxicity effects, while data related to Green S are not available.
Other Synthetic Food Colors
Indigotine or Indigo carmine is a synthetic color, although it
used to be extracted from plants of the genus Indigofera.
Together with Patent Blue V, it is used in order to confer
blue coloration to foodstuffs. Both blue colors are mainly
used in coatings, ice creams, and confectionary. Erythrosine is
exclusively used for cherry products in the EU and used also for
sweets in other countries. In general, these food colors have
low absorption and limited availability and are excreted almost
completely via feces. Health effects of these colors are not
worth mentioning as they are recognized as safe under proper
manufacture practices and normal intakes.
See also: Caramel: Methods of Manufacture; Caramel: Properties and
Analysis; Carotenoids: Physiology; Chlorophyll; Codex Alimentarius
Commission: Role in International Food Standards Setting; Colors:
Properties and Determination of Natural Pigments; Colors: Properties
and Determination of Synthetic Pigments; Food Additives:
Classification, Uses and Regulation; Food Allergies; Nutrition and
Health Claims for Food: Regulatory Controls, Consumer Perception,
and Nutrition Labeling; Riboflavin: Physiology; Riboflavin: Properties
and Determination.
Further Reading
Commission Regulation (EU) No 1129/2011 of 11 November 2011 Amending Annex II
to Regulation (EC) No 1333/2008 of the European Parliament and of the Council by
Establishing a Union list of Food Additives.
De Pascual- Teresa S, Moreno DA, and Garcia-Viguera C (2010) Flavanols and
anthocyanins in cardiovascular health: a review of current evidence. International
Journal of Molecular Sciences 11: 1679–1703.
Fernández-Garcı́a E, Carvajal-Lérida I, Jarén-Galán M, Garrido-Fernández J, Pérez-
Gálvez A, and Hornero-Méndez D (2012) Carotenoids bioavailability from foods:
from plant pigments to efficient biological activities. Food Research International
46: 438–450.
Lok KYW, Chung YW, Benzie IFF, and Woo J (2011) Synthetic colourings of some
snack foods consumed by primary school children aged 8–9 years in Hong Kong.
Food Additives and Contaminants: Part B Surveillance 4: 162–167.
Mazza G and Mimiati E (1993) Anthocyanins in fruits vegetables and grains. London:
CRC Press.
McCann D, Barrett A, Cooper A, et al. (2007) Food additives and hyperactive behaviour
in 3-year-old and 8/9-year-old children in the community: a randomised, double-
blinded, placebo-controlled trial. Lancet 370: 1560–1567.
Moreira ASP, Nunes FM, Domingues R, and Coimbra MA (2012) Coffee melanoidins:
structures, mechanisms of formation and potential health impacts. Food and
Function 3: 903–915.
Moreno DA, Garcia-Viguera C, Gil JI, and Gil-Izquierdo I (2008) Betalains in the era of
global agri-food science, technology and nutritional health. Phytochemistry Review
7: 261–280.
Ordway GA and Garry DJ (2004) Myoglobin: an essential hemoprotein in striated
muscle. Journal of Experimental Biology 207: 3441–3446.
Patakova P (2013) Monascus secondary metabolites: production and biological activity.
Journal of Industrial Microbiology and Biotechnology 40: 169–181.
Sant’Anna V, Gurak PD, Marczak LDF, and Tessaro IC (2013) Tracking bioactive
compounds with colour changes in foods – a review. Dyes and Pigments
98: 601–608.
Shehzad A, Ha T, Subhan F, and Lee YS (2011) New mechanisms and the anti-
inflammatory role of curcumin in obesity and obesity-related metabolic diseases.
European Journal of Nutrition 50: 151–161.
Relevant websites
http://www.efsa.europa.eu – European Food Safety Authority (EFSA).
http://www.fda.gov/ – US Food and Drug Administration (FDA), Code of Federal
Regulations – Title 21 – Food and Drugs.
http://www.food.gov.uk/ – U.K. Food Standards Agency (FSA).
http://www.mhlw.go.jp/english/index.html – Japanese Ministry of Health, Labour and
Welfare.
http://www.nih.gov/ – US Department of Health & Human Services, National Institutes
of Health (NIH).
 Colors: Properties and Determination of Natural Pigments
A Giuliani, University “G. d’Annunzio”, Chieti-Pescara, Italy
L Cerretani, Pizzoli S.p.A., Budrio, Italy
A Cichelli, University “G. d’Annunzio”, Chieti-Pescara, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Abbreviations
APCI Atmospheric pressure chemical ionization
DAD Diode array detector
ESI Electrospray ionization
Anthocyanins
Anthocyanins are water-soluble vacuolar flavonoid pigments.
They are responsible for the orange, magenta, violet, and blue
color of plant organs, including leaves, stems, roots, flowers,
and fruits. Until now, 600 different anthocyanins are known
mainly in the form of heterosides. The aglycone form, or
anthocyanidin, is a polyhydroxy and polymethoxy derivative
of flavylium (2-phenyl-1-benzopyrylium), which is usually
bonded to a sugar moiety and can be possibly acylated by a
phenolic or aliphatic acid (Figure 1). Among the 23 types of
known anthocyanidins, only six are common in higher plants:
pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and
malvidin.
Many factors, that is, pH, heat, light, metals, and
copigmentation, may affect their stability, color, hue, and den-
sity. Under acidic conditions, the eight conjugated bonds in
anthocyanin structures carrying a positive charge on the het-
erocyclic oxygen ring are responsible for the intense red-orange
to blue-violet color. They show a wavelength of maximum
adsorption between 465 and 550 nm and a significant absorp-
tion in the UV range between 270 and 280 nm.
In vitro, under mildly acidic conditions (pH 3–6), the red
and stable flavylium cation form predominates, while at pH 6
and above, it changes into the colorless carbinol pseudobase or
chalcone forms and finally into the bluer and unstable quino-
noidal base form (Figure 2). The larger the number of hydroxyl
groups on the B-ring, the bluer the color is. Aromatic acylation
causes a blueshift and stabilizes anthocyanins, whereas ali-
phatic acylation increases the stability and solubility. Self-
association with flavonoids stabilizes anthocyanins and causes
bluing and intensifying of color.
Sources and Production
Cyanidin, delphinidin, and pelargonidin glycosides are the
most common in nature, being present in 80% of pigmented
leaves, 69% of fruits, and 50% of flowers. The distribution of
the six more common anthocyanidins in fruits and vegetables
is the following: cyanidin (50%), delphinidin (12%), pelargo-
nidin (12%), peonidin (12%), petunidin (7%), and malvidin
(7%). 3-Glycosides occur about two and a half times more
frequently than 3,5-diglycosides.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00189-6
HPLC High-performance liquid chromatography
MS Mass spectroscopy
SCC Sodium copper chlorophyllin
The major sources of anthocyanins are red fruits, some
cereals, and root vegetables (aubergines, beans, cabbages,
radishes, and onions).
Berries such as black currants and blackberries contain up
to 2–4 g kg
1 fresh weight anthocyanins mainly in the skin.
They are also present in apples, plums, and pears and in the
flesh of cherries and strawberries. In particular, red grapes can
contain up to 250 mg/100 g, while the concentration in red
wines varies in accordance with the varieties of grapes
employed and with the type of vinification and, especially,
with aging. However, a medium value of around 500 mg l
1
of anthocyanins could be established in young wines.
Actually, the European Union allows the use of anthocya-
nins as food colorants named E163 in acidic food or beverages
including soft drinks, fruit preserves, sugar confectioneries,
dairy products, frozen products, dry mixes, and alcoholic
drinks. In particular, grape skins, a by-product of the wine
industries, are the most common source for economic reasons
along with the concentrated juice of black currants, straw-
berries, cranberries, elderberries, cherries, and red cabbages.
Other less familiar sources include radishes, black carrots,
and red and purple sweet potatoes.
Moreover, berry, grape skin, and seed extracts are commer-
cialized as nutraceuticals and as dietary supplements for their
antiaging effect.
Despite their great potential of application, anthocyanin
extraction from natural sources is complex due to their relative
instability.
Determination of Anthocyanins
Usually, anthocyanins are extracted under mild conditions
with cold acidified polar organic solvents (i.e., methanol,
ethanol, and acetone) at a composition of 70–80% in water.
The acid employed is usually acetic acid, trifluoroacetic acid, or
sulfured water in order to minimize the pH effects on the
flavylium equilibriums. Then, the concentrated extract is puri-
fied by washing with hexane, petroleum ether, or diethyl ether
to remove unwanted lipophilic compounds. Alternative tech-
niques that reduce the use of organic solvents and maximize
recovery with minimal degradation of the pigments are solid
phase extraction and elution by solvent with increasing
polarity, supercritical fluid extraction using CO2 and polar
organic modifiers as cosolvents, pressurized hot water
273
274 Colors: Properties and Determination of Natural Pigments

R1

OH
3′
4′
+ 5′
HO 7 O
1 R2

3
5
R3

OH

Anthocyanins R1 R2

Pelargonidin-3-glucoside H H

Peonidin-3-glucoside OCH3 H

Cyanidin-3-glucoside OH H

Malvidin-3-glucoside OCH3 OCH3

Petunidin-3-glucoside OCH3 OH

Delphidin-3-glucoside OH OH

Figure 1 Chemical structure of the six most common dietary anthocyanin 3-O-glucosides. Structural classification of the six anthocyanins (flavylium
form). R3 is a sugar moiety.

R1 R1
OH OH

O O HO O+
R2 R2

R3 R3 pH 1
pH 7 +
–H
OH Quinoidal base OH Flavylium cation
blue Red to orange

R1 H2O/-H+
R1
OH OH
OH OH
HO O
HO O
R2 R2

R3 R3
OH pH 6 OH pH 5
Chalcone Carbinol pseudobases
Yellow to colorless Colorless

Figure 2 Structural transformations of anthocyanins upon pH changes.

 Colors: Properties and Determination of Natural Pigments
extraction, microwave-assisted extraction with polar solvents,
and countercurrent chromatography that allows fractionation
and isolation of pure compounds for direct identification by
mass spectroscopy (MS) and nuclear magnetic resonance.
Recently, enzyme-assisted extraction has been carried out
for anthocyanin extraction from cherry, red grape, strawberry,
black currant, and blueberry juice processing wastes and grape
residues. Nowadays, some researchers have also developed cell
cultures within plant species for the production of
anthocyanins.
The analysis of anthocyanins is commonly carried out
through high-performance liquid chromatography (HPLC)
on octadecylsilane, polystyrene, or phenyl-bonded columns
in reversed phase using gradient solvent systems of
acetonitrile–water or methanol–water with a small amount of
acid and a UV detector. Anthocyanins have a unique maximum
absorption at 520 nm, and a diode array detector (DAD) per-
forms a complete spectrophotometric scan on each peak as it
elutes and provides a unique chromatogram for each com-
pound. Recently, electrospray ionization–MS (ESI–MS) detec-
tion has been used allowing very low fragmentation and exact
molecular weight determinations. Another common technique
used for the separation of anthocyanins is capillary zone elec-
trophoresis (CZE) with UV or ESI–MS detection using acidic
media optimized via a phosphate buffer and a cationic
surfactant.
Patterns of Consumption
Depending on the diet, the daily intake of anthocyanins in
humans has been estimated to range between a few and
100 mg. The average anthocyanin intake in adults within the
United States is about 12.5 mg day
1. A higher consumption
has been reported in European countries with a North–South
gradient. This result is consistent with the Mediterranean diet
including berries, other red- and blue-colored fruits, and red
wine. Considerably, enhanced anthocyanin consumption of
commercially available extracts is also suggested.
Availability, Absorption, and Metabolism
Anthocyanins are rapidly absorbed in the stomach and small
intestine reaching a maximum plasma concentration (Cmax) of
between 1.4 and 200 nm for doses of 10–720 mg of anthocy-
anins. The Cmax is reached between 45 min and 4 h after inges-
tion of an anthocyanin-containing meal depending on its
composition. In addition, anthocyanins are rapidly eliminated
in urine after 1.5–6 h of ingestion. Since the proportion of
absorbed and excreted anthocyanins is < 0.1% of the ingested
amount, there is a paradox about the metabolic fate of a very
high percentage of the ingested anthocyanins that needs to be
explained.
In both blood and urine, they appear as intact, methylated,
glucuronide derivatives and in sulfoconjugated form. Interest-
ingly, the glycosides do not appear to be easily hydrolyzed to
aglycones.
The reasoning of the paradox is that the bioavailability of
anthocyanins compared with other flavonoids is influenced by
physicochemical characteristics such as size, lipid/water
solubility, and pKa; by food matrix effects like the presence of
275
lipids; or by interaction with other compounds in the meal.
Moreover, the changes of pH and the presence of digestive
enzymes, biliary acids, and microbiota along the gastrointesti-
nal tract can greatly affect the chemical stability of anthocya-
nins and their metabolism.
Anthocyanins occur under cationic form in the gastric
lumen, but whether they are adsorbed through binding to
some specific protein transporters, it is still unknown. On the
other hand, the absorption of anthocyanin glycosides in the
small intestine, mainly in the form of carbinol pseudobase,
may involve specific glucose transporters. Another possible
mechanism could be the hydrolyzation of anthocyanins by
brush border enzymes prior to passive diffusion of the agly-
cone. Finally, large amounts of unabsorbed anthocyanins and
derivatives excreted in the bile from the enterohepatic circula-
tion can reach the colon. Enzymes of the gut microbiota can
also induce metabolization including methylation, sulfation,
and conjugation with glucuronic acid and breaking of glyco-
side linkages. These compounds may undergo adsorption and
further metabolization in the liver.
Moreover, the low stability of anthocyanins in the pH of the
intestine and the activity of the intestinal microbiota are at
least partially responsible for the conversion of anthocyanins
into more stable small phenolic acids, such as protocatechuic
acid, or other molecules of unknown structure.
Therefore, the overall anthocyanin bioavailability should
result from the contribution of the amount that crosses all
physiological barriers in all their possible forms: native, degra-
dation products, metabolites, and anthocyanin derivatives.
Moreover, several intact anthocyanins and some metabo-
lites have been detected in many animal tissues including the
liver, the kidneys, the brain, the lungs, and the eyes.
As a consequence, what is left to explore is whether the
biological effects associated with anthocyanins are due to
their native forms or to some specific metabolites arising after
adsorption.
Betalains
Betalains are water-soluble nitrogen-containing pigments
responsible for the yellow-orange-red colors of flowers and
fruits belonging to the angiosperm order Caryophyllales. How-
ever, in Caryophyllaceae and Molluginaceae, they are replaced
by anthocyanins, and interestingly, the two pigment families
are mutually exclusive. Betalains comprise 55 structures: the
red-violet betacyanins and the yellow-orange betaxanthins that
are ammonium conjugates of betalamic acid with cyclo-DOPA
and amino acids and amines, respectively. Although more than
50 types of betacyanins are known, betanidin is the most
common aglycone that can be glycosylated or acetylated on
one or both C5 or C6 hydroxyl groups (Figure 3). Further
glycosylation of the 5-O-glucoside and esterification with
hydroxycinnamic acids are also very common.
Betacyanins display a wavelength of maximum absorption
in the UV range between 270 and 280 nm due to the cyclo-
DOPA structure and a second adsorption maximum in the
visible range at about 535–538 nm depending on the solvent
system. Acylation with hydroxycinnamic acids produces a third
maximum at about 300–330 nm, whereas aliphatic acid
276 Colors: Properties and Determination of Natural Pigments
R1O
5
6
R2O
2
N+ COOH
12
13
15
HOOC
N HOOC
H COOH
Betacyanins
Betanidin: R1 and R2 =
Betanin: R1=glucose R2=OH
Figure 3 Basic structure of betacyanins (left) and betaxanthins (right).
derivatives are not distinguishable from their corresponding
betanidin glycosides. Betacyanins and betaxanthins are cations
below pH 2.0, zwitterions at pH 2.0, monoanions between pH
2.0 and 3.5, and stable bisanions above pH 3.5 and 7.5.
Sources and Production
The major dietary sources of betalains are not only cactus pear
and red and yellow beetroot but also colored Swiss chard and
grain or leafy amaranth (Table 1). The major commercially
exploited betalain crop is red beetroot (Beta vulgaris) because
the extract contains mainly betanin, which is approved as a red
food colorant E162 in different low-acid and neutral food
commodities (pH 3–7) from beverages to candies and from
dairy to cattle products by both the European Union and the
United States. Betalains are more stable as food colorants over
a broader pH range than anthocyanins, although their use is
still limited, being not always edible.
For example, cactus fruit betalains cover a broader color
spectrum without negative flavor impact as compared with red
beet. An additional advantage to cactus fruits is their minimal
soil and water requirements, as it is regarded as an alternative
culture for the agricultural economy of arid and semiarid
regions.
Determination of Betalains
Plant cells and tissue cultures are alternative sources of beta-
lains that ensure continuous supplies of easily extractable
products with uniform quality and yield, independently from
their geographic location and minimizing risks of political
interference. The most widely used plant in vitro systems are
cell suspensions, hairy roots, and tissue cultures. Extraction of
betalains is commonly performed with an aqueous organic
solvent (i.e., methanol); however, the addition of ascorbic
acid in the extraction medium is recommended to slightly
H R3
N+
N COOH
H
Betaxanthins
OH R3 = aminoacid or ammine
Indicaxanthin: proline-betaxanthin
Vulgaxanthin: glutamine-betaxanthin
lower the pH, which stabilizes betacyanins and inhibits possi-
ble oxidation by polyphenol oxidases.
HPLC with C18 in reversed phases and UV detection is the
method of choice for the analysis of betalains. Elution is per-
formed by a gradient solvent system using methanol and phos-
phoric acid at pH 2.75 in ion suppression mode. Two different
wavelengths were used to monitor selectively the red and yel-
low pigments (538 and 476 nm). Another common method of
detection is ESI–MS in positive mode.
Patterns of Consumption
Betalains represent a group of phytochemicals with a restricted
occurrence in the diet since their plant food sources are quite
reduced. Only red beet, Swiss chard, amaranth, cactus pear,
pitaya, and some tubers and their derived products provide
betalains to our diet.
However, the use of betanin as a food colorant and the
plant betalain-enriched extract in functional foods may
increase their consumption.
Availability, Absorption, and Metabolism
Despite the scarce studies on bioavailability and metabolism of
betalains, they all show that these pigments are bioavailable. In
particular, cactus pear fruit or red beet juice indicaxanthin and
betanin, which represent the adducts of betalamic acid with
proline and cyclo-DOPA-5-O-b-glucoside, respectively, are
reported to reach plasma concentrations of mM order, which
is quite higher compared with that of polyphenols. Simulated
gastrointestinal adsorption studies have indicated that indicax-
anthin and betanin are absorbed passively by solvent drag
through paracellular junctions of intestinal epithelial cells
without any metabolic transformation and circulate as uncon-
jugated molecules. However, in the case of betanin, its bio-
availability appears to be limited by a digestive instability and
 Colors: Properties and Determination of Natural Pigments 277

Table 1 Dietary natural pigments and their occurrence in food flowers, fruits, roots, and seeds. The carotenoid backbone con-
expressed as mg of compound per 100 g of fresh weight (FW) of food sists of eight isoprenoid units linked covalently to create a
symmetrical polyene chain with multiple conjugated double
Concentration
bonds (Figure 4). Cyclization of the end groups occurs in
Natural pigments Dietary sources mg/100 g FW
hydrocarbon carotenes, whereas the introduction of oxygen
Anthocyanins functions gives rise to xanthophylls. Carotenoids are highly
Malvidin glycoside Blueberry 115–148 prone to chemical degradation during food processing and
Red grape 2–19 storage due to the effects of light, oxygen, and heat.
Cyanidin glycoside Blackberry 176–312
Red grape 2–5
Red cabbage 281–363 Sources and Production
Petunidin glycoside Blueberry 58–86
Green vegetables, such as broccoli, spinach, and lettuce, con-
Red grape 0.2–2
tain lutein as their main pigment (45%), b-carotene
Peonidin glycoside Cranberry 59–86
Red grape 6–15 (35–30%), violaxanthin (15%), neoxanthin (10%), and
Pelargonidin glycoside Strawberry 17–23 small amounts of a-carotene, b-cryptoxanthin, zeaxanthin,
Red radish 71–130 and antheraxanthin. Yellow and red fruits and vegetables
Delphinidin glycoside Black currant 255–411 (carrots, watermelon, potatoes, and tomatoes) have a more
Red grape 0.3–2 distinctive carotenoid distribution with high amounts of
Betalains b-carotene. Tomatoes and watermelon are rich in red lycopene.
Betanin, isobetanin, vulgaxanthin Red beet 40–60 Yellow and orange fruits (pumpkins, oranges, and peaches)
I contain primarily xanthophyll esters and b-carotene.
Betaxanthin Opuntia ficus- 11–54
Vegetable oils, in particular palm oil, and some spices like
indica
red pepper (capsanthin and capsorubin) and saffron (crocin
Betacyanin Opuntia ficus- 5–25
indica and crocetin) are good sources of carotenoids.
Carotenoids Animal tissues (salmon, trout, and shrimp) contain the
Lycopene Watermelon 5–14 orange-pink astaxanthin depending on their intake of phyto-
Tomato 1–13 plankton (macro- and microalgae), which biosynthesize carot-
a-Carotene Pumpkin 0–7 enoids. Egg yolk is also rich in lutein and zeaxanthin depending
Carrot 3–6 on their foraging.
b-Carotene Carrot 4–21 Actually, few carotenoids are used as food colorants: a-, b-,
Pumpkin 1–15 and g-carotene (E160a), bixin, norbixin, annatto (E160b),
Spinach 3–5
capsanthin, capsorubin, paprika (E160c), lycopene (E160d),
Apricot 0.5–4
b-apo-8-carotenal (E160e), ethyl ester of b-apo-8’-carotenal
Red pepper 1–2
Lutein Spinach 6–8 (E160f), lutein, and astaxanthin.
Virgin olive oil 0.4–1 Some plant and seafood by-products, algae, and microor-
Lettuce 1–5 ganisms are eco-friendly and low-cost sources of carotenoids.
Red pepper 0–8 Moreover, production from algae and microalgae has many
b-Cryptoxanthin Red pepper 0.2–0.4 advantages, such as easier extraction with higher yields and no
Chlorophylls lack of raw materials or limited seasonal variation. The main
Chlorophyll a and b Spinach 79–125 carotenoids derived from algae are listed in Table 2.
Green bean 5–7
Green pea 5–13
Pheophytin a Virgin olive oil 0.2–4 Determination of Carotenoids
The extraction of carotenoids is generally carried out with
organic solvents or supercritical fluid extractions. In recent
by an intestinal efflux mechanism, thus reducing the absorp- years, in order to increase the recovery efficiency, a novel
tion by around 35% of betanin intake. Moreover, its intestinal approach based on enzymatic extraction has been developed
permeation may be influenced negatively by the food matrix. for marigold (lutein) and tomato products (lycopene).
Since unknown metabolites that may fit with conjugates or Recently, biotechnological productions or metabolic engineer-
microbial metabolites are also detected in urine, there is the ing of carotenoids using microorganisms and crop plant’s cell
need to develop further studies on their effects on health. cultures such as potatoes, tomatoes, rice, and maize has also
been exploited.
The most common method for the analysis of carotenoids
is HPLC with UV detection on a C18 or C30 reversed phase in
Carotenoids isocratic or gradient elution mode using various mixtures of
solvents (methanol, acetonitrile, and tetrahydrofuran) with the
Carotenoids comprise more than 700 lipid-soluble, yellow- addition of butylated hydroxytoluene. Carotenoids absorb
orange-red pigments with a broad distribution from bacteria strongly between 400 and 500 nm (all-trans isomers), while
to higher plants. They are found in chloroplasts of leaves and cis and cis–trans isomers also exhibit absorption at around
green fruits along with chlorophylls and in chromoplasts of 320 nm. Another sensitive detection technique employed is
278 Colors: Properties and Determination of Natural Pigments

CH3 CH3 CH3 CH3

1 3 7 11 15 14′ 12′ 10′ 8′ 6′ 4′ 2′
5′ CH3
H3C 5 9 13
2 4 6 8 10 12 14 15′ 13′ 11′ 9′ 7′ 3′ 1′
CH3 CH3 CH3 CH3
lycopene

H 3C
H 3C CH3 CH3 CH3

CH 3 CH 3 H 3C CH 3
CH 3
a–carotene

H 3C
H3C CH3 CH3 CH3

CH3 CH3 H3 C CH3

CH3 b –carotene

H3C
H3C CH3 CH3 CH3

CH3 CH3 H3C CH3

HO CH3
b –cryptoxanthin

H3C OH
H3C CH3 CH3 CH3

CH3 CH3 H3C CH3

HO CH3
zeaxanthin

H3C OH
H3C CH3 CH3 CH3

CH3 CH3 H 3C CH3

HO CH3 lutein
Figure 4 Chemical structure of the most common dietary carotenoids.

the atmospheric pressure chemical ionization (APCI)–MS in
positive and negative mode. A very promising method for fast
analysis of carotenoids is capillary electrochromatography on
stationary phases similar to those of HPLC.
among European countries has been reported since southern
(Mediterranean) countries (i.e., Greece, Italy, Portugal, and
Spain) consume greater amounts of fruits and vegetables than
northern countries (i.e., the United Kingdom, Ireland, and
Scandinavian countries).

Patterns of Consumption
In developed countries, 80–90% of carotenoid intake comes
from vegetable and fruit consumption. A European North–
South gradient for the intake of some carotenoids within and
Availability, Absorption, and Metabolism
The carotenoid bioavailability is affected by the food matrix,
because fruit carotenoids seem to be more bioavailable than
 Colors: Properties and Determination of Natural Pigments 279

Table 2 Natural pigments (mg/kg of dry weight (DW)) in seaweed The chemical structure of carotenoids plays an important
role in micellization. As a matter of fact, xanthophyll esters
Concentration exhibit more fat solubility and are more available than free
Natural pigments Sources mg/kg DW
xanthophylls and apolar carotenes.
Carotenoids The intestinal assimilation of carotenoids seems to occur by
b-Carotene Laminaria digitata 63–336 simple or facilitated diffusion involving a membrane receptor.
Fucus serratus 80–173 The bioavailability of lutein (79%) was found to be higher
Fucus vesiculosus 80–95 than that of lycopene (40%) and b-carotene (27%), and it
Fucoxanthin Laminaria digitata 468 was suggested that about 90% of b-carotene, lutein, and lyco-
Laminaria japonica 178–213 pene contained in fruits and vegetables are adsorbed. The
Fucus serratus 495–720 increase of bioaccessibility of carotenoid formulations in
Zeaxanthin and lutein Ascophyllum nodosum Up to 100
hydrophilic matrices has also been developed.
Chlorophylls
After the intestinal uptake, carotenoids are packed into
Chlorophyll a and b Laminaria digitata 1250
Ascophyllum 565–1030 chylomicrons, which are then excreted into lymphs. Once
nodosum taken up by the liver, carotenoids are exported to various
tissues by lipoproteins like low density lipoproteins (LDLs)
or high density lipoproteins (HDLs). In particular, carotenes
those in vegetables where they are linked to proteins. More- are transported by LDLs, whereas xanthophylls by HDLs, so
over, ripening, food processing, and cooking increase their they will accumulate differentially in certain tissues depending
availability due to the disruption of the food matrix. Heating on the receptor density for LDLs or HDLs.
also induces isomerization from the trans-form, which is
mostly found in raw vegetable materials, to the cis one that is
easily adsorbed. Chlorophylls
Being lipophilic compounds, carotenoid absorption takes
place in the small intestine and requires the formation of Chlorophylls are green lipid-soluble pigments consisting of a
mixed micelles with biliary salts. As a consequence, carotenoid substituted tetrapyrrole with coordinated magnesium in the
bioavailability is enhanced by dietary fat, while it is decreased center. The skeleton of chlorophyll derives from further ester-
by soluble fiber. It is estimated that the consumption of at least ification of the porphyrin macrocycle by phytol, a diterpene
5 g of fat in a meal is required for suitable adsorption. alcohol (Figure 5). Chlorophyll a and b are predominant in

H2C
R
3 5 7
8
H3C 4 6
2
N N CH3
1 9
20 Mg 10
19 11 Metal substitution: Pheophytinization
N N
18 14 12

H3C 16 CH3
17 15 13

O
O O
OCH3 Cleavage of the macrocycle
and loss of the phytol chain
O
CH3
Chlorophyll a R=CH3

CH3 Chlorophyll b R=CHO

CH3

CH3

CH3

Figure 5 Chemical structure of dietary chlorophyll derivatives.

280 Colors: Properties and Determination of Natural Pigments
higher plants, whereas chlorophyll c, d, and e derivatives are
found throughout various photosynthetic algae and diatomic
species (brown, red, and yellow-green algae). Furthermore,
several additional classes of bacteriochlorophylls have been
isolated in photosynthetic bacteria. In plants, chlorophyll a is
typically found in higher amounts than chlorophyll b by
a 3-to-1 margin. The difference between chlorophyll b and a is
the presence of an aldehyde residue instead of a methyl group at
position 7. As a consequence, it results in chlorophyll a being a
blue-green pigment with a lmax of 660–665 nm and chlorophyll
b being a green-yellow pigment lmax of 642–652 nm. Chloro-
phylls occur in chloroplasts where they act as light-harvesting
pigments during photosynthesis. Their distribution and con-
tent in fruits and vegetables depend on several factors includ-
ing species, agroclimatic conditions, pre- and postharvest
treatments, and type and degree of food processing. In fact,
chlorophylls are relatively unstable and easily oxidized and
converted to numerous derivatives responsible for the yellow
discoloration of processed products.
Sources and Production
Commonly consumed green vegetables contain chlorophyll
concentrations by up to fivefold higher than carotenoids. In
some species, chlorophylls can exceed 1000–2000 mg kg
1
wet weight. Dietary chlorophylls include a large number of
derivatives with structural differences affecting the color and
the commercial and nutritive values of foodstuffs.
Fresh fruits and vegetables contain mainly chlorophylls a
and b, which are both converted to olive-brown Mg-free pheo-
phytin and yellow pyropheophytin during severe thermal pro-
cessing or acidification common in canning operations.
During blanching operations, chlorophyll undergoes the
endogenous enzymatic cleavage of phytol forming the water-
soluble chlorophyllide that can further degrade to Mg-free
pheophorbide. However, in order to overcome the yellow
discoloration of vegetables before thermal processing, metal-
free derivatives can be converted to the stable Zn chlorophylls
via the regreening process.
Chlorophyll extracts can be added as food colorants in jam,
jelly, candy, ice cream, and several other products, although its
application is limited because of the lability of the coordinated
magnesium and the associated color change. To overcome this
problem, a commercial-grade sodium copper chlorophyllin
(SCC) has been developed. This is a mixture of Cu–chlorine
derivatives prepared by the saponification of chlorophyll with
NaOH, followed by a replacement of the Mg2þ atom by Cu.
Since the copper complex is not absorbed by the body and is
removed in its entirety as an excretion product, it is considered
to be safe and is permitted to be used in most countries as the
food additive E140. However, the concentration of free ioni-
zable copper in the coloring must be kept below 200 mg kg
1
under current regulations.
Determination of Chlorophylls
Chlorophylls are commonly extracted from edible plants, net-
tle, grass or alfalfa, silkworm droppings and mulberry leaves,
and, recently, marine microalgae. Traditionally, chlorophyll is
extracted with organic solvents (methanol, ethanol, and
acetone) under mild conditions. The chlorophyll extract is
then mixed with dioxane and then precipitated through a
dropwise addition of water in order to remove carotenoids.
An alternative and safer technique is the supercritical fluid
extraction with carbon dioxide, which is suitable for the isola-
tion of thermolabile compounds and provides a solvent-free
highly pure extract.
Chlorophyll pigment separation and identification are com-
monly achieved by HPLC with DAD (660 nm) and fluorescent
detection (excitation 440 nm and emission 660 nm) on a C18
reversed phase using a gradient solvent system starting with
methanol and ending with an ether–methanol mixture. Ammo-
nium acetate is usually added as a suppression ion and ion-
pairing reagent for the analysis of polar chlorophyll derivatives.
An improved technique of detection is APCI–MS in both positive
and negative modes. CZE with laser-induced fluorescence detec-
tion offers selectivity and sensitivity for the identification of the
fraudulent addition of E140 to refined olive oil.
Patterns of Consumption
Compared with other food pigments, the dietary consumption
of chlorophyll is really significant if we consider its natural
abundance in green fruits and vegetables, the derivatives
formed through food processing and cooking, and the increas-
ing use of commercial-grade chlorophylls. In Mediterranean
countries, a good source of chlorophylls is also virgin olive oil,
although its chlorophyll content and profile are affected by
both agronomic and technological factors, whereas, in East
Asian culture, edible marine algae are an important source
accounting for more than 10% of the Japanese diet with an
average consumption of 1.4 kg per person per year.
Availability, Adsorption, and Metabolism
Considering that chlorophyll derivatives span a broad range of
polarity from lipophilic (natural chlorophylls) to highly water-
soluble (SCC) and have variable sensitivities to pH, they have
different digestive behaviors and routes of absorption.
In vitro digestion models revealed that, due to the acid pH of
the gastric lumen, both chlorophyll a and b are converted into
pheophytins as the rate of pheophytinization is higher for
chlorophyll a (97% of pheophytin a and 61% of pheophytin
b). However, the type of food matrix also affects the digestive
stability, and it seems that oily matrix exerts a slightly protec-
tive effect against pheophytinization. During the gastric phase,
allomerization reactions involving C13 oxidation by triplet
molecular oxygen can also take place, whereas de-esterification
of pheophytins to give pheophorbides does not occur.
As for carotenoids, the adsorption of lipophilic chloro-
phylls by the small intestinal epithelium requires dispersion
and solubilization into micelles for transport and incorpora-
tion through enterocytes. The bioavailability of the dephyt-
ylated chlorophyll derivatives is up 65-fold higher than that
of the phytylated pigments. Where phytylated chlorophyll
derivatives show passive absorption by simple diffusion,
the dephytylated ones are passively absorbed by facilitated
diffusion in the lower range of concentrations tested. In
contrast to the lipophilic chlorophyll derivatives, the water-
soluble SCC derivatives do not require association with bile
 Colors: Properties and Determination of Natural Pigments
salt micelles, and the subsequent intestinal cell uptake seems
to proceed predominantly by a facilitated process with
absorbed SCC derivatives efficiently effluxed back to the
luminal compartment.
The extent in which both natural lipophilic chlorophyll and
SCC derivatives are metabolized and secreted into circulation
is, however, currently unknown.
Curcumin
Curcumin is a yellow oil-soluble pigment found in the popular
Indian spice turmeric deriving from the rhizomes of Curcuma
longa. Curcumin, along with desmethoxycurcumin and bis-
desmethoxycurcumin, belongs to curcuminoid, a group of
polyphenolic pigments distributed not only in the Curcuma
but also in the Zingiber species. Curcumin is a bis-a,
b-unsaturated b-diketone and exists in at least two tautomeric
forms, keto in acidic and neutral solutions and stable enol in
alkaline medium. It is practically insoluble in water at acidic
and neutral pH but soluble in alkali. It is stable at high tem-
peratures and in acids but unstable in alkaline conditions and
in the presence of light.
At pH 3–7, curcumin acts as an extraordinarily potent H-
atom donor and it is a potent antioxidant, while above pH 8,
curcumin acts mainly as an electron donor, a more typical
mechanism for the scavenging activity of phenolic antioxi-
dants. Due to the presence of two phenolic groups and one
active methylene group, it also binds metals, particularly iron
and copper, and can function as an iron chelator.
Sources and Production
Curcumin (diferuloylmethane) has been identified as the most
bioactive constituent (2–8%) of the turmeric herb extract
along with the other two curcuminoids. Curcumin is used as
a food preservative and as a yellowish colorant. Turmeric
has been also used in traditional medicine as a household
remedy for various diseases. Nowadays, the health properties
(neuroprotection and chemoprevention and cancer preven-
tion) of curcumin and curcuminoids are gaining an ever-
growing attention also in medicinal and clinical industries.
Commercial curcumin, generally extracted from the dried
root of Curcuma longa, contains  77% diferuloylmethane,
17% desmethoxycurcumin, and 6% bisdesmethoxycurcumin.
Determination of Curcumin
The extraction of curcuminoids is generally carried out using
organic solvents. In particular, the efficiency of the solvent
extraction presents the following pattern: methanol >
acetonitrile > ethyl acetate. The efficient extraction of curcumi-
noids is obtained by also using supercritical CO2 modified
with 10% ethanol. On a massive scale, the microwave-assisted
extraction technique allows the rapid and selective recovery of
curcuminoids in a few minutes. Recently, there is an increased
interest in the isolation of the individual curcuminoids and
study of their various biological activities.
In this context, a high-speed countercurrent chromatog-
raphy preparation with or without pH zoning has been
281
developed in order to separate multigram quantities of curcu-
min and other curcuminoids from curcumin or turmeric pow-
der while maintaining a high level of purity.
The main methods for the quantitative determination of
curcumin include UV spectrophotometry (curcuminoids
exhibit strong absorption between 420 and 430 nm in organic
solvents), HPLC, and capillary electrophoresis. HPLC with
diode array detection at 420 nm on C18 reversed phase in a
gradient elution mode is the most widely used method without
considering its limited sensitivity and low selectivity.
HPLC–MS is very sensitive and has advantages over other
methods in obtaining the structural information of analytes.
However, it requires nebulization and vaporization steps, ion-
ization of samples, and expensive instruments, which make
this procedure unavailable in many laboratories. Therefore, a
simple method with high sensitivity and low costs is still
needed for the determination of these curcuminoids. HPLC
coupled with electrochemical detection (ECD) is a highly sen-
sitive and selective technique for the determination of redox
substances. In recent years, the ECD method has also been
reported for curcuminoid detection.
Patterns of Consumption
Curcuminoids have a restricted occurrence in nature; however,
a higher consumption has been reported in Korean diets. Curry
(37.24–617.98 mg g
1), especially curry powder, mustard,
candy, pickle, and snack food types, exhibits the highest curcu-
minoid content among various food items in Korean markets.
In Europe, curcumin is known as E100 food additive (tur-
meric or curcumin), which is widely applied to several food
items (e.g., smoked fish, savory snacks, sauce, mustard, and
seasoning) and to fabrics.
Availability, Absorption, and Metabolism
Many clinical studies have shown that curcumin has a very low
bioavailability after oral assumption because of its poor solu-
bility in aqueous media and its rapid metabolism and elimi-
nation. When orally administered, curcumin is absorbed in the
gut and is present in the general blood circulation after being
largely metabolized as glucuronide/sulfate conjugates. Unfor-
tunately, whether curcumin metabolites are as active as curcu-
min itself remains unclear. In order to improve the
bioavailability, novel delivery strategies such as lipid and poly-
meric nanoparticles, liposomes, and phospholipid complexes
have been developed. Concomitant administration of
adjuvants like piperine that interferes with glucuronidation
has been undertaken.
Hemoglobin and Myoglobin
Myoglobin and hemoglobin are both hemeproteins responsi-
ble for the storage and transport of oxygen in many aerobic
organisms, respectively. The characteristic red color of these
proteins is due to the heme group (iron protoporphyrin IX)
consisting of a porphyrin ring with a centrally located iron
atom (Fe2þ). Myoglobin is a globular protein of about 153
amino acids (about 17 000 g mol
1), holding the heme in a
282 Colors: Properties and Determination of Natural Pigments
protein pocket, while hemoglobin consists of four myoglobin-
like subunits each containing a heme. The porphyrin ring
structure held in the confines of the protein accounts for four
of the six coordination sites available on the iron atom. These
four bonds are with pyrrole nitrogens, while the fifth coordi-
nates with the proximal histidine 93. A sixth site is available to
reversibly bind oxygen and other small ligands. The presence
of the ligand and the valence of iron dictate the pigment color
of myoglobin and hemoglobin. A distal histidine 64 also influ-
ences the color dynamics by affecting space relations within the
hydrophobic heme pocket.
Myoglobin is the dominant pigment in muscles and hence
responsible for most lean meat color of land mammals. There-
fore, four major chemical forms of myoglobin influence meat
color: oxymyoglobin, deoxymyoglobin, metmyoglobin, and
carboxymyoglobin.
Deoxymyoglobin occurs when no ligand is present at the
sixth coordination site and the heme iron is ferrous (Fe2þ).
This results in the purplish-red or purplish-pink color typically
associated with vacuum-packaged products and muscles
immediately after being cut.
Oxygenation occurs when myoglobin is exposed to oxygen
and is characterized by the development of a bright cherry-red
color.
Discoloration or browning results from oxidation of both
ferrous myoglobin derivatives to ferric iron (Fe3þ) myoglobin
called metmyoglobin. Reduction of metmyoglobin is crucial to
meat color life and greatly depends on muscle oxygen scaveng-
ing enzymes, thus reducing enzyme systems, and the NADH
pool, which is limited in postmortem muscles. Carboxymyo-
globin is a relevant chemical state of myoglobin because of the
current increased interest in packaging with low levels of CO.
Moreover, combustion during cooking in a gas oven generates
small quantities of CO or NO that bind to the myoglobin,
forming stable pink compounds.
Sources and Production
The myoglobin concentration varies widely among different
types of muscles, and it correlates with the oxidative capacity of
the muscle cells within the species. Muscles involved in sus-
tained repetitive actions, like breathing (diaphragm), contain
higher concentrations of myoglobin than less-used muscles.
The lateral line muscles of fish, which are used for sustained,
low-power movement, are redder than the muscle groups used
for short bursts of high-power movement. The pectoralis mus-
cles of chickens, used for power takeoffs, are paler than the leg
muscles. Moreover, the myoglobin concentration differs from
species to species according to the age of the animals. The meat
from older animals will be darker in color because of the
myoglobin level increase with age due to the myoglobin loss
of its affinity for oxygen.
Moreover, the processing and packaging conditions of meat
also affect the concentration of myoglobin, thus the color of
meat.
Determination of Hemoglobin and Myoglobin
The main methods for the quantitative determination of myo-
globin and hemoglobin include UV spectrophotometry
(myoglobin and hemoglobin exhibit absorption peaks
between 400 and 600 nm) and colorimetric analysis. Colori-
metric CIE (L*, a*, and b*) values are largely used to monitor
color changes on meat surfaces. Moreover, size-exclusion
HPLC combined with reversed-phase HPLC has been applied
in order to obtain the separation of hemoglobin and myoglo-
bin peptic hydrolysates.
See also: Authenticity of Food; Carotenoids: Occurrence, Properties
and Determination; Carotenoids: Physiology; Chlorophyll; Colors:
Health Effects; Colors: Properties and Determination of Synthetic
Pigments; Mass Spectrometry: Principles and Instrumentation.
Further Reading
Anand P, Kunnumakkara AB, Newman RA, and Aggarwal BB (2007)
Bioavailability of curcumin: problems and promises. Molecular Pharmaceutics
4: 807–818.
Azeredo HMC (2009) Betalains: properties, sources, application and stability – a review.
International Journal of Food Science and Technology 44: 2365–2376.
Christaki E, Bonos E, Giannenas I, and Florou-Paneri P (2013) Functional properties of
carotenoids originating from algae. Journal of the Science of Food and Agriculture
93: 5–11.
Deroles S (2009) Anthocyanins biosynthesis in plant cell cultures: a potential source of
natural colorants. In: Winefield C, Davies K, and Gould K (eds.) Anthocyanins:
biosynthesis, functions, and applications, pp. 108–167. Palmerston North, New
Zealand: Springer.
Fernandez-Garcia E, Carvajal-Lerida I, Jaren-Galan M, et al. (2012) Carotenoids
bioavailability from foods: from plant pigments to efficient biological activities. Food
Research International 46: 438–450.
Ferruzzi MG and Blakeslee J (2007) Digestion, absorption, and cancer preventive
activity of dietary chlorophyll derivatives. Nutrition Research 27: 1–12.
Gandul-Rojas B, Gallardo-Guerrero L, and Minguez-Mosquera MI (2009) Influence of
the chlorophyll pigment structure on its transfer from an oily food matrix to
intestinal epithelium cells. Journal of Agricultural and Food Chemistry
57: 5306–5314.
Hosikian A, Lum S, Halim R, and Danquah MK (2010) Chlorophyll extraction from
microalgae: a review on the process engineering aspects. International Journal of
Chemical Engineering 2010: 1–11.
Long Y, Zhang W, Wang F, and Chen Z (2014) Simultaneous determination of three
curcuminoids in Curcuma longa L. by high performance liquid chromatography
coupled with electrochemical detection. Journal of Pharmaceutical Analysis
4: 325–330.
Mancini RA and Hunt MC (2005) Current research in meat science. Meat Science
71: 100–121.
Martin Bueno J, Saez-Plaza P, Ramos-Escudero F, et al. (2012) Analysis and
antioxidant capacity of anthocyanins pigments. Part II: Chemical structure,
color, and intake of anthocyanins. Critical Reviews in Analytical Chemistry
42: 126–151.
Moreno DA, Garcia-Viguera C, Gil JI, and Gil-Izquierdo A (2008) Betalains in the era of
global agri-food science, technology and nutritional health. Phytochemistry Reviews
7: 261–280.
Naksuriya O, Okonogi S, Schiffelers RM, and Hennink WE (2014) Curcumin
nanoformulations: a review of pharmaceutical properties and
preclinical studies and clinical data related to cancer treatment. Biomaterials
35: 3365–3383.
Namitha KK and Negi PS (2010) Chemistry and biotechnology of carotenoids. Critical
Reviews in Food Science and Nutrition 50: 728–760.
Navas MJ, Jimenez-Moreno AM, Bueno JM, Saez-Plaza P, and Asuero AG (2012)
Analysis and antioxidant capacity of anthocyanin pigments. Part
IV: Extraction of anthocyanins. Critical Reviews in Analytical Chemistry
42: 313–342.
Pojer E, Mattivi F, Johnson D, and Stockley CS (2013) The case of anthocyanin
consumption to promote human health: a review. Comprehensive Reviews in Food
Science and Food Safety 12: 483–508.
 Colors: Properties and Determination of Natural Pigments
Rymbai H, Sharma RR, and Srivastav M (2011) Biocolorants and its implication in
health and food industry – a review. International Journal of PharmTech Research
3: 2228–2244.
Scotter MJ (2011) Methods for the determination of EU permitted added natural colours
in foods: a review. Food Additives and Contaminants. Part A, Chemistry, Analysis,
Control, Exposure and Risk Assessment 28: 527–596.
Sowbhagya HB and Chitra VN (2010) Enzyme-assisted extraction of flavorings and
colorants from plant material. Critical Reviews in Food Science and Nutrition
50: 146–161.
Young OA, Rogers RW, Hui YH, and Nip WK (2001) Meat science and applications.
Boca Raton, FL: CRC Press.
283
Relevant Websites
http://www.buzzle.com/articles/food-coloring-chart.html – Buzzle, Food Coloring
Chart.
http://en.wikipedia.org/wiki/Food_coloring – Food coloring, From Wikipedia, the free
encyclopedia.
http://www.food-info.net/uk/colour/natcolour.htm – Food-Info.net the food technology
and food safety programmes of Wageningen University.
http://www.natcol.org/ – NATCOL the new EU Guidance Notes on Colouring Foods.
 Colors: Properties and Determination of Synthetic Pigments
E Diacu, University “Politehnica” of Bucharest, Bucharest, Romania
ã 2016 Elsevier Ltd. All rights reserved.
General Considerations
Food industry has developed rapidly in recent years by pro-
ducing a large amount of processed foods for consumers. These
consumers select processed foods, having little time to prepare
them. The main challenge today for the food industry is to
satisfy the growing consumers’ demands, who want food to be
attractive, to taste good, to be healthy, and to be at a reasonable
price. This has led food manufacturers to develop technologies,
in which there is almost no food recipe that does not contain
food additives.
Synthetic pigments (SPs) are among the first listed food
additives, standing out as one of the widest used additive class-
es for food industry. Apart from providing important proper-
ties to the finished product, such as color uniformity and
stability during processing and storage, SPs are used to enhance
sensory response in concordance with consumers’ desire. This is
leading to a state of good mood, with the desire to consume
more. SPs have been introduced as food colorants once they
were synthesized by chemists, at first without any suspicion of
their negative health effects. Produced on industrial scale, they
could be used in a variety of purposes and areas, including food
processing, often without taking into account the possible
threat posed to human health, especially when they are present
in excess or consumed for a long period. Until a few decades
ago, there was no a rigorous control on the use of these pig-
ments in foods. Over the years, due to health side effects arising
from the consumption of foods containing pigments, in the
population appeared a great concern over their possible adverse
effects. Inevitably, laws were issued to protect the consumer and
to regulate the addition of SPs in foods. Therefore, only a short
list of SPs was compiled under certain condition and limits in
use; a maximum daily intake was also established, either for
each SP alone or in their binary mixtures.
To check the fulfillment of the laws relating to the addition
of SPs in food and to take action when problems arise,
advanced analytic methods able to determine with accuracy
these compounds at low concentrations are needed. This
article will present an overview of properties and determina-
tion of SPs, where the term ‘pigment’ refers to any synthetic
compound that can give color to food and nonfood.
Short History
Pigments have been known to people since prehistoric times,
at that time being used to decorate the walls or to mark their
passing. In the very beginning, the number of pigments was
limited, because most of them were obtained only from natu-
ral sources (earth, plants, animals, etc.). Earth pigments, yellow
ochre, red ochre, white chalk, and black, were the most famous
ones, being easily accessible from their environment and with
a very simple technology for their preparation. Only by
284 Encyclopedia of Food and Health
grinding these colored earths to a fine powder and then mixing
them with some animal fats, people could apply these mate-
rials on different surfaces, resulting in latter paintings.
Even around 3000 BC in Egypt, pigments were used for
painting. Also mineral malachite, azurite, and cinnabar pigments
followed by a new color obtained by mixing more natural
minerals, Egyptian blue. The Egyptians also were able to produce
insoluble pigments by mixing organic dyes extracted from plants
with hydrated clay or tannin, the procedure that was called lake
making. Almost at the same time, the Chinese civilization devel-
oped vermilion (mercury sulfide), a new bright red pigment
produced by burning mercury and sulfur.
In antiquity, Greeks manufactured two new pigments based
on lead: first full white opaque color, the white lead, and the red
lead, which was used also in cosmetics as powder. Today, lead
pigments were banned for use by the general public due to
their toxic potential. Romans have benefited from the existing
pigments obtained before, fortifying the palette with a very
expensive pigment obtained from small mollusks, the Tyrian
purple, affordable only by the very rich people, being also used
to dye the togas of Roman emperors. In the same period of
time, the cinnabar was brought from the Spanish mined and
used by Romans.
During the Middle Ages, the palette of pigments was enriched
by discovering innovative combination and processing for other
ores and rocks. Thus, a wide range of pigments were created, such
as bone white, green malachite or green earth, and lazulite (Lapis
Lazuli). In this period, the most important blue in the Middle
Ages was ultramarine and also the most expensive pigment
known so far. Due to its price and having a strong blueness,
ultramarine has been predominantly used to decorate sacred
subjects, especially for the Virgin Mary who was graced in blue.
In the eighteenth century, the synthetic chemistry revolution-
ized the field of pigments. The chemically synthetic pigment
Prussian blue (blue lake) manufactured in 1704 by Heinrich Dies-
bach can be considered as the first modern pigment. However, it
must be also related the synthesis of picric acid in laboratory
almost in the same time (1771), used as yellow dye in a silk fabric.
Since then, the chemistry has spent almost three centuries and
succeeded to synthesize thousands of organic pigments in the
finest quality ranges, which are very much used in a variety of
industries. So, early in nineteenth century, once the Industrial
Revolution started, the pigment industry has flourished and new
permanent pigments were produced: cobalt pigments (in blue,
green, violet, yellow, and red colors), synthetic ultramarine (less
expensive than genuine ultramarine, but with identical proper-
ties), chrome yellow, genuine emerald green (a copper aceto-
arsenite compound, potentially lethal), synthetic iron oxides
(known as Mars colors), and synthetic organic pigments
mauveine, discovered by Perkin in 1856, alizarin crimson, and
others.
Throughout the twentieth century, continuing apace until
today, many new pigments – based on the discovery of new
http://dx.doi.org/10.1016/B978-0-12-384947-2.00191-4
 Colors: Properties and Determination of Synthetic Pigments
chemical elements and on complex organic synthesis – were
produced. An important place is taken by azo pigments,
known especially by commercial names (Hansa Yellows, Win-
sor Yellow, Monastral Blue, Allura Red, etc.), because the sci-
entific definition has been quite difficult to practice.
With strict reference only for foods, they were colored since
ancient times by using naturally occurring pigments from veg-
etable and mineral sources, Egyptians, around 1500 BC being
the first who used plant extracts as pigments in order to
improve the appearance of candy. Three millennia ago, saffron,
spice with a high nutritional value, was used to color certain
foods and to cure some diseases in Greece. In early 300 BC,
wine was also colored with saffron, and in the next period,
many other food have improved their appearance by adding
various natural pigments, like flower petals, marigold, spinach,
parsley, and indigo. Starting with the twentieth century, SPs
began to be produced on industrial scale and gradually started
to be also used for food. At the very beginning, SPs were used
without any regulation, although some of them presented a
great toxic potential and were even poisonous (pigments based
on lead, arsenic, and mercury complexes). As a result of such
findings, a careful assessment of the SPs used in food started,
their use was regulated, and only some of them were consid-
ered safe and approved for use in food. First foods for which
the use of artificial pigments was authorized (yellow pigments)
were butter and cheese in 1881 in the United States. Today,
worldwide, more than ever in history, SPs are used for all kinds
of food, drugs, and cosmetics. Nevertheless, as chemical sub-
stances, they are strictly regulated and monitored, being the
subject of many clinical studies.
Synthetic Food Pigments Used in Food Industry
Color is the attribute most commonly used as base for the
quality assessment, a natural color showing to the consumer
a high-quality food product, while a pale color may indicate an
inferior-quality product. For example, orange in its various
forms is associated with aroma, pleasant taste, and energy.
Therefore, SPs, provided by industry in a full range of colors,
have gained great importance. Making food more attractive for
consumers, SPs became always present components in almost
every processed food, starting with fruit juices, fast food, dress-
ings, ice cream, cakes, and so on. More than that, added color
serves as a kind of code that allows to identify in the easiest way
the food products such as candy flavors.
Standing out as one of the widest food additive classes,
synthetic food pigments are used in food processing for many
specialized applications, especially to keep or to improve the
perceived color of fresh and processed foods, being known that
colors are a clue to the flavor to be savored. In particular,
synthetic food pigments are used not only to provide color to
colorless food but also to produce ‘fun’ foods for children, who
are thus exposed, without being aware, to the most undesirable
effects of this type of food consumption.
In food processing, manufacturers are tempted to use syn-
thetic food pigments because they are more attractive than
natural ones, with a number of advantages: high stability dur-
ing food processes, exposure to light or storage, more intense
and uniform color, easy production by synthesis, a wide
285
variation of colors, and convenient prices. Unfortunately,
these colors were sometimes used not in a reasonable quantity,
without taking into consideration the possible harmful effects
on human health, in particular to most vulnerable members of
the population (children). Moreover, cases have been identi-
fied when the synthetic dyes were used to disguise grave defects
of the low-quality food.
Despite possible adverse effects on human health and on
the environment, today, SPs are widely used in the food pro-
duction and other goods, representing more than 60% of all
the dyes produced annually.
Effects of Synthetic Food Pigments on Human Health
The dangers on human health through using SPs in food have
been a source of controversy for decades, which continues
today. On one hand, nowadays, the use of SPs by food industry
has grown constantly, becoming a necessity with the increasing
need for processed food. The manufacturers’ opportunity to
use a wider range of SPs considerably increased due to high
complexity of food matrices, notwithstanding possible harm-
ful effects on the health of humans. On the other hand, a
growing concern of all consumers regarding food safety relates
to the presence of the SPs in food and drinks, because these
chemical compounds have been suspected to cause serious
health troubles, complaints regarding their mutagenic and
carcinogenic risks being also reported.
The most incriminated synthetic compounds as carcino-
genic are azo pigments, due to the presence in their molecule
of azo linkage, which do not occur naturally. This linkage is the
most labile portion of azo dye molecule and can be easily
broken enzymatically (i.e., metabolic cleavage), inducing the
oxidative stress that leads to formation of free radicals in mam-
mals, including man. Therefore, numerous toxicological and
metabolic researches on animals and humans have been per-
formed to assess the effects of food colors on human health,
and almost all these studies have shown that synthetic food
pigments are unhealthy substances for consumers, especially
for children. Many research results show that SPs may cause
mainly DNA damage, which then manifests differently. Thus,
allergies and changes in hepatic and renal parameters, behav-
ioral problems and attention disorder (particularly in chil-
dren), respiratory problems (asthma), and heart troubles
have been reported especially if azo dyes were present in food
(Tartrazine-E102, Sunset Yellow-E110, Allura Red AC-E129,
Ponceau 4R–E124, etc.). Azo dyes are also incriminated for
their mutagenic potential, especially in the development of
intestinal and kidney cancers. Also, there are some SPs that
have been banned for food use due to toxic side effects of the
breakdown products of these chemicals.
Studies on the dangerous effects of the presence of synthetic
food pigments on humans are nonconclusive, and some of
them are contradictory. Most toxicological studies showed
that adverse health effects are observed when foods containing
SPs are consumed in large quantities or when there is an
extended exposure over a period of time. But effects on certain
sensitive groups of children cannot be excluded.
Although a lot of issues still remain unknown, the majority
of toxicological research results agree that the presence of color
286 Colors: Properties and Determination of Synthetic Pigments
additives in foods, in limited amounts, as required by law, does
not present serious health hazard. On the other hand, there are
highly sensitive people to the presence of unnatural food
ingredients that often exhibit severe negative reactions, even
in a very limited exposure to such chemicals. Therefore, it is
necessary to create consumer awareness regarding the possible
ill effects of these food azo dyes and, generally, to pay attention
to the type and concentration of each chemical substance
added to food, which is compulsory to be written clearly on
the respective label.
Current Legislation
Globally, there is a growing consumers’ concern surrounding
food quality, the food naturalness, the ecological origin of raw
materials, the processing technology, packaging, labeling,
transportation, and storage. However, in recent decades, there
has been an increasing demand of processed foods, and along
with it is a high increase in the use of food additives, including
SPs. Under the circumstances, the consumer’s anxieties regard-
ing the presence of SPs in food were stressed, and the legisla-
tion came into force to prevent their unsafe and fraudulent
uses. Strict rules were imposed, and the most dangerous SPs
were banned in most countries. It was established that the
introduction of any SP in food must be subject to premarket
approval, and only a limited number of permitted SPs were
certified. For each food color, a maximum admitted limit per
unit of food was established, and furthermore, limits for simul-
taneous presence of two or more pigments were foreseen.
There is no common legal framework valid in all countries
for the use of SPs in food. In the European Union, the legisla-
tion for the use of food additives has been harmonized across
all countries, the Council Directive 94/36/EC (1994) being the
major law on food colors. Among other food safety regula-
tions, in this directive, the color additives are termed as E
numbers (from E 102 to E181), abbreviation given to their
easy usage as shorter than chemical denominations. A list of
permitted colors with maximum allowable levels in various
food categories and of colors that have restricted application
is annexed here. For example, the maximum accepted level in
food for tartrazine is of 100 ppm (mg ml
1) and for Sunset
Yellow is 50 ppm, when used individually and no more than
100 ppm in binary mixtures. The purity criteria for colors must
also be taken into account by law, these aspects being stipu-
lated in Commission Directive 95/45/EC. In the United States,
the Code of Federal Regulations is the body that restricted the
use of color food additives in a number of seven water-soluble
dyes and six insoluble lakes. Here, SPs are denoted as FD&C
colors; for example, tartrazine is known as FD&C Yellow 5.
It should be mentioned that most other countries have their
own rules and regulations for the use of synthetic food colors
in various applications. For example, Sunset Yellow and Allura
Red AC are permitted food pigments by European law, but
there are European countries where these colors are banned
due to their potential allergic reaction.
Moreover, the synergetic effect due to the simultaneous
presence of all types of additives in food has not been investi-
gated yet. Nor the prolonged consumption of synthetic foods
containing multiple additives with disastrous consequences on
the health of humans, an effect that may occur immediately or
over time, was not taken in the study to date.
The establishment of a general legal framework for the use
of SPs in foods to be available around the world is a difficult
task, although it would be particularly useful. Also, a cumula-
tive additive food content regulation (in terms of synergy
effects) is not yet covered so far.
SP Classification and Properties
SPs are substances that absorb the light in the visible spectrum
and have the ability to uniformly impart color to a food or
nonfood due to the presence in their molecule of at least one
chromophore group and of conjugated system with double
and single bonds. They are liquid or powdered chemical sub-
stances, many of which are petroleum by-products. For thou-
sands of pigments produced through chemical synthesis, in
literature, there are reported several types of classification,
taking into account various criteria.
As chemical compounds, SPs may be inorganic or organic
compounds. These two categories can be further subdivided
into other classes. Thus, organic SPs, as the main topic of this
work, can be classified in four main classes, according to their
chemical structure: azo, nonazo, fluorescent, and miscella-
neous pigments. SPs can be also divided into ‘lakes’ or ‘dyes.’
But the most common classification system for SPs is drawn
according to their use: artwork and industrial paints, architec-
tural, automotives, textiles, cosmetics, printing inks, plastics,
pharmaceuticals, foods, etc.
Synthetic organic pigments display a wide variety of phys-
ical and chemical properties such as color, light and heat
stability, solubility in water or organic solvents, and reactivity;
depending on the class they belong to, but generally, SPs
within a chemical class display similar properties. However,
in the same class, there is the possibility to create some differ-
entiations intentionally through fine synthesis for quality
improvement, especially for artwork.
Synthetic Food Pigments
Synthetic food pigments, also called dyestuffs, have the capac-
ity to give color to the food in a stronger and longer-lasting
manner than natural pigments. They can be easily identified in
food in several ways. Each pigment is listed at international
level with five-digit C.I., according to Color Index Generic
Numbers, and at European level, through 94/36/EC Directive,
by an E number, which has the correspondent FD&C colors in
the United States. Synthetic food pigments are also represented
by their meaningful commercial names, such as Patent Blue V
and Indigo carmine.
Synthetic food pigments could be subdivided into two
major classes: azo food pigments and nonazo food pigments,
but often, they are categorized by their predominant shade,
such as pigments yellow, red, or blue.
Azo food pigments
Azo pigments are synthetic compounds that contain in their
molecular structure one or more azo groups (dN]Nd),
being the oldest and most used pigments in food. The base
 Colors: Properties and Determination of Synthetic Pigments 287

for the development of azo pigments was the discovery of
diazo reaction in the laboratory of Peter Griess in 1858, when
the first azo compound was produced, and very rapidly after
these, organic chemists have discovered new azo pigments.
The most representative food monoazo pigments permitted
by the EU are the following: tartrazine E102, C.I. 19140; Sunset
Yellow E110, C.I. 15985; carmoisine E122, C.I. 14720, amaranth
E123, C.I. 16185, Ponceau 4R E124, C.I. 16255; Red 2G E128,
C.I. 18050; and Allura Red AC E129, C.I. 16035 (Figure 1).
Azo pigments are purely of anthropogenic origin, being
generally obtained through a relatively simple organic syn-
thesis carried out in two steps, when two organic compounds
are required for the coupling and azo linkage formation,
respectively, the diazotization of a primary aromatic amine
is followed by coupling. Because they have a long scientific
name, in most of the cases, azo pigments are known by
their market name. For example, the azo pigment 3-carboxy-5-
hydroxy-1-(40 -sulphophenyl)-4-(40 -sulfophenylazo) pyrazole
trisodium salt is known as tartrazine, disodium 6-hydroxy-5-
[(4-sulfophenyl)azo]-2-naphthalenesulfonate is Sunset Yellow,
disodium 6-hydroxy-5-((2-methoxy-5-methyl-4-sulfophenyl)
azo)-2-naphthalenesulfonate is Allura Red AC, and so on.
Class of nonazo food pigments allowed in the European
Union (EU) includes four main groups: the triarylmethanes
(with Patent Blue V E131, C.l. 42051; Brilliant Blue FCF E133, C.
l. 42090; and Green S E142, C.l. 44090), the chinophthalon
derivative (Quinoline Yellow E 104, C.I. 47005), the xanthenes
(Erythrosine E127, C.I. 45430), and the indigo colorants (Indigo
Carmine E132, 73015). But the representative class for food
colorants is azo food pigments, and they are currently the
market leaders.
Synthetic food pigments can be used either alone or in
binary mixtures for obtaining a large spectrum of colors, such
as tartrazine and Sunset Yellow, tartrazine and Erythrosine, and
Allura Red AC and Ponceau 4R. Usually, manufacturers choose
a pigment that is very effective, or they choose a pigment as a
base for other color combinations, as appropriate for the type
of food product.
Synthetic Food Pigment Determination
Potential health hazards due to the presence of SPs in food
have attracted considerable attention, and much effort has
been devoted to the development of methods for their detec-
tion and analysis. These issues have become very topical in
terms of current legislative constraints, the literature reporting
an impressive number of papers dealing with the determina-
tion methods for synthetic food pigments. From the outset, it
must be said that the determination of SPs is often a difficult
task not only because they are complex chemical compounds
of relatively low concentration in food but also because they
are in a very complex food matrix sample. The analysis
becomes more difficult, or even impossible, for those synthetic

COONa
N NaO3S SO3Na
NaO3S N
N N
N N
HO
OH
SO3Na
Tartrazine E102 Lemon yellow Sunset Yellow E110 Orange yellow

SO3Na

HO HO

NaO3S N
N
N N

SO3Na NaO3S SO3Na

Carmoisine E122 Red to maroon Amaranth E123 Red to purple

SO3Na CH3
O CH3
SO3Na
OH
HO HN SO3Na
N
N SO3Na N
N HO
O
CH3
N
N

SO3Na
SO3Na SO3Na

Ponceau 4R E124 Brilliant red Allura Red AC E129 Dark red Red 2G E128 Red

Figure 1 Chemical structures and predominate color for seven most important food azo pigments.
288 Colors: Properties and Determination of Synthetic Pigments
organic pigments that differ only in terms of structure, with a
different position of their substituents on the benzene ring.
Nevertheless, a variety of analytic techniques for the identifica-
tion and determination of SPs have been developed, tradi-
tional analytic techniques and most advanced ones being
reported in the literature. In the following, the most commonly
used of these methods in SPs analysis will be briefly reviewed.
Spectral Methods
Spectral methods were the first methods used in SP
examination; the analysis of pigments from tube paints was
and is commonly performed by ultraviolet–visible spectrome-
try. Due to the possible presence in the same sample of the
natural pigments and of other numerous interferences, absorp-
tion molecular spectrometry, in its classical form, was applied
mainly to individual pigment analysis and had a relatively
small number of applications for analyzing mixtures of SPs.
Thereafter, this method has supported numerous improve-
ments in terms of selectivity, especially by the application of
chemometrics, and simultaneous analysis of two or more SPs
has been reported. From the beginning, infrared spectroscopy
had a bigger potential to be used in the analysis of SPs, dry or
in solution, although the presence of fillers and extenders as
main components in sample matrix makes analysis particularly
difficult. Therefore, more important results have been reported
after improving sensitivity of IR method by using, as sample,
the extracts of SPs. Undoubtedly, the most important achieve-
ment in the field was the development of new FTIR technique,
by which nearly all SPs could be identified against the spectra
of pure compounds that have a characteristic molecular
fingerprint. Further studies have recommended Raman spec-
troscopy as a good technique when analyzing synthetic organic
pigments from artworks, either individually or in mixtures (up
to twenty-one dry azo pigments can be analyzed simulta-
neously). Compared with FTIR, Raman spectroscopy has the
advantage of sharper peaks that do not overlap each other,
making much easier the identification of each pigment from
a complex sample. However, FTIR and Raman spectroscopies
2.0x10−5
1.0x10−5
2.0 x 10−5
i (A)
−1.0x10
−2.0x10−5
0.0
i (A)
2c
−2.0 x 10−5
−2
Figure 2 DPV curves for tartrazine; upper inset – dependences of anodic (i1a) and cathodic peaks currents (i1c) on tartrazine concentration (c).
are complementary techniques, together succeeding to over-
come almost any problem that may arise in the pigment
analysis.
Spectral methods described in the preceding text have
reported just few applications in the determination of SPs in
real food samples, which include especially the analyses in soft
drinks. The reason for this is the high complexity of food
samples; almost always, an additional extractive step in sample
preparation is needed in order to avoid the spectral overlap,
and therefore, more time for analysis is required. New achieve-
ments in the simultaneous determination of SPs, such as Sun-
set Yellow, tartrazine, and Allura Red AC, in soft drink powders
have been recently reported by the use of derivative signals of
the ratio spectra and the involvement of chemometrics. The
obtained linear response covers tens of ppm (mg ml
1) starting
with 2 mg ml
1 up to 60 mg ml
1, so the spectral methods may
be used for SP control in soft drinks.
Electrochemical Methods
Many SPs possessing electroactive properties have led
researchers to develop electrochemical methods for their deter-
mination, especially for azo pigments. Differential pulse vol-
tammetry (DPV) and cyclic voltammetry, using different
working electrodes, are the most common electrochemical
techniques used for the investigation of SPs. Depending on
the respective pigment, voltammograms show specific anodic
or cathodic peaks that allow the identification and quantifica-
tion, the peak current being in directly proportionality to the
pigment concentration. Figure 2 presents the DPV curves for
tartrazine at different concentrations (mmol l
1) on glassy
carbon electrode at pH ¼ 3, where linear dependence of anodic
and cathodic peaks with the tartrazine concentration is
observed in the upper inset.
Numerous studies revealed that voltammetric methods are
selective and sensitive techniques, with very good detection
limits of 0.80 mmol l
1 for common azo pigments. Also, vol-
tammetric techniques have proved to be not only powerful
i1a
i1c
i2c
1a
0.0
−5
0.0 0.5 1.0 1.5 2.0
c (mM)
0 mM
0.5 mM
1.0 mM
1.5 mM
1c 2.0 mM
−1 0 1 2
E (V)
 Colors: Properties and Determination of Synthetic Pigments
Figure 3 Chromatogram for an orange juice sample on Hypersil 5 m MOS C8 column. HPLC system Agilent Series 1100-DAD detection.
tools in the SP investigation that offer analytic options for their
analysis but also good way to determine the degradation prod-
ucts because they are able to establish certain degradation
processes that are likely. Moreover, they can make distinction
between synthetic and natural pigments of the same color
because the latter are not electroactive (do not have azo
group in their molecule).
Although voltammetric methods are simple, sensitive, and
less expensive than spectrometric ones, they do not have a
broad use in the control of SPs in food, mainly due the same
reasons as mentioned in the preceding text. However, the latest
breakthroughs in the use of new materials for electrodes are a
promising outlook in simultaneous determination of SP in
food, particularly in beverages, and these methods are a hope-
ful alternative to classical approaches to the analysis of SPs.
Chromatographic Methods
Compared with other analytic methods used in the analysis of
SPs in food, chromatographic methods are by far the most
attractive for achieving the requested performance criteria,
either under research requirements or under routine analysis.
These methods offer the possibility to analyze SPs with high
accuracy and precision, with a very good detection limit
(mg ml
1), without a prior step of sample preparation. In
order to detect the presence and content of SPs in foodstuff,
many separation techniques have been developed and applied,
such as thin-layer chromatography (TLC), high-performance
thin-layer chromatography (HPTLC) capillary electrophoresis,
gas chromatography, high-performance liquid chromatogra-
phy (HPLC), including ion chromatography and ion-pair
chromatography, reporting valuable results. Among these
methods, TLC and HPLC have been widely used for food
pigment analysis in various food samples, such as fruit-
flavored drinks, alcoholic drinks, fruit yogurts, jams, sweets,
sugar confectionery, spice, and brightly colored foods. Mainly,
TLC aimed to develop rapid methods for the separation and
identification of complex mixtures of pigments. This method,
with new improved version of reversed-phase HPTLC, has been
289
successfully applied in the analysis for mixture up to 9 pig-
ments on the same chromatographic plate. Recently, HPTLC
has been used in the investigation of chili, paprika, and curry
powders for the presence of illegal pigments, such as Sudan
I–IV, Sudan Red B, Sudan Red G, Para Red, Butter Yellow, and
Toluidine Red. All these pigments are classified as carcinogenic
substances and are unauthorized substances in foodstuffs
under the EU Food Regulations. Reversed-phase HPLC method
with diode-array detection in the isocratic elution mode
became one of the most valuable approaches for simulta-
neously determination of complex mixtures of SPs from food
matrices and only requires a simple pretreatment of the sam-
ple. This can consists in a solvent extraction or in a filtration
and sonication. A chromatogram for tartrazine and Sunset
Yellow determination in an orange soft drink sample is pre-
sented in Figure 3.
HPLC method has reached today a new level of perfor-
mance by further advances in instrumentation and column
technology, this method being known as ultrahigh-
performance liquid chromatography (UHPLC). This chro-
matographic technique brought significant increases in speed,
with a very good resolution and sensitivity, UHPLC technology
having the capacity to solve almost every problem raised in
food pigment analysis.
In this brief overview of methods for determining the SPs, a
special role is played by analytic methods based on liquid
chromatography coupled with mass spectrometry, considered
as unrivaled techniques in the identification and quantifica-
tion of SPs in food. Performance criteria of these coupled
methods, especially the selectivity, provide unambiguous iden-
tification and accurate determination of SPs in complex matri-
ces at trace levels, without the need of laborious pretreatment
procedures.
Conclusions
Dangers to human health caused by synthetic food pigments
are in the spotlight, food safety experts raising numerous
290 Colors: Properties and Determination of Synthetic Pigments
concerns regarding the use of SPs as food colorants. Conse-
quently, the European authorities have established the legal
framework for reevaluation of azo pigments as food additives.
The aspects raised in this article have highlighted few issues
regarding safety and quality of food and drinks that are of high
priority worldwide:
1. The need to reduce as much as possible the human health
risks due to the presence of chemical ingredients in food by
using natural ones. More and more people have become
increasingly aware of the dangers of foods that contain
artificial pigments, and such foods became less popular,
polls indicating global preference for natural food colors.
2. The requirement to test and evaluate the safety of any SP
before being used in food with the strict revision of already
permitted SPs in food and the law enforcement for taking
action if problems are found.
3. The necessity to review the food additive science by running
projects on the subject and to develop advanced analytic
techniques for the presence of the colors additives in food.
It is unanimously recognized that determination of syn-
thetic organic pigments is not easy to perform because of
their high coloring power and of similarity in the structure;
therefore, there is a critical demand for new development of
analytic procedures addressed to improve the determina-
tion of SPs in food. Thus, an accurate assessment of SPs in
foods can ensure the compliance of legal requirements, in
many countries, synthetic food colors being banned in the
interest of public health. However, it should be understood
that actually, food technology passes through colors, but at
the same time, it must go to people’s health. Therefore, it is
necessary to try to make sense of SPs by setting universal
standards.
See also: Allergies: Public health; Amaranth; Butter: Properties and
Analysis; Cancer: Diet in Cancer Prevention; Carcinogenic:
Carcinogenic Substances in Food; Colors: Health Effects; Colors:
Properties and Determination of Natural Pigments; Consumer
Protection Legislation; Food Additives: Classification, Uses and
Regulation; Food Allergies: Occurrence and Analysis; Food Allergies;
Mutagens; Quality Control in Food Processing; Storage Stability:
Mechanisms of Degradation.
Further Reading
Adam Burrows JD (2009) Palette of our palates: a brief history of food coloring and its
regulation. Comprehensive Reviews in Food Science and Food Safety 8: 394–408.
Diacu E and Ene CP (2009) Simultaneous determination of tartrazine and sunset yellow
in soft drinks by liquid chromatography. Revista De Chimie 60: 745–749.
Downham A and Collins P (2000) Colouring our foods in the last and next millennium.
International Journal of Food Science and Technology 35: 5–22.
Food Solutions with HPLC (2006). Agilent Solutions Guide, Publ. No. 5989-5674EN.
Herbst W and Hunger K (2004) Industrial organic pigments, 3rd ed. Weinheim: WILEY-
VCH Verlag.
Katz SH and Weaver WW (2003) Encyclopedia of food and culture. New York: Scribner.
Lomax SQ and Learner T (2006) A review of the classes, structures, and methods of
analysis of synthetic organic pigments. Journal of the American Institute for
Conservation 45: 107–125.
Moldoveanu SC and David V (2012) Essentials in modern HPLC separations.
Amsterdam: Elsevier.
Pathare PB, Opara UL, and Al-Said FAl-J (2013) Colour measurement and analysis in
fresh and processed foods: a review. Food and Bioprocess Technology 6: 36–60.
Zollinger H (2003) Color chemistry: syntheses, properties, and applications of organic
dyes and pigments, 3rd ed. Weinheim: Wiley-VCH Verlag.
Relevant Websites
http://ec.europa.eu/food/food/chemicalsafety/additives/comm_legisl_en.htm.
http://www.efsa.europa.eu/en/efsajournal/pub/1327.htm – EFSA.
http://www.efsa.europa.eu/en/efsajournal/pub/1331.htm – EFSA.
www.understandingfoodadditives.org/ – Food Colours – Origins and Chemistry.
http://www.winsornewton.com/about-us/our-history/history-of-pigments/ – The
History of Pigments.
 Condensed Milk
SD Kalyankar and MA Deshmukh, Government College of Dairy Technology, Udgir, India
CD Khedkar, SS Deosarkar, and AR Sarode, College of Dairy Technology, Pusad, India
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Since ancient times, people have been aware that milk could
only be kept fresh for a short while and that it was only
available in the immediate vicinity of a cow. In the thirteenth
century, Marco Polo reported that the Tatars were able to
condense milk. He stated that 10 pounds (4.5 kg) of milk
paste was carried by each man, who would subsequently mix
the product with water. However, this report probably refers to
the soft Tatar curd katyk, which can be made into a drink
(ayran) by diluting it, and therefore it refers to a fermented,
not fresh, milk concentrate. Nicolas Appert condensed milk in
France in 1820, and Gail Borden Jr. did the same in the United
States in 1853, in reaction to the difficulty of storing fresh milk
for more than a few hours. While returning from a trip to
England in 1851, Borden was devastated by the death of several
of his children, apparently from poor quality milk obtained
from shipboard cows. Borden was inspired by the vacuum pan
he had seen being used by Shakers to condense fruit juice, and
he was at last able to reduce milk without scorching or curdling
it. Even then, his first two factories failed, and only the third,
built in New York with his new partner, Jeremiah Milbank,
produced a usable milk derivative that was long-lasting and
needed no refrigeration.
The first person to preserve milk in a concentrated form was
Nicolas Appert, who, in the early nineteenth century, concen-
trated milk by boiling it in a water bath over a fire, then pour-
ing it into glass bottles after cooling, and sterilizing the final
product by heating the bottles for 2 h in a boiling water bath.
Two inventions made in the second half of the nineteenth
century resulted, essentially, in the process that is still used
today. Gail Borden (1856) patented the process to evaporate
milk, which uses low pressure to make sweetened condensed
milk. In 1884, John B. Meyenberg patented a process for ster-
ilizing concentrated milk in tinned cans, which were rotated
under pressurized steam, allowing for a relatively short sterili-
zation time. A brief account of the historical developments of
condensed milk manufacturing is conveyed in Table 1.
Condensed Milk
Condensed dairy products are value-added milk products with
extended shelf life. Fresh milk is clarified and standardized to a
suitable level of fat, and it is then heat treated at 85–90  C for
several seconds. This heating process acts as a hurdle, which
destroys the majority of microorganisms. It also decreases fat
separation and inhibits oxidation. The water content of the
milk is reduced due to evaporation. Condensed milk products
have several advantages over fresh milk, such as they require
less storage space, they retain high quality, they preserve milk’s
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00192-6
valuable surplus nutrients, and they reduce transportation
costs. These products can be used under adverse conditions
such as wars, epidemics, or earthquakes or other natural disas-
ters when fresh milk is unavailable. They are also suitable for
specialty food products designed for people such as sportsmen,
convalescents, or older individuals.
Evaporated milk (EM) is sterilized unsweetened con-
densed milk, and this product is available commercially. It
is prepared from fresh milk from which a sizable (almost
half) amount of water is evaporated. Then the product is
homogenized and canned, and the cans are subjected to the
thermal process of sterilization (Figure 1). In the event of
limited availability of fresh milk, milk powder is used in part
or all of the solids-not-fat (SNF). Similarly, the fat portion
can be obtained from anhydrous milk fat (AMF). If the
product is prepared using only milk powder and AMF, it is
called recombined evaporated milk (REM). In addition to milk,
skim milk, and cream, sweet cream buttermilk is also fre-
quently used as a source of some of the milk solids. Because
sweet cream buttermilk contains a large portion of the fat
globule membrane lipids in milk, it improves the heat stabil-
ity and the flavor of products, particularly REM. The proper-
ties of REM resemble those of EM.
There is no provision under the Codex Alimentarius stan-
dards to distinguish a product made from milk powders and
AMF from that made from fresh milk. But legislation in some
nations has set limits on the use of powders (e.g., EU legisla-
tion requires that no more than 25% of the milk solids in
regular unsweetened condensed milk are derived from milk
powder). There is an emerging trend in some countries to
prepare EM using vegetable fats (most frequently, palm oil)
instead of milk fats; this trend could be due to an attempt to
reduce costs or simply a local preference to not use milk fats.
The product thus prepared is called ‘filled’ EM. Oils high in
polyunsaturated fatty acids, such as soybean or sunflower oil,
are used in food products to lower blood cholesterol. The
oxidation stability of the fats used in REM is of paramount
importance.
To improve milk’s heat stability during thermal processing,
sodium orthophosphate is added. In addition, calcium carbon-
ate, soy lecithin, or calcium chloride is added to help improve
milk’s heat stability. Polyphosphates are also added to ultra-
high-temperature (UHT)-sterilized evaporated milk to control
defects such as age gelation of the product. The Codex sets a
maximum limit for an addition of 0.2% on an individual basis,
or 0.3% if a combination is used.
When the liquid product is mixed with a proportionate
amount of water, EM becomes the rough equivalent of fresh
milk. However, it requires half the space of its nutritional
equivalent (fresh milk), which makes EM attractive for trans-
portation purposes as it can have a shelf life of months or even
years, depending on the fat and sugar content. EM is a safe and
291
292 Condensed Milk

Table 1 Historical developments in condensed milk manufacture

Year Developments in condensed milk manufacturing

1357 Marco Polo (Mongolia) described the method for preparing a pasty milk concentrate, which was mixed with water
1809 Nicolas Appert (France) described a method for preserving milk by condensing it
1856 Gail Borden Jr. commercially developed a method of condensing milk; he is known as ‘the Father of the process of milk condensing’
1858 Borden’s milk, sold under the Eagle Brand, gained popularity for its purity, durability, and affordability
1864 Borden’s New York Condensed Milk Co. constructed the New York Milk Condensery, the largest and most advanced condensery, in Brewster,
New York
1883 The vacuum pan was markedly improved due to advancements in scientific knowledge in the field of sterilization
1884 John B. Meyenbarg (Switzerland) experimented to create the process to sterilize condensed milk using steam under pressure. He conceived the
idea of making condensed milk without the addition of sugar or other preservatives and without storing it under refrigeration
1906 The Federal Food & Drug Act of 1906 changed the name ‘evaporated cream’ to ‘evaporated milk’
1911 Nestlé constructed the world’s largest condensed milk plant in Dennington, Victoria, Australia
1914 Professor O. F. Hunziker, Head of the Department of Dairy at Purdue University published a book titled Condensed Milk and Milk Powder.
Professor Hunziker and others with the American Dairy Science Association standardized and improved condensery operations in the United
States and other countries
1958 Plate-type evaporators were developed
1961 Anand Milk Producer’s Union Ltd. (AMUL), in India, started the first ever commercial production of sweetened condensed milk in that country

Milk lactose, milk permeate, or milk retentate. However, additions

that change the ratio of casein-to-whey protein are not allowed,
Receiving and selection with the local legislations in some countries still forbidding
standardization of the protein.
Preliminary treatment
(Clarification, Cream separation/Standardization)
Method of Evaporated Milk Preparation
Pre-heating
(115–128 °C, 1–6 min) The method to manufacture sterilized and UHT-sterilized EM
is shown in Figure 1. The major processing steps are discussed
Vacuum evaporation in the following sections.
(45–70 °C)
Preheating
Homogenization
(P1=15–20 MPa; P2=5-10 MPa) Fresh milk is heat treated before it is concentrated. This treat-
ment is mainly to increase the concentrated milk’s heat
Packaging stability. The homogenized full-cream EM cannot be sterilized
if the milk is not subjected to preheating. Generally, milk is
Sterilization preheated at 110–130  C for about 1–3 min in a continuous
(100–120 °C, 15–20 min or 140 °C, 3 s) flow to yield EM with the highest heat stability. Preheating also
acts as an important hurdle for microbial growth as it inacti-
Storage
(20 °C)
vates microbial spores. For REM, preheating is carried out by
the powder manufacturer, before concentration and drying.
Figure 1 Flow diagram for the preparation of condensed milk. This allows the powder to be reconstituted at the predeter-
mined concentration for REM.

reliable substitute for perishable fresh milk, and it was very
popular before the invention of refrigeration. It can be easily
shipped to locations devoid of resources for safely producing
or storaging milk. Today, households across the world use it
most often to prepare desserts and in baked goods due to its
characteristic flavor.
The Codex Alimentarius standards, which are the most
common in international trade, prescribe a minimum of
7.5% milk fat and 25% total milk solids (TMS) in EM. Simi-
larly, legislation in various nations has set limits for the com-
position of EM. According to the traditional British standard,
EM requires a minimum of 9% milk fat and a minimum of
31% TMS, while the U.S. standard prescribes a minimum of
7.9% milk fat and 25.9% TMS. Thus, the concentration of EM
ranges from 2.0 to 2.5. The Codex allows the protein content in
the SNF to be adjusted, with a minimum of 34%, by adding
Concentration of milk
Modern multistage falling film evaporators with energy
efficient designs are invariably used for milk concentration.
Overconcentration should be avoided to prevent the risk of
lowering the milk’s heat stability. Similarly, overconcentration
also requires higher energy use and lowers the production
capacity. Based on the continuous measurement of the refrac-
tive index or density of the concentrate, the total solids (TS)
content is adjusted at the point where the product leaves the
evaporator. The final standardization of fat and TS is usually
carried out between the concentrating and sterilizing processes.
The milk, which is concentrated by reverse osmosis (RO), can
also be used to prepare unsweetened condensed milk. EM
produced by RO has virtually the same composition and prop-
erties as that produced by evaporation, but the industrial
application of RO for this purpose appears to be very limited.

Homogenization
The milk is homogenized as this process prevents coalescence
of fat globules and reduces the rate of creaming while the
product is stored. The homogenization pressures up to about
5 MPa generally have little effect on or slightly increase heat
stability, but higher pressures result in a large decrease in heat
stability. The concentrated milk is homogenized immediately
after it leaves the evaporator. A higher homogenization tem-
perature results in a smaller fat globule size at the same pres-
sure with better heat stability.
Heat stability test
During preparation of EM, it is obvious that the heat stability
of the product varies from batch to batch. Stabilizers are added
to regulate the heat stability. The amount of stabilizer needed
also varies to a great extent. The quantity of stabilizers to be
added is determined by adding different amounts of the stabi-
lizers to a series of cans containing the product. The cans are
subsequently sterilized, and the contents are checked for heat
stability. The amount of stabilizer that results in the optimum
properties is added to the total batch.
Cooling and cold storage
After homogenization, EM is cooled and stored, and then a
predetermined quantity of stabilizer (dissolved in water) is
added; this quantity is determined by the results of the stability
test. At this stage, prolonged storage should be avoided to
prevent microbial growth. Final standardization of fat and TS
can be carried out at this stage, taking the amount of water that
is added with the stabilizer into account. The cold storage of
unsterilized EM for more than 24 h considerably increases the
risk of the sterilized product’s age gelation.
EM is expected to be stored for longer period, which
requires various conditions so that the product’s quality does
not deteriorate during storage. The storage temperature is one
major factor determining the shelf life of the product.
Condensed milk and EM are stored at about 10–15  C. Storage
at low temperatures such as 0  C or below may lead to sugar
separation in the condensed milk, leading to sandiness; this
sugar separation is caused by the formation of very large lactose
crystals. A very low storage temperature also increases the
viscosity of the product, which may be beneficial up to a
certain point, but it also affects the product’s body and texture
characteristics. It has been shown that commercial EM remains
acceptable even after 2 years when stored below 15  C, but it
deteriorates rapidly when stored at 21  C or above. The humid-
ity of the storage space should also be kept low (below 50%) to
check the spoilage of cans and labels. Inversion of the cans
during storage minimizes fat separation in EM.
Packaging
One of the characteristic features of EM is its long shelf life at
ambient temperatures; this feature places high demands on the
packaging materials. The packaging materials should meet the
criteria of mechanical resistance and permeability to water,
light, gasses, and hydrophobic components. Those parts that
are in direct contact with the product must be made of food-
grade materials. The can is the most widely used container for
EM, with a standard content of 170 or 411 g (6 or 14.5 oz,
Condensed Milk 293
respectively). Mechanically sealed cans are widely used for the
packaging of concentrated milk products. Modern tinned plate
is coated with a layer of a polymer to prevent the dissolution of
the tin and iron into the product. In some Western European
countries, glass bottles with twist-off caps are used to package
EM used as a coffee creamer. Aluminum foil-lined milk cartons
and single-portion cups made of aluminum or polystyrene are
also widely used. The EM packaged in translucent (white)
polystyrene single-portion cups usually develops an oxidized
flavor due to the permeability of this material to both oxygen
and light.
Sterilization
Sterilization not only kills all microorganisms but also inacti-
vates all microbial spores that may germinate during the stor-
age of EM. The native milk enzymes are inactivated during
preheating. While selecting the milk to prepare as concentrated
milks, its excellent microbiological quality should be noted.
The enzymes produced by microorganisms (especially psy-
chrotrophs) should be absent because they are thermostable,
and even sterilization is insufficient to inactivate them. Heating
is carried out in continuous sterilizers. Several machines have
been designed to fill cans with concentrated milk. In a com-
monly used system, known as ‘vent hole type,’ concentrated
milk flows through a small aperture (3 mm or 1/8 in.
diameter) into cans. After filling, the cans are sealed carefully
because the seal has to withstand the heat of the sterilization
process. It is also essential that the cans be filled as quickly as
possible to avoid contaminating the milk. The opening in the
cans is usually soldered by a mechanical finger. The filled and
sealed cans are then tested for leaks by plunging them into a
hot water tank. If any of the cans rise or give off air bubbles,
they are discarded as unfit for sterilization. Modern sealing
processes are carried out so rapidly that a large number of
cans may be sealed in 1 min. In this process, open or sanitary-
type cans are used. The concentrated milk fills the open can,
one end of which is already seamed, and, after filling, the other
end of the can is seamed by an automatic seaming machine.
Cans are sterilized in a horizontal rotating system with rotary
air locks, while the bottles are sterilized in hydrostatic steril-
izers. These are highly energy-efficient systems because they
have considerable heat regeneration.
Defects in Evaporated Milk
Condensed milk and EM are products with a prolonged shelf
life. They have typical body, texture, and sensory properties by
which they are identified. These properties must be such that
the product is fit for sale immediately after production and
does not alter during a reasonable period of storage. The prod-
uct should be physically, chemically, and microbiologically fit
for human consumption until the end of the storage period.
Routine examination of the product soon after its manufacture
as well as during its storage may be carried out to judge the
quality of the product. If a defect is noted, care should be taken
to eliminate the defect in subsequent batches. Therefore, it is
important to know the expected defects, the probable reasons
for their occurrence, and the preventive measures used to avoid
these defects. The defects that may occur in condensed milk are
classified as microbial defects and nonmicrobial defects.
294 Condensed Milk
Microbial defects
For all sterilized milk products, there is continuous risk of
postprocessing contamination. It may be through microleaks
in the cans or during the filling process. The growth of Bacillus
stearothermophilus, a thermophilic spore-forming bacteria, in
an evaporator is one of the most important causes of product
defects. Prolonged running time of the evaporator or improper
cleaning of the equipment may lead to an appreciable spore
count in the concentrated milk. Inactivation of these spores
during sterilization is limited, and the residual spores may
germinate and grow if the product is sold in tropical countries,
where storage temperatures may exceed 40  C. A brief account
of microbial defects follows.
Gassy fermentation/bloats: Gas can form in cans and barrels
of condensed milk, causing them to bulge or burst. Gas-
producing yeasts are the cause of many types of gaseous fer-
mentations. The source of the contamination may be the raw
milk, inferior quality sugar, or unhygienic factory conditions
(e.g., improperly cleaned and sanitized equipment and filling
machines). To avoid this microbiological defect, good quality
raw milk and a proper preheating temperature should be used.
In addition, only good quality sugar without any yeast con-
tamination should be used. Proper sanitary conditions should
be maintained during the manufacture and packaging of con-
densed milk. The containers should be filled fully, with little
space for air or oxygen.
Bacterial thickening: Condensed milk thickens progressively
during storage because of microorganisms, which produce
rennet-like enzymes. These organisms are easily destroyed dur-
ing the preheating process. An optimum sugar ratio also
inhibits the growth of the microorganisms. In addition, low-
temperature storage helps to reduce the bacterial thickening.
Mold buttons: This defect occurs during storage due to mold
contamination. Small reddish-brown pieces of curd about
¼ in. to 3/4 in. in diameter form on the product’s surface,
causing localized coagulation. These pieces of curd are caused
by the mold Aspergillus repeno, which produces a rennet-like
enzyme that causes localized clotting. High-temperature stor-
age promotes the growth of the organism. The milk may be
infected with the organism during the concentration process.
To avoid this defect, scrupulous cleaning and care of dairy
equipment is essential. Because this organism does not grow
at low temperatures, storing condensed milk at a lower tem-
perature will prevent this defect.
Nonmicrobial defects
These defects are of chemical or physical origin, and they are
briefly described in the following paragraphs.
Sandiness: Good quality condensed milk should possess a
smooth homogenous texture and be pleasant to the palate.
Sometimes, however, the milk may be gritty and contain a
large number of large lactose crystals. The solid particles are
so large that the product lacks smoothness. This defect is
readily detected by an average consumer. Sandy, rough, grainy,
granular, and gritty are the terms used to describe this defect. If
an excess amount of sugar is used to manufacture condensed
milk, the sugar particles may also crystallize out and cause
sandiness. To check this defect, condensed milk must be
cooled so that a large number of minute sugar crystals are
formed, which results in a smooth texture. An optimum stor-
age temperature is also essential to avoid this defect. High
viscosity also delays crystal formation; therefore, it is necessary
to rapidly cool the condensed milk in the initial stages. Correct
cooling and induced rapid crystallization with the correct
amount of seed lactose will help to avoid this defect.
Age thickening: Thickening of the condensed milk is the
most common defect seen in sweetened condensed milk.
This defect varies markedly in its intensity from slight
gelation to a firm and thick consistency. The defect becomes
progressively more intense with storage, especially at room
temperature or above. The preheating temperature of the
milk and the degree to which the milk is concentrated have
been observed to have profound effects on age thickening.
With high packaging and heating temperatures, there is a
greater tendency for the product to thicken early. With
increasing concentration of milk solids, the thickening ten-
dency becomes more marked. To avoid early thickening of
condensed milk, an optimum preheating temperature should
be maintained. Sugar should only be added at the end of the
milk condensing process, and the product should preferably
be stored at a temperature below 15  C. With the addition
of the proper type of stabilizers, age thickening may be
decreased to a great extent.
Brown color: Usually brown color discoloration is associated
with age thickening, both of which become progressively more
intense with storage. The acidity and storage temperature
determine the rapidity of this change. This defect may be
avoided by storing the condensed milk at reasonably low
temperatures.
Other defects in condensed milk include some flavor
defects such as rancid, tallowy, and metallic. Following good
manufacturing practices; using good quality raw materials,
good quality milk, and good packaging; and storing the prod-
uct at a low temperature may help to avoid these defects.
Uses of Condensed Milk
Milk is converted into EM to preserve its nutrients, which
should not perish during months or even years of storage
without refrigeration, and EM can be easily transported over
long distances. For countries with low milk production, espe-
cially in the tropics, EM is a general-use milk product. In other
markets, EM is used for specific purposes such as in coffee and
tea or for cooking. As fluid milk, EM is consumed after 1:1
dilution with boiled water. This yields a product with a slightly
higher SNF content and fat:SNF ratio than those of the regular
3.5% fat full-cream milk. Some consumers prefer the flavor of
sterilized milk to that of pasteurized or UHT-sterilized milk.
The quantity of EM required to give coffee, cocoa, or tea a
milky flavor and a white appearance is relatively small. The
products of the Maillard reaction in EM give coffee, cocoa, or
tea a yellowish hue, which is preferred over the grayish hue
obtained if UHT-sterilized or pasteurized milk is added. EM is
obtained from homogenized milk, so it is recommended for
infant feeding because of the formation of a soft curd in the
stomach. The smooth consistency of EM makes it an important
ingredient in several types of puddings, sauces, and gravies.
Owing to its high TMS content, it is used to manufacture ice

cream. It is also used to prepare chocolate, as well as bakery
and confectionery products. In addition, it is diluted with milk
and cream to produce coffee creamer.
See also: Dairy Products: Dietary and Medical Importance; Milk
Powder; Milk: Role in the Diet; Preservation of Foods; Protein: Food
Sources; Sterilization of Foods.
Further Reading
Alvarez de FA, Melcon B, and Zapico J (1991) Structural changes in sweetened
condensed milk during storage: an electron microscopy study. Journal of Dairy
Research 58: 337–344.
Caric M (1994) Concentrated and dried dairy products. New York: VCH Publishers.
Clarke PT (1999) Recombined sweetened condensed milk: the survivor. In: Proceedings
of the 3rd international symposium on recombined milk and milk products,
pp. 35–40. Brussels, Belgium: IDF, IDF Spl. Issue No. 9902.
Codex Alimentarius Commission (2011) Milk and milk products, 2nd ed. Rome: Food
& Agriculture Organization (FAO)/World Health Organization (WHO) Publishers.
Hunziker OF (1947) Condensed milk and milk powder, 7th ed. La Grange, IL: OF
Hunziker.
Kieseker FG (1982) Recombined evaporated milk. In: Proceedings of the of seminar an
recombined milk and milk products, pp. 79–88. Brussels: IDF.
Condensed Milk 295
Newstead DF (1999) Sweet-cream buttermilk powders: key functional ingredients for
recombined milk products. In: Proceedings of the 3rd international symposium on
recombined milk and milk products, pp. 55–60. Brussels: IDF.
Nieuwenhuijse JA and van Boekel MAJS (2003) Protein stability in sterilised milk
and milk products. In: Fox PF and McSweeney PLH (eds.) 3rd ed.,
Advanced dairy chemistry: proteins, 3rd ed., vol. 1, pp. 947–974. New York: Kluwer,
Part B.
Patton S (1952) Studies of heated milk. IV. Observations on browning. Journal of Dairy
Science 35: 1053.
Singh H, Creamer LK, and Newstead DF (1995) Heat stability of concentrated milk.
In: Fox PF (ed.) Heat-induced changes in milk, 2nd ed., pp. 256–278.
Brussels: IDF.
Relevant Websites
http://www.cabdirect.org/abstracts/19800463869.html – Cab Direct - Abstract.
http://ebook.worldlibrary.net/articles/Condensed_milk – World eBook Library -
Condensed Milk.
http://www.uoguelph.ca/foodscience/book-page/evaporated-skim-or-whole-milk –
University of Guelph - Evaporated Skim or Whole Milk.
http://www.uoguelph.ca/foodscience/book-page/concentrated-dairy-products –
University of Guelph - Concentrated Dairy Products.
http://www.uoguelph.ca/foodscience/book-page/concentrated-and-dried-dairy-
products – University of Guelph - Concentrated and dried dairy products.
 Consumer Protection Legislation
K Purnhagen and B van der Meulen, Wageningen University, Wageningen, The Netherlands
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Access to safe food is an essential precondition for the life of
every human being. In this respect, the United Nations (UN),
the Food and Agriculture Organization (FAO), and the World
Health Organization (WHO) recognized that access to safe and
adequate food in terms of nutritional values forms a right of
each individual (Article 11, International Covenant on Eco-
nomic, Social and Cultural Rights). Complex legal frameworks
are needed to ensure the functioning of food markets and
protect consumers from risks associated with food.
As exposure to health risks in food as well as consumer
expectations varies vertically (e.g., from nation to nation, cul-
ture to culture, and region to region) and horizontally (accord-
ing to the scientific knowledge on risks available), the amount
of regulation needed for the effective realization of the right to
food largely depends on the circumstances of the respective
food markets and available scientific knowledge. Consumer
protection regulation on food and health has to be different
in different countries and regions and react to new insights
from science. In addition, different legal cultures with a view of
enforcement and application of the law require nuanced solu-
tions according to the respective legal context.
Food safety is becoming more and more a global challenge
by virtue of its economic implications as well as its public
health impact. Effective regulation of consumer protection on
markets is hence under threat, as consumer protection regula-
tion designed for a specific regional food market increasingly
governs other food markets, which are not necessarily
embedded in the same prerequisites.
In order to realize the right to food, states and increasingly
private standard setters have on the one hand to find an
optimal level of regulation and on the other hand to design
regulation that attracts safe foodstuffs to their markets.
Food Law and Consumer Protection
Consumer protection legislation takes various forms, which
cut across commonly known distinctions in law, and are
applied according to their own distinct economic policies
and access to justice policies. When protecting from risks
involved with foods, this legislation forms consumer protec-
tion legislation in the functional area of food law:
(1) Classical state-centered regulation encompasses civil law,
criminal law, and administrative law. Civil law protection
provides consumers rights within a contractual relation-
ship, for example, by allowing consumers to remedy a
contract for unsafe foods. Criminal law imposes sanctions,
for example, on food producers for selling unsafe foods.
Administrative law typically imposes market entry
restriction, for example, by demanding an authorization
procedure for foodstuff.
296 Encyclopedia of Food and Health
(2) In a market-driven global food sector, the importance of
governments as regulators decreases and is increasingly
replaced by private standard setters and enforcers. The law
of private standards has hence become an increasingly
important area for consumer protection legislation in the
global food chain.
(3) In consumer protection legislation in federal,
supranational, or international legislation such as in the
EU or World Trade Organization (WTO), consumer protec-
tion on food markets typically follows a subordinate role.
In these regimes, protection of the consumer is used pri-
marily to satisfy other objectives such as the fostering of
cross-country trade or forming the product of a spillover
from legislation of the supply side of markets. However, the
more these legislative systems mature into autonomous
systems, the more state-centered their regulation becomes.
Consumer Protection in Food Law
As consumer protection legislation differs according to the legal
regime in foods to be applied, consumer protection food
legislation needs to be viewed according to the logics of the
underlying legal regime. According to constraints of space, this
piece will hence present an overview of consumer protection
legislation of what we consider to be the most important legal
food regimes in the world as well as the area of private standards.
Consumer Protection Legislation in International
Food Law
In International Food Law, consumer protection in food
markets is mainly sought by establishing food safety standards.
These are then incorporated and enforced in other regulatory
regimes such as national law or private standards. To this end,
the FAO and the WHO established in 1963 the Codex Alimen-
tarius Commission (CAC). The work of the CAC has resulted in
a vast collection of internationally agreed food standards that
are presented in a uniform format, the ‘Codex Alimentarius.’
Most of these standards are of a vertical (product-specific)
nature. They address all principal foods, whether processed,
semi-processed, or raw. Standards of a horizontal nature are
often called ‘general standards,’ like the General Standard for
the Labelling of Prepackaged Foods.
According to this general standard, the following informa-
tion shall appear on the labeling of prepackaged foods:
• The name of the food (this name shall indicate the true
nature of the food)
• List of ingredients (in particular if one of a list of eight
allergens is present)
• Net content
• Name and address of the business
http://dx.doi.org/10.1016/B978-0-12-384947-2.00194-X

• Country of origin where omission could mislead the
consumer
• Lot identification
• Date marking and storage instructions
• Instructions for use
In addition to the formally accepted standards, the Codex
Alimentarius includes recommended provisions called codes
of practice or guidelines. Most notably are, for example, the
‘Code of Ethics for International Trade in Food’ and a set of
hygiene codes like the ‘Recommended International Code of
Practice, General Principles of Food Hygiene’ and the ‘Hazard
Analysis and Critical Control Point System and Guidelines for
its Application.’
The ‘constitution’ of the Codex Alimentarius is the
procedural manual. The procedural manual gives not only
the procedures and format for setting Codex Alimentarius
standards and guidelines but also some general principles
and definitions. The principles relate among other things to
the scientific substantiation of the work of the Codex Alimen-
tarius and the use of risk analysis for food safety.
Consumer Protection Legislation in WTO Law
Concerning Food
The WTO is primarily concerned with removing barriers to
trade between its member countries. (The WTO was estab-
lished on 1 January 1995 by the Agreement Establishing the
WTO as the result of the so-called Uruguay Round of trade
negotiations and signed in Marrakesh on 15 April 1994 (WTO
Agreement). The WTO is the institutional continuation of the
General Agreement on Tariffs and Trade (GATT, 1947).) It does
so by establishing equal treatment of all trading partners as the
norm. As different consumer protection legislations form bar-
riers to trade, they require an exception from the general appli-
cation of liberalizing the WTO law. To this end, Art. XX(b) of
the GATT recognizes that certain exceptions to free trade can be
necessary to protect social values like health and (food) safety.
This opens up a leeway for members of the WTO to legiti-
mately regulate consumers’ health and safety of foods. The
Agreement on Technical Barriers to Trade (the TBT Agreement)
and the Agreement on the Application of Sanitary and Phyto-
sanitary Measures (the SPS Agreement) further substantiate
these requirements. The SPS Agreement ensures that countries
only apply measures to protect human and animal health
(sanitary measures) and plant health (phytosanitary measures)
based on the assessment of risk or, in other words, based on
science. The measures must be scientifically justified and they
may neither be discriminating nor constitute disguised barriers
to international trade. The SPS Agreement incorporates, there-
fore, the main safety aspects of foods in trade. The TBT Agree-
ment covers all technical requirements and standards (applied
to all commodities), such as labeling, that are not covered by
the SPS Agreement. Therefore, the SPS and TBT Agreements can
be seen as complementing each other.
If the measures are in conformity with international stan-
dards, no scientific proof of their necessity is required. These
measures are by definition considered to be necessary. The
most important international standards regarding SPS are set
Consumer Protection Legislation 297
by the so-called three sisters of the SPS Agreement: the Codex
Alimentarius Commission, the International Office of Epizo-
otics (OIE) (the abbreviation follows the French spelling), and
the Secretariat of the International Plant Protection
Convention. The standards on food and food safety are mainly
found in the Codex Alimentarius. (The agreement on TBT
treaty has similar articles.)
Consumer Protection in Private Food Law
Private standards stipulate rules that food businesses impose
upon each other in contractual relations (from a strategic busi-
ness viewpoint). Legal food safety systems limited by national
borders and political compromise are often ill-suited to reflect
consumers’ and suppliers’ demands in the food chain. For this
reason, corporations increasingly choose to develop their own
food safety standards. These standards form ‘uniform’ food
safety management systems, which lay down detailed require-
ments for service providers and producers. Such private food
standards have been so widely adopted by retailers in particular
that they have emerged as the primary mode of governance in
global food supply chains. While voluntary, the widespread
adoption of private food standards means that these standards
have become de facto mandatory as a precondition for partici-
pation in the majority of global supply chains. This initially
applied primarily to export markets. In the developing world
retailer capture of food markets in recent years has left few
alternatives, even in a domestic context.
Under such private standard regimes, suppliers have to
obtain certification and demonstrate compliance with the
respective quality management system, verified by indepen-
dent audits. The variety of standards demanded by customers
often requires suppliers to obtain multiple certificates. As their
differences are mainly bureaucratic in nature, this is an unnec-
essary and costly burden. In order to cope with this shortfall,
the Global Food Safety Initiative (GFSI) makes a move to level
or mutually recognize these different standards as far as possi-
ble. To this end, the GFSI approves certification regimes, which
then lead to worldwide acceptance (‘certified once, accepted
everywhere’).
Consumer Protection Legislation in American Food Law
The United States of America
In the United States, consumer protection legislation in food
law is shared between the federal and the state level. However,
since President Roosevelt signed the Pure Food and Drug Act
and the Meat Inspection Act into law in 1906, consumer pro-
tection in food law became increasingly federalized. Along
with the federalization of consumer protection law, the
federal Food and Drug Administration (FDA) matured into
an ever more powerful regulative body in the United States.
However, as the power of criminal law remains with the States,
federalization of consumer protection food laws embraced
private and administrative laws only.
Consumer protection food legislation in the United States
was to a large extent triggered by the cheating practices of the
food industry. As a reaction to this, US consumer protection
298 Consumer Protection Legislation
food legislation is largely occupied with the prevention of
adulteration of foods in order to ‘promote honesty and fair
dealing in the interest of consumers’ (21 U.S.C. Sec. 341). This
is why the concept of ‘adulteration,’ unlike, for example, in EU
law, takes a prominent position in consumer protection legis-
lation in the United States (21 U.S.C. Sec. 342). The large
bunch of legislation hence seeks to prevent such adulteration
by ensuring that consumers can trust in the information they
receive about the properties of the respective food. To this end,
the FDA first and foremost regulates the labeling of food (see
especially 21 U.S.C. Sec. 343 and the health claims
requirements). Adulteration shall also be prevented by regulat-
ing, for example, the manufacture, processing, or packing of
foods (see, e.g., 21 U.S.C. Sec. 348–350).
Important legislation (selection): Federal Food, Drug, and
Cosmetic Act (FD&C Act); Federal Meat Inspection Act; Federal
Trade Commission Act; Filled Milk Act; Import Milk Act; Public
Health Service Act; Trademark Act of 1946; Reorganization
Plan 1 of 1953; Poultry Products Inspection Act; Fair Packaging
and Labeling Act; National Environmental Policy Act of 1969;
Controlled Substances Act; Controlled Substances Import and
Export Act; Egg Products Inspection Act; Lead-Based Paint Poi-
soning Prevention Act; Federal Advisory Committee Act; Gov-
ernment in the Sunshine Act; Federal Anti-Tampering Act;
Sanitary Food Transportation Act; Mammography Quality
Standards Act (MQSA); Bioterrorism Act of 2002; Project
BioShield Act of 2004; FDA Food Safety Modernization Act
(FSMA) (still pending).
Internet reference address: http://www.fda.gov/RegulatoryIn-
formation/Legislation/default.htm.
Canada
As Canada is a federal system, it is in this respect in many ways
comparable to the US legal system. Consumer protection leg-
islation in food law is likewise shared between the federal and
the state level. However, with the enactment of several federal
laws with regard to consumer protection in food law (most
notably, the Canadian Food and Drugs Act (CFDA) and the
Safe Food for Canadians Act; for an overview, see list at the end
of this subarticle), food legislation became increasingly
federalized. Along with the federalization of consumer protec-
tion law, the Canadian Food Inspection Agency matured into
an ever more powerful regulative body in Canada. Unlike in
the United States, the federalization of consumer protection
food legislation embraces also criminal law.
The main concern of the early consumer protection food
legislation was adulteration of liquor. Hence, like US con-
sumer protection food legislation but for a different reason,
Canadian consumer protection food legislation is historically
likewise occupied with the prevention of adulteration of foods.
However, after modernization, Sec. 3 and Sec. 4 of the CFDA
(R.S.C., 1985, c. F-27) are much more up to date than its US
counterpart: such as in many other jurisdictions, Sec. 4 obliges
food sellers as a general rule to ensure that the food on the
market shows certain properties. In particular, food needs to be
not poisonous or harmful or unfit for human consumption. If
it is, Sec. 22 et seqq. of the act empowers several Canadian
authorities to take action.
Important legislation (selection): Agriculture and Agri-Food
Administrative Monetary Penalties Act; Appropriation Acts;
Canada Agricultural Products Act; Canadian Food Inspection
Agency Act; Consumer Packaging and Labelling Act; Feeds Act;
Fertilizers Act; Fish Inspection Act; Food and Drugs Act; Health
of Animals Act; Meat Inspection Act; Plant Breeders’ Rights Act;
Plant Protection Act; Safe Food for Canadians Act; Seeds Act.
Internet reference address: http://www.inspection.gc.ca/
about-the-cfia/acts-and-regulations/eng/1299846777345/
1299847442232.
Latin America
Consumer protection legislation in food law in Latin America
is as diverse and vivid as the continent. Although there have
been several attempts made in the past decade, however, until
the time of writing, only some countries such as Brazil have
developed a fully fledged food regulation. However, an all-
encompassing Latin American approach to consumer protec-
tion food law that one could present in an encyclopedia such
as this is nonexistent.
Consumer Protection Legislation in European Food Law
In Europe, consumer protection food legislation has to be
evaluated in view of the specific features of different legal
entities. Food law is largely harmonized at the supranational
level of the EU with the result that member states of the EU do
not have a strong say in consumer protection food legislation.
Countries such as Switzerland, which are not part of the EU,
also increasingly follow the example of EU regulation in food.
The European Union
There is no general authorization procedure for foodstuffs in
the EU. Art. 14 (1) of the General Food Law (Regulation (EC)
178/2002, GFL), however, stipulates that “food shall not be
placed on the market if it is unsafe.” Hence, liability of pro-
ducers as well as legitimate intervention of authorities depends
on whether the respective food passes an ‘unsafety’ test. The
Unfair Commercial Practices Directive 2005/29/EC ensures
that consumers are not subject to fraudulent or deceptive prac-
tices, adulteration, and other practices, which may mislead the
consumer (Art. 8 GFL). Product liability for foodstuffs is regu-
lated by Directive 85/374/EEC (see Art. 21 GFL). If there is
scientific uncertainty about the unsafety of foods, authorities
may nonetheless take precautionary measures according to the
precautionary principle (see Art. 7 GFL).
Compliance with numerous food processing and other
market regulation is generally monitored by member state
authorities. They are, however, interwoven in a dense commu-
nication network between member state authorities and the
European Food Safety Authority (EFSA). The EFSA monitors
the work of member state authorities. Together with the Com-
mission, it has also extended competences in food crisis man-
agement and information regulation.
According to the principle of mutual recognition (also
known as the Cassis de Dijon principle), foods that are lawfully
put on the market in one member state can and should be

allowed access to the markets in other member states if mem-
ber states cannot provide good justifications why they should
not. In this sense, consumer protection food law is local and
travels with the food. However, with the enactment of the
horizontal General Food Law, the principle of mutual recogni-
tion is increasingly on the decline. In case intervention is
needed in order to enforce consumer protection rules, the
Court of Justice of the European Union has established a
preference for information-related over content-related
measures. That is why a wide array of measures available to
authorities are information-related in nature. Health claims
and novel foods even need authorization.
Genetically modified foods and other novel foods fall under
a different regulatory regime. They need authorization as a
general rule. The authorization decision is taken by the Euro-
pean Commission. It is based on risk assessment by the EFSA.
Important legislation (selection): Regulation (EC) No 178/
2002 laying down the general principles and requirements of
food law; Regulation (EC) No 510/2006 of 20 March 2006 on
the protection of geographical indications and designations of
origin for agricultural products and foodstuffs; Regulation
(EC) No 852/2004 on the hygiene of foodstuffs; Regulation
(EC) No 853/2004 laying down specific hygiene rules for food
of animal origin; Regulation (EC) No 854/2004 laying down
specific rules for the organization of official controls on prod-
ucts of animal origin intended for human consumption; Direc-
tive 2002/99/EC laying down the animal health rules
governing the production, processing, distribution and intro-
duction of products of animal origin for human consumption;
Regulation (EC) No 882/2004 on official controls performed
to ensure the verification of compliance with feed and food
law, animal health and animal welfare rules; Regulation (EC)
No 1829/2003 on genetically modified food and feed; Regula-
tion (EC) 1924/2006 on nutrition and health claims made on
foods.
Internet reference address: http://europa.eu/legislation_-
summaries/food_safety/general_provisions/index_en.htm.
Switzerland
Since 2010 and pursuant to Art. 16a (1) of the Loi fédérale sur
les entraves techniques au commerce, products lawfully mar-
keted in the EU or the European Economic Area may also be
marketed in Switzerland. Switzerland has thereby autono-
mously expanded the Cassis de Dijon principle to their home
country. However, in order to successfully market foodstuffs
marketed in the EU in Switzerland, they still require authori-
zation by the competent Swiss authority.
Internet reference address: http://www.bag.admin.ch/the-
men/lebensmittel/10380/index.html?lang¼en.
Consumer Protection Legislation in Asian Food Law
China
China is a multilevel system, whereby state laws interplay with
local regulation. However, there are strong desires and prepa-
ratory works for a basic food law modeled after EU or US
standards. Although since 2009 China has enacted horizontal
food laws after the model of Western legal systems, at the time
Consumer Protection Legislation 299
of writing, food legislation in China is nonetheless still primar-
ily scattered along the different levels of Chinese legislation.
Although the food legislative system in China is modernizing
fast, consumer safety regulation still mainly follows the classi-
cal approach of administrative control.
China knows three different levels of regulation. The first and
highest level concerns laws passed by the National People’s
Congress. Here, the main act concerned with consumer protec-
tion in foods is the horizontal Food Safety Law of the People’s
Republic of China. The China FDA is the main focal point of the
administration and supervision of food (including food addi-
tives and health food, the same in the succeeding text) safety at
this level. Other important legislations include the Food
Hygiene Law, Product Quality Law, Agricultural Product Quality
and Safety Law, and Consumer Rights Protection Law. The
second level embraces legal instruments issued by the State
Council or the provincial People’s Congress. They are primarily
concerned with the administration of pesticides and veterinary
drugs, inspections of slaughterhouses and abattoirs, and the
regulation of genetically modified organisms. Third-level regu-
lation embraces decrees or provisions formulated by ministries
of the national or provincial government(s). The most impor-
tant decrees concern practices on food processing, decrees on
foods for particular populations, and decrees specific to the
management of food poisoning and street foods.
Important legislation (selection): Food Hygiene Law; Product
Quality Law; Agricultural Product Quality and Safety Law;
Consumer Rights Protection Law.
Internet reference address: http://eng.sfda.gov.cn/WS03/
CL0756/ (CFDA); http://www.fas.usda.gov/gainfiles/200903/
146327461.pdf (unofficial translation of the Food Safety Law
of the People’s Republic of China).
India
Indian food law focuses on strengthening both food security
and food safety. In 2013, the Indian National Food Security Act
was signed into law. This act creates legal entitlements to
subsidized food grains for large parts of the Indian population.
In 2006, a new Food Safety Standards Act was put in place.
The Ministry of Health and Family Welfare implemented this
act through the Food Safety and Standards Regulation, 2011.
The act and the regulation are the domain of the Food Safety
and Standards Authority of India (FSSAI).
The law covers topics such as prevention of adulteration,
hygiene, and labeling for packaged food including health
claims, permitted food additives and colors, and microbiolog-
ical requirements.
All domestic food operators are required to be licensed by
the FSSAI.
Important legislation (selection): Food Safety Standards Act,
2006; Food Safety and Standards Regulation, 2011; Indian
National Food Security Act, 2013.
Internet reference address: http://www.fssai.gov.in/
Japan
Consumer protection food regulation is based on the Food
Safety Basic Act such as other more specific legislations. Food
safety policy in Japan is conducted in accordance with the
300 Consumer Protection Legislation
general internationally recognized deviation of risk manage-
ment and risk assessment. While the Food Safety Commission
is responsible for scientific risk assessment, the Ministry of
Health, Labour, and Welfare and the Ministry of Agriculture
are in charge of risk management.
Important legislation (selection): Food Safety Basic Act, 2003;
Food and Sanitation Law; Business Control and Poultry
Inspection Law.
Internet reference address: http://www.fsc.go.jp/english/
index.html
Consumer Protection Legislation in South African
Food Law
South Africa is governed by not only a pluralist system,
whereby state laws interplay with local regulation at a vertical
level, but also by the laws of different societies, tribes, and
governmental regulation, which interplay horizontally. This
is reflected in legal acts as well as control of consumer protec-
tion food legislation. The most important act is the Foodstuffs,
Cosmetics and Disinfectants Act, 1972. Several other acts
accompany the regulatory framework in this respect.
Important legislation (selection): Foodstuffs, Cosmetics and
Disinfectants Act, 1972; Meat Safety Act, 2000; Agricultural
Products Act, 1990; Liquor Products Act, 1989; Health Act, 1977.
Internet reference address: http://www.doh.gov.za/healthto-
pics.php?t¼Food%20Control
See also: Allergies: Public health; Cured Foods: Health Effects; Food
and Agriculture Organization of the United Nations; HACCP and
ISO22000: Risk Assessment in Conjunction with Other Food Safety
Tools Such as FMEA, Ishikawa Diagrams and Pareto; Legal
Requirements for Food Hygiene; Quality Control in Food Processing;
Risk Assessment of Foods and Chemicals in Foods.
Further Reading
Anelich L (2010) South Africa. In: Boisrobert C, Stjepanovic A, Sangsuk O, and
Lelieveld H (eds.) Ensuring global food safety. London: Academic Press.
Appelhof T and Van den Heuvel R (2011) In: Scholten-Verheijen I, Appelhof T, Van den
Heuvel R, and Van der Meulen B (eds.) Roadmap to EU Food Law The Hague:
Eleven Publishing.
Arcuri A (2000) Product safety regulation. In: Bouckaert B and De Geest G (eds.)
Encyclopedia of law and economics, volume III. The regulation of contracts.
Cheltenham: Edward Elgar. Available at, http://encyclo.findlaw.com/5130book.pdf.
Boisrobert C, Stjepanovic A, Oh S, and Lelieveld H (eds.) (2010) Ensuring global food
safety. Amsterdam: Elsevier.
Bradford A (2012) The Brussels effect. Northwestern University Law Review 107: 1–68.
Bremmers H, van der Meulen B, and Purnhagen K (2013) Multi-stakeholders-responses
to the European health claims requirements. Journal on Chain and Network
Sciences 13: 161–172.
Cafaggi, F. (2010). Private Regulation, Supply Chain and Contractual Networks: The
Case of Food Safety. RSCAS Working Paper Series 2010/10 (Robert Schuman
Centre for Advanced Studies – Private Regulation Series).
Downes C (2014) The impact of WTO SPS law on EU Food Regulation. Dordrecht:
Springer.
Forte W (1966) The Food and Drug Administration and the economic adulteration of
foods. Indiana Law Journal 41: 346–402.
Fortin N (2009) Food regulation. Hoboken: Wiley.
Fulponi L (2007) Private voluntary standards in the food system: the perspective of
major food retailers in OECD countries. Food Policy 31.
Gnirrs G (2008) A history of food law in Canada. Food in Canada 38.
Hasler C (2008) Health claims in the United States: an aid to the public or a source of
confusion? The Journal of Nutrition 138: 1216S–1220S.
Henson S (2007) The role of public and private standards in regulating
international food markets. Journal of International Agricultural Trade and
Development 4(1).
Henson, S. and Humprey, J. (2009). The Impacts of Private Food Safety Standards on
the Food Chain and on Public Standard-Setting Processes. Paper Prepared for FAO/
WHO. Codex Alimentarius Commission. Thirty-second Session. FAO Headquarters,
Rome.
Howells G and Weatherill S (2005) Consumer protection law, 2nd ed. Farnham:
Ashgate.
Joerges C, Falke J, Micklitz H-W, and Brüggemeier G (1988) Die Sicherheit von
Konsumgütern in der Europäischen Gemeinschaft. Baden-Baden: Nomos.
López-Garcı́a R (2010) Latin America. In: Boisrobert C, Stjepanovic A, Sangsuk O, and
Lelieveld H (eds.) Ensuring global food safety. London: Academic Press.
Pollak, M. (2013). A Truce in the Transatlantic Food Fight: The United States, the
European Union, and Genetically Modified Foods in the Obama Years, available at
http://ssrn.com/abstract=2295197.
Ponte S and Gibbon P (2005) Quality standards, conventions and the governance of
global value chains. Economy and Society 34: 1–31.
Purnhagen K (2013a) Systematization of EU Product Safety Regulation. Dordrecht:
Springer.
Purnhagen K (2013b) Beyond threats to health: may consumers’ interest in safety trump
fundamental freedoms in information on foodstuffs? European Law Review
38: 711–719.
Purnhagen K (2015) Mapping private regulation – Classification, market access and
market closure policy, and law’s response. Journal of World Trade 49: 2
(forthcoming).
Purnhagen K (2014) Why there is no ‘principle of mutual recognition’ in EU law (and
why that matters to consumer lawyers). In: Purnhagen K, and Rott P (eds.), Varieties
of European economic law and regulation. Dordrecht: Springer.
Reardon T, Timmer P, and Berdegue J (2004) The rapid rise of supermarkets in developing
countries: induced organisational, institutional and technological change in agri-food
systems. Journal of Agricultural and Development Economics 1: 168–183.
Van der Meulen BMJ (2010) Global arena of food law: emerging context of a meta-
framework. Erasmus Law Review 3: 217–240.
Van der Meulen B (ed.) (2011) Private food law. Governing food chains through
contract law, self-regulation, private standards, audits and certification schemes.
Wageningen: Academic Publishers.
Van der Meulen BMJ and Van der Velde M (2014) EU food law handbook. Wageningen:
Wageningen Academic Publishers.
Vos E (1999) Health and safety regulation. Oxford: Hart.
Weatherill SK (2014) The virtue of Cassis de Dijon 25 years later - It is not dead, it just
smells funny. In: Purnhagen K, and Rott P (eds.), Varieties of European economic
law and regulation. Dordrecht: Springer.
Zhou J and Jin S (2013) Food safety management in China: a perspective
from food quality control system. Singapore: World Scientific Publishing
Company.
 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs
Z Escobedo-Avellaneda and J Welti-Chanes, Tecnológico de Monterrey, Monterrey, Mexico
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Fruits and vegetables are biological systems that once harvested
deteriorate rapidly because they continue metabolic processes.
To diminish the rate of senescence, vegetable products must
be handled adequately. Low-temperature storage is the most
effective and simplest way to reduce metabolic processes caus-
ing deterioration. Nevertheless, the combination of low tem-
peratures with preservation factors that avoid the action of
oxygen involved in many enzymatic reactions is recommended
to extend shelf life while preserving the quality of foods.
Although the use of packages under normal atmospheric con-
ditions protects foods from environmental factors such as
light, physical, and biological contaminants, vegetable prod-
ucts are still in contact with oxygen.
Controlled atmosphere (CA) and modified atmosphere (MA)
are two technologies used to change the gas composition of the
surrounding environment with the aim of reducing metabolic
reactions, maintaining the freshness, and extending the shelf life
of foods. In these technologies, the concentration of oxygen,
carbon dioxide, ethylene, and/or nitrogen of the surrounding
atmosphere of foods is changed. While in CA the CO2 and O2
levels are constantly monitored and adjusted, in MA, the gas
concentration changes due to metabolic processes of the fruits
and vegetables, and this concentration is not controlled during
storage. As a consequence, in MA, the rate of change depends on
the respiration rate of the product, package permeability, and
storage conditions. Due to the gas control in CA, it is a more
costly method, but it is more appropriate for long-term storage
products.
CA and MA can be applied once the product is packaged
or also be used during the bulk storage where the gas com-
position inside the storage rooms is changed according to the
requirements of foods stored. The use of CA in bulk storage
extends the shelf life of foods, avoids microbiological reac-
tions, and controls pests including insects, rats, and mice.
Some of these effects are achieved due to the capacity of CA
to reduce ethylene biosynthesis retarding ripening. During
the storage of fruits and vegetables, the level of O2 is generally
lowered to diminish ethylene production, and the level of
CO2 is often increased to decrease the action of ethylene. The
concentration of gases in CA storage rooms is monitored by
infrared gas analyzer to measure CO2 and by a paramagnetic
analyzer for O2.
The use of inadequate preservation conditions in CA stor-
age can cause negative effects on the quality of foods; very low
levels of O2 can result in fermentation causing off-flavors
related with ethanol and acetaldehyde production. This can
also decrease the synthesis of volatile compounds affecting the
sensory quality of foods. This contribution is focussed on the
preservation of foods by CA bulk storage.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00196-3
Metabolic Processes of Fruits and Vegetables
Affecting Their Shelf Life
After harvesting, fruits and vegetables continue having meta-
bolic processes like respiration, transpiration, and production
of ethylene. These physiological activities are responsible for
the ripening and senescence of vegetable products; thus, ade-
quate postharvest handing is necessary to delay changes main-
taining quality. The rate of these processes determines the shelf
life, and they depend on food composition, physiological
characteristics, and storage conditions such as temperature
and relative humidity. Respiration oxidizes carbohydrates by
using the atmospheric oxygen generating ATP, CO2, and water.
The ATP produced is used in many ripening reactions; thus, the
control of the respiration rate is a way of controlling the rate of
senescence. Storage at low temperatures decreases the activity
of enzymes involved in ripening. Diminished levels of oxygen
reduce respiration and the action of enzymes like polyphenol
oxidase and peroxidase. The application of CA at low O2 levels
combined with reduced temperature is a way of decreasing
senescence of foods. Transpiration is the process of water loss
causing wilting, shriveling, shrinkage, softening, flaccidity,
limpness, loss of crispness, juiciness, and loss of nutritional
quality in fruits and vegetables. Ethylene regulates many
aspects of growth, development, and senescence of vegetable
products and can be physiologically active at very low concen-
trations. Transpiration and ethylene production can be con-
trolled by adequate storage conditions.
CA Storage Definition and Principles
The desired atmosphere in CA storage is first achieved by
replacing the normal gas composition by purging some gas or
by the metabolism of fruits and vegetables, and then, it is
maintained by lowering the rate of gas input. The gas input
rate is determined by the room capacity, its degree of gas
tightness, humidity, temperature, and metabolism of the
stored foods.
CA technology generates products that remain turgid because
the lower O2 concentration inhibits or retards the activity of
enzymes that act on cellular membranes avoiding texture
changes. CA also expands the climacteric and postclimacteric
periods; delays senescence during storage due to the reaction
between O2 and ethylene; reduces microbial degradation due
to the decreased O2 levels and the increased CO2 concentrations;
improves the color stability due to a low effect on chlorophylls;
allows obtaining foods with similar nutritional and sensory
characteristics than the fresh product due to the low effect on
acidity, sugars, and vitamins; and controls proliferation of pests
and molds at low O2 or high CO2 atmospheres.
301
302 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs
CA constraints are related with the high costs of storage, the
special seals required in the storage room to avoid gas leakage,
and the longtime of exposure needed (8–12 days). Another
disadvantage is the variability in the concentration of gases
required for the storage of foods that depends on many factors
such as the type of fruit or vegetable, species, stage of maturity,
medium, and climate in which they were grown. Biochemical
and anatomical differences, including the size of intracellular
spaces and the rate of gas diffusing through the food structure,
also influence the variability in gas concentrations. Another
disadvantage of CA is that at inadequate storage conditions,
undesirable changes like fermentation can appear. For example,
apples and pears stored at very low O2 concentrations presented
changes in flavor and color and changes in hardness and dark-
ness of skin due to high concentrations of CO2. The need for
low temperature and high relative humidity to enhance CA
effectiveness is also considered as constraints of CA storage.
Main Gases Used in the CA Storage of Foods
There are many gases to be controlled in order to reduce the
metabolic processes of foods, microorganisms, or pests in CA
storage. The control of both O2 and of CO2 levels can be used
in order to have synergetic or additive effects to extend the shelf
life of foods. The election of either low level of O2 or high
concentrations of CO2 depends on factors such as the time of
treatment, design of the room used, cost of gases, and systems
available to control them.
The use of CO2 prevents the growth of aerobic bacteria and
fungi and avoids the oxidation of some compounds. The
increase in CO2 level has different effects in fruits and vegetables
such as decrease of synthetic reactions in climacteric fruit, delay
in the initiation of ripening, inhibition of some enzymatic
reactions, decrease of production of some organic volatiles,
modification of the metabolism of some organic acids, reduc-
tion in the rate of pectin breakdown, inhibition of chlorophyll
breakdown, production of off-flavor, induction of physiological
disorders, retarding of fungal growth, inhibition of the effects of
ethylene, inhibition of postharvest development, retention of
tenderness, and decrease of color loss. Oxygen is necessary for
the growth of aerobic bacteria and enzymatic or chemical reac-
tions in foods. Therefore, its concentration may affect the prod-
uct quality. However, it should not be completely eliminated
because it would cause the growth of anaerobic bacteria causing
fermentation. Decreasing O2 levels reduces respiration rate,
decreases oxidation, delays ripening of climacteric fruit, extends
shelf life, delays breakdown of chlorophyll, reduces rate of
ethylene synthesis, changes fatty acids synthesis, reduces degra-
dation rate of soluble pectin, decreases off-flavors, and alters
texture. Nitrogen is another gas used in CA, normally as filler
instead of CO2. It is innocuous and when it is used in packages,
it may prevent the collapsing. It is also used in some cases
instead of oxygen as a nonreactive gas to avoid decomposition
and oxidation of lipophilic compounds.
Methods Used to Control Gas Levels in CA Storage
CA storage rooms are built with structures capable of support-
ing the gases used, and they are provided with the adequate
system to produce the atmosphere and the best-available
method to maintain the atmosphere composition for the
required time. In CA storage, foods are placed into an insulated
store room whose walls and doors are gastight. CA rooms are
made from metal-faced insulated panels, which are fitted
together with gastight locking devices. Doors are usually sealed
by having rubber gaskets around the perimeter, which corre-
spond to another rubber gasket around the door jamb of frame
to avoid leaks. Static and dynamic are the two ways used to
produce the atmosphere needed in CA storage rooms. In the
static method, the product generates the atmosphere and it is
maintained by ventilation and scrubbing. In the dynamic
system, the gas concentrations are supplied by flushing gases.
There are also different ways to control the gas levels in CA
rooms. Systems such as external burners, liquid or gaseous
nitrogen, gas separator systems, and hypobaric storage can be
used to control the O2 levels. For the removal of CO2 scrubbing
systems based on NaOH, hydrated lime (Ca(OH)2), water,
activated charcoal, and molecular sieves have been implemen-
ted. Heated catalyst scrubbers, ethylene-absorbing beads, and
UV light are used to control the ethylene levels (Table 1).
CA Storage of Fruits and Vegetables
The recommended storage conditions for fruits and vegetables
under CA are highly variable because they depend on many
factors. Factors such as species, cultivars, anatomical differences,
initial gas concentration in the storage room, temperature, humid-
ity, degree of ripeness, ethylene concentration, and pre- and post-
harvest conditions influence the level of gases used. Thus, there are
many recommended conditions for the storage of foods.
Temperature is one of the factors that more influence the
required concentration of CO2 and O2 for the CA storage
of foods. The effect of temperature on delaying the ripening of
foods by decreasing the rate of reactions is well known. Never-
theless, due to the diversity of factors influencing, when select-
ing the conditions for the storage or certain food, it will be
necessary to evaluate their effects on food quality in terms on
color, texture, nutritionals changes, and microbiological safety.
Increasing the CO2 levels can have different effects on
the biochemical and physiological changes associated with rip-
ening such as the production of ethylene that can be inhibited,
increased, or not altered. In the first case, CO2 acts by displacing
ethylene from its receptor side, delaying fruit ripening; CO2
concentration lower than 1% favors ethylene production. The
oxygen levels also influence the effects of CA storage on ripening
of fruits by inhibiting or lowering the action of ethylene and also
altering its synthesis. Lowering O2 to more than 8% reduces the
binding of ethylene to its receptor influencing its actions and
therefore ripening. O2 is required in the final step of ethylene
biosynthesis from methionine. Oxygen levels below 5% greatly
reduce ethylene production of fruits by inhibiting the ACC
oxidase enzyme activity. Very low levels of O2 can cause fermen-
tation of foods with consequent development of off-flavors,
while very high levels of CO2 can cause texture changes. It was
reported that apple (Delicious) stored at 1
C in 0.5% O2 þ 0.2%
CO2 for 30 days developed high concentrations of ethanol and
acetaldehyde and that at 0% O2, the respiration rate of cucumber
stored at 10
C or 20
C increased because of fermentation. CA
storage of mango in 1% O2 produces off-flavors and skin
 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs
Table 1 Systems used to control the O2, CO2, and ethylene levels
in CA storage
System Characteristics
O2 control
External burners The level of O2 in the storage room is
reduced by the combustion of propane
or natural gas generating CO2 and
water vapor. They usually require a
CO2 scrubber to control the levels of
this gas and cooling systems to reduce
the temperature of air before and after
combustion. They are classified as
open flame or catalytic burners. The
open flame burner does not allow air
recirculation because of the risk of
extinguishing the flame by the O2
depleted recirculating airstream.
Catalytic burners provide more
complete combustion reducing O2
levels to 3%; in addition, they have
lower operation cost despite being
more expensive to install
Flushing with liquid or O2 levels are reduced by directly flushing
gaseous N2 N2 in the storage room until the
desired O2 level is achieved. Liquid
N2 is injected into the room
through spray headers that atomize
the N2
Gas separator systems Gas separator systems decrease the O2
levels while increasing the N2
concentration from 97% to 99%. They
are classified as pressure swing
absorption (PSA), hollow fiber
membrane separators (HFMS), and
high-temperature ammonia cracking
systems (HTAC)
PSA system compresses dry air and
once filtered forces it through a bed of
pelletized carbon material, which
absorbs O2 and enhances the N2 levels
HFMS systems are based in the
different diffusion rates of gases
through membranes. CO2 and O2 have
much higher permeation rates than N2;
thus, the levels of CO2 and O2
decrease, while the level of N2
increases
In the HTAC system, high-temperature
anhydrous NH3 gas reacts with air
from the CA room, yielding hydrogen
and inert N2 gas. Further, hydrogen
reacts with the O2 to yield water vapor
Hypobaric storage Hypobaric pumps are used to reduce the
pressure of the room with consequent
decreasing in O2 levels
CO2 control
NaOH NaOH dissolved in water can be used to
remove CO2 in the storage room by
circulating the air in open tubes. The
amount of CO2 removed is
proportional to the NaOH exposure
time
303
Table 1 (Continued)
System Characteristics
Hydrated lime scrubber (Ca Ca(OH)2 is one of the simplest and most
(OH)2) effective systems for controlling the
level of CO2. Ca(OH)2 is placed in an
insulated and airtight box outside the
storage room to which it is connected
by two pipes in which one of them
allows air from the storage room to
enter the box that contains the lime
Ca(OH)2 can also be placed on bags or
pellets that react with CO2 yielding
calcium carbonate, water, and heat
maintaining it at level lower than 1%.
This methods required space to place
the bags and is inefficient because
80% of the lime is not used
Water CO2 scrubbers A brine solution is pumped or sprayed
over the evaporator coils in the CA
room. The brine absorbs CO2, due to
the pressure difference between the
incoming water and the atmosphere in
the CA room. The corrosion caused by
the brine solution may be prevented by
using dry cooling units
Activated charcoal and The air from the CA room is blow
molecular sieve scrubbers through an absorbing unit reducing the
CO2 levels, and then it is forced back to
the CA room. When saturated with
CO2, the absorbent must be reactivated
by pursing it with air
Ethylene control
Heated catalyst scrubber The air is forced thought ceramic
pickings used as heat exchangers
removing up to 87% of the ethylene
from the air. The catalyst is electrically
heated and the high temperature
promotes the oxidation of ethylene to
CO2 and water vapor. This system
requires energy for heating and cooling
Ethylene absorbing beads Small particles of aluminum silicate
impregnates with potassium
permanganate are placed in a sealed
unit. Air from the CA room is circulated
through the unit and the ethylene
reacts with the permanganate
changing the color of the beds when
saturated with ethylene
UV light The UV light reacts with O2 to form
ozone, which eliminates ethylene
Source: Thompson, A. K. (2010). CA technology. In: Thompson, A. K. (ed.) Controlled
atmosphere storage of fruits and vegetables (2nd ed.), pp. 26–42. London: CAB
International; Rennie, T. J., Vigneault, C., Deell, J. R. and Raghavan, S. G. (2003).
Cooling and storage. In: Ramaswamy, H. S., Vijaya Raghavan, G. S., Chakraverty, A. and
Mujumdar, A. S. (eds.) Handbook of postharvest technology: cereals, fruits, vegetables,
tea, and spices, pp. 505–538. New York: CRC Press.
discoloration, but the storage at 12
C in 5% CO2 þ 5% O2 was
possible for 20 days. With 3% O2 þ 6% CO2, mango did not
present significant fermentation.
CA including either reduced O2 or elevated CO2 not only
generally decreases the loss of provitamin A but also inhibits the
304 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs

biosynthesis of carotenoids. Concentration of 5% CO2 caused
loss of carotenes, while 7.5% CO2 or higher favors their
synthesis. CA storage with 2% O2, air þ 12% CO2, and 2%
O2 þ 12% CO2 had no effect on quality attributes of sliced
peaches over 7 days of storage, while the visual quality in
color of persimmon slices was slightly enhanced by the treat-
ments containing 12% CO2. Peach slices stored in 12% CO2
had a lower content of b-carotene and b-cryptoxanthin. The
various carotenoids found in persimmon slices responded dif-
ferently to the tested atmospheres; storage in 2% O2 or 12%
CO2 tended to result in lower retinol equivalents after 8 days
storage at 5
C, but the loss was not significant for fruit stored
under 2% O2 þ 12% CO2. In the case of other vitamins like
vitamin C, it was noticed that 2% O2, air þ 12% CO2, or 2%
O2 þ 12% CO2 had no significant effect on total ascorbate
content for both fresh-cut strawberries and persimmons.
The effects of CA on texture and other sensorial attributes of
some fruits and vegetables have also been evaluated. CO2 con-
centrations above 4% with 15–20% O2 retard the softening of
kiwifruit, and this effect increased as the CO2 content increased
from 4% to 10%, but without additional effects at CO2 content
above 10%. Low O2 levels (2–3%) with 3–5% CO2 further
delayed the rate of kiwifruit softening and increased storage life
up to 3–4 months. Although CA increases storage life of kiwifruit,
the magnitude of the effects varied from year to year. Contami-
nation of the storage atmosphere by as little as 0.1 ml1 ethylene
severely reduced the effectiveness of controlled atmosphere stor-
age in maintaining kiwifruit firmness, even at 0
C. It was showed
that Valencia oranges can be treated with 0.5%, 0.25%, or 0.02%
O2 at 5
C or 10
C for up to 20 days without detrimental effects
on their appearance, nutritional value, and flavor, while 60%
CO2 at 5
C could injure oranges, and 7–12% O2 þ 0–5% CO2
retards decay and toughening of asparagus. Elevated CO2
concentrations resulted in firmer strawberries, while low O2 did
not affect texture demonstrating that although strawberry is
highly perishable, under high CO2 condition, the firmness and
shelf life could be maintained. Off-flavor developed after 3 days
of storage at 20% CO2, but it decreased when fruit was subse-
quently held for 24 h at 20
C. Similar results were obtained
using low O2 (2%, 1%, and 0.5%) or elevated CO2 (10%, 15%,
and 20%) concentrations, and their combinations reduced respi-
ration and ethylene production rates of strawberry stored at 2
C
and fruits maintained their color and firmness. The rate of color
change in cucumber could be delayed with high CO2 and low O2
in the storage atmosphere, but 10% CO2 could impair their
flavor.
The storage of mamey at 15
C in 1% CO2 þ 5.6%
O2 þ 89.3% N2 for 2 weeks retarded their maturation, and it
was recommended to store onion at 5% CO2 þ 3% to reduce
sprouting and root growth. The use of 1–5% O2 þ 14% CO2
allows to reduce the levels of crown rot in bananas as com-
pared with those stored in air, and this effect continued even
during subsequent ripening in air at 25
C, and the storage of
cauliflower at 10%, 15%, or 20% O2 the leaf yellowing and rot
caused by Alternaria brassicicola is delayed, but it caused injury
inside the stem and vegetables developed off-flavors and odors
after cooking. Some other recommended CA storage condi-
tions for fruits and vegetables are present in Table 2.
Some products are also classified according to the minimum
concentrations of O2 and maximum level of CO2 required for
storing. Most fruits and vegetables have a threshold level of O2
and CO2 to cause injury that includes problems such as off-flavor
development, discoloration, off odors, alcoholic taste, browning,
and softening. Some minimum concentrations of O2 and maxi-
mum level of CO2 required for storage of some foods are pre-
sented in Table 3.

Table 2 Recommended CA storage conditions for some fruits and vegetables

Product O2 (%) CO2 (%) Temperature (
C)/relative humidity (%) Shelf life Variety

Apple 0.02 12 days

1.5 Up to 3
1.5 <0.5 0 9 months Gala
<1 2 3.5–4 23 weeks Gala
3 3 0 9 months Golden Delicious
1.5 Up to 3 0 Golden Delicious
3 1 Golden Delicious
3 3 0 8.5 months Golden Delicious
0.8–2.5 0.8–5 0.5–2 8–10 months Granny Smith
1.5 1.5 0.5 9 months Red Delicious
1–2.5 1–3 0.5 to 1 8–10 months Red Delicious
Blueberry 0 14–21 days
Cherry 0.02 25 days
Mango 0.1–0.2 5 days
5 5
5 10 10/90
3–5 5–10 10–15
Nectarine 0.02 14 days
Orange 0.02 16 days
5–10 0–5 5–10
5–10 0–5 5–10
Papaya 0.2–0.4 3 days
10 18
 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs 305

Table 2 (Continued)

Product O2 (%) CO2 (%) Temperature (
C)/relative humidity (%) Shelf life Variety

2–5 5–8 10–15

3–5 5–10 10–15
2–5 5–8
Peach 0.02 40 days
2 5
1–2 3–5
Pear 0.02 14 days
>2 <5 Bartlett
2–2.5 0.8–1
1–1.5 <0.1
Strawberry 0.25 10 days
10 15–20
5–10 15–20 0–5
1.5 1.5 3 weeks
Apricot 2–3 2–3
0.3 0 or 6 37 days Reale d’Imola
Avocado 2 10 5.5 4 weeks
2–5 3–10 10–13
Banana 3 5 14
10–13.5 Green bananas
2 8 15 Green bananas
3 5–8 15 Green bananas
Lemon 5 5–10
5–10 0–10 10–15
0 10 6 months
Asparagus 5–10 5–15 20 4.5 days
5 9 0/95
>10 <10 5
20 5–10
Cabbage 3 3 0 Red and Savoy
3 0–3 0 White
2.5–5 2.5–5 0
3 5 0 White
Celery 2–4 2–5
3 5 0
Cucumber 3–5 5–7
Lettuce 3 2 1 month
2–5 0
1–3 0
1–3 0
Onion 1–2 0
3 5 0/65–75
0–1 0–5 0–5
Tomato 3–5 3–5 10–15
1.5 0

Source: Ke, D. and Kader, A. A. (1990). Tolerance of ‘Valencia’ oranges to controlled atmospheres as determined by physiological responses and quality attributes. Journal of
American Society of Horticultural and Science 115(5), 779–783; Ke, D. and Kader, A. A. (1992). Potential of controlled atmospheres for postharvest insect disinfestation of
fruits and vegetables. Postharvest News and Information 3(2), 31N–37N; Ke, D., Goldstein, L., O’Mahony, M. and Kader, A. A. (1991). Effects of short term exposure to low O2 and high
CO2 atmosphere on quality attributes of strawberries. Journal of Food Science 56, 50–54; Ke, D., Van Gorsel, H. and Kader, A. A. (1990). Physiological and quality
responses of “Bartlett” pears to reduced O2 and enhanced-CO2 atmospheres and storage temperatures. Journal of the American Science for Horticultural Science 155, 435–439;
Thompson, A. K. (2010). Recommended CA storage conditions for selected crops. In: Thompson, A. K. (ed.) Controlled atmosphere storage of fruits and vegetables (2nd ed.),
pp. 116–191. London: CAB International.

CA Storage of Grains and Animal Products
CA can be also used to store grains and cereals for the control
of insect’s proliferation. Similar to CA storage of vegetable
products, CA storage of grain involves the alteration of the
proportion of normal atmospheric gases to give an atmosphere
inadequate for the development of insects. CA in grain can
cause lethal and sublethal effects. Sublethal effects include
delayed or disturbed development, other deleterious pheno-
typic effects, and behavioral phenomena. Thus, the low levels
of O2 and high concentrations of CO2 used in CA represent an
alternative way to the use of poisonous gases for the control of
306 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs
Table 3 Minimum O2 level and maximum CO2 concentration (%)
recommended for the CA storage of some foods
Minimum O2 Examples
level
0.5 Nuts, dried fruits, and vegetables
1.0 Pear, apple, broccoli, mushroom, garlic, onion, and
minimally processed vegetables
2.0 Apple, pear, kiwi, apricot, plum, cabbage, peach,
cherry, nectarine, papaya, strawberry, pineapple,
melon, cabbage, lettuce, and cauliflower
3.0 Avocado, carrot, tomato, persimmon, pepper,
cucumber, and artichoke
5.0 Citrus fruits, pea, asparagus, potato, and sweet potato
10 Potato
Maximum CO2 Examples
level
2 Apple (Golden Delicious), pear, grapes, tomato,
pepper, and lettuce
5 Apple, peach, nectarine, orange, avocado, banana,
mango, kiwi, couliflower, pear, and papaya
10 Grapefruit, lemon, lime, pineapple, cucumber,
zucchini, broccoli, asparagus, potato, onion, and
garlic
15 Strawberry, raspberry, blueberry, cherry, mushrooms,
brocoli, and spinach
Source: Fellows, P. J. (1996). Food processing technology. Cambridge: Woodhead
Publishing; Kader, A. A. and Ke, D. (1994). Controlled atmospheres. In: Paull, R. E. and
Armstrong, J. W. (eds.) Insect pests and fresh horticultural products, pp. 223–236.
Wallingford: CAB International.
insects. CO2 in high concentrations kills insects acting directly
as fumigant, while low O2 levels have asphyxiate effects acting
on all the development stages of the insect without detrimental
effects on foods but with dangerous results to human,
especially low levels of oxygen and high contents of carbon
dioxide. High levels of CO2 are often more effective to control
insect than low levels of O2 because they act more quickly and
in addition require a less stringent standard of gastightness.
The time required to kill insects depends on the species and
developmental stage, temperature, O2 and CO2 levels, and air
humidity; an initial level of 70% CO2 maintained above 35%
for 10 days is appropriate for complete insect control at tem-
peratures above 20
C. Oxygen level below 1.2% for 1 week at
temperatures above 35
C or more than 24 weeks at 15
C has
been proposed for complete insect control in Hass avocados.
Sunrise papayas and Keitt mangoes can be stored at 0.5% O2
and 50% CO2 for 1, 2, and 5 years at 20
C. These treatments
could have some potential application as insecticides for quar-
antine insect control, on the basis of fruit tolerance, insect
mortality, and cost. There are some studies about the CA
storage of animal products; for whole pork loins divided into
roasts and subjected CO2 at 1
C for up to 21 days, a strong
inhibition on microbial growth as well as a marked residual
effect during posttreatment storage in air was observed. In the
evaluation of the quality of whole ungutted bigeye tuna during
bulk storage in CA with 60% CO2 þ 15% O2 þ 25% N2 and
40% CO2 þ 40% O2 þ 20% N2, at day 33 of storage, none of
the lots were rejected based on pH, trimethylamine nitrogen,
total volatile base nitrogen and histamine, or the tasting panel
scores. The control and CA lots were rejected after inspection
(sensorial analysis) at 13 and 22 days, respectively. Fish muscle
undergoes a series of changes during storage under atmo-
spheres and conventional chilled storage, resulting in deterio-
ration and loss of quality.
Legislation
Act No. 228 of 1959 for fruit and vegetable storage by CAs from
the State of Michigan, the United States, indicates some defi-
nitions, specifications for the construction of storage room,
and general conditions for the adequate handling of fruits
and vegetables stored under CA. The regulation No. 530 of
this act specifies the conditions for the CA storage of apples.
The Ontario Regulation 95/97 also gives some specifications
for the CA storage of apples. Section 3–502.12 of the Food
Code (FDA Food Code 2009: Annex 6 – Food Processing
Criteria) provides the criteria that are to be met in the
HACCP plans of those operators who are conducting reduced
oxygen packaging operations. According to the European
Union directive No. 95/2/EEC, food stored under CA must
have that indicated in the label and gases used must be treated
as additives.
See also: Fumigants; Fungicides; Insect Pests; Spoilage: Bacterial
Spoilage; Spoilage: Yeast Spoilage of Food and Beverages.
Further Reading
Aharoni N, Rodov V, Fallik E, Porat R, Pesis E, and Lurie S (2008) Controlling humidity
improves efficacy of modified atmosphere packaging of fruits and vegetables. Acta
Horticulturae 804: 121–228.
Bailey SW and Banks HJ (1980) A review of recent studies of the effects of controlled
atmospheres on stored product pets. In: Shejbab J (ed.) Developments in
agricultural engineering 1: controlled atmosphere storage of grains, pp. 101–118.
Amsterdam: Elsevier.
Hardenburg RE, Watada AE, and Wang CY (1990) The commercial storage of fruits,
vegetables and florist and nursey stocks. Agriculture handbook 66. Washington,
DC: United States Department of Agriculture, Agricultural Research Service.
Kader AA (1980) Prevention of ripening in fruits by use of controlled atmospheres. Food
Technology 34: 51–54.
Kader, A. A. (1989). A summary of CA requirements and recommendations for fruit
other than pome fruits. In: Proceeding of the Fifth International Controlled
Atmosphere Research Conference, Wenatchee, WA, vol. 2, pp. 303–328.
Kader AA (1994) Modified and controlled atmosphere storage of tropical fruits.
In: Champ BR, et al. (ed.) Postharvest handling of tropical fruits. ACIAR Proceedings
50, pp. 239–249. Canberra, Australia: ACIAR.
Kader AA (1995) Regulation of fruit physiology by controlled/modified atmosphere.
Acta Horticulturae 398: 59–70.
Lalel HJD and Singh Z (2006) Controlled atmosphere storage of “Delta R2E2” mango
fruit affects production of aroma volatile compounds. Journal of Horticultural
Science and Biotechnology 81: 449–457.
Manzano-Mendez JE and Dris R (2001) Effect of storage atmosphere and temperature
on soluble solids in mamey amarillo (Mammea americana L.) fruits. Acta
Horticulturae 553: 675–676.
Mattheis JP, Buchanan DA, and Fellman JK (1991) Change in apple fruit volatiles after
storage in atmospheres inducing anaerobic metabolism. Journal of Agricultural and
Food Chemistry 39: 1602–1605.
Mcdonald B and Harman JE (1982) Controlled atmosphere storage of kiwifruit. I. Effect
on fruit firmness and storage life. Scientia Horticulturae 17(2): 113–123.
Pantastico EB (1975) Postharvest physiology, handling and utilization of tropical and
sub-tropical fruits and vegetables. Westport, CT: AVI Publishing Co.
Peppelenbos HW, Deell JR, and Prange RK (2003) Postharvest physiology of fresh
fruits and vegetables. In: Ramaswamy HS, Vijaya Raghavan GS, Chakraverty A, and
 Controlled Atmosphere Storage: Applications for Bulk Storage of Foodstuffs 307

Mujumdar AS (eds.) Handbook of postharvest technology: cereals, fruits, Relevant Websites

vegetables, tea, and spices, pp. 455–484. New York: CRC Press.
Silliker JH, Woodruff RE, Lugg JR, Wolfe SK, and Brown WD (1997) Preservation of
refrigerated meats with controlled atmospheres: treatment and post-treatment effects
of carbon dioxide on pork and beef. Meat Science 1(3): 195–204.
Thompson AK (2010) Controlled atmosphere storage of fruits and vegetables, 2nd ed.
London: CAB International.
http://atmosferaprotectora.es/qc-qa-of-modified-atmosphere-packaging-for-extended-
food-shelf-life/regulations – Modified atmosphere packaging. Regulations covering
modified atmosphere packaging.
http://www.fda.gov/Food/GuidanceRegulation/RetailFoodProtection/FoodCode/
ucm188201.htm – FDA Food Code 2009. Annex 6-Food processing criteria.
 Controlled Atmosphere Storage: Effect on Fruit and Vegetables
A Valdez Fragoso and H Mújica-Paz, Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, Mexico
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Controlled atmosphere (CA) storage of fruits and vegetables
refers to storage in atmospheres with reduced levels of oxygen
and/or relatively high carbon dioxide concentrations, at low
temperature and high relative humidity (RH). The modified
atmospheres are created in an air tight room, and they are
continuously monitored and maintained throughout the stor-
age period.
CA storage is an appropriate and practical technology for
prolonging the shelf life and maintaining the quality of fruits
and vegetables on a large scale. CA storage reduces the meta-
bolic activity of the produce, allowing the control of ripening
and senescence, which reduces weight loss, delays softening
and anthocyanin formation, inhibits browning and decay, and
favors flavor and chlorophyll retention.
CA storage is most effective on climacteric fruit such as
apples, pears, apricots, mangos, tomatoes, bananas, and
papayas, and it has great potential for preserving vegetables
with high respiration rates. It is less effective on nonclimacteric
fruit and horticultural products with slow respiration rates.
Climacteric fruits should be subjected to CA at the earliest
possible stage after harvest. Nonclimacteric fruits, such as pine-
apples, grapes, and limes, have greater flexibility in the after-
ripening process.
Oxygen concentration is lowered to the desired level in the
storage room by first flushing with nitrogen gas or liquid
nitrogen, and then injecting carbon dioxide from a gas gener-
ator. The overall result is that oxygen concentration declines
and carbon dioxide concentration increases in the atmosphere
around the stored commodities.
CA Storage Variables
The response of fruits and vegetables to CA storage varies with
species, cultivar, growing conditions before harvest, prestorage
treatments, maturity or ripeness stage at harvest, ethylene sen-
sitivity, concentration of gasses, temperature in the storage
room, and storage time.
The distinctive characteristic of the CA technique is that the
storage variables must be rigorously controlled. Thus, to
achieve optimum storage conditions for fruits and vegetables,
the main variables to be considered are temperature; RH; the
concentrations of oxygen, carbon dioxide, and ethylene; and
storage duration (Figure 1).
Temperature
Cold storage is the most used method for maintaining the
quality of fruits and vegetables. Lower temperatures lead to
slower respiration rates and reduce other metabolic or degra-
dation reactions. Temperatures are usually kept between 1 and
3  C in CA storage, but the accurate optimal temperature
308 Encyclopedia of Food and Health
depends on the produce and cultivar, as well as other charac-
teristics of the produce.
The storage of fruits and vegetables at the lowest tem-
peratures would seem to be the most beneficial for long-
term storage. However, some products, especially tropical
and subtropical fruits, show cold stress (termed ‘chilling
injury’) when exposed to low but not freezing temperatures.
Chilling injury implies physiological and biochemical dis-
orders. It causes membrane breakdown and the release of
metabolites, such as amino acids, sugars, and mineral salts,
and enzymes from cells, resulting in enzymatic reactions
and the development of harmful microorganisms. Some
chilling injury symptoms are surface pitting, discoloration,
internal breakdown, flesh browning, failure to ripen, loss of
flavor, and decay (Table 1). The severity of the injury
increases with lower temperatures and longer durations of
chilling exposure.
The beneficial effects of using low-oxygen and high-carbon
dioxide atmospheres to reduce the development of chilling
injury have been reported. The effectiveness of CAs in reducing
chilling injury varies with the commodity, maturity, gas com-
position, and, above all, the storage temperature. The critical
temperature for chilling injury varies with the commodity and
cultivar. For instance, it occurs at temperatures below about
12–13  C in tropical produce and close to 0  C in the majority
of the other products.
Relative Humidity
The RH is also an essential CA storage factor to maintain the
quality of fruits and vegetables. Most of these products should be
maintained at high RH in the CA room, close to the saturation
point (95%), to delay the ripening of different commodities; to
maintain the freshness, flavor, and commercial weight of the
commodities; and to restrict the development of chilling injury
symptoms in sensitive tropical fruits.
However, low RH in the storage room induces the loss of
moisture from living tissues (transpiration) that, in turn, causes
weight loss in the stored fruits and vegetables. These changes
deteriorate the produce appearance (color), texture (shrinking,
wrinkling), juiciness, flavor, and nutritional quality. The impact
of RH on the water loss of the stored product is strongly related to
the temperature and air velocity in the storage room. Other
factors, such as variety and maturity, are also important, however.
On the other hand, at high RH (100%), water condensation
on the product may occur, providing an ideal environment for
the growth of decay organisms. An RH between 85% and 95%
is recommended for the storage of fresh produce, in order to
restrict weight loss and microbial spoilage. Apples, cherries,
peaches, pears, plums, apricots, lemons, berries, litchi, and
avocados are among the fruits that need high humidity levels,
and the vegetables that require these conditions include aspar-
agus, broccoli, cabbage, carrots, cauliflower, celery, beans,
peas, radishes, and corn.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00197-5
 Controlled Atmosphere Storage: Effect on Fruit and Vegetables 309

0 −10 °C
T controller
08 °C
Ethylene CO2
absorber scrubber
scrubber

90−98%
RH controller
93.3
95.5 %

Gas
controller

O2

Air
Nitrogen generator
Figure 1 Cold storage room.

Table 1 Possible effects of CA storage conditions on fruits or vegetables

Effect of storage condition

Storage condition Optimal conditiona Out-of-range condition

Decrease temperature • Minimizes metabolic activity Very low temperature causes:

• Minimizes respiration rate • Chilling damage
• Freezing damage
• Browning
• Decay
Decrease O2 level • Reduces aerobic respiration rate Very low concentrations induce:
• Reduces degradation of color, texture, flavor, and vitamins • Fermentation
• Reduces production rate and action of ethylene • Off-flavor
• Delays ripening of climacteric fruit • Alcoholic taste
• Failure to ripen
Increase CO2 level • Minimizes aerobic respiration Very high concentrations induce:
• Inhibits the effect of ethylene • Fermentation
• Delays ripening • Drowning of tissue
• Reduces discoloration due to enzymatic reactions • Softening
• Decreases the production of some organic volatiles • Off flavor
• Reduces the rate of breakdown of pectin and chlorophyll • Decay
• Retards fungal growth on the crop
a
Optimal and out-of-range O2 and/or CO2 concentrations depend on the commodity, cultivar, duration of storage, and interactions between O2 and CO2.

To assure the beneficial effects of CA storage, the RH needs to

be monitored and controlled. Humidity is monitored with a
hygrometer or psychrometer, and it is controlled by different
means: operating a humidifier in the produce environment,
wetting the storage room floor, regulating air movement, main-
taining a difference between the air and the evaporator of
0.5–1  C. At high RH, a small fluctuation in temperature can
induce water condensation on the product surface and promote
decay.
Oxygen and Carbon Dioxide
It has been demonstrated that, when the storage conditions of
fruits and vegetables change from the normal air composition
(78% N2, 21% O2, and 0.03% CO2) to an environment with
low concentrations of O2 (1–10%) and high concentrations of
CO2 (1–20%), a reduction in the rates of metabolic activity,
respiration, and ethylene production is induced. The effects of
reduced oxygen and elevated carbon dioxide on metabolic
310 Controlled Atmosphere Storage: Effect on Fruit and Vegetables

changes and respiratory activity are additive. The prime result
of the metabolic modification is that the stored commodity
maintains the quality characteristics (color, firmness, soluble
solids, acidity, vitamins, and freshness) for a longer storage
time. In addition, properly maintained levels of O2 slow
down microbial deterioration and inhibit enzymatic browning
for some products, such as apples, lychees, and bananas.
However, storage in a CA environment may have adverse
effects at too high carbon dioxide concentration (e.g., >3% for
red delicious apples and apricots; >30% for cherries) or too
low O2 concentration (e.g., <1% for avocados and bananas). If
the O2 and CO2 levels are not tolerated by fruits and vegeta-
bles, physiological disorders and loss of product may occur.
For instance, low O2 concentration changes the respiration of
apples, pears, and tomatoes from aerobic to anaerobic, pro-
ducing an accumulation of alcohols and aldehydes, which are
responsible for undesirable flavors. The extent of these injury
symptoms is also a function of exposure time and temperature.
On the other hand, elevated CO2 concentrations (>5%)
may affect the anthocyanin stability and color in red fruits
such as strawberries, or it may inhibit the enzymatic activity,
due to the high solubility of CO2 in water and the consequent
generation of carbonic acid and lowering of pH. Thus, the
success of CA storage technology depends on convenient levels
of gas being achieved and maintained in room storage.
of leaves in Brassica species; softening, pitting, and develop-
ment of off-flavor in peppers and watermelons; and browning
and discoloration in eggplant pulp.
The removal of ethylene from the CA room can retard the
development of undesirable disorders. Preventing ethylene
accumulation or ensuring an environment free of ethylene
can be accomplished through the ventilation of the cold
room, through the use of oxidizing agents (potassium perman-
ganate or ozone), or through the filtration of the air.
Recommended CA Storage Conditions
The inherent variability of fresh commodities and their
responses to storage conditions complicate the establishment
of the optimal CA. Tables 2 and 3 show recommended tem-
perature, RH, and oxygen and carbon dioxide concentrations
for the storage of several fruits and vegetables. However, the
recommended CA storage conditions found in literature for a
specific fresh produce should be refined through experimenta-
tion, empirical observations, and mathematical modeling.
Table 2 Controlled atmosphere conditions for some fruits

Fruits T ( C) RH (%) O2 (%) CO2 (%)

Ethylene
Apricot 0.5 to 0 90–95 2–3 2–3
Ethylene (C2H4) is a natural gaseous plant hormone, which is Apple 0–1 90–95 1–3 1–4
produced by fresh fruit and vegetables and released into the Avocado 5–13 90–95 2–5 3–10
atmosphere around the fresh product. It increases the respira- Banana 12–15 90–95 2–5 2–5
tory activity of most fruits and vegetables, which, in turn, Fig 1 to 0 90–95 5–10 15–20
Guava 5–15 90–95 5–8 2.5–5
determines the evolution of their physical and chemical char-
Kiwifruit 0 90–95 1–2 3–5
acteristics (appearance, texture, taste, and color) and, conse-
Lemon 12–14 90–95 5–10 0–10
quently, their storage-life. Mamey 1–14 90–95 5–10
Controlling ethylene gas in the storage room preserves the Mango 12–14 90–95 5–10 0–10
commodity quality parameters and freshness, and it extends Orange 3–8 90–95 5–10 0–5
the life cycle, allowing the produce to be maintained for a Pineapple 7–13 85–90 3–5 5–8
longer storage time. Prickly pear 6–8 90–95 2 2–5
The rate of ethylene production in fruits undergoing ripen- Mamey sapote 10–15 70–80 10–15 5–10
ing is determined by the ability of the fruit to synthesize 1- Strawberry 4 90–95 5 15
aminocyclopropane-1-carboxylic acid (ACC) and to convert
ACC to ethylene, which is controlled by the activity of ACC
synthase and ACC-oxidase. Table 3 Controlled atmosphere conditions for some vegetables
The ethylene-releasing rate of commodities is highly depen-
dent on the temperature and the concentrations of oxygen and Vegetables T ( C) RH (%) O2 (%) CO2 (%)
carbon dioxide. For instance, the optimum temperature for
ethylene production is 30  C for apples, 32  C for some plum Asparagus 0–1.5 95–100 7 7
Beans, green 8 95 3 3
cultivars, and 20  C for peaches, nectarines, and pears.
Bell peppers 7–14 90–95 2–3 5
The O2 and CO2 concentrations in the fruit play important
Broccoli 0 95–100 1–2 5–10
roles in the biosynthesis and action of ethylene. Ultra-low Cauliflower 0 95–98 2 5
oxygen and elevated carbon dioxide concentrations inhibit Celery 0 98–100 2–4 3–5
the activity of ACC synthase and ACC oxidase and suppress Cucumbers 10–13 95 3–5 10
ethylene production in fruits such as apples and pears. Garlic 0 65–70 1–3 5–15
Ethylene induces the quality attributes of fruits and vegeta- Jicama 13–18 65–70 5–10
bles: color development and softening in apples and pears, the Lettuce 0–5 98–100 1–3 7–10
greening of citrus, and increases in soluble solids, organic Mushrooms 0 95–98 3 10
acids, aroma, and flavor in honeydew melons. However, Onions 0 65–70 3 5–7
Peas 0 95–98 2–3 2–3
reduced produce quality can be related to the ethylene hor-
Radish 0 95–100 1–2 2–3
mone. For example, this hormone promotes chlorophyll Tomatoes 13–15 90–95 4 5
destruction in lettuce and cucumbers; yellowing and dropping
 Controlled Atmosphere Storage: Effect on Fruit and Vegetables
Conclusions
To date, controlled atmosphere storage is the unique technol-
ogy that can assure long-term storage of fresh fruits and vege-
tables. Quality of fruits and vegetables is maintained through
the application of specific CA storage conditions to each com-
modity, and control of the gas composition, temperature, and
relative humidity of the environment. In addition to these
factors, metabolism changes of fruits and vegetables are been
considered to establish the optimal storage conditions. This is
leading to the development of interactive storage systems, the
next generation of storage systems.
See also: Apples; Ascorbic Acid: Properties, Determination and Uses;
Avocado; Controlled Atmosphere Storage: Applications for Bulk
Storage of Foodstuffs; Flavor Enhancers: Characteristics and Uses; pH:
Principles and Measurement.
Further Reading
Herregods MC (1999) Storage of fruits and vegetables. In: Gerasopoulos D (ed.)
Post-harvest losses of perishable horticultural products in the Mediterranean
region, pp. 3–9. Chania: CIHEAM. (Cahiers Options Méditerranéennes; n. 42).
http://om.ciheam.org/om/pdf/c42/CI020452.pdf.
Kole NK and Prasad S (1994) Respiration rate and heat of respiration of some fruits
under controlled atmosphere conditions. International Journal of Refrigeration
17(3): 199–204.
311
Nguyen TA, Verboven P, Schenk A, and Nicolaı̈ BM (2007) Prediction of water loss
from pears (Pyrus communis cv. Conference) during controlled atmosphere
storage as affected by relative humidity. Journal of Food Engineering 83(2):
149–155.
Paull ER (1999) Effect of temperature and relative humidity on fresh commodity quality.
Postharvest Biology and Technology 15(3): 263–277.
Payasi A and Sanwal GG (2010) Ripening of climacteric fruits and their control. Journal
of Food Biochemistry 34: 679–710.
Polderdijk JJ, Boerrigter HAM, Wilkinson EC, Meijer JG, and Janssens MFM (1993)
The effects of controlled atmosphere storage at varying levels of relative humidity on
weight loss, softening and decay of red bell peppers. Scientia Horticulturae
55: 315–321.
Saltveit ME (2003) Is it possible to find an optimal controlled atmosphere? Postharvest
Biology and Technology 27: 3–13.
Sánchez-Mata MC, Cámara M, and Dı́ez-Marqués C (2003) Extending shelf-life and
nutritive value of green beans (Phaseolus vulgaris L.), by controlled atmosphere
storage: macronutrients. Food Chemistry 80: 309–315.
Sevillano L, Sanchez-Ballesta MT, Romojaro F, and Floresc FB (2009) Physiological,
hormonal and molecular mechanisms regulating chilling injury in horticultural
species. Postharvest technologies applied to reduce its impact. Journal of the
Science of Food and Agriculture 89(4): 555–573.
Thompson AK (2010) Controlled atmosphere storage of fruits and vegetables, 2nd ed.,
pp. 11–24. UK: CAB International.
Relevant Website
http://postharvest.ucdavis.edu/research/ – University of California: Postharvest
Technology.
http://www.ca-storage.com/ – Fruitong Controlled Atmosphere Storage Solution
Provider.
http://www.van-amerongen.com/ – Van Amerongen: CA Technology.
http://www.agroripe.com/ – Agroripe: Advanced Post-Harvesting Technologies.
 Convenience Food
TA Brunner, Bern University of Applied Sciences, Zollikofen, Switzerland
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Convenience food is one of the major food trends in Western
societies. However, it is not a new phenomenon. In Ancient
Rome, the urban population mainly ate convenience food. The
Romans did not even have a choice; their apartments did not
include a kitchen so they had to obtain their food from cook
shops in the streets. One reason for such a lifestyle was the high
cost of firewood; a more practical reason was to prevent fires that
could destroy whole quarters or cities. Today, there are different
reasons for using convenience food: People want to save time
and minimize their physical and mental efforts, and an increas-
ing number of people no longer know how to cook a meal from
scratch, so they are dependent on convenience food products.
Definition
There are many definitions of convenience food in the litera-
ture. However, most of them include the three aspects of time,
effort, and skills. An early definition from 1979 by Traub and
Odland defines convenience food as: “Any fully or partially
prepared foods in which significant preparation time, culinary
skills, or energy inputs have been transferred from home
kitchen to the food processor or distributor.” Today, the defi-
nition of convenience food not only includes the preparation
stage of planning, buying, and preparing meals but also incor-
porates the following stages of consumption and cleanup.
The meaning of using convenience food products has chan-
ged dramatically over the decades. In 1950, for example, Haire
presented two identical shopping lists to his participants, with
the exception of ground coffee on one list and instant coffee on
the other. Participants had to characterize the women who
bought the items on the lists. Women who bought instant
coffee were described as lazy, unable to manage their house-
hold, unthrifty, and just not good wives in general. It would be
very interesting to replicate this study today; the results would
certainly be quite different.
Classification Systems
Several different classification systems for convenience foods
have been proposed, for example, classifying them on a scale
ranging from zero convenience to full-service convenience, on
the basis of the level of prior processing, or on the level of
preparation required after industrial processing. Later classifi-
cation systems have become more complex, for example, a
matrix with two levels of prior processing and three levels of
preparation and an even more complex matrix with four levels
of shelf life and four levels of preparation. All of these classifi-
cation systems were developed theoretically, and some of them
were subsequently validated with consumer data. In the
312 Encyclopedia of Food and Health
author’s own research, the opposite approach was used. They
started with food frequency data from consumers and con-
ducted a principal component analysis to identify convenience
food categories. Four components emerged: (1) highly pro-
cessed food products (e.g., frozen ready-made meals), (2)
moderately processed food products (e.g., chilled fresh tortel-
lini), (3) single components (e.g., marinated meat), and (4)
salads (e.g., cut and washed salad in bags).
Most of these classification systems show that the overall
category of convenience foods is very heterogeneous, with
some products being only slightly processed and others being
heavily processed. There is a continuum of products being pro-
cessed, and every convenience food product can be indexed at
some level of processing. What level of processing is required for
consumers to label a product a convenience food is not clearly
defined. Today, hardly anybody would label bread, yogurt, and
cheese convenience foods, although according to most defini-
tions, they fall into this category. There are other products, such
as gravies and soups, that some consumers would call conve-
nience foods, while other consumers would not. And then, of
course, there are products like ready-to-heat meals or frozen
pizzas that every consumer considers convenience foods.
Overriding Trends
A major trend fostering convenience food consumption that is
often discussed in the literature is the increasing participation of
women in the workforce. The reason seems plausible: dual-
income households have higher incomes but less time. Thus,
they are willing to trade some of their money back into time by
purchasing convenience food products. Surprisingly, most stud-
ies investigating the relationship between female participation
in the workforce and convenience food consumption have
found no evidence for a positive correlation. Only by applying
attitudinal measures it was possible to find some evidence. For
example, it was shown that meal preparers with a workload of
more than 30 h per week were more convenience-oriented than
those with a workload of less than 9 h a week.
Another overriding trend that results in higher convenience
food intake is the increasing number of single-person house-
holds that can be observed in most Western countries.
Although single people have more time available, they are
not inclined to use that time cooking for themselves. An
unpublished analysis by the author confirms that people living
in single-person households consume more highly and mod-
erately processed convenience food products than people liv-
ing together with at least one other person. For salads, no
significant difference was found, indicating that almost every-
one buys cut and washed salads in bags. Finally, people from
multiperson households consume more single components
than those living in single-person households. This seems
plausible, since with single components, one still has to cook
http://dx.doi.org/10.1016/B978-0-12-384947-2.00198-7

and prepare a meal. Together with less-structured mealtimes
and the replacement of traditional meals with snacks for all
households, the increase in single-person households gives
convenience foods a strong boost.
Whereas the causalities for the effects of the increasing
female participation in the workforce and the increasing num-
ber of single-person households are straightforward, the cau-
sality for the decline of cooking skills is not clear. Do people
use convenience foods because they lack cooking skills, or did
the emergence of convenience foods cause the decline in cook-
ing skills? The fact is that today, many consumers lack cooking
skills and therefore depend on convenience foods. The author’s
own research shows that a lack of cooking skills is indeed a
significant driver for convenience food consumption. The
more people lack cooking skills, the more they consume highly
and moderately processed food items. For salads and single
components, cooking skills did not reach significance, which
makes sense, as foods from these two categories require much
less skill if one wants to prepare them from scratch than foods
from the former two categories.
Drivers of Convenience Consumption
To identify the drivers of convenience food, the author con-
ducted a comprehensive study. A sample of N ¼ 918 participants
completed a survey that included 17 common convenience food
items and 14 potential drivers and seven sociodemographic
questions. Usage of the various convenience food items differed
greatly ranging from salad cut and/or washed, which was con-
sumed on average 73 times per year, to instant pasta, which was
only used 2.6 times per year. As mentioned in the preceding text,
convenience food items were grouped into four categories based
on a principal component analysis: highly processed items,
moderately processed items, single components, and salads.
For each category, the potential drivers were analyzed separately
using regression analysis.
Age is the most important driver for convenience food
consumption. The older the consumer, the less convenience
food is eaten. This was true for all four categories. There might
be two reasons for this: First, after retirement, people have
more time available and might spend that time cooking, and
second, older people are more accustomed to cooking from
scratch from their younger days and may simply continue this
habit. Another important driver is an affinity for naturalness.
People who pay attention to the naturalness of food do not like
processed food items. Naturalness was significant for all
categories but salads. This seems plausible since cutting and
washing do not affect the naturalness of a food item and, thus,
people concerned with food’s naturalness can buy these
convenience products. Another strong driver is nutritional
knowledge. This was significant for highly processed foods,
single components, and salads. People with a high level of
nutritional knowledge are capable of evaluating what is good
for their health. Most highly processed foods and single com-
ponents contain many additives and preservatives that con-
cerned consumers might judge as unhealthy. For salads, the
reason might be the cut and/or washed salad’s sensitivity to
fungi and bacteria. Most moderately processed foods, on the
other hand, are fresh products (e.g., readily bought sandwiches
Convenience Food 313
and warm pizza bought/delivered) that are equivalent to self-
prepared foods. Therefore, consumers with a high level of
nutritional knowledge have no reason not to buy them.
Three more drivers turned out to be significant for two of the
four categories: the presence of children in a household, a desire
to avoid waste, and cooking skills. Having children reduces the
consumption of highly and moderately processed food items. In
these categories, one finds the typical ready-made meals that are
convenient for single-person households. In a family house-
hold, it might be more efficient to prepare meals from scratch
because of the higher number of persons living in these house-
holds. Another reason might be that parents are concerned
about their children having health issues and therefore try to
avoid processed foods containing a great deal of additives and
preservatives. People who try to avoid food and packaging waste
consume less moderately processed food items and less single
components. For moderately processed foods, the issue of food
waste may be the motivating factor, as these foods usually come
in portions that are too large for one person. For single compo-
nents, it might be the packaging waste, as most of these items are
extensively wrapped. A lack of cooking skills is again a driver for
highly and moderately processed food items. People with a
lower level of cooking skills are not able to prepare the foods
in these two categories by themselves and therefore depend on
processed foods.
Each category features several drivers that are specific to it.
For highly processed food items, these are working status and
sociability. People working only part-time or not working at all
consume more highly processed foods than people working
full-time. This phenomenon has been observed in other stud-
ies as well. For example, a snacking consumer segment was
identified that was less likely to be well educated and more
likely to be unemployed. Sociability refers to an attitude of
enjoying eating together with others. The more people display
such an attitude, the fewer highly processed food products they
consume. This is because in most households, it is still not
appropriate to serve ready-made meals when inviting, for
example, friends for dinner. For moderately processed food
items, education is a specific driver. The more well educated
people are, the more moderately processed food products they
consume. This seems plausible since well-educated people are
likely to have busy jobs but still want to eat rather healthy.
Therefore, they go for the moderately processed foods that are
freshly prepared, like a sandwich or other to-go foods. For
single components, there are three specific drivers: value for
money, household size, and physical effort. Value for money
refers to a smart shopper mentality, that is, consumers who try
to get the best value for their money. It seems that single
components, such as marinated or crumbled meat and fish,
have good value for their money since smart shoppers buy
more of these food items. Household size is also positively
related to the consumption of single components. This cate-
gory also contains foods like frozen french fries and croquettes,
things that are prepared easily and things that children really
like. This might explain the correlation with household size.
Physical effort is the third specific driver for single compo-
nents. Everyone who has prepared french fries from scratch
can understand why people who want to minimize the phys-
ical effort of preparing meals buy more foods from the single
components category. Finally, two specific drivers emerged for
314 Convenience Food
salads. Consumers who think that convenience foods are too
expensive for what one gets buy fewer convenience salads. It
seems that, particularly for salads, the surcharge does not seem
justified. Some people are not willing to pay extra for merely
having their salad already cut and washed. The second driver
for salads is an attitude of considering the kitchen to be the
woman’s domain. The more people think that housekeeping is
the woman’s responsibility, the more convenience salads they
consume. Salads might be regarded as a rather female food
product, and therefore, people with such an attitude might
want to avoid preparing these foods.
It is also just as interesting to look at the potential drivers that
did not turn out to be significant. Surprisingly, variables related
to time and effort, such as time-saving, time pressure, and
mental effort, did not predict convenience food consumption,
with the exception of the physical effort for single components,
as discussed earlier. The reason for this might be that conve-
nience foods are now omnipresent. They have crept into our
daily lives without us noticing; think of gravy, for example.
Today, everyone uses convenience food products. Therefore,
these time- and effort-related variables do not differentiate
consumers’ convenience consumption. Of course, this does
not mean that time and effort are not important reasons to use
convenience foods; it is just that they do not differentiate con-
sumers and therefore do not turn out to be significant predic-
tors. Instead of having reasons to use convenience foods, it
seems that all consumers use them and only consumers who
have explicit reasons not to use these items, such as an affinity
for naturalness and concerns resulting from a high level of
nutritional knowledge, will abstain from consuming them. In
other words, the standard is to use them, and people only avoid
using such products if they have reasons not to consume them.
Besides these time- and effort-related variables, three more
potential drivers did not prove significant. For cooking involve-
ment, the same reasoning mentioned earlier might apply.
Regardless of the level of cooking involvement, convenience
food products are used. Income was also insignificant. One
explanation could be that many convenience foods are not
really that expensive. For example, a ready-made frozen pizza
is usually even cheaper than a self-made pizza when adding up
the costs of the ingredients. If one includes the preparation time
required to cook a pizza from scratch, it is even worse. More
surprising is that concerns about a healthy diet did not predict
convenience food consumption. Many researchers found that
consumers have an unfavorable image about convenience food.
However, it seems that when it comes to a single convenience
food product, consumers might not judge it to be unhealthy;
they only have an unhealthy image when they think about the
marketing category of convenience food in general.
Consumer Segments
In 2007, Buckley and colleagues conducted a comprehensive
survey to identify convenience food lifestyle segments. A total of
N ¼ 779 respondents were included in the cluster analysis that
was run over 20 factors related to convenience food consump-
tion. The authors identified four convenience food lifestyle
segments: (1) food connoisseurs, (2) home meal preparers, (3)
kitchen evaders, and (4) convenience-seeking grazers.
For food connoisseurs, convenience is not important. They
like to experiment in the kitchen, and eating for them is a social
event that is done together with family or friends. They eat
proper meals and do not snack in between these meals. They
like to take their time when cooking, they are not in a hurry,
and they do not feel time pressure. Therefore, they are less
likely to buy and consume convenience foods. Food connois-
seurs are not very price-sensitive; in fact, they like to spend
money on food. For them, cooking is not only a woman’s task;
male food connoisseurs like to cook either alone or together
with their wives. Consumers belonging to this segment think
that convenience foods are rather unhealthy and not very
attractive in terms of their value for the price. This segment
comprises 26% of all consumers, has an average age of about
40 years, and includes the highest proportion of men.
Home meal preparers are similar to food connoisseurs but
even less convenience-oriented. They have plenty of time
resources to shop and prepare foods and to clean up after a
meal. This is the segment that purchases the fewest convenience
food products; they prefer preparing a meal from scratch. Home
meal preparers are well organized when it comes to shopping
for food. They pay attention to freshness, and they try to get
good value for the price they ought to pay. Meal preparation
activities are a family endeavor as is the intake of meals. A
microwave is usually not found in these households. As with
food connoisseurs, home meal preparers regard convenience
food products as rather unhealthy and expensive. This segment
makes up 25% of consumers, most of whom are older and
retired, and is more or less balanced in regard to gender.
Kitchen evaders comprise the first of two highly convenience-
oriented segments. They use convenience foods to make their
lives easier and to avoid food waste. With convenience food, they
just buy what they need so nothing is spoiled. If they cooked
from scratch, they would have ingredients left that they would
have to then throw away. Kitchen evaders are busy people who
are always under time pressure. Therefore, they do not want to
plan their meal preparation activities; they decide what to eat
spontaneously while they are shopping. They are neither inter-
ested in product information, nor do they want to try new foods.
Instead of having proper meals, they often snack during the day.
They are used to eating alone and display a high level of indi-
vidualism in their consumption behavior. Kitchen evaders have a
positive attitude toward convenience food products and think
that these products offer good value for their price, are healthy,
and save them a lot of time. This segment consists of 16% of all
consumers, is below the population’s average age, and is rather
balanced when it comes to gender.
The last segment is the convenience-seeking grazers. Similar
to kitchen evaders, they are very convenience-oriented and are
also similar in many other aspects. Unlike kitchen evaders,
they are organized in their meal preparation activities and
more price-sensitive. Although this segment has the highest
proportion of females, convenience-seeking grazers are more
likely to think that the kitchen is the woman’s task. When it
comes to the trade-off between freshness and convenience,
they opt for convenience. Similar to kitchen evaders, they are
time-stressed, use the microwave, often eat alone, and snack in
between meals. Interestingly, convenience-seeking grazers per-
ceive convenience foods as the least beneficial for time-saving.
However, they have a positive attitude toward convenience

foods and think that they have good value for their money.
One-third of consumers fall into this segment, and most of
them are between 30 and 50 years of age.
Healthy Convenience Food
Convenience food has a reputation of being unhealthy. How-
ever, the food industry has recently started combining the
convenience food trend with the trend of healthy eating by
producing healthy convenience foods and healthy ready-made
meals. In a tasting test of two rather healthy ready-to-heat
meals – one with chicken and rice and the other with salmon
and pasta and both with a large portion of vegetables – overall
liking was found to be the strongest predictor of the likelihood
of buying for both meals. Sociodemographic variables were
only relevant for the salmon meal, whereas for the chicken
meal, no such effects were observed. It seems that consumers
are not ready to make trade-offs when it comes to taste. The
food industry’s challenge will be to develop healthy and tasty
convenience foods, not just one or the other.
Another study segmented consumers based on their health-
related motive orientations and found two segments to be
particularly inclined to choose ready-made meals. For the
first segment, vitality and energy were the main terms used to
describe health. They want to keep their bodies in good con-
dition to live active lives and to explore new things. The second
segment perceives health mainly in terms of achievement and
outward appearance. They want to look good, stay slim, and be
successful. Individuals from both segments were rather young,
with most of them having no children. This paints a picture of
who will be buying healthy convenience food products. It is
the young, busy consumers who are successful in what they are
doing and who lead active lives.
Convenience food products will surely become healthier in
the years and decades to come. Consumers’ demand for
healthy food is steadily rising, and the trend toward increased
convenience will not stop. The ability to bring these two major
trends together will be decisive for the success of a particular
convenience food producer. Consumers will benefit from this
development by obtaining healthy food and saving time,
which will improve their work–life balance.
See also: Adolescent Nutrition; Appetite Control in Humans: A
Psychobiological Approach; Chilled Foods: Effects on Shelf-life and
Sensory Quality; Chilled Foods: Modified Atmosphere Packaging;
Chilled Foods: Packaging Under Vacuum; Chilled Foods: Principles;
Convenience Food 315
Consumer Protection Legislation; Controlled Atmosphere Storage:
Applications for Bulk Storage of Foodstuffs; Controlled Atmosphere
Storage: Effect on Fruit and Vegetables; Cooking: Domestic
Techniques; Obesity: The Role of Diet; Preservation of Foods;
Preservatives: Food Use; Stabilizers: Types and Function.
Further Reading
Ball SD (1992) Fast-food operations and their management. Cheltenham: S. Thornes.
Brewis J and Jack G (2005) Pushing speed? The marketing of fast and convenience
food. Consumption, Markets and Culture 8: 49–67.
Brunner T, Van der Horst K, and Siegrist M (2010) Convenience food products. Drivers
for consumption. Appetite 55: 498–506.
Buckley M, Cowan C, McCarthy M, and O’Sullivan C (2005) The convenience consumer
and food-related lifestyles in Great Britain. Journal of Food Products Marketing
11: 3–25.
Buckley M, Cowan C, and McCarthy M (2007) The convenience food market in Great
Britain: convenience food lifestyle (CFL) segments. Appetite 49: 600–617.
Candel M (2001) Consumer’s convenience orientation towards meal preparation.
Conceptualization and measurement. Appetite 36: 15–28.
De Boer M, McCarthy M, Cowan C, and Ryan I (2004) The influence of lifestyle
characteristics and beliefs about convenience food on the demand for convenience
food in the Irish market. Food Quality and Preference 15: 155–165.
Geeroms N, Verbeke W, and Van Kenhove P (2008) Consumers’ health-related motive
orientations and ready meal consumption behaviour. Appetite 51: 704–712.
Hwang A (2010) Ready-to-eat foods: microbial concerns and control measures. Boca
Raton, FL: Taylor & Francis.
Kerry JP (2011) Processed meats: improving safety, nutrition and quality. Woodhead
Publishing series in food science, technology and nutrition, no. 211. Oxford, UK:
Woodhead Publishing.
Knipe CL and Rust RE (2010) Thermal processing of ready-to-eat meat products. Ames,
IA: Wiley-Blackwell.
Menlove A (2002) Ready meal technology. Surrey, UK: Leatherhead.
Olsen NV, Menichelli E, Sorheim O, and Naes T (2012) Likelihood of buying healthy
convenience food: an at-home testing procedure for ready-to-heat meals. Food
Quality and Preference 24: 171–178.
Van der Horst K, Brunner T, and Siegrist M (2010) Ready-meal consumption:
associations with weight status and cooking skills. Public Health Nutrition
14: 239–245.
Van der Horst K, Brunner T, and Siegrist M (2011) Fast food and take-away food
consumption are associated with different lifestyle characteristics. Journal of Human
Nutrition and Dietetics 24: 596–602.
Relevant Websites
http://www.bbc.com/news/?ocid¼global-news-pinned-ie9 – BBC News.
http://www.bbc.com/news/?ocid¼global-news-pinned-ie9 – BBC News France.
http://www.bbc.com/news/?ocid¼global-news-pinned-ie9 – BBC News India.
http://www.oxforddictionaries.com/definition/english/convenience-food – Oxford
Dictionaries.
http://www.sfa.org/ – Snack Food Association.
http://www.swissconvenience.ch/ – Convenience Food Association.
http://en.wikipedia.org/wiki/Convenience_food – Wikipedia.
 Cooking: Domestic Techniques
AJ Rosenthal, Oxford Brookes University, Oxford, UK
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from Encyclopedia of Food Sciences and Nutrition, volume 3, pp. 1622–1627, ã 2003, Elsevier Ltd.
Introduction
As with all forms of cooking, domestic cooking is intended to
improve the palatability of the food, making it more appetizing.
Unlike industrial food preparation and catering or food service,
people who may not have any technical knowledge of what is
happening from an engineering or biochemical point of view
carry out domestic cooking in the home. By definition, cooking
raises the temperature of the food, which results in a number of
simultaneous and interrelated processes that influence flavor,
texture, appearance, nutrient content, and food safety. The dif-
ferent techniques for domestic cooking reflect the way in which
the temperature of the food is raised (Figure 1).
Clearly, there are two basic ways in which energy can be
applied to a foodstuff to achieve a rise in temperature. The
traditional route is by contact with a heated medium, which
causes heat to flow to the surface of the food and, subse-
quently, to the center through conduction. An alternative
route is to apply electromagnetic radiation. Of the two types
of electromagnetic radiation commonly used in domestic
cooking, infrared radiant heating (grilling) employs short-
wavelength radiation that is only able to penetrate a couple
of millimeters below the surface of the food. The inner regions
of the food are heated by conduction. Microwaves have longer
wavelengths that are able to penetrate deep into foods, gener-
ating heat in situ, albeit in nodes requiring food to be rotated
during cooking to ensure even distribution of heat.
In many cases, energy transfer during cooking is not via a
single mechanism. For example, ovens absorb and emit infra-
red energy and baked food heat through a combination of
convection and radiation. Similarly, barbecues emit infrared
radiation and generate hot combustion gases, which flow
around the food, heating it by convection.
General Considerations
Surface Heat Transfer
The crucial difference between the different techniques is what
happens at the surface of the food.
When a solid food is placed in a hot fluid, there is a stagnant
layer of fluid around the food. This boundary layer acts as an
insulating barrier that slows the flow of heat from the fluid to
the food.
Air is a good insulator (thermal conductivity, 0.024 W
m 1 K 1); thus, a boundary layer consisting of air slows signifi-
cantly the flow of heat from an oven to the food (Figure 2(a)).
One way of reducing the thickness of this boundary layer is to
agitate the heating medium. In the case of fan-assisted ovens, air is
forced to circulate, thereby impinging on the surface of the food
and physically reducing the thickness of the boundary layer
316 Encyclopedia of Food and Health
(Figure 2(d)). The surface heat transfer coefficient in a fan-assisted
oven is about 10 times greater than in a conventional oven.
Heat travels by conduction through static boundary layers.
Heating media that are better conductors of heat than air will
increase the rate of heat flow to the surface of the food. Water
has a thermal conductivity of 0.573 W m 1 K 1, and the rates
of convective heat transfer during boiling and simmering are,
therefore, considerably faster than in ovens at the same tem-
perature (Figure 2(e)). However, the maximum temperature
attainable with water is limited by its boiling point. Alternative
liquid heating media, such as oil, have thermal conductivities
in the same region and can operate at temperatures around
180  C. Hence, rates of heat flow to the surface in frying are
considerably greater than in boiling. This is also attributable in
part to physical agitation of the boundary layer caused by
generation of water vapor at the surface of the food, which is
in contact with the hot oil (Figure 2(g)).
When saturated steam is used as the heating medium, heat
flow to the surface of the food is simultaneously accompanied
by condensing of the steam. The dramatic volume change dur-
ing condensation results in fresh steam flowing to occupy the
void, thereby maintaining a virtually negligible boundary layer
(Figure 2). However, if the steam is not saturated, the non-
condensing air accumulates at the surface of the food, forming
an insulating layer. In fact, as little as 6% air in steam reduces
surface heat transfer by 90%.
Rate of Heating
During heating, the rate at which heat flows into any food
depends on its shape and size and the temperature difference
between the food and the heating medium as well as the rate of
heat transfer between the food and the heating medium.
Obviously, large surface areas allow more heat flow than
small surfaces. Shapes with a higher surface-to-bulk ratio, such
as spherical foods, will heat quicker than other shapes (e.g.,
slabs). Consequently, considering how rapidly heat penetrates
to the center of two ‘spherical’ potatoes of different sizes,
evidently, it takes longer for heat to penetrate to the center of
a larger potato. There are two reasons for this: greater distance
for heat to flow and a smaller area to mass ratio.
The driving force in any heating operation is the difference
in temperature between the food and the heating medium. The
greater the differences, the faster the rates of heat transfer. In
the case of domestic cooking, foods heat by unsteady-state heat
transfer. That is to say, the temperature difference reduces as
the temperature of the food rises, and hence, the driving force
lessens as the process ensues.
The physical properties of the food and the heating system
contribute to the heating rate. Factors such as how easily the
heat is able to flow through the insulating surface boundary
http://dx.doi.org/10.1016/B978-0-12-384947-2.00199-9
 Cooking: Domestic Techniques 317

Energy transferred by
Energy transferred by contact with a heated:
electromagnetic radiation

Solid Liquid Gas or vapor Typical wavelength

Oil Water Steam Air convection 0.03 mm 300 mm

Atmospheric Pressure Natural Forced

Boiling
Griddling Frying Steaming Pressure Roasting/baking Grilling Microwave
Simmering cooking

All these techniques achieve surface heating: Heat generated

heat flow to center by conduction throughout

Figure 1 Classification of domestic cooking techniques.

Key Convection Conduction through

boundary layer 14
Potato Conduction
Boundary through Electromagnetic 12
layer potato radiation
10
Time (min)

8
6
4
(a) (b) Conventional oven
2
Boiling water
Oil layer 0 Condensing steam
100

Fan-assisted oven
150

200

250

Temperature (°C)

(c) (d) Figure 3 Predicted time for the center of 2 cm diameter potatoes to
reach 80  C.

layer and through the bulk of the food by conduction toward

the center are influential.
There are methods for predicting rates of heating based
(e) (f)
on the physical properties of materials and heating systems.
Vapor Figure 3 illustrates factors that influence the rate of heating.
bubbles
Time taken for a food of fixed shape and size to reach a partic-
ular temperature has been used as a measure of heating rate. In
Figure 3, 2 cm diameter spherical potatoes starting at 5  C have
been used. Identical potatoes are placed into different heating
(g) (h)
media at designated temperatures and the time taken for the
Figure 2 Eight ways to cook a potato: (a) baking, (b) baking with a good core temperature to reach 80  C predicted. The temperature
conductor (e.g., knife) inserted, (c) roasting, (d) forced convection (80  C) has been chosen as a realistic temperature for many
(fan-assisted oven), (e) boiling, (f) steaming or pressure cooking; (g) types of meat and vegetables and one that will achieve gelatini-
frying (chipped), (h) microwaving. zation of starch in a food like a potato.
318 Cooking: Domestic Techniques
Clearly, short cooking times can be achieved in a number of
ways; working at higher temperature achieves more rapid cook-
ing, as does selecting a cooking process with a high surface heat
transfer coefficient. Hence, a fan-assisted oven is faster than
conventional ovens because they reduce the thickness of the
insulating surface boundary layer around the food.
Cooking by Electromagnetic Radiation
Infrared
As with the other traditional forms of cooking, grilling (broil-
ing) causes the surface of the food to heat, and the center heats
by conduction. Absorption of infrared radiation at the surface
is proportional to the difference between the fourth power of
the radiation source temperature and the surface of the food.
However, foods absorb infrared radiation to differing extents
depending on their absorptive capacity (e.g., black body, 1;
perfect reflector, 0; and water, 0.96).
Conventional grilling and barbecuing are restricted by the
fact that radiant energy is supplied from one direction; cooking
of all sides is only achieved by frequently turning the food, for
example, on a spit.
In practice, infrared heating is involved in many cooking
techniques, such as baking and roasting. In these cases, oven
walls heat up by convective heat transfer and then emit infrared
radiation.
Microwaves
Microwaves penetrate deep into most foods; their sinusoidal
electromagnetic waveform causes dipoles (e.g., water) to oscil-
late, which in turn generates heat (Figure 2(h)). As microwaves
pass through a food material, energy is lost; although heating
occurs within the food, it is greatest at the surface. The actual
temperature achieved within the food depends on several fac-
tors, including the frequency of the radiation, strength of the
field, dielectric loss factor of the food, free moisture present,
and the shape of the food.
Quality Changes during Cooking
General Considerations
Arguably, there are only two basic cooking methods: wet and
dry. Wet cooking includes boiling, simmering, steaming,
pressure cooking, stewing, and poaching. During wet cooking,
the surface temperature of the food does not exceed the boiling
point of water (usually 100  C, but up to about 120  C in
domestic pressure cookers); consequently, the surface remains
moist. Dry cooking includes frying (deep and shallow), bak-
ing, roasting (in an oven with natural or added fat), grilling
(broiling), barbecuing, and griddling. The surface temperature
during dry cooking may well exceed 100  C, leading to evapo-
ration of moisture and a dry or crisp surface.
Whatever is being cooked, it is worth bearing in mind that
most foods are biological in origin and many are still respiring
up to the time of cooking. The rise in temperature during
cooking can result in a disruption of structural integrity and
the termination of normal metabolic activity.
We eat a diverse range of foods, each with unique quality
criteria. Since changes that occur during cooking are dependent
on both the temperature achieved and how long food is held at
each temperature, product quality is constantly changing. Most
of the changes taking place during domestic cooking can be
described by first-order Arrhenius kinetics (i.e., after a finite
activation energy is reached, rate of change is proportional to a
rate constant, which is affected exponentially by temperature).
Activation energy and the rate constant vary for different qual-
ity factors; moreover, any particular factor may behave differ-
ently in each food (e.g., thiamine is destroyed more rapidly
during cooking of rainbow trout than in a buffered solution
with the same time–temperature profile). If a food is cooked
on the basis of one quality characteristic (e.g., color or texture)
and if the time–temperature treatment is varied from an estab-
lished procedure, other quality parameters may be suboptimal.
For example, microwaved meat may not develop the same
flavor as conventionally roasted meats.
Textural Changes
No single mechanism is responsible for the texture of foods,
yet changes in protein structure and solubilization of polysac-
charides (and some proteins) are the two primary phenomena
involved.
Heat causes the tertiary structure of proteins to unwind.
Heating for long periods or at high temperatures frequently
results in irreversible changes in tertiary structure, termed dena-
turation, which causes changes in functional properties that may
or may not be desirable. If the protein is highly charged, the
uncoiled amino acid chains tend to repel one another increasing
the affinity for water and enhancing solubility.
If the protein is reasonably close to its isoelectric point, the
amino acid chains tend to attract one another, with hydropho-
bic interactions and hydrogen bonds, forming a network of
chains. This association behavior results in a reduction in the
amount of water associated with protein and the following
consequences: a loss of protein solubility; precipitation of the
protein from solution, a solid structure (thermal setting or
gelation); loss of water-holding capacity accompanied by an
aqueous exudate from the product (often observed when cook-
ing meats); shrinkage as both the aforementioned occur; and
increased opacity of the food (e.g., egg white).
Some proteins undergo reversible thermal transitions when
heated. For example, collagen (and its partially hydrolyzed
derivative, gelatin) is solubilized when heated. Its prevalence
in connective tissue means this has important implications in
the tenderness of cooked meat and is responsible in part for the
softening action of long-duration stewing.
Solubilization is also a process that affects polysaccharide
material between cell walls of plants. Dissolution of pectin
results in softening of plant tissues. Addition of sodium bicar-
bonate enhances solubilization of pectin, through production
of the sodium salt, and displaces calcium ions that are natu-
rally chelated in the structure. Another mechanism commonly
involved in providing rigidity in plant tissues is turgor. Dena-
turation of proteins present in the cell membranes causes
termination of osmoregulation and softening of the tissues.
Deliberate protein denaturation is the aim of blanching, brief

exposure to temperatures around 85  C with the intention of
destroying flavor-modifying enzymes prior to home freezing.
Starch is present in many plant foods, and heating starch
with an ample supply of water leads to gelatinization. The
granules swell as they absorb water and the crystalline regions
are disrupted. This has both a softening effect and a thickening
effect, which is why gelatinized starch provides the viscosity of
many sauces. The light, open texture of baked flour products is
due to the presence of gas or air cells in the prebaked dough or
batter. During heating, the gases expand aided by the genera-
tion of additional gas from chemical leavening agents (e.g.,
sodium bicarbonate) and increased vapor pressure of the water
present. Finally, the liquid matrix is heat-set by denaturation of
proteins and starch gelatinization. In the case of popcorn, the
impervious grain coat acts like the wall of a pressure vessel. As
the grain is heated, the water vapor pressure increases until the
wall ruptures. The vapor flashes off, opening out the endo-
sperm, which then heat-sets.
A crisp outer surface is often associated with dry cooking
because temperatures in excess of 100  C result in moisture
being lost from the surface.
Color and Flavor Changes
With the exception of added colors sometimes used in domes-
tic cooking (e.g., saffron or cochineal), two types of color
change result from domestic cooking: modification of natural
pigments present in the raw materials and browning reactions.
Most natural colors tend to be relatively unstable when
cooked. In addition to these chemical reactions, in wet cook-
ing, loss of natural color may result from leaching of water-
soluble pigments into the cooking medium.
Metalloporphyrin colors are found in foods of both animal
origin and plant origin. Myoglobin, the pigment found in
muscle, undergoes changes color from red purple to brown
when heated; this is as a result of certain amino acids in the
globin protein becoming coordinated with the central iron
atom. Chlorophylls are also prone to lose their color when
heated in acid conditions. The change from bright green to a
grayish green is the result of demineralization of the central
magnesium atom from the molecule. Other natural pigments
exhibit sensitivity to pH. Anthocyanins, for example, change
from red (in acid conditions), to colorless (when neutral), to
blue (when alkaline). This can result in color changes when
heat disrupts cells and allows these water-soluble pigments to
mix with the cooking water.
Browning reactions tend to occur at low levels of available
water. They are, therefore, common in dry cooking where the
surface layers may dry out. Caramelization occurs when sugars
are heated, particularly in the presence of acids or alkalis, and is
important in both sugar and flour confectionery. The most
famous of these reactions is the Maillard browning, which
comprises the reaction between carbonyl groups (as found in
reducing sugars) and amino groups. The reaction gives rise to a
variety of brown compounds and characteristic aromas. Simple
mixtures of one amino acid and one reducing sugar have been
shown to produce distinctive aromas, reminiscent of particular
foods (e.g., glucose and cysteine heated at 180  C for 30 s
smells of puffed wheat, yet after 3 min, it smells of overroasted
Cooking: Domestic Techniques 319
meat). Generally speaking, volatile components are lost during
cooking, and this can result in a loss of the uncooked flavor.
Since the end user carries out domestic cooking, loss of vola-
tiles is not completely wasted and may act as an appetizer.
Sustained heating of dry surfaces can result in carboni-
zation, usually accompanied by smoke. In such circumstances,
the surface browns and then blackens as the food burns; such
products are usually regarded as spoiled.
Nutritional Changes
Although the act of cooking involves raising the temperature of
the food, the term is frequently undifferentiated from other food
preparation procedures, many of which commonly precede
cooking. Peeling and trimming are two such operations that
can lead to substantial loss of available nutrients, by cutting
unsightly portions off the food and throwing them away.
Nutrient loss during cooking is attributable to two basic
routes: thermally induced chemical reactions and leaching of
nutrients into the cooking medium.
Many nutrients are thermally unstable, and when heated,
concentrations fall exponentially with time. Obviously, differ-
ent nutrients have unique rates of destruction. The most sensi-
tive are ascorbic acid (vitamin C) and folic acid, both of which
can be completely destroyed by domestic cooking. Of the
essential amino acids, lysine is the least stable and up to 40%
may be lost by domestic cooking practices. In general, rapid
cooking methods, using high temperatures for short periods or
microwaves, cause less nutrient destruction than long-
duration, low-temperature cooking methods such as stewing.
High-temperature short-time processes, such as frying, can
result in similar levels of vitamin C as in raw potatoes.
In addition to thermal decomposition, nutrients can be lost
through reactions with each other. For example, proteins
will participate in the Maillard reactions, particularly when
e-amino groups are present.
Sodium bicarbonate is sometimes added to vegetables for
its softening effect. Unfortunately, its addition leads to the
destruction of vitamin C as well as chemically modifying pro-
teins, lowering their biological value.
Cooking processes are not entirely detrimental from a nutri-
tional point of view. For example, frying can result in an uptake
of oil, resulting in increased energy density of foods.
Safety Aspects
A common misconception is that domestic cooking is per-
formed purely to improve the gastronomic experience to the
detriment of biological value. In fact, domestic cooking can
make safe products that would otherwise contain harmful or
toxic components.
Harmful components in foods arise in the form of naturally
present toxins (e.g., cyanides in kidney beans and cassava) or
compounds that interfere with digestion, effectively making
the food less nutritious (e.g., trypsin inhibitors found in
many legumes). The presence of pathogenic microorganisms
may lead to infections, while other microorganisms produce
toxins, both of which result in food poisoning. Cooking is
320 Cooking: Domestic Techniques

effective at destroying or removing many of these harmful See also: Arsenic: Properties and Determination; Browning: Non-
components. However, some toxins are relatively heat-stable enzymatic browning; Colors: Properties and Determination of Natural
(e.g., the toxin produced by Staphylococcus aureus) and may not Pigments; Starch: Structure, Property, and Determination.
be destroyed by temperatures and times incurred during
domestic cooking. Domestic cooks without formal training
may be unaware of the risks involved in eating particular
foods or to the neutralizing effects of cooking. A further risk
Further Reading
arises from cross contamination of cooked food by raw ingre-
dients, resulting in the potential for fresh growth of pathogenic McGee H (1991) On food and cooking: the science and lore of the kitchen, 2nd ed.
microorganisms. London: Harper Collins.
 Copper: Physiology
J Bertinato, Health Canada, Ottawa, ON, Canada; University of Ottawa, Ottawa, ON, Canada
Crown Copyright ã 2016 Published by Elsevier Ltd. All rights reserved.
Properties, Production, and Industrial Uses of Copper
Copper (Cu) is a transition metal with atomic number 29. Cu
has 29 isotopes, two stable isotopes (63Cu and 65Cu), and 27
radioisotopes. The most abundant isotope is 63Cu that
accounts for approximately 69% of naturally occurring Cu.
Cu has a face-centered cubic crystal structure. Pure Cu is
reddish orange in color, malleable, and a good conductor of
heat and electricity.
Cu is naturally present in the Earth’s crust and is found in
the environment as a metal and in various minerals (e.g.,
cuprite and malachite). Concentrations can vary from less
than 50 ppm to more than several thousand ppm in certain
areas. Most of the extracted Cu comes from sulfide ores. Both
natural and anthropogenic activities release Cu into the envi-
ronment. Natural events include volcanic eruptions, wind-
blown dusts, forest fires, and decaying vegetations. Mining,
milling, and metal productions are common human activities
that discharge Cu into the environment. Mean Cu concentra-
tions in air range from 5 to 200 ng per cubic meter in rural and
urban areas. Combustion of fossil fuels is a major contributor
to airborne Cu.
In 2013, the largest producers of Cu were Chile, China,
Peru, the United States, and Australia. Chile was the top pro-
ducer with an estimated production of 5 700 000 tons. Cu is
used in a variety of industrial applications. Major uses are in
electrical wires, plumbing, roofing, and industrial machinery.
Cu is also combined with other elements such as zinc and tin
to make the alloys brass and bronze, respectively. These alloys
are used in applications that require a harder material than
pure Cu.
Analytic Measurement of Copper
The most commonly used methods for determining Cu con-
tent in food and environmental and biological samples are
flame or graphite furnace atomic absorption spectrometry
(AAS) and inductively coupled plasma–atomic emission spec-
troscopy (ICP-AES). In AAS, the sample is atomized by a flame
or graphite tube (electrothermal). Quantification of Cu (or
other elements) is made by determining the absorption of
element-specific optical radiation (light) by free atoms in the
gaseous state. ICP-AES produces excited atoms and ions using
inductively coupled plasma. The atoms emit electromagnetic
radiation at characteristic wavelengths and the intensity of the
emission is related to the concentration of the atoms in the
sample. For both AAS and ICP-AES, standards with known
analyte concentration are used to derive the concentration of
the analyte in the sample. Prior to Cu determination by AAS or
ICP-AES, samples often need to be prepared for analysis. The
sample preparation procedure depends on the specific sample
and can involve acid digestion, filtration, and acidification.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00201-4
Additional methods that can be used for the measurement of
Cu include inductively coupled plasma-mass spectrometry
(ICP-MS), anodic stripping voltammetry, x-ray fluorescence,
neutron activation analysis, photon-induced x-ray emission,
and gas/liquid chromatography.
Reference Values for Copper
Nutrient reference values for Cu have been established by
scientific bodies. Table 1 shows recommended intakes and
tolerable upper intake levels (UL) for Cu by life stage group
established for North America (i.e., Canada and the United
States) and the European Union. Depression in Cu status
induces a number of quantifiable physiologic changes. Despite
this, no single indicator was deemed sufficient for deriving the
estimated average requirement (EAR) for Cu as part of the
North American Dietary Reference Intakes due to inconsis-
tencies in results from studies measuring similar biomarkers.
For adults aged
19 years, four indicators were used to estab-
lish the EAR: serum Cu concentration, serum ceruloplasmin
concentration, erythrocyte Cu/Zn superoxide dismutase
(SOD1) activity, and platelet Cu concentration. The EAR for
adults range from 0.7 to 1 mg day1 and the recommended
dietary allowances (RDA) range from 0.9 to 1.3 mg day1. EAR
and RDA are the same for males and females and these refer-
ence values are higher for pregnancy and lactation. It was
determined that there were inadequate data to separately estab-
lish EAR for infants, children, and adolescents. For infants
0–12 months, adequate intakes were established based on
mean Cu intake of infants fed with mainly human milk. For
children and adolescents aged 1–18 years, the EAR were
derived by extrapolation from the adult EAR. The European
Union established population reference intakes that are
slightly higher than the North American RDA.
There are limited data on the effects of high intakes of Cu in
healthy people. Chronic high intakes of Cu result in the accu-
mulation of Cu in the liver that can lead to liver damage. Acute
ingestion of large amounts of Cu salts in drinking water has
been reported to cause gastrointestinal symptoms; however,
liver damage was selected as the more relevant endpoint to
establish the UL for Cu. A no observed adverse effect level
(NOAEL) of 10 mg day1 was established for adults based on
data showing no liver injury in adults consuming 10 mg of
supplemental Cu daily. North America applied an uncertainty
factor (UF) of 1 to the NOAEL to derive a UL of 10 mg day1
because of the low prevalence of liver damage from Cu expo-
sure in human populations with normal Cu homeostasis and
other evidences indicating the absence of adverse effects with
Cu intakes of 10–12 mg day1 from foods. The European
Union set a more conservative UL of 5 mg day1, applying a
UF of 2 to account for variability to Cu toxicity within the
normal population. It was also concluded that the UL is not
321
322 Copper: Physiology

Table 1 Reference values for coppera

North Americab European Unionc

Life stage group AI (mg day1) EAR (mg day1) RDA (mg day1) UL (mg day1) PRI (mg day1) UL (mg day1)

0–6 months 0.2 – – – – –

6–11 months – – – – 0.3 –
7–12 months 0.22 – – – – –
1–3 years – 0.26 0.34 1 0.4 1
4–6 years – – – – 0.6 2
4–8 years – 0.34 0.44 3 – –
7–10 years – – – – 0.7 3
9–13 years – 0.54 0.7 5 – –
11–14 years – – – – 0.8 4
14–18 years – 0.685 0.89 8 – –
15–17 years – – – – 1 4
19–50 years – 0.7 0.9 10 1.1 5
51–70 years – 0.7 0.9 10 1.1 5
>70 years – 0.7 0.9 10 1.1 5
14–18 years (P) – 0.785 1 8 1.1 –
19–50 years (P) – 0.8 1 10 1.1 –
14–18 years (L) – 0.985 1.3 8 1.4 –
19–50 years (L) – 1 1.3 10 1.4 –
a
AI, adequate intake; EAR, estimated average requirement; L, lactation; P, pregnancy; PRI, population reference intake; RDA, recommended dietary allowance; UL, tolerable upper
intake level.
b
Values obtained from Food and Nutrition Board, Institute of Medicine (2001). Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron,
manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press.
c
Values obtained from the Scientific Committee on Food, European Food Safety Authority (2006). Tolerable upper intake levels for vitamins and minerals. Parma, Italy: European
Commission Publications Office; Scientific Committee for Food, Commission of the European Communities. (1993). Nutrient and energy intakes for the European community.
Luxembourg: Office for Official Publications of the European Communities.

applicable during pregnancy and lactation because of limited
data relating to these life stages. Given the paucity of data on
Cu toxicity in healthy infants, children, and adolescents, a UL
was not set for infants and UL for children and adolescents
were derived by extrapolation from the adult UL.
Sources of Copper Exposure
For humans, the main sources of Cu exposure are food, Cu-
containing supplements, and drinking water. Intake of Cu
through dermal routes or inhalation is minimal for most peo-
ple. Table 2 shows the Cu content of foods rich in Cu obtained
from the Canadian Nutrient File. Foods have been separated
into different food categories, and the Cu content per serving
size and 100 g of the food source have been provided. Organ
meats, seafood, nuts, and seeds are rich in Cu. Consumption of
one serving of foods highest in Cu content is sufficient to meet
the daily Cu requirements for an adult.
Usual Cu intakes from food and supplements for the US
population were determined in the third National Health and
Nutrition Examination Survey (1988–1994). For men and
women aged 19–70 years, mean Cu intakes from food alone
ranged from 1.54 to 1.70 and 1.13 to 1.18 mg day1, respec-
tively. Mean Cu intakes increased to 1.79–1.85 mg day1 for
men and 1.32–1.45 mg day1 for women when Cu intake
from supplements was also considered. Cu intakes for most
people were above recommended intakes and below the upper
limits. However, usual intake of Cu from food and supple-
ments at the 5th percentile was below the EAR for women of
several age groups. Mean Cu intakes from food for adults in
European countries range from 1.0 to 2.3 mg day1 for men
and 0.9 to 1.8 mg day1 for women.
Health-based and aesthetics-based guidelines for Cu in
drinking water have been established. The US Environmental
Protection Agency (US EPA) has set a maximum contaminant
level goal of 1.3 mg l1 to protect against gastrointestinal
symptoms. The World Health Organization (WHO) has set a
guideline value of 2.0 mg l1 to protect against acute gastroin-
testinal effects and to provide a margin of safety for popula-
tions with normal Cu homeostasis. Canada has set an
aesthetics-based guideline for Cu in drinking water of
1.0 mg l1 to ensure palatability and to minimize staining
of laundry and plumbing fixtures. The European Union
standard for maximum concentration of Cu in drinking water
is 2 mg l1.
Cu concentrations in natural water (e.g., rivers, lakes, and
oceans) are relatively low (median: 4–10 mg l1). Drinking
water in some instances can make a substantial contribution
to total Cu intake. Tap water from dwellings with Cu piping
can contain much higher concentrations of Cu. A number of
factors can affect the amount of Cu dissolution from Cu piping
accounting for the large variability in Cu concentrations. Tap
water of a first-draw sample is more likely to contain higher
amounts of Cu than a flush sample since the water has sat in
the plumbing for some time. In a monitoring survey carried
out in Ontario, Canada, communities, the Cu concentration in
consumed water averaged 0.176 mg l1 with a range of
0.020–2.02 mg l1. It can be estimated that an adult consum-
ing 1.5 l of water would have a daily Cu intake of 0.264 mg
 Copper: Physiology 323

Table 2 Copper content of foods

Food Serving size Copper per serving (mg) Copper per 100 g (mg)

Organ meats
Veal/beef/lamb/pork/chicken liver (cooked) 75 g 0.476–11.3 0.635–15.1
Pork kidney (braised) 75 g 0.500 0.683
Beef kidney (simmered) 75 g 0.423 0.564
Liver pate 75 g 0.300 0.400
Seafood
Oyster (boiled or steamed) 75 g 1.10–5.68 1.47–7.57
Lobster (boiled or steamed) 75 g 1.46 1.94
Squid (breaded and fried) 75 g 1.41 1.89
Crab, octopus, clam, crayfish (boiled or steamed) 75 g 0.466–0.886 0.621–1.18
Nuts and seeds
Sesame seeds (whole, dried) 60 ml 1.5 4.08
Flaxseeds (whole and ground) 60 ml 0.809 1.90
Cashew nuts (dry roasted) 60 ml 0.771 2.219
Pumpkin/squash/sunflower seeds 60 ml 0.623–0.734 1.08–1.28
Brazil nuts, hazelnuts, walnuts, pine nuts 60 ml 0.413–0.619 1.16–1.74
Legumes
Meat substitutes 1 serving 1.10–1.34 0.733–0.893
Fermented soybean products (natto, tempeh) 175 ml 0.863 0.667
Pink lentils (boiled) 175 ml 0.714 0.399
Soy/white/kidney/navy beans 175 ml 0.283–0.507 0.166–0.283
Peanuts (oil-roasted) 60 ml 0.438 1.30
Meats
Quail (cooked) 75 g 0.444 0.592
Veal (varied cuts) 75 g 0.161–0.360 0.215–0.480
Cured pork (ham, bacon) 75 g 0.115–0.313 0.153–0.417
Vegetables
Mushrooms (cooked) 125 ml 0.415–0.686 0.504–0.895
Heart of palm (raw) 85 g 0.547 0.644
Potato with skin (boiled or baked) 1 potato 0.379 0.253
Fruit
Prunes 60 ml 0.205 0.612
Avocado ½ fruit 0.191 0.190
Raisins 60 ml 0.133 0.362
Milk and alternatives
Soy beverages 250 ml 0.424 0.165
Goat cheese 50 g 0.366 0.732
Chocolate milk 250 ml 0.172 0.065
Grains
All bran ready-to-eat cereal 30 g 0.318–0.343 1.06–1.14
Kamut (cooked) 125 ml 0.225 0.248
Quinoa (cooked) 125 ml 0.14 0.191
Others
Dark chocolate 40 g 0.408–0.704 1.02–1.760

Source: Data obtained from Health Canada. Canadian Nutrient File. 2010. Available at www.hc-sc.gc.ca/fn-an/nutrition/fiche-nutri-data/index-eng.php.

from drinking water. A Swedish study found that the median
daily intake of Cu from drinking water for young children was
0.32 mg. It should be noted that considerably higher Cu con-
centrations that exceed standards have been reported in drink-
ing water from a small percentage of dwellings. Consuming
water with high concentrations of Cu concomitant with a Cu-
rich diet could result in total Cu intakes exceeding the UL.
Copper Bioavailability
Dietary interactions of Cu with a number of food constituents
have been described, yet under most circumstances, Cu
bioavailability is minimally affected by the composition of
the diet. It is well established, however, that consumption of
large quantities of zinc can decrease Cu absorption and lead to
Cu deficiency. In fact, decreased Cu status was the endpoint
selected by the Institute of Medicine in establishing the UL for
zinc for adults. High iron intakes may also interfere with Cu
absorption in infants. The amount of Cu ingested greatly
affects Cu absorption. When a diet is low in Cu (0.4 mg
day1), up to 75% of the Cu is absorbed, whereas only 12%
is absorbed when the diet is high in Cu (7.5 mg day1). Nota-
bly, even though fractional Cu absorption decreases with
higher Cu intakes, the absolute amount of Cu absorbed is
greater.
324 Copper: Physiology
Whole-Body Copper Metabolism
In healthy people consuming a balanced diet, overt Cu deficiency
and toxicity are rare because of the body’s elaborate systems that
maintain Cu homeostasis over a moderate range (10-fold) of
exposures. The intestine is the primary site for Cu absorption.
Ingested Cu from foods and Cu from digestive fluids (e.g., pan-
creatic and biliary secretions) present in the intestinal lumen are
absorbed by enterocytes by the Cu permease, Cu transporter 1
(Ctr1). Ctr1 is present on the brush-border surface and transports
reduced Cu1þ. The reduction of Cu2þ to Cu1þ prior to transport
by Ctr1 is not well understood, but may involve Dcytb or Steap2.
Cu exits the basolateral side of absorptive enterocytes in a process
mediated by the action of the P-type ATPase, ATP7A. Once
absorbed, Cu is transported bound to carrier proteins (albumin
and a2-macroglobulin) to the liver where it is subsequently
released into the circulation for transport to tissues.
The liver is a main organ for controlling Cu balance and
maintaining circulating Cu concentrations. The body responds
to increases in Cu by eliminating endogenous Cu into the bile
in a processed mediated by the P-type ATPase, ATP7B. Biliary
Cu excretion is a key homeostatic mechanism for ridding the
body of excess Cu and preventing Cu overload. Renal excretion
plays a minor role. In healthy people consuming a normal Cu
diet, urinary Cu excretion is low (<0.1 mg day1).
Intracellular Copper Trafficking
Cu is a redox-active metal. Free Cu can participate in Fenton-
type reactions and lead to the production of reactive oxygen
species that can damage cellular components. The cell has
evolved an overcapacity to sequester Cu and it has been esti-
mated that there is less than one free Cu atom per cell. Ctr1
plays a major role in the uptake of cellular Cu, although Ctr1-
independent Cu uptake systems exist. Once inside the cell, Cu
is transferred to a family of proteins called Cu chaperones and
possibly other small Cu-binding proteins (e.g., metallothio-
neins and GSH). Cu chaperones deliver Cu to specific
Cu-requiring enzymes by directly transferring the Cu to the
apo-cuproprotein by protein–protein interaction. This direct
transfer prevents the accumulation of free Cu that can other-
wise engage in deleterious reactions. Metallothioneins provide
the cell with additional capacity to bind Cu under conditions
of high Cu exposure.
The Cu chaperone for SOD1 (CCS) transfers Cu to SOD1 in
the cytoplasm and intermitochondrial space. COX17 is
required for incorporating Cu into cytochrome c oxidase
(CCO) in the mitochondria along with other Cu chaperones
including Cox11, Sco1, and Sco2. ATOX1 delivers Cu to ATP7A
and ATP7B. When cellular Cu levels are normal, ATP7A and
ATP7B are largely localized to the trans-Golgi network and
function to transport Cu into the lumen of the trans-Golgi
network for incorporation into nascent secretory cuproen-
zymes such as ceruloplasmin, lysyl oxidase, peptidylglycine
a-amidating monooxygenase, and dopamine b-hydroxylase.
When cellular Cu levels rise, ATP7A and ATP7B translocate in
cytoplasmic vesicles from the trans-Golgi network to the cell
surface to expel Cu from the cell.
ATP7A is expressed in the intestine and most other tissues.
The tissue expression pattern of ATP7B is more restricted, with
high expression in the liver. ATP7A mediates Cu efflux from
enterocytes into the plasma by a process that involves fusion
of Cu-loaded vesicles with the basolateral membrane of enter-
ocytes. ATP7B mediates Cu excretion into the bile by a
mechanism involving fusion of Cu-loaded vesicles with
the canalicular membrane of hepatocytes. Cu metabolism
MURR1 domain-containing protein 1 (COMMD1) has been
shown to interact with ATP7B and may facilitate the excretion
of Cu by ATP7B into the bile canaliculus. Mutation of the
COMMD1 gene in dogs has been associated with Cu toxicosis.
Cu transporter 2 (Ctr2) shares sequence similarity with Ctr1
although its function in mammalian cells is less understood.
Ctr2 is localized to cytoplasmic vesicles and a small proportion
has been detected on the plasma membrane. Studies in yeast
support a role for Ctr2 in the mobilization of Cu from intra-
cellular vesicles to the cytosol. Recently, Ctr2 has been shown
to play an important role in regulating the function of Ctr1 by
controlling the accumulation of a truncated form of Ctr1 lack-
ing the Cu-binding ectodomain. Ctr2/ mice were found to
be defective in the accumulation of the truncated form of Ctr1
and showed increased tissue Cu that accumulated as intracel-
lular foci.
The role of other Cu-binding proteins such as the prion
protein (PrPc) and amyloid precursor protein (APP) in Cu
metabolism is not well understood. Some data suggest that
the APP may be involved in regulating Cu levels in the brain.
Copper-Dependent Enzymes
Cu has been known to be an essential nutrient for almost a
century. Cu is required by organisms in small quantities and
therefore considered a trace mineral. Total body Cu content in
an adult is approximately 80 mg and most (half to two-thirds)
is located in the skeleton and muscle because of their relative
size. The brain, liver, heart, and kidneys have the highest Cu
concentrations.
The ability of Cu to cycle between its oxidized cupric (Cu2þ)
and reduced cuprous (Cu1þ) forms is critical for its biological
function. As an essential component of Cu-dependent enzymes
(also called cuproenzymes), Cu functions as a catalytic cofactor
carrying out single-electron transfer reactions. Cuproenzymes pre-
sent in mammals and their physiological function are listed in
Table 3. Cuproenzymes function in various physiological
processes such as antioxidant defense (SOD1 and extracellular
superoxide dismutase (SOD3)), iron homeostasis (ceruloplas-
min, hephaestin, and zyklopen), mitochondrial respiration
(CCO), development of connective tissue (lysyl oxidase), melanin
biosynthesis (tyrosinase), catecholamine production (dopamine
b-hydroxylase), peptide hormone processing (peptidylglycine
a-amidating monooxygenase), and oxidative deamination
(amine oxidases).
Copper Deficiency and Toxicity
Symptoms of Cu deficiency are a result of reduced activity of
cuproenzymes. Symptoms of overt Cu deficiency in mammals

Table 3 Physiological function of mammalian cuproenzymes
Cuproenzyme Physiological function
Amine oxidases Oxidative deamination
Ceruloplasmin (ferroxidase) Ferrous iron oxidation
Cytochrome c oxidase Reduction of molecular
oxygen
Dopamine b-hydroxylase Norepinephrine production
Hephaestin Ferrous iron oxidation
Lysyl oxidase Elastin cross-linking
Peptidylglycine a-amidating Peptide C-terminal
monooxygenase a-amidation
Superoxide dismutases (SOD1 and Superoxide
SOD3) disproportionation
Tyrosinase Dopaquinone production
Zyklopen Ferrous iron oxidation
include hypochromic anemia, neutropenia, thrombocytopenia,
and hypopigmentation. Structural and functional abnormalities
occur in the skeleton and cardiovascular system and depressed
immune function and neurological abnormalities are also
observed. Overt Cu deficiency is rare in humans consuming a
balanced diet. Subpopulations at increased risk for Cu deficiency
included preterm infants fed with cow’s milk (or other milk-
based formulas low in Cu), individuals with malabsorption
syndromes (e.g., celiac disease), and gastric bypass patients. Cu
deficiency has also been observed in patients receiving total
parenteral nutrition. Consumption of high amount of zinc can
also predispose individuals to Cu deficiency. Cu toxicity has
been observed with overzealous use of Cu-containing supple-
ments and following ingestion of contaminated water with high
concentrations of Cu.
Genetic Disorders of Copper Metabolism
The best characterized genetic disorders of Cu metabolism are
Menkes syndrome and Wilson’s disease. Menkes syndrome is an
X-linked recessive disorder that results from mutations in the
ATP7A gene leading to impaired intestinal Cu absorption and a
systemic Cu deficiency. Symptoms include growth failure, mental
retardation, weak muscle tone, and brittle hair. A milder form of
the disease termed occipital horn syndrome has been described.
Occipital horn syndrome is characterized by calcium deposits in
the occipital bone, coarse hair, and loose skin and joints.
Wilson’s disease is an autosomal recessive disorder that is
caused by mutations in the ATP7B gene. In Wilson’s disease,
Cu accumulates in tissues, namely, the brain and liver, result-
ing in neuropsychiatric symptoms and liver damage. Indian
childhood cirrhosis, idiopathic Cu toxicosis, and endemic
Tyrolean infantile cirrhosis are rarer diseases of hepatic Cu
accumulation in infancy and childhood that have a genetic
predisposition. Manifestation of these diseases is often associ-
ated with high intakes of Cu from contaminated food or water.
Evaluation of Copper Status
The most commonly used biomarkers for routine assessment
of Cu deficiency are plasma Cu concentration and plasma
Copper: Physiology 325
ceruloplasmin (Cp) protein concentration or activity. Most
Cu in the circulation is bound to Cp (>90%) and therefore
changes in plasma Cu closely follow changes in Cp. A limita-
tion of both these biomarkers is that they are affected by
conditions unrelated to Cu status including inflammation,
age, gender, hormone use, and pregnancy. In addition, strong
homeostatic mechanisms act to maintain plasma Cu and Cp
within a normal range, which likely makes these indicators
insensitive to mild reductions in Cu status. Several studies
have shown changes in biochemical indicators in response to
Cu restriction in the absence of changes in plasma Cu or Cp.
Erythrocyte SOD1 enzymatic activity and protein content
decrease in experimental animals with Cu deficiency. Enzy-
matic activity is a better measure of Cu deficiency than protein
level given that inactive apo-SOD1 accumulates in Cu-deficient
cells. Erythrocyte SOD1 activity has been shown to be lower in
Cu-deficient patients, in subjects consuming a low Cu diet and
in people taking high-dose zinc (a Cu absorption antagonist)
supplements. Limitations of erythrocyte SOD1 activity as an
indicator of Cu deficiency are the absence of a simple, stan-
dardized assay with a reference interval and the high intra-
individual variation. Erythrocyte SOD1 activity is also affected
by certain diseases and conditions.
Platelet Cu concentrations may response more quickly to
change in Cu intakes and be more sensitive to changes in Cu
status compared to other conventional biomarkers. In estab-
lishing requirements for Cu in North America, diets that
decreased serum Cu and Cp concentrations were considered
deficient in Cu. If the diet did not alter serum Cu and Cp, but
reduced platelet Cu concentration, the diet was considered to
be marginally adequate in Cu.
The search for more sensitive, specific, and noninvasive bio-
markers of Cu status that can be easily measured in humans is
ongoing given that traditional biomarkers may be insensitive to
small changes in Cu status and have other disadvantages. Cu
deficiency induces many biochemical changes. Changes in
cuproenzymes, Cu-binding proteins, and markers of immune
function and bone metabolism have been proposed as potential
indicators for assessing Cu status. One promising biomarker is
CCS. Studies in rodents and cattle have shown that CCS protein
increases in cells in response to Cu deficiency. The mechanism
by which CCS is regulated by Cu has been elucidated. Cu
binding to CCS decreases its stability and increases its rate of
degradation by the 26 S proteasome. Under Cu-deficient
conditions, CCS is more stable and protein levels increase.
Since CCS is responsive in erythrocytes, it can be measured in
a small blood sample and is therefore appealing for use in
humans. Since SOD1 protein decreases and CCS protein
increases with Cu deficiency, the CCS:SOD1 protein ratio has
been proposed as a measure of Cu deficiency. The CCS:SOD1
ratio has the advantage of increasing the magnitude of the
change in some situations compared to assessing CCS alone. A
study in rats has shown that erythrocyte CCS protein decreases
with Cu overload suggesting that CCS may also be useful as a
biomarker for Cu excess. Furthermore, CCS mRNA in peripheral
blood mononuclear cells has been shown to decrease in
response to Cu supplementation in people with high Cp con-
centration. Other promising biomarkers for assessing Cu defi-
ciency include serum and tissue activity of peptidylglycine
a-amidating monooxygenase and CCO activity in platelets.
326 Copper: Physiology
Presently, liver Cu concentration is considered the ‘gold
standard’ for diagnosing Cu overload, but the invasiveness of
a liver biopsy precludes its use in routine assessment. The non-
Cp-bound Cu and protein-free Cu in plasma are promising
indicators for assessing Cu excess. Protein-free Cu in serum or
plasma was found to be higher in patients with untreated
Wilson’s disease compared to treated patients. New research
has revealed that the relative exchangeable Cu, defined as the
exchangeable Cu to total serum Cu ratio, is a promising test for
diagnosis of Wilson’s disease.
Acknowledgment
I am grateful to Josephine Deeks (Bureau of Nutritional Sci-
ences, Health Canada) for the assistance in generating the food
composition data from the Canadian Nutrient File.
Further Reading
Festa RA and Thiele DJ (2011) Copper: an essential metal in biology. Current Biology
21: R877–R883.
Grubman A and White AR (2014) Copper as a key regulator of cell signalling pathways.
Expert Reviews in Molecular Medicine 16: e11.
Howell SB, Safaei R, Larson CA, and Sailor MJ (2010) Copper transporters and the
cellular pharmacology of the platinum-containing cancer drugs. Molecular
Pharmacology 77: 887–894.
Lutsenko S (2010) Human copper homeostasis: a network of interconnected pathways.
Current Opinion in Chemical Biology 14: 211–217.
Prohaska JR (2012) Copper. In: Erdman Jr. JW Jr., Macdonald IA, and Zeisel SH (eds.)
Present knowledge in nutrition, 10th ed., pp. 540–553. International Life Sciences
Institute, Wiley.
Robinson NJ and Winge DR (2010) Copper metallochaperones. Annual Review of
Biochemistry 79: 537–562.
Scheiber IF, Mercer JF, and Dringen R (2014) Metabolism and functions of copper in
brain. Progress in Neurobiology 116: 33–57.
van den Berghe PV and Klomp LW (2009) New developments in the regulation of
intestinal copper absorption. Nutrition Reviews 67: 658–672.
Relevant Websites
http://www.atsdr.cdc.gov/ToxProfiles/tp.asp?id¼206&tid¼37 – Agency for Toxic
Substances & Disease Registry.
www.hc-sc.gc.ca/fn-an/nutrition/fiche-nutri-data/index-eng.php – Health Canada.
http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/copper-cuivre/index-eng.php –
Health Canada.
http://minerals.usgs.gov/minerals/pubs/commodity/copper/mcs-2014-coppe.pdf –
U.S. Geological Survey.
http://www.who.int/water_sanitation_health/publications/2011/dwq_guidelines/en/ –
World Health Organization.
 Cream: Clotted Cream
RS Chavan and A Kumar, National Institute of Food Technology & Entrepreneurship Management (NIFTEM), Kundli, India
S Bhatt, Anand Agricultural University, India
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Clotted cream is a thick dairy cream with a nutty flavor, a
golden crust of grainy texture, and high fat content (a mini-
mum of 55%, but an average of 64%). Historical records have
it that the clotted cream was first produced in Afyon province
(Western Turkey) known as Afyon Kaymag in which buffalo
milk was used to manufacture the same. British clotted cream
also has a long and intriguing history, and it was suggested by
Alan Davidson that the arrival of Phoenician traders to Corn-
wall around 2000 years ago may have introduced the tradition
of clotted cream. In the nineteenth century clotted cream was
considered a better source of nourishment as compared to ‘raw’
cream because the latter was liable to go sour easily and when
consumed without any heat treatment caused several health
problems. Consumers used to prefer clotted cream over raw
cream due to its nutritive value, characteristic aroma, and
flavor. The European Union made an application in 1993
called ‘Cornish clotted cream’ to obtain a protected
designation of origin for the production of cream by the tradi-
tional method in Cornwall. Clotted cream is produced on a
day-to-day basis in many parts of European countries, which
gives a regular income to many small producers. Clotted cream
is creamier than ordinary cream, with a unique flavor devel-
oped as a result of milk sugar caramelization and perhaps a
slight butteriness. Buffalo milk is preferred over cow’s milk for
manufacturing of clotted cream as it is high in fat, protein,
lactose, total solids, mineral, and vitamin contents, which gives
the final products its characteristic rich aroma and flavor.
Qashta is a type of thick cream from the Eastern Mediterranean
region where, due to its higher amount of fat and protein than
buffalo milk, ewe’s milk is preferred.
Traditionally, clotted cream was produced by straining
cow’s or buffalo milk. After filtration, the milk was left to
stand in a shallow pan in a cool place for several hours,
which facilitated the cream to rise to the surface, which is
followed by heating either over hot cinders or in a water bath
for hours and then cooled. Yellow clots that formed on the
surface of the milk were then skimmed off with the help of a
long-handled cream-skimmer known as a reamer or raimer in
Devon. By the mid-1930s, the modern processing technique
had become famous in Devon because using a cream separator
actively separated the cream from the milk using centrifugal
force, which helped to produce a larger quantity of clotted
cream than the traditional method from the same amount of
milk in much less time. English luxury clotted cream has a
lower fat content of 48% and is generally used as a topping
for desserts, cakes, and muffins, but it is also suited for savory
recipes, including pasta and sauces. In bakery products like
‘butter shortbread’ and ‘clotted cream biscuits’ clotted cream
along with butter is used in the formulation to give the bread
and biscuits their distinctive rich and soft texture.
The characteristic flavor of clotted cream is developed due to
slow heating of cream for longer duration. The color of the
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00206-3
clotted cream can vary from a pale off-white to golden yellow
color, with a smooth texture similar to cream cheese. Clotted
cream production provides a lot of employment. Clotted cream
is called ‘malai’ in India and ‘Devonshire cream’ in the Western
world. Malai is made by the heating of nonhomogenized milk
to about 80  C (180  F) for about an hour and then cooled a
long time in undisturbed condition. Finally, a thick yellowish
layer of fat and coagulated proteins are formed on the surface,
which are skimmed off, and the process is repeated to recover
most of the fat until it reaches a minimum fat content of
about 55%.
Regular consumption of clotted cream was usually believed
to be bad for health due to its higher amount of fat. According
to the United Kingdom’s Food Standards Agency, a 100 g cup
of clotted cream provides 586 kcal, roughly equivalent to a
200 g cheeseburger.
Manufacturing Methods of Clotted Cream
In the traditional manufacturing technique, raw buffalo milk,
which is preferred for producing clotted cream, is bolted by
cheesecloth twice and transferred into copper containers. The
milk is heated on a low flame and is continued until the milk
reaches temperature of about 90  C and the milk is held for
30 min; this stage is called ‘göbek baglama.’ After heating and
holding of milk at 90  C for 30 min, the milk is cooled at room
temperature overnight. The cream layer formed on the top of
the milk is collected by releasing the milk by perforating the
side of the container with a pin. Fresh milk is again added to
the previous day’s heat-treated cream, which is again slowly
heated to 90  C for 45 min and again slowly cooled to room
temperature and left in the cool room for about 4–6 h, fol-
lowed by refrigeration for 6–8 h. The clotted cream layer
formed on the surface is separated out and then inverted into
a plastic can, which is packed and sealed before sale.
Today, manufacturers are following two methods for pro-
duction of clotted cream, that is, (1) farmhouse production
method and (2) commercial production method. In the farm-
house production method, milk is usually strained into a
rectangular stainless steel tray followed by centrifugal separa-
tion of cream with the help of a centrifugal separator. The
cream obtained after centrifugal separation is then subjected
to heat treatment either by using a forced air convection oven
maintained at 80–90  C for 50–60 min or by scalding the
cream in a ‘bain marie’ type equipment, which operates like a
huge water bath in which the hot water is circulated until the
final temperature of about 77–88  C is reached with a holding
period of 45–50 min. After slow heating of cream, the cream is
allowed to cool for the next 24 h without any disturbance.
Finally a golden-color crust that has a fat content of 55–60%
is formed on the surface and is separated out with great caution
(Figure 1).
327
328 Cream: Clotted Cream

Buffalo Milk

Straining of milk into rectangular stainless steel trays

Cream Separation using centrifugal separator

Heating of cream

Forced air convection oven Scalding in bain marie

(80–90 ⬚C for 50–60 min) (77–88 ⬚C for 45–50 min)

Cooling of cream for 24 h

Scrapping of golden color crust having 55–60% fat

Packaging
Figure 1 Farmhouse production method for manufacturing of clotted cream.

Commercial manufacturers of clotted cream produce the
product by using either a ‘float method’ or ‘scald method.’
In the float method, double cream is floated on milk or
skim milk in large jacketed trays and are heated up to 80–85  C
by circulating steam or hot water into the jackets, and the
cream is given a holding period of 40–60 min. After slow
heating of cream, it is allowed to cool followed by refrigeration
to set a golden-color crust, which is scrapped off and packaged
into containers (Figure 2).
Cream with a fat content of about 56% is usually used as a
starting material for producing clotted cream by the ‘scald
method.’ The cream is heated at 80  C for 40–60 min with
the help of a plate heat exchanger and cooled to below 7  C
and left undisturbed for 12–14 h. It is then packaged for sale in
polystyrene or polypropylene pots (Figure 3).
The scald cream method is very similar to float method, but
the milk layer is removed and a layer of cream that has been
mechanically separated to a minimum fat level is used. In the
United Kingdom the resultant cream is allowed to be equiva-
lent to pasteurized cream for legal purposes. A high level of
hygiene standards is to be maintained when the processing
temperatures are lower than those used in the standard
pasteurization process.
Chemistry of Flavor and Golden Crust
As clotted cream is a traditional product, the literature available
is limited, but several scientists have studied different aspects,
which include microbiological quality, chemical properties,
and suitability of packaging materials for enhancing the shelf-
life. One interesting study on clotted cream was reported by
Senel in which he discussed the effect of carbonyl compounds
and free fatty acids on aroma and flavor. The formation of
aroma and flavor compounds is the result of the chemical
Floating the double cream on milk/skim milk in jacketed trays
Heating the cream at 80–85 ⬚C for 40–60 min
Cooling
Golden color crust formation at refrigeration temperature
Scraping off the golden-color clotted cream
Packaging
Figure 2 Float method for manufacturing of clotted cream.
and biochemical transformations of milk components and
may be divided into four categories: (1) nonvolatile acids
(lactic and pyruvic); (2) volatile acids (formic, acetic, and
butyric); (3) carbonyl compounds (acetaldehyde, acetone,
and diacetyl); and (4) miscellaneous compounds (certain
amino acids, constituents formed by thermal degradation of
protein, fat, or lactose). Apart from imparting characteristic
flavor to the clotted cream, carbonyls, lactic acid, and free
fatty acids also play a vital role in imparting shelf-life of the
product. Carbonyl compounds and free fatty acids in clotted
cream produced from buffalo milk were found to be much
different when compared with other dairy products. The char-
acteristic flavor of clotted cream may also be attributed to the
slow heating treatment of cream for a long time, which causes
caramelization of milk sugar. Golden-color crust of clotted
cream may be attributed to high carotene levels in the milk,
which is normally present in the grass eaten by buffalo.
 Cream: Clotted Cream 329

Cream (56% fat)

Heating the cream at 80 ⬚C for 40–60 min

Cooling the cream at below 7 ⬚C for 12–14 hr

Golden color crust formation at refrigeration temperature

Careful cutting off the golden-color clotted cream

Packaging in polystyrene or polypropylene pots

Figure 3 Scald method for manufacturing of clotted cream.

imperial gallons of skimmed milk are produced, which can be

further used to manufacture different dairy products.

Uses of Clotted Cream

Clotted cream is also known as ‘baked cream’ and is mostly

used in the bakery industry as a topping and also in culinary
dishes.

Figure 4 MasoSine pump ideal for clotted cream.
Cream Tea
In most of the countries, an essential part of the ‘cream tea’ is a
clotted cream. In Cornwall, clotted cream is served on scones
with different fruit flavors of jam, along with a cup of tea.
Langage Farm in Devon has started a campaign for the
protection of origin of ‘Devon cream tea’ as similar to ‘Cornish
clotted cream,’ where scones are spread with jam or lemon curd
and then topped with clotted cream, which is served with after-
noon tea and known as ‘Devonshire tea.’

Recent Status of Clotted Cream

The largest commercial producer of clotted cream in United
Kingdom (Cornwall) is Rodda’s, founded in 1890, which is
now able to produce more than 25 tons per day. Clotted cream
is believed to have quality as it requires longer manufacturing
time. Commercial manufacturers of clotted cream are using
modern equipment to manufacture the product under
hygienic conditions, both at a large and small scale. Rodda’s
company in 2012 installed a MasoSine sanitary process pump
(Figure 4) supplied by Watson Marlow Pumps Group to carry
out the production of clotted cream under hygienic conditions.
As a by-product, from every 100 imperial gallons of milk, 94
Confectionery
On many occasion clotted cream is used as a supplement to
hot or cold desserts. Clotted cream, especially Devon clotted
cream, which is less yellow in color due to the lower carotene
levels, is regularly used in different baked products. Clotted
cream is used in most of the southwest part of England for ice
cream and fudge production. One of the products, in which
bread is topped with clotted cream and golden syrup honey, is
called ‘thunder and lightning.’ Clotted cream can be spread as a
topping over any baked treat, like English muffins, biscuits,
croissants or muffins, ice cream, cake, trifle or pudding, or
crusty bread, and it also serves a purpose of filling in cakes.
330 Cream: Clotted Cream
Savory Dishes
Clotted cream is used in savory dishes and can also be incor-
porated into mashed potatoes or scrambled eggs. Clotted
cream, along with rich cheddar sauce and mustard, can be
used as a dip with its unique richness of flavor and texture. A
moist scone can be split like a shortcake biscuit, topped with
berries and clotted cream, and served as a dessert. Clotted
cream is also used in some of the creamy savory dishes like
risotto, pasta, curry, quiche, or gratins. In some parts of
England, barley is used instead of rice for preparing risotto or
rice pudding, which adds a unique texture and slightly nutty
flavor. The addition of clotted cream to this dish enriches and
enhances the creamy texture.
Risks Associated with Consumption of Clotted Cream
Clotted cream might be quite delicious, but on health grounds it
cannot be suggested as a daily diet due to its high amount of
saturated fat. The market for clotted cream has not expanded
beyond certain parts of the world as it has a very short shelf-life
and hence is difficult to export. Clotted cream is often associated
with a high number of microbiological counts, which makes it
vulnerable for its consumers. To obtain a good quality of clotted
cream apart from hygienic conditions, factors like status of
healthy animals, hygienic milking, manufacturing tools and
equipment, and hygienic practices during production and stor-
age of clotted cream must be considered. Brucella abortus was first
reported in March 1966 from a market sample of clotted cream,
and aerobic spore-forming bacteria like Bacillus subtilis are more
prominent. Researchers have found that cooling for a long time
during manufacturing of clotted cream and contamination dur-
ing handling of packing are the main reasons for such a high
number of microbial load. To deliver a safe product to the
consumers and to reduce the level of spoilage bacteria, acceptable
heat treatment must be applied during the production of clotted
cream, and apart from this the clotted cream must be produced
in a clean environment under hygienic conditions.
See also: Cream: Types of Cream; Ethnic Foods; HACCP and
ISO22000: Risk Assessment in Conjunction with Other Food Safety
Tools Such as FMEA, Ishikawa Diagrams and Pareto; Heat Treatment:
Effect on Microbiological Changes and Shelf Life; Heat Treatment:
Principles and Techniques; Lactose; Milk: Processing of Milk; Milk:
Role in the Diet; Spoilage: Bacterial Spoilage; Traditional Foods.
Further Reading
Ahmad S (2013) Buffalo milk. In: Milk and dairy products in human nutrition:
production, composition and health, pp. 519–553. Oxford: Wiley-Blackwell.
Budhkar YA, Bankar SB, and Singhal RS (2014) Milk and milk products | Microbiology
of cream and butter. In: Encyclopaedia of food microbiology, 2nd ed., 728–737.
New York: Academic Press.
De S (1980) Outlines of dairy technology. Outlines of dairy technology. Oxford: Oxford
University Press 539 p.
Komorovski ES and Early R (1992) Liquid milk and cream. In: The technology of dairy
products, pp. 1–23. London: Blackie Academic & Professional.
Nalbantoglu S, Sari B, Cicek H, and Karaer Z (2008) Prevalence of coccidian species in
the water buffalo (Bubalus bubalis) in the province of Afyon, Turkey. Acta Veterinaria
Brno 77: 111–116.
Sadlep W (1917) Clotted cream. Journal of Dairy Science 1: 291–302.
Senel E (2011) Some carbonyl compounds and free fatty acid composition of Afyon
Kaymagı (clotted cream) and their effects on aroma and flavor. Grasas Y Aceites
62: 418–427.
Siriken B and Erol I (2009) Microbiological and chemical quality of Afyon clotted cream.
Journal of Animal and Veterinary Advances 8: 2022–2026.
Relevant Websites
http://medlibrary.org/medwiki/Cream_tea – Med Library Open Source Encyclopedia -
Cream Tea.
https://www.pinterest.com/explore/clotted-cream/ – Pinterest - Clotted Cream.
http://www.watson-marlow.com/us-en/range/masosine/ – Watson Marlow Fluid
Technology Group - Masosine Process Pumps.
http://en.wikipedia.org/wiki/Clotted_cream – Wikipedia - Clotted Cream.
 Cream: Types of Cream
SS Deosarkar and CD Khedkar, College of Dairy Technology, Pusad, India
SD Kalyankar, Government College of Dairy Technology, Udgir, India
AR Sarode, College of Dairy Technology, Pusad, India
ã 2016 Elsevier Ltd. All rights reserved.

Introduction stratify or separate from one another by virtue of their differing

densities. Separating the cream phase from the milk, standard-
Cream is one of the most important dairy products. It was izing the cream to the desired fat content, and heating the
traditionally considered a luxury product, but it is now readily cream to increase its shelf-life are universal to the manufacture
used in many forms and for a variety of purposes. For example, it of (almost) all industrially processed cream products.
is a primary raw material for the manufacture of table butter and
for preparing ghee, a fat-rich Indian dairy product. It is also used
as an ingredient in sweet and savory dishes, such as ice cream,
Gravity Methods
soup, custard bases, and cakes. Cream is a concentrated emulsion
of milk lipid globules in skimmed milk, and it is separated from When milk is allowed to stand for some time, there is a ten-
milk either by gravity or centrifugal force. Different types dency for the fat to rise. The velocity, or rate, at which the fat
of creams are primarily classified according to their fat content globules rise is described by the following equation, which is
(g/100 g) as double cream (45–50%), cream or full cream known as Stoke’s Law
(30–40%), single or half cream (15–25%), coffee cream
(15–18%), and light coffee cream (<10%). According to the V ¼ 2G=9*ðds  df Þr2=n
standards of the United Nations Food and Agriculture Organiza- Where,
tion (FAO), cream is classified as followed, based on fat content: V¼velocity or rate at which a single fat globule rises,
(a) Cream: 18–26% G¼acceleration due to gravity, ds¼density of skim milk,
(b) Light cream (or coffee cream): > 10% df¼density of fat, r¼radius of fat globule and n¼viscosity of
(c) Whipping cream: >28% skim milk.
(d) Heavy cream: > 35% According to Stoke’s Law, the velocity is increased by:
(e) Double cream: > 45% (i) An increase in the radius of the fat globule
The fat-based standards for various types of cream vary in (ii) An increase in the difference in the densities of skim milk
individual countries, and diverse names are used to describe and fat
different creams. So, it is not possible to provide a uniform (iii) A decrease in the viscosity of skim milk
international definition or universally accepted classification However, in practice, the important factors affecting the rate of
system. However, Table 1 provides the regulations for the fat cream rise using gravity methods are as follows:
content of cream products in major cream-producing
countries. Cream products are also classified according to Table 1 Standards for the lipid content of cream in some countries
their final uses (e.g., whipping cream, coffee cream, or cream
liqueur) or by processing method (e.g., pasteurized cream, Lipid content
ultra-high-temperature (UHT)-treated cream, frozen cream, Country Cream type (g/100 g)
dried cream, and cultured or sour cream).
Australia and New Cream 18–40
The physicochemical properties of cream are affected by Zealand
several factors, such as the state of the lipid globules and the France Light cream 12–30
milk lipid globule membrane, the concentration of lipid glob- Cream 30–40
ules, the type and concentration of nonfat milk solids in cream Germany Coffee cream 10–30
(e.g., proteins, salts, and added emulsifiers and stabilizers), Whipping cream 30–40
the temperature of the cream, and the physical handling of The Netherlands Cream 10–30
the cream (e.g., pumping, aeration, and agitation), which can Whipping cream 30–40
cause the disruption or agglomeration of the globules. Table 2 United Kingdom Half cream 12–18
Single cream 18–35
provides the chemical composition of cream.
Whipping cream 35–48
Double cream 48–55
Technology of Cream Separation United States Half and half cream 10–18
Light cream 18–30
Light whipping 30–36
The basic principle of cream separation, whether by gravity or
cream
centrifugal methods, is based on the fact that milk fat is lighter
Heavy cream 36–45
than the skim milk portion. At 16
C, the average density of
milk fat is 0.93 and skim milk 1.036. Thus, when milk, which Source: Hoffmann, W. (2002). Cream products. In: Roginski, H., Fox, P. F. and
is a mixture of fat (as cream) and skim milk, is subjected to Fuquay, J. W. (eds.) Encyclopaedia of dairy sciences, pp. 551–557. London:
either gravity or centrifugal force, the cream and skim milk Academic Press.

Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00205-1 331

332 Cream: Types of Cream

Table 2 Chemical composition of cream

Constituents Percentage

Fat 30 25.00 50.00

Water 64 68.20 45.45
Serum: protein 02.4 02.54 01.69
Lactose 03.5 03.71 02.47
Minerals/ash 00.4 00.56 00.37
Total solids 36 31.80 54.55
Nonfat solids 06 06.80 04.55

(i) Size of fat globules: As the size of the fat globules increases,
the rate at which the cream rises also increases. (Thus
gravity creaming occurs faster for buffalo milk than it
does for cow milk because of the larger size of fat globules
in buffalo milk.)
(ii) Temperature: As temperature increases, viscosity decreases
and, in turn, velocity increases.
(iii) Clumping: A clump or cluster acts like a single globule, in
so far as movement through skim milk is concerned.
Therefore, the effective r2 is increased, which then
increases velocity.
(iv) Addition of adhesives: Adhesives ultimately help increase
the rate at which fat globules rise.

Centrifugal Methods
When milk enters the rapidly revolving bowl of the cream
separator, it is immediately subjected to a tremendous centrif- Figure 1 Paring chamber and disk of a semienclosed separator.
ugal force, which is 3000–6000 times greater than gravitational
force. While both the fat and skim milk are subjected to the
centrifugal force, the difference in density affects the heavier
portion (i.e., skim milk) more intensely than the lighter portion
(i.e., cream). Centrifugal separators contain stacks of conical
disks with vertically aligned distribution holes through which
whole raw milk is introduced. Under the influence of a centrif-
ugal force, the sediment and lipid globules in the milk settle
radially outward or inward, respectively, in the separation
channels. Solid milk impurities of high density rapidly settle
out to the outside of the separator, and they are then collected
in the sediment space. Because the cream is less dense than
skimmed milk, it moves inward toward the axis of rotation,
and it is removed through an axial outlet. Skimmed milk moves
outward beyond the disk stack and is removed to a skimmed
milk outlet through a channel between the top of the disk stack
and the conical hood of the separator bowl. Therefore, the
separation of cream from whole milk involves the concentra-
tion of lipid globules, followed by the removal of the cream
from the skim phase to produce two streams, skimmed milk
and cream, the latter of which commonly amounts to 10% of
the total throughput. This method allows rapid and efficient
separation, and although very high centrifugal forces are
applied, not all lipid globules are removed. Skim milk usually
has a residual fat content of 0.1 g/100g, which primarily con- Figure 2 Open-design cream separator.
sists of lipid globules <1 mm in diameter (Figures 1–3).
(a) Position of the cream screw: The cream screw/outlet consists
Factors Influencing the Fat Percentage of Cream of a small, threaded, hollow screw pierced by a circular
orifice through which the cream emerges. This screw can
A brief summary of the factors influencing the fat percentage of be driven IN or OUT, thus bringing it nearer to, or away
cream by centrifugal separation is given below: from, the center of rotation. Under normal conditions, the

Figure 3 Hermetic-design cream separator.
ratio of skim milk to cream is 90:10. Changes in the
separation procedures affect the fat percentage in the
cream. Altering the position of the cream screw can change
the ratio of skim milk to cream. Therefore, when the cream
screw is moved IN toward the axis of rotation, a higher fat
percentage in the cream is obtained, and vice versa; this is
because the force that tends to discharge cream through
the orifice is decreased.
(b) Fat percentage in milk: Because all of the fat in milk is
present in cream, the cream from the separation of high-
fat milk has a higher fat content than that from low-fat
milk. Higher fat content in cream is obviously consistent
with higher fat percentage, and vice versa.
(c) Speed of the bowl: Higher bowl speeds exert a greater cen-
trifugal force, resulting in the rapid flow of skim milk from
the bowl. An increase in bowl speed increases the skim
milk discharge, leading to a higher fat percentage in the
cream.
(d) Rate of inflow of milk: When the rate of inflow increases, the
discharge from the cream outlet increases, as the skim milk
discharge remains constant (with constant centrifugal
force); more cream containing the same amount of fat
results in a lower fat percentage and vice versa.
(e) Temperature of milk: Lowering the temperature increases
the viscosity of both cream and skim milk, but the viscosity
of cream increases more than that of skim milk. Thus, the
Cream: Types of Cream 333
quantity of cream discharged is reduced because of bowl-
clogging, resulting in a higher fat percentage.
(f) Amount of water or skim milk added to flush the bowl: The
addition of more water or skim milk will cause an increase
in the amount of cream produced, which, with the same fat
content, will show a lower fat percentage.
Standardization of Cream
Because traditional cream separators cannot be accurately
operated to produce a cream of specific fat content, it is usually
necessary to produce cream with a higher fat content. This
cream is collected and mixed in cream tanks from which the
cream was sampled for fat percentage. The fat percentage is
determined and then reduced via the addition of skimmed
milk in a process called standardization, which is necessary
for producing cream with a fat content close to a predefined
value. Direct in-line standardization is usually combined with
separation, and equipment that automatically monitors and
controls the fat content of cream is used for this purpose. On
discharge from the separator, the skim and cream streams are
mixed, and the proportion of cream included determines the
fat content of the cream prepared.
Fat content must be as close to the stipulated value as
possible; too high a fat content results in economic losses for
the processor, whereas too low a fat content may result in
cream products not meeting legal requirements. During stan-
dardization, the temperature of cream can be over 40
C, so
bacterial growth may occur. Thus, it is essential that standard-
ization is carried out rapidly, to prevent cream being held at
these temperatures for unnecessarily long periods. Subsequent
pasteurization and cooling steps must also follow rapidly.
Pasteurization of Cream
Cream is pasteurized chiefly to inactivate enzymes and
microorganisms, thus extending the shelf-life of cream. It ren-
ders a safe product for the consumer. The effects of heating
largely depend on the intensity of the treatment. To pasteurize
cream, either high-temperature short-time (HTST) pasteuriza-
tion or UHT sterilization is commonly applied. The high fat
content of cream protects microbes during heating, necessitat-
ing a more severe heat treatment than that required for the
pasteurization of milk. For the HTST pasteurization of cream,
the International Diary Federation recommends heating at
75
C, for 15 s for cream containing < 20 g fat/100 g, or to
>80
C for 15 s, for cream containing >20 g fat/100 g.
When cream is UHT-treated, the quality of the raw material
is particularly important, owing to the longer shelf-life of this
product. The UHT processing of cream involves heating the
product to 135–150
C for a few seconds, whereby minimal
chemical, physical, and organoleptic changes occur in the
cream, but spoilage and pathogenic microorganisms are inac-
tivated. The UHT treatment of cream does not necessarily
produce a sterile product, as some bacterial spores, especially
those of Bacillus spp., can survive UHT treatment. The indige-
nous milk enzymes, lipoprotein lipase and proteinase, cause
lipolysis and proteolysis, respectively, during storage.
334 Cream: Types of Cream
Neutralization of Cream
The neutralization of sour cream refers to the partial reduction
of its acidity. It is done to avoid the excessive fat loss in butter
milk that results from churning highly acid pasteurized cream.
When pasteurizing sour cream, the casein curdles, thereby
entrapping fat globules, and as a bulk of curd goes in butter-
milk, this causes high fat loss. Neutralization also guards
against the production of an undesirable off-flavor in cream.
In addition, neutralization improves the storage quality of
the product. The neutralizer used should never be dried when
added to cream. So it is dissolved in clean potable water and
then diluted to a 10% solution, before being distributed with
vigorous steering at 29–32
C. The cream acidity is checked to
know whether it is correctly neutralized or not.
Homogenization of Cream
The main objective of homogenization is to prevent, or at least
minimize, the creaming phenomenon. However, the alteration
of the characteristics of cream products or the production of
new product structures is also possible by selecting the appro-
priate homogenizing parameters. Although homogenization is
essential for all types of single and half cream, and may be
applied to double cream, it can have negative effects on cream
functionality.
Ripening of Cream
Ripening is a process for fermenting cream, and it may be
coupled between pasteurization and churning, during which
cream undergoes changes in flavor, aroma, and texture. Ripen-
ing cream results in the improved and uniform flavor of butter
and enhances churning. Diacetyl is a major flavor compound
in the ripened cream, and a large number of other compounds
also contribute at levels above and below the individual sen-
sory threshold where synergistic interaction may occur.
Cream Products
Whipping Cream
Whipped cream is valued by consumers for its taste and
texture, and it is often considered a luxury product. The
whipped product is created by beating air into cream to form
a stiff-foamed product, which has many applications, includ-
ing those in desserts and cakes.
Cream is first standardized to the desired fat content, which
is generally between 30 and 40 g/100 g. Stabilizers may be
added to the standardized cream before heat treatment. The
pasteurization of cream is commonly carried out at 80
C, and
then the cream is cooled, aseptically packaged, and distributed.
Pasteurized cream has a shelf-life of <3 weeks at refrigeration
temperature. The need for a cream with an extended shelf-life
has led to the production of UHT whipping cream, which, after
the optional addition of stabilizers, is heated at >135
C for a
few seconds. As a result, a shelf-life of several months can be
obtained.
However, the physical shelf-life of such cream is limited,
owing to the fact that the creaming of lipid globules occurs, so
a homogenization step needs to be applied to reduce lipid
globule size and minimize the separation of the globules dur-
ing storage. However, homogenization impairs the whipping
properties of cream, so choosing the optimal conditions for the
homogenization of UHT whipping cream represents a trade-off
between maintaining optimal whipping characteristics and
minimizing the separation of lipid globules during storage.
Following homogenization, UHT-treated whipping cream is
cooled, packaged aseptically, and distributed.
Process of whipping
Whipping involves beating air into the cream, resulting in the
formation of a coarse foam, which contains air bubbles with an
average diameter of 150 mm. These are rapidly covered by milk
proteins, which stabilize them against collapse. A large propor-
tion of the protein absorbed on the bubble surface is formed by
the surface-active b-casein, which is present in large quantities
in nonmicellar form at the low temperatures at which cream is
commonly whipped, but other caseins and whey proteins are
also found at the bubble interface. On further whipping, air
bubble size is reduced approximately threefold, and milk lipid
globules displace some of the proteins from the bubble
interface. On adsorption at the bubble interface, the globules
shed their globule membrane material from the contact area
with the air bubble in the process, thereby creating an air–lipid
interface.
Prolonged whipping leads to the stiffening of the cream, as
a result of the creation of a network of partially coalesced lipid
globules. Partial coalescence may be induced as a result of
mechanical damage to the milk lipid globules during whip-
ping. This process is enhanced by the presence of large lipid
crystals in the globules. The partial coalescence of the lipid
globules can also result from the collapse of air bubbles in
the foam. Because the globules have shed their original mem-
brane material on the area that interacts with the bubble inter-
face, the collapse of air bubbles leads to partially uncovered
globules, which are extremely susceptible to partial coales-
cence. Eventually, a stiff foam results, in which the air bubbles
are surrounded and stabilized by a network of coalesced lipid
globules. A network of coalesced lipid globules in the serum
phase of the foam adds structure and stability and prevents the
collapse of the whipped cream.
Effect of processing parameters on whipping characteristics
Various processing steps can significantly affect the whipping
characteristics of cream. These characteristics of cream
obtained from the same milk can vary considerably, just by
use of a different separator for separating the cream, and the
overrun of whipped cream increases with the increasing tem-
perature of separation in the range 25–50
C. Homogenization
is required to prevent the creaming of lipid globules in UHT
whipping creams. It increases the whipping time of the cream
and reduces the overrun and stiffness of the whipped cream.
These changes are related to the membrane of homogenized
lipid globules primarily composed of caseins. Compared to
globules, they are less susceptible to absorption onto the air

bubble interface and shear-induced aggregation, however. A
thickening agent such as K-carrageenan is generally added at
around 0.01% to prevent creaming during storage.
To improve the whipping characteristics of homogenized
cream, emulsifiers may be added to the cream. Heat treatment
does not drastically affect the whipping properties of cream per
se, but it is commonly applied in conjunction with homoge-
nization because highly heat-treated creams generally have
poor whipping properties.
Sour Cream
Sour cream is a heavy-bodied, ripened cream of high acidity
(0.6% lactic acid), clean flavor, and smooth texture made by
inoculating sweet, pasteurized, and homogenized cream with a
culture of lactic acid bacteria and allowing fermentation to
proceed until these desired qualities are obtained. It should
have a white-to-yellowish, slightly creamy appearance; clean,
slightly acidic, rich flavor; and clean, milk-sour, flavorful taste.
Method of manufacture
Sweet cream is standardized to get 18–20% milk-fat. It is then
pasteurized, homogenized, and chilled to 15–20
C, and the
final fat content is set. It is inoculated with a lactic acid bacte-
rial starter (i.e., lactic acid/butter culture) at 2–4% and 24
C,
before being fermented until the desired qualities are obtained.
During the acid production, the homogenization clusters floc-
culate, resulting in a highly viscous cream. To increase the
firmness, rennet or permitted thickening agents are sometimes
added to the sweet cream. When the pH reaches 4.5, the cream
is further cooled with gentle stirring and then chilled to 2–4
C
and packed. Alternatively, souring may be applied to the
package. Fermentation, which takes 14–24 h, may occur in
filled cream containers to produce set-style cultured cream, or
in a fermentation tank from which cream containers are sub-
sequently filled. Both types of fermentation have disadvan-
tages. Following in-tank fermentation, the cream has to come
in contact with the production equipment again, because it has
to be further processed, thereby increasing the possibility of
recontamination and reduction in the viscosity of the cream.
Consequently, the need for the addition of hydrocolloids
increases. Although set-style cultured cream is thicker, it tends
to become inhomogeneous during the long fermentation at
ambient temperatures.
Cream may also be chemically acidified, using glucono-
d-lactone or food-grade acids. This method has several
advantages over conventional culturing practices, including
the elimination of culture-handling problems, improved
production efficiency, and quality control. Chemically acid-
ified sour creams have a similar appearance and texture to
those of cultured sour creams, but the latter have superior
flavor characteristics. Sour cream is mainly used in prepared
foods.
Coffee Cream
Coffee cream is a shelf-stable product with a fat content of
>10%. It is homogenized and UHT-processed, filled
aseptically, or sterilized in the container. It is a popular product
that is mainly used for whitening coffee, as well as for
Cream: Types of Cream 335
imparting a pleasant flavor to coffee. It is also used in the
preparation of food and drinks, and for direct consumption.
Coffee cream has a minimum shelf-life of 4 months at room
temperature, and it normally contains 10–12 g fat/100 g, and,
less often, 15–20 g fat/100 g. Its shelf life is similar to the shelf
life of UHT milk. The important quality attributes of coffee
cream are taste, whitening power, and stability in hot coffee.
Following separation and standardization, the cream is
heat-treated at 90–95
C and cooled to 6
C. Stabilizers such
as phosphates or citrates may be added to increase the pH and
to reduce the concentration of ionic calcium, thereby reducing
the susceptibility of casein micelles to aggregation during the
sterilization process or on addition to hot coffee. Cream is
subjected to two-stage homogenization and sterilization. Tra-
ditionally, coffee cream was sterilized in cans or bottles, but
since 1996, continuous-flow sterilization in a UHT plant, fol-
lowed by aseptic packaging, has largely replaced the former
process. Both homogenization steps operate at a total pressure
of 20 MPa and a temperature of 70
C. Flow-sterilized coffee
cream is subsequently cooled to 25
C and aseptically filled,
usually into plastic cups (7.5–15 g capacity) or preformed cups
or cans (100–200 g capacity).
Importance of homogenization
Coffee cream must be homogenized. This prevents a fat layer or
fat plug from forming in the container, thus improving taste,
whipping power, and stability. It has a direct influence on the
flocculation stability of coffee cream. When sterilizing cream in
the pack, homogenization has to take place before the sterili-
zation, which again is a double stage process (20 MPa) using
the same pressure. Flocculation of cream in hot coffee is due to
casein precipitation.
Desirable attributes of coffee cream
In addition to good sensory properties, the two main criteria
for assessing the quality of coffee creams are as follows:
Stability of the emulsion: This is the ability of the particulate
constituents (primarily lipid and protein particles) to
remain evenly dispersed throughout the coffee cream dur-
ing its long shelf-life.
Whitening effect: This is the ability to form a milky, homoge-
nous mixture on addition to coffee, irrespective of the
composition, brewing conditions, and temperature of the
coffee.
Like UHT milk, homogenized UHT creams exhibit a gelation
phenomenon, generally attributed to the activity of heat-
resistant proteolytic enzymes, after prolonged storage.
One important defect with respect to coffee creams is the
phenomenon known as ‘feathering.’ Feathering is the forma-
tion of macroscopic aggregates containing proteins and lipids
through the acid-induced flocculation of partially protein-
covered lipid globules in hot coffee drinks. Although aggre-
gates always form in hot coffee, the visible aggregates (i.e.,
100 mm) render the coffee cream defective, because the con-
sumer commonly perceives such aggregates as an indication of
the sourness of the product. The susceptibility of coffee cream
to feathering is often referred to as its coffee stability.
336 Cream: Types of Cream
Cream Liqueur
Cream liqueurs are a group of liqueurs that contain milk cream
as an ingredient. The roots of cream liqueur lie in home-made
products that can be traced back for centuries, such as the
Scottish product Atholl Brose. Atholl Brose consists of milk
cream and honey, to which whisky, steeped with oatmeal to
impart a pleasant nutty flavor, is added. In the second half of
the twentieth century, commercial-scale attempts to produce
Atholl Brose and other home-made cream liqueurs largely
failed, until 1974, when Baileys Irish Cream Liqueur became
the first commercialized cream liqueur. The most famous
cream liqueurs are the Irish product Baileys Irish Cream and
the South African product Amarula.
Composition of cream liqueur
In terms of commercial practice, a standard cream liqueur does
not exist. Each manufacturer decides the preferred components
and their concentrations. This flexibility enables a product of
desired organoleptic properties and a sufficiently long shelf-
life. Table 3 shows the composition of a standard cream
liqueur, which appears to represent most commercially avail-
able products. The 14 g/100 g concentration of ethanol in the
product equates to 17 g/100 ml ethanol. Of the ingredients,
sodium caseinate is used as an emulsifier, and citrate is added
to prevent serum separation during storage.
Technology of cream liqueur
Although there is no universal manufacturing process for
cream liqueur, the principle steps of mixing the ingredients
and homogenizing the cream base are universal. The sodium
caseinate can be dissolved in hot water (85
C), after which
time the other ingredients are added to produce the cream
base. Ethanol may be added to the cream base either before
or after homogenization. The function of homogenization is to
reduce the lipid globule size, thereby prolonging the physical
stability of the product by preventing creaming and/or the
formation of the cream plug on storage. A general rule-of-
thumb for homogenization efficiency is that >98% of the
milk lipid globules should have a diameter <0.8 mm.
Maintaining the quality of cream liqueur
As with other cream products, the shelf-life of cream liqueurs
has two dimensions, the microbiological shelf-life and the
physicochemical shelf-life. The microbial shelf-life of cream
Table 3 The composition of a standard cream liqueur
Component Concentration (g kg1)
Milk fat 160
Sucrose 195
Sodium caseinate 30
Nonfat milk solids 14
Total solids 399
Ethanol 140
Water 461
Source: Banks, W. and Muir, D. D. (1988). Stability of alcohol-containing emulsions.
In: Dickinson, E. and Stainsby, G. (eds.) Advances in food emulsions and foams.
London: Elsevier Applied Science.
liqueurs causes little concern, because human pathogens are
unable to grow in this medium of high ethanol and sugar
content.
Physicochemical defects in cream liqueurs
Different types of physicochemical defects have been noted in
cream liqueurs:
(a) Cream plug formation: Cream plug formation refers to the
accumulation of fat in the neck of the bottle during stor-
age, with the fat unable to be redispersed into the product
through shaking or agitation. Cream plug formation
results from insufficient homogenization. As a result,
lipid globules rise to the top, which may ultimately lead
to a cream plug. Because the plug cannot be redispersed on
shaking or agitation, it is likely that the creamed lipid
globules have aggregated or coalesced. The extent of
cream plug formation correlates positively to the propor-
tion of large lipid globules in the product, and it can be
prevented by increasing the homogenization pressure.
Homogenization efficiency may also be increased,
reducing creaming, by the inclusion of low levels (0.5 g/
100 g) of low-molecular-weight surfactants in the cream
liqueur. Cream plug formation is likely enhanced at low
pH levels (<6.0), high concentrations of ionic calcium,
and low concentrations of emulsifier, and it can also be
increased by temperature fluctuations during storage.
(b) Serum separation: The gelation of cream liqueur results in
syneresis, leading to the separation of a serum layer. The
shelf-life of cream liqueur is the number of days the prod-
uct can be stored at 45
C before noticeable serum separa-
tion, and it increases in a sigmoidal manner with
increasing pH. The use of washed cream or anhydrous
milk fat improves the shelf-life of cream liqueurs, and
the same is true for the addition of the calcium-chelating
agents such as trisodium citrate. The addition of citrate
reduces the concentration of ionic calcium and
accordingly increases the shelf-life of cream liqueurs by
up to two orders of magnitude. Replacing caseinate with
whey protein concentrate also increases product stability.
Furthermore, the use of sorbitol as a carbohydrate compo-
nent significantly increases the shelf-life of cream liqueurs.
(c) Precipitation: Although the addition of citrate to cream
liqueur enhances its stability against serum separation, it
also causes a defect in the form of a slightly granular
precipitate, consisting primarily of calcium and citrate, at
the bottom of the bottle. The precipitate is particularly
noticeable on storage at above ambient temperatures,
because the solubility of calcium citrate decreases with
increasing temperature. As a result, the amount of citrate
added to cream liqueur becomes a trade-off between pre-
venting serum separation and minimizing the risk of pre-
cipitate formation.
Other Cream Products
Clotted Cream
Clotted cream is prepared by heating cream to 77–88
C
(170–190
F) in shallow pans and then allowing it to cool

slowly. The surface layer consists of dotted cream, which is
skimmed off and strained. Clotted cream is exceedingly rich,
containing 60–70% milk fat. This fat is in a finely emulsified
condition, which renders it easily digestible. The product has a
peculiar boiled taste and rough appearance, and it exhibits a
white-flaked surface. The average composition of clotted cream
is 67.50% milk fat, 4.90% protein; 1.00% lactose, 0.50% ash,
and 26.10% water.
There is no standardized method for preparing clotted
cream. Several systems differ in obtaining the raw cream, thus
resulting in considerable variation in the texture, flavor, and
appearance of the finished product. The flavor and physical
consistency of cream depend on the acidity of the original
milk, the temperature of scalding, and the time allowed for
scalding.
Canned or Sterilized Cream
Canned or sterilized cream generally possesses a peculiar flavor
because of its processing, as well as a high viscosity due to
homogenization. The texture should be smooth, and the
cream should be free from lumpiness and separation of
serum. Sterilization spoils its whipping quality. The fat content
is about 20–25%, and the nonfat solids content may vary
between 6.5% and 9.5%. The various steps in manufacture
are as follows:
(i) Fresh, sweet cream is standardized to 20% fat.
(ii) Pre-Standardized cream is heated to 80
C, without
holding.
(iii) Cream is double-homogenized at 80
C, using
2500–3000 psi in the first stage and 500 psi in the second
stage.
(iv) Cream is immediately cooled to 16
C, preferably over a
surface cooler using brine.
(v) Cream is filled into tin cans (or bottles) and immediately
sealed.
(vi) Filled cream is sterilized in retorts employing 15 min for
coming-up, 12–15 min for holding at 118
C, and 15 min
for cooling to room temperature.
Plastic Cream
Plastic cream is more viscous than any other type of cream. Its
texture resembles paste. Its fat content is between 65% and
85%, and it can be used directly for the manufacture of butter-
oil. Plastic cream is obtained by (i) reseparating normal cream
(30–40%) in a normal cream separator or (ii) separating milk
in a specially designed plastic cream separator. In both of the
above cases, the initial product is pasteurized at about
71–77
C for 15 min and cooled to 60–66
C before
separation.
Cream: Types of Cream 337
Conclusion
Cream is an extremely versatile product with applications that
extend far beyond the dairy sector. Cream products have been
traditionally regarded as luxury products, and their popularity
may be affected in the current climate by consumer concerns
about high fat intake. Given recent advances in our under-
standing of milk and cream constituents and their interactions
with other ingredients, the development of reduced fat cream
products with properties equivalent to their traditional high-fat
counterparts should be possible.
See also: Buffalo Milk; Butter: Manufacture; Coffee: Health Effects;
Coffee: Types and Production; Cream: Clotted Cream; Dairy Products:
Dietary and Medical Importance; Ghee.
Further Reading
Anonymous (2003) Centrifugal separators and milk standardization. In: Dairy processing
handbook, 2nd ed., pp. 99–122. Lund: Tetra Pak Processing Systems AB.
Banks W and Muir DD (1988) Stability of alcohol-containing emulsions. In: Dickinson E
and Stainsby G (eds.) Advances in food emulsions and foams London: Elsevier
Applied Science.
Brooker BE (1993) The stabilization of air in foods containing fat – review. Food
Structure 12: 115–122.
Hoffman W, Moltzen B, and Buchheim W (1996) Photometric measurement of coffee
cream stability in hot coffee solutions. Milchwissenschaft 51: 191–194.
Hoffmann W (2002) Cream products. In: Roginski H, Fox PF, and Fuquay JW (eds.)
Encyclopaedia of dairy sciences, pp. 551–557. London: Academic Press.
Hoffmann W and Buchheim W (2006) Significance of milk fat in cream products.
In: Fox PF and McSweeney PLH (eds.) Advanced dairy chemistry. 3rd ed., Lipids,
3rd ed., vol. 2, pp. 365–375. New York: Springer.
Huppertz T and Kelly AL (2006) Physical chemistry of milk fat globules. In: Fox PF and
McSweeney PLH (eds.) Advanced dairy chemistry. 3rd ed., Lipids, 3rd ed., vol. 2,
pp. 173–212. New York: Springer.
IDF (1982) Technical guide for the packaging of milk and milk products. Brussels:
International Dairy Federation, Document no. 143.
IDF (1996) UHT cream. Brussels: International Dairy Federation, Document no. 315, pp.
4–34.
Smiddy MA, Kelly AL, and Huppertz T (2009) Cream and related products.
In: Tamime AY (ed.) Dairy fats and related products, pp. 61–85. West Sussex:
Wiley-Blackwell.
Tamime AY, Saarela M, Korslund Søndergaard AA, Mistry VV, and Shah NP (2005)
Production and maintenance of viability of probiotic micro-organisms in dairy
products. In: Tamime AY (ed.) Probiotic dairy products, pp. 39–72. Oxford:
Blackwell Publishing.
Walstra P, Wouters JTM, and Guerts TJ (2006) Cream products. In: Dairy science and
technology, 2nd ed., pp. 447–466. Boca Raton, FL: CRC Press.
Relevant Websites
http://anp.sagepub.com/content/31/1/131.abstract – Sage Publications.
http://ebook.worldlibrary.net/articles/Whipped_cream – World eBook Library.
http://en.wikipedia.org/wiki/Dairy_product – Cambridge Journals.
http://journals.cambridge.org/action/displayAbstract?
fromPage¼online&aid¼5142848&fileId¼S0022029900025887 – World Library,
India.
 Cured Foods: Health Effects
J Ruiz-Carrascal, University of Copenhagen, Copenhagen, Denmark
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Curing refers to the technological process involving the addi-
tion of salt and nitrates and/or nitrites to different types of raw
materials to produce the so-called cured food products. The
origin of the word ‘curing’ roots in the Latin word ‘cure,’
translating as ‘means of healing, remedy,’ and was used for
products processed that way because they were known to be
safer and no longer capable of transmitting any disease. Curing
is no doubt one of the earliest processing technologies for
preservation of different foodstuff, whose origin probably
dates backs to ancient Egypt, being well known and described
by the ancient Greeks and Romans. In even earlier times, basic
processing technologies consisted of adding salt to different
raw food materials, such as fish, cheese, and meats, thereby
leading to a stable food product that could be preserved at
room temperature. The main preserving effect of salt relies on
the decrease of water activity; however, in the past, the addition
of sea salt involved the unintentional addition of nitrates,
whose effects on preservation were not understood until
later. Nevertheless, ‘saltpeter,’ a salt efflorescence very rich in
nitrates, was used in ancient China and India, and its effects on
producing a stable meat color were also well known.
Nowadays, curing is undoubtedly the technological process
most commonly used for transforming meats, including fer-
mented, dry-cured, smoked, cooked, or canned products.
The technological and safety effect of curing salts is well
known and appreciated, and it is thoroughly explained else-
where in this encyclopedia. To synopsize, curing leads to the
development of a stable red color in meats due to the forma-
tion of a stable pigment through the reaction of myoglobin
with nitric oxide (NO), which in turn is formed from nitrite.
Furthermore, the antioxidant effect of nitrites is well known,
and its addition helps to control the formation of compounds
showing rancid flavors involved in warmed-over off-flavor
development in cooked and reheated meats. In addition to
this effect on flavor, nitrates and nitrites are known to posi-
tively influence the overall flavor of cured products, although
their exact involvement has not been entirely elucidated yet.
But it is perhaps the antimicrobial effect associated with
nitrate/nitrite usage that is the most outstanding property.
The presence of a certain amount of nitrites inhibits the germi-
nation of Clostridium botulinum spores and also helps in the
control of the growth of other pathogenic and spoilage bacteria
in meat products. Therefore, nitrate and nitrite addition is
among the most effective and reliable hurdles for avoiding
some of the most serious microbial poisonings.
However, in spite of all these positive effects, the use of
nitrate and nitrite in food processing has been controversial
over the past 40 years, and this has been mainly due to the
toxicity of nitrite itself and to the further reaction with amino
compounds to form N-nitroso compounds, some of which,
like nitrosamines (NAs), have been shown to be potential
338 Encyclopedia of Food and Health
carcinogens. On the other hand, the intake of nitrate and nitrite,
through their transformation into NO, has also been shown to
have a protective role in some cardiovascular diseases.
The balance between all these effects has been thoroughly
researched for quite some years, but still, there is no clear final
scientific outcome supporting either the total avoidance of
these preservatives or their indiscriminate use. Accordingly,
food legislations in most countries have restricted their use to
some specific products and to limited amounts. While meat
companies claim that their use is absolutely necessary for
obtaining safe products, other groups ask for their total ban-
ning from food processing.
This article attempts to collect evidence pertaining to the
potential harmful or health-promoting effects of the addition
of nitrate and nitrite to food (especially meat) products.
Formation of N-Nitroso Compounds
The major concern for the use of nitrate/nitrite in food is
related to the involvement of nitrite in the formation of carci-
nogenic N-nitroso compounds, especially NAs, and their
subsequent effect as known carcinogenic compounds. These
N-nitroso compounds can be formed either in the food during
processing and prior to consumption or in the human body,
due to the ingestion of nitrates or nitrites. The basic chemistry
for the formation of NAs is explained elsewhere, but basically,
it involves the formation of a nitrosating agent, typically
formed from nitrites, which further reacts with a secondary or
a tertiary amine (Figure 1). This takes place faster in acidic
conditions, although it can also happen in an alkaline medium
in the presence of some carbonyl compounds. In the case of
added nitrates, it would involve a previous reduction of
nitrates to nitrites, which is also favored not only under acidic
conditions but also by microbial activity. Such kind of routes
can be produced not only in the food itself but also in the
human body, as a consequence of the ingestion of amines and
the nitrite from the food or through interaction with saliva.
The concern about the involvement of curing salts in the
formation of carcinogenic compounds commenced in the
1950s and 1960s, with several studies highlighting the poten-
tial implication of nitrite in the formation on NAs. This fact
was also linked to the occurrence of different types of cancer. In
1970, a report paper published in Nature by Lijinsky and
Epstein reviewed the role of NAs in the development of cancer.
Due to the implication of nitrite and amino compounds in the
formation of these carcinogenic compounds, the authors con-
cluded that the best way to avoid the risk of NA intake was to
eliminate any of these precursors. Since amino compounds are
hardly avoidable in most foods, the final indication toward a
banning of curing salts was quite clear.
Thus, there are different aspects that should be considered
within the controversy of nitrates/nitrites as food additives.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00207-5

NaNO2 + H+ HNO2 + Na+
HNO2 + H+ NO+ + H2O
2HNO2 N2O3 + H2O
N2O3 NO + NO2
NO + M+ NO+ + M
R2NH + NO+ R2N-N=O + H+
Figure 1 Formation of nitrosamines (M/Mþ are transition metals ions
like Fe2þ/Fe3þ or others). Adapted from Honikel, K. O. (2008). The use
and control of nitrate and nitrite for the processing of meat products.
Meat Science 78, 68–76, with permission.
First, it seems clear that several N-nitroso compounds have
been characterized as being potential carcinogenic com-
pounds, but it is important to know which ones and at what
level. Then, the actual involvement of nitrates and nitrites in
the formation of NAs, both in the food and in the human
organism, and other factors affecting their formation in the
food, should also be considered as very few compounds can be
considered totally harmless. It is the dose and the intake con-
ditions that make them more or less dangerous. Furthermore,
the actual contribution of cured foods as a source of nitrates,
nitrites, and N-nitroso compounds in the diet should also be
taken into consideration. Finally, there is the question about
the link between consumption of cured products and the
incidence of cancer.
As far as the role of NAs in the development of different types
of cancer, their high carcinogenic potency has been proven in
animal experiments on many different species, and virtually no
species has been found to be resistant to their cancer-inducing
potential. In fact, the metabolic activation of NAs into DNA-
damaging electrophilic agents is very similar in all animals
and tissues, resulting in the induction of similar mutagenic
DNA lesions. So, although direct proof of carcinogenicity
to humans is not available, the evidence for these compounds to
be considered human carcinogens is overwhelming. However,
each NA seems to have a different ability to induce cancer.
In fact, the volatile NAs seem to be more carcinogenic,
while nonvolatile ones appear to be not as carcinogenic, or
not directly carcinogenic; nevertheless, some of them can be
further transformed into volatile versions. Among those fre-
quently found in cured products, N-nitrosodimethylamine
(NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine
(NPYR), N-nitrosopiperidine (NPIP), N-nitrosomorpholine,
N-nitrosomethylbenzylamine, and N-nitrososarcosine have
shown strong evidence of being carcinogenic, while
N-nitrosothiazolidine, N-nitrosoproline, or N-nitrosothiazoli-
dine-4-carboxylic acid seems to either be noncarcinogenic or
show a low carcinogenic potential. Despite the type of compound,
the maximum allowed level for the sum of all NAs in cured meat
products has been set to a limit of 10 mg kg
1 of total volatile NAs
in the United States, while no maximum limits have been estab-
lished within the EU as of yet. Nevertheless, the actual levels
leading to the development of a cancerous lesion can be very
different from one NA to another, and in the case of humans, it
Cured Foods: Health Effects 339
is mostly unknown. To make things even more complex, it seems
that NAs show a higher effect as carcinogens in laboratory animals
when applied in repeated small doses as compared with larger
single applications. This situation would in fact mimic the expo-
sure of humans to traces of NAs on a daily basis.
The presence of nitrate and nitrite enables the formation of
NAs in cured products, not only cured meats but also cheese
and some fish products. The level and type of NAs found in
cured products are very variable. In cooked cured meat prod-
ucts, the amount of secondary amines produced when the
product is heated is rather low, since they are processed using
fresh meat. The presence of free secondary amines in meat is
mainly due to microbial metabolism, and thus, their forma-
tion involves high microbial counts, the presence of specific
conditions, and the factor of time. On the other hand, due to
the high reactivity of nitrite, in raw cured meat products, the
amount of residual nitrite is rather low, and moreover, there is
no heating step during processing, which would otherwise
boost nitrosation reactions. As a consequence of all this, in
meat products, the two most frequently determined NAs
(NDMA and NPYR), with the production conditions and addi-
tive levels allowed nowadays, are found at levels rarely higher
than 5 mg kg
1. Other common NAs in cured meat products
are NDEA and NPIP, and they also reach similar or slightly
lower levels. Cured fish products and cheese usually show
lower levels of these same NAs, even though some specific
fermented and salted fish products, especially from Southeast
Asia, may show very high amounts of NDMA, NPIP, and
NPYR, most likely due to their elevated content of biogenic
amines. There have been several attempts to address how much
the different types of food consumed in a regular diet contrib-
ute to the total intake of nitrate, nitrite, and N-nitroso com-
pounds (or NAs). Beer, cured meats, and fish products have
been highlighted as the main sources of NAs in several coun-
tries. Nevertheless, the levels found in cured products are usu-
ally low and within established safe limits, except for
exceptional circumstances (whether these limits are actually
safe in the long term for humans is largely unknown, but
they have been established using animal model systems).
Different processing factors may enhance the NA content in
cured products: nitrate and nitrite content, pH, cooking tem-
perature, presence of secondary amines or presence of ascor-
bate or tocopherols, etc. A deep knowledge about how these
factors influence the formation of NAs may enable the devel-
opment of processing protocols aimed at controlling the for-
mation of such carcinogenic compounds.
It seems clear that the amount of nitrate and especially
nitrite positively influences the formation of NAs, and in fact,
it has been suggested that their formation is proportional to the
square of the nitrite concentration. As far as nitrate is con-
cerned, an almost linear relationship between the amount of
added nitrate and the content of NDMA and other volatile NAs
has been found in several cured meat products and in cured
cheese. Such a relationship between nitrate and nitrite content
and amount of NAs is the reason behind the limitation in the
use of these curing salts in most food products all around the
globe and also the voluntary reduction by many food manu-
facturers. These controls have led, over the past 20 years, to a
reduction in the NA content of many common cured meat
340 Cured Foods: Health Effects
products. Nevertheless, when comparing the actual levels of
NAs in commercial cured meat products in Denmark, where
the national normative only allows a maximum level of
60 mg kg
1, with those in meat products from other EU mem-
bers, in which the maximum allowed level is more than twice
the Danish level (Table 1), differences were very scarce. In fact,
some studies have indicated that there is a positive, but not
necessarily linear, correlation between the amount of nitrite
added into the food and the amount of NAs formed in the final
product.
In some countries, the legislation for organic products does
not allow the use of nitrates and/or nitrites in the processing of
cured meat products. In fact, consumer preferences for organic
and natural foods are largely based on concerns about addi-
tives used in conventionally produced foods. Consumers are
willing to pay significantly higher prices for organic foods.
Strangely, many of these organic processed meats (the term
‘cured’ is not allowed in US legislation, because of the lack of
nitrate and nitrite addition) show similar properties to the
conventional ones in terms of color and oxidative stability. In
most of these products, producers are using spices or plant
extracts naturally rich in nitrates, like celery, spinach, and
beetroot. Among these, celery is perhaps the most popular,
due primarily to its lack of negative impact in relation to
color and flavor tainting. The nitrate content of celery powder
may be higher than 20 g kg
1. Nevertheless, in this type of
product, the actual final amount of curing salts is usually
lower than in conventional cures, which could be a concern
relative to microbiological safety.
Considering that nowadays the addition of nitrite to cured
products remains one of the only plausible strategies to ensure
the inhibition of C. botulinum spore germination, a further
reduction in allowed nitrite levels for cured meat could imply
a safety risk. Therefore, other strategies for reducing the forma-
tion of NAs would be of enormous interest from a public
health perspective. Indeed, ascorbic acid, erythorbic acid, and
tocopherols have shown to have a remarkable decreasing effect
on the formation of NAs in different types of cured meats. They
are capable of inhibiting NA formation by competing with the
precursor amine for available nitrosating agents. During curing
at a regular meat pH, ascorbic acid reacts faster than secondary
Table 1 Maximum allowed levels for nitrate and nitrite (mg kg
1) in some cured products by the EU legislation (EU Commission Regulation,
No. 1129/2011)
Type of product Nitrite (E249/E250)
Non-heat-treated processed meat 150
Heat-treated processed meat 150
Heat-treated processed meat 100
Wiltshire bacon and similar products 175
Dry-cured bacon and similar products 175
Dry-cured ham 100
Salchichón, chorizo, saucisson sec
Ripened cheese
Whey cheese
Cheese products
Dairy analogues
Pickled herring and sprat
In all cases, it refers to the maximum added amount, except when otherwise specified.
amines, such as proline, with the nitrosating agent N2O3,
thereby preventing the manner in which NA formation occurs.
Ascorbate reacts with nitrite 240 times more rapidly than
ascorbic acid, and it is, therefore, the preferred candidate of
the two. In fact, ascorbate is commonly used in the processing
of cured meat products, with the aim of helping both the
formation of a stable red pigment through its positive effect
on the nitrosation of myoglobin and the prevention of the
formation of NAs during processing and subsequent storage.
Some lipid-soluble salts of ascorbic acid have been shown to
have an even better effectiveness in preventing the formation of
NPYR during the frying of bacon. Gamma irradiation has also
been shown to have promising effectiveness in reducing the
NA content in cured meat products. However, this type of
technology is far from being popular in most countries right
now, particularly within the EU.
Heating is well known to be one of the factors boosting the
formation of NAs during the processing of cured meat products
and also during cooking before consumption. For example,
raw bacon is mostly free of NAs, but after cooking, and espe-
cially after frying, their levels considerably rise. In fact, when
analyzing the effect of heating on the NA content of different
foodstuffs, a general trend of higher levels of NAs in products
with higher processing temperatures has been found. However,
in a recent study in which NA content was corrected for mois-
ture loss, the trend was not the same for all NAs: while NPIP
increased in almost all cured meat products due to heating,
other volatile NAs showed either an increase or a decrease
depending on the product. Such varying results could be due
to the specific temperatures used for cooking, since very high
temperatures could lead to a decrease in the content of certain
NAs due to evaporation.
Cured products with a high initial level of secondary
amines, usually present due to microbial metabolism, run a
higher risk for the formation of NAs during processing. In
many of these products, such as meat, cheese, or fish, there is
no additional heating of the product before consumption,
which in a way limits the formation of NAs. However, it should
be considered carefully that some of these products are actually
heated when being used as part of the recipe of another food-
stuff, such as pizza, pasty, and soups. In some fish products in
Nitrate (E251/E252) Observations
150
Except sterilized meat products (Fo > 3.00)
Only sterilized meat products (Fo > 3.00)
250 Maximum residual amounts
250 Maximum residual amounts
250 Maximum residual amounts
250
150 Only hard, semihard, and semisoft cheese
150 Only cheese milk of hard, semihard, and semisoft cheese
150 Only hard, semihard, and semisoft ripened products
150 Only dairy-based cheese analogue
500

which the initial content of free amines is enormous, curing
implies a very high risk for the formation of unacceptable
amounts of NAs. In fact, addition of nitrites to fish and seafood
is banned or severely restricted by most food legislation.
Some unusual NAs, such as N-nitrosodi-n-butylamine and
N-nitrosodibenzylamine, have also been detected in cured
meat products packaged in elastic rubber nettings, formed as
a result of the interaction of the residual nitrite in the cured
meats with amine additives in the rubber nettings. NPIP in
cured products due to the simultaneous presence of nitrite and
pepper can occur either by the oxidative cleavage of the amide
bond of piperine and a subsequent nitrosation of piperidine or
by a direct nitrosation of the already available piperidine.
In addition to the formation of NA in the food, nitrates and
nitrites are also involved in their formation in vivo in the
human body, mainly due to the acidic conditions of the stom-
ach. Gastric nitrosation proceeds through reaction between
amines from the food and nitrosating agents derived from
the diet, both nitrites present directly in the food and nitrates
reduced to nitrites in the mouth. In fact, nitrate is resorbed in
the intestine and excreted through the saliva; thereafter, it is
partially reduced to nitrite by nitrate reductase-containing bac-
teria in the oral cavity. Some studies have suggested that
around 90% of ingested nitrite comes from the saliva. Nitrite
intake in most countries mainly comes from cured meats,
while the main source of nitrates is green leafy vegetables. In
fact, most global food legislation has limited the use of nitrates
for cooked cured meat products, so that the actual amount of
nitrite can be more precisely controlled (Table 1). Moreover,
there has been a trend toward a reduction in allowed levels of
nitrate and nitrite in cured meats, so that nowadays, the resid-
ual levels are much lower than they used to be 30 years ago.
Nevertheless, EU legislation still allows the use of nitrate in
several traditional cured meat products and in cured cheese
(Table 1). In the case of traditional cured meat products, pro-
cessors claim that without the addition of nitrates, it would be
difficult to achieve the features that consumers expect in this
type of products, while in cheeses, the allowance is based in the
prevention of early blowing during the processing of less acidic
varieties. The use of such high levels of nitrates allowed in
pickled herring by the EU normative is remarkable, and as
such, usage entails a risk for NA formation.
Nitrosation in the intestinal track seems to be markedly
diminished by the presence of ascorbate, so that its presence
in the food would limit the in vivo formation of NAs. However,
some findings suggest that in the presence of fat, ascorbate
could actually enhance the formation of NA in the stomach.
The parallel intake of polyphenols or other plant compounds
with antioxidant activity could also help in the reduction of
in vivo nitrosation, while other studies suggest that heme from
meat could play a role in boosting nitrosation in the large
intestine. Without question, more research is needed in this
area, which will potentially allow designing cured food prod-
uct formulas targeted at reducing nitrosation of amines, both
in the food and in vivo.
As discussed previously, the potential involvement of cured
products in the development of cancer caused by N-nitroso
compounds could be due to both the formation of NAs (and
other N-nitroso compounds) during product processing and
storage and their contribution to the total nitrate and nitrite
Cured Foods: Health Effects 341
intake, which would be in turn related to in vivo nitrosation.
Indeed, the role of NAs in cancer was already described in the
1950s. Later, in the 1960s and 1970s, several papers linked the
consumption of cured meats to the development of different
types of cancer. Although subsequent studies failed to confirm
that nitrite intake caused cancer, the Assembly of Life Sciences in
the United States was prompted to commission a report on the
toxicity of nitrate and nitrite, which was only partially reassur-
ing. In 2003, the Joint FAO/WHO Expert Committee on Food
Additives stated that the epidemiological studies showed no
consistently increased risk for cancer with increasing consump-
tion of nitrate. Subsequent epidemiological studies have simi-
larly failed to show a convincing link between nitrate or nitrite
intake and cancer. Nevertheless, the International Agency for
Research on Cancer (IARC) published a monograph in 2010
in which it was concluded that ingested nitrate or nitrite under
conditions that result in endogenous nitrosation is probably
carcinogenic to humans. However, this statement does not spe-
cifically refer to cured products. In fact, and as explained earlier,
the major sources for nitrate and nitrite intake in most countries
are saliva, vegetables, and water.
More specifically related to cured products, different epide-
miological studies relating the consumption of cured meats or
fish with a higher risk for the development of different types of
cancer have been published and have been widely communi-
cated in the media. Moreover, the World Cancer Research Fund
published guidelines for cancer prevention, one recommenda-
tion being to limit the consumption of red meat and eliminate
processed meat consumption entirely. Although not specifi-
cally mentioned, it is assumed that such a risk is attributed to
curing, although other factors specifically linked to the proces-
sing of meats could also be involved. Such claims have been
very controversial, and a number of papers and articles have
been published highlighting the fact that such published
claims have lacked epidemiological evidence. In fact, it has
been pointed out that studies linking the consumption of
processed meat to cancer seriously failed to completely con-
sider several additional factors that can contribute to chronic
disease, including participants’ behavior as to alcohol and
tobacco use, exercise, weight, and access to health care.
At any rate, if processed meats are truly involved in a higher
risk of colorectal cancer, it is unlikely that this is due to residual
nitrate or nitrite, because green leafy vegetables show much
higher levels and seem to show no epidemiological relation-
ship to cancer. Moreover, both nitrate and nitrite are formed
endogenously and recycled with the saliva. Nevertheless, some
studies suggest that the concomitant ingestion of heme and
nitrosation agents could boost the endogenous formation on
N-nitroso compounds in the colon. Others have hypothesized
that N-nitrosamides, rather than NAs, could be related to the
development of some types of cancers, but little is known
about their actual content or their specific involvement in
this type of disease.
Other Potential Harmful Effects of Cured Foods
Cases of infant methemoglobinemia as a consequence of well-
water with a high nitrate content occurred in the past, and that
is the reason why the maximum allowed amount of nitrates in
342 Cured Foods: Health Effects
water is regulated in most countries. In fact, it is nitrite that
causes methemoglobinemia, and in this sense, it could be
thought that the consumption of food containing residual
nitrite, like cured foods, could somehow be related to this
type of disease. Nevertheless, the levels of dietary nitrites caus-
ing this condition are much higher than those that can be
found in cured foods. Yet, there are reports linking cases of
methemoglobinemia to the consumption of cured products
with high levels of nitrites. For example, in one such incidence
dating from the 1950s, an outbreak of methemoglobinemia
occurred after consuming wieners and bologna containing
more than 5000 ppm of nitrite. At any rate, episodes of cured
food involved in cases of acute nitrite poisoning are most likely
due to processing mistake.
Potential Beneficial Effects of Nitrate and Nitrite
Nitrate conversion into nitrite, and subsequently into NO, is
behind the potential positive health effects of intake of these
inorganic salts. Most of these possible effects have been shown
for vegetable extracts, very rich in nitrates, and it is unlikely that
any significant effect could be due to the residual levels of
nitrate or nitrite found in cured products. Nevertheless, as a
part of the diet, cured products no doubt contribute to the total
intake of nitrate and specially nitrite, since they are estimated
to be the third source of these salts, after vegetables and water.
NO is an endogenously produced modulator of vascular
tone. Indeed, as a consequence of eating a meal rich in nitrates,
a subsequent fall in both systolic pressure and diastolic pres-
sure has been described. It seems clear that the reduction of
ingested nitrate into nitrite is behind this effect, since in an
experiment in which subjects had to eliminate their saliva after
nitrate ingestion, the nitrite peak in blood disappeared and so
too did the effect on blood pressure. NO also plays a key role in
vascular homeostasis by maintaining vessels in their relaxed
state; in experiments carried out in humans, dietary nitrate
supplementation attenuated the impairment in endothelial
function seen in ischemia reperfusion injury. NO also plays a
role in the inhibition of platelet adhesion and aggregation. In
this sense, dietary supplementation with either nitrate or beet-
root juice has resulted in significant inhibition of platelet
aggregation.
Widespread media attention has focussed on the positive
effect of nitrate on muscle efficiency and fatigue resistance. In
different experiments, a short-term dietary supplementation
with sodium nitrate resulted in improved muscular efficiency
and a reduction in oxygen consumption during submaximal
exercise in healthy subjects. NO has numerous physiological
signaling functions that may impact on muscle fatigue and
performance: regulation of blood flow, muscle contractility,
glucose and calcium homeostasis, and mitochondrial oxygen
consumption. The reduction of nitrite to NO is facilitated
when oxygen availability is limited, which agrees with the
fact that nitrate supplementation appears to be particularly
effective in enhancing performance in hypoxia and ischemia.
Recent studies suggest that nitrate supplementation may spe-
cifically improve calcium handling and force production in
type II muscle fibers and result in preferential distribution of
blood flow to type II muscles. Given the importance of type II
fiber recruitment and metabolism to performance in high-
intensity intermittent exercise, and the likelihood that tissue
hypoxia develops to a greater extent during such exercise com-
pared to continuous low-intensity exercise, these recent studies
therefore provide a clear rationale for nitrate supplementation
to enhance performance during multiple sprint sport.
See also: Cancer: Diet in Cancer Prevention; Food Additives:
Classification, Uses and Regulation; Nitrites and Nitrates.
Further Reading
Cross AJ, Pollock JRA, and Bingham SA (2003) Haem, not protein or inorganic iron,
is responsible for endogenous intestinal N-nitrosation arising from red meat.
Cancer Research 63: 2358–2360.
Dellisanti A, Cerutti G, and Airoldi L (1996) Volatile N-nitrosamines in selected Italian
cheeses. Bulletin of Environmental Contamination and Toxicology 57: 16–21.
Drabik-Markiewicz G, Dejaegher B, De Mey E, Kowalska T, Paelinck H, and Vander
Heyden Y (2011) Influence of putrescine, cadaverine, spermidine or spermine on the
formation of N-nitrosamine in heated cured pork meat. Food Chemistry
126: 1539–1545.
Gangolli SD, van den Brandt P, Feron V, et al. (1994) Nitrate, nitrite, and N-nitroso
compounds. European Journal of Pharmacology 1: 1–38.
Herrmann SS, Duedahl-Olesen L, and Granby K (2015a) Occurrence of volatile and
non-volatile N-nitrosamines in processed meat products and the role of heat
treatment. Food Control 48: 163–169.
Herrmann SS, Duedahl-Olesen L, and Granby K (2015b) Formation and mitigation of
N-nitrosamines in nitrite preserved cooked sausages. Food Chemistry
174: 516–526.
Honikel KO (2008) The use and control of nitrate and nitrite for the processing of meat
products. Meat Science 78: 68–76.
Hord NG, Tang Y, and Bryan NS (2009) Food sources of nitrates and nitrites: the
physiologic context for potential health benefits. American Journal of Clinical
Nutrition 90: 1–10.
IARC (1978) IARC monographs on the evaluation of carcinogenic risks to humans, vol.
17: some N-nitroso compounds. Lyon: IARC.
Lijinsky W and Epstein SS (1970) Nitrosamines as environmental carcinogens. Nature
225: 21–23.
Pegg RB and Shahidi F (2008) Nitrite curing of meat: the N-nitrosamine problem and
nitrite alternatives. Trumbull: Wiley-Blackwell.
Pierre FHF, Martin OCB, Santarelli RL, et al. (2013) Calcium and a-tocopherol suppress
cured-meat promotion of chemically induced colon carcinogenesis in rats and
reduce associated biomarkers in human volunteers. American Journal of Clinical
Nutrition 98: 1255–1262.
Skibsted LH (2011) Nitric oxide and quality and safety of muscle based foods. Nitric
Oxide 24: 176–183.
Stuff J, Goh ET, Barrera SL, Bondy ML, and Forman MR (2009) Construction of an N-
nitroso database for assessing dietary intake. Journal of Food Composition and
Analysis 22(S): S42–S47.
Tricker AR and Preussmann R (1991) Carcinogenic N-nitrosamines in the diet:
occurrence, formation, mechanisms and carcinogenic potential. Mutation Research
259: 277–289.
Yurchenko S and Molder U (2006) Volatile N-nitrosamines in various fish products.
Food Chemistry 96: 325–333.
Relevant Websites
http://www.cmc-cvc.com/en/nutrition-health/nitrite-cured-meat-products – Canadian
Meat Council.
http://www.efsa.europa.eu/ – EFSA.
http://epic.iarc.fr/ – EPIC study.
http://www.meatami.com/ – North American Meat Institute.
 Cystic Fibrosis, Nutrition in
S Sabharwal, Boston Children’s Hospital, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Definition
Cystic fibrosis (CF) is a multiple organ system disease, affecting
the lungs and pancreas. Nutritional status is impaired by inad-
equate intake, increased energy needs, and pancreatic insuffi-
ciency. Pancreatic insufficiency results in maldigestion and
malabsorption of nutrients and fat-soluble vitamins. Pancre-
atic enzyme supplementation and optimizing nutritional defi-
ciencies prevent growth failure and improve other outcomes in
patients with CF, including quality of life, resistance to infec-
tion, and chronic lung disease prevention, leading to longer
life expectancy in patients with this disease.
CF is a result of the defects in the CF transmembrane
conductance regulator (CFTR), which is responsible for the
excretion of salt, in turn creating viscous secretions in multiple
organ systems. For decades, CF was thought to be a disease
only of childhood given the lower life expectancy. Largely
because of improvements in nutrition, the average life expec-
tancy of patients with CF has advanced well into adulthood.
Clinical Manifestations
Defects in the CFTR result in multisystem disease including
lung, liver, and gastrointestinal (GI) diseases, notably pancre-
atic insufficiency. While the majority of CF mutations cause
lung disease, growth and nutritional status are closely tied to
lung disease and affected by the type of CF mutation. Body
mass index (BMI) being maintained at  50% for age has been
shown to correlate with the percent predicted forced expiratory
volume in 1 s of greater than or equal to 90%, which is an
excellent marker of lung function. For CF patients ages greater
than or equal to 20 years, it is recommended that women
maintain a BMI at or above 22 and that men maintain a BMI
at or above 23.
Nutritional failure in CF is multifactorial. Insufficient pro-
duction of pancreatic enzymes leads to fat malabsorption,
which can be exacerbated by bile salt abnormalities if there is
concurrent liver disease. Progressive pulmonary infection leads
to increased work of breathing and therefore increased caloric
needs. Chronic lung infection can reduce appetite and lead to
an inflammatory catabolic process in the body. Other factors
affecting nutrition include CF-related diabetes mellitus, altered
motility of the GI tract, and small bowel bacterial overgrowth.
Management
CF derived its name from the cysts and fibrosis noted in the
pancreas of patients with CF. The pancreas is responsible for
secreting enzymes that aid in the absorption of nutrients
including fat-soluble vitamins A, D, E, and K. In order to
counteract this malabsorption, pancreatic enzyme replacement
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00211-7
therapy is used. Lipase is the enzyme responsible for fat absorp-
tion. Pancreatic enzyme replacement is based on the dosage of
lipase in the supplement (500–2500 lipase units per kilogram
of body weight per meal or <10 000 lipase units per kilogram
of body weight per day). In addition, fat-soluble vitamin sup-
plements are routinely given. The CF Foundation (CFF) sup-
ports that higher energy intake is needed for improved weight
gain. In order to achieve energy intake of 110–200% of the
healthy population, the CFF makes several recommendations
with the goal being a high-calorie unlimited fat meal.
For children ages 1–12 with CF, intensive behavioral and
nutritional counseling is recommended to promote weight
gain. For those with growth deficits and adults who are having
difficulty with maintaining weight gain, oral or enteral nutri-
tional supplements are recommended.
Fat-soluble vitamins A, D, E, and K are supplemented in all
children with CF. Supplements are started at diagnosis includ-
ing asymptomatic infants and those without pancreatic insuf-
ficiency. Standard ADEK supplements tend to overestimate
vitamin A and underestimate vitamins D and K. This can be
problematic, for example, in CF patients who also have CF-
related liver disease, as vitamin A toxicity can affect the liver.
Vitamin A deficiency is rare except at the time of diagnosis.
Vitamin A is necessary for vision, gene expression, growth, and
immune function. Available forms of vitamin A include pre-
formed vitamin A (retinol and retinoic acid) and provitamin A
(alpha-carotene and beta-carotene). Vitamin A levels are mon-
itored via serum retinol and retinol-binding protein. Vitamin A
toxicity is possible in CF patients and is marked by bone
mineral loss and liver abnormalities.
Vitamin D deficiency in common in CF. Vitamin D helps the
body use calcium from the diet and deficiency can lead to poor
improper bone mineralization. Vitamin D3 (cholecalciferol) is
contained in most supplements and is the form produced in the
skin by sunlight. The CFF recommends vitamin D3 versus vita-
min D2 (ergocalciferol) because a small study showed it
achieved better target 25-hydroxyvitamin D. Levels are checked
annually at the end of winter. Bone disease in CF results from
decreased mineral density, which worsens with age, severe lung
disease, and malnutrition. There is also an increase in fracture
rates and kyphosis in young adults with CF. In addition to
vitamin D deficiency, chronic corticosteroid use and reduced
weight-bearing activity can contribute to bone disease.
Vitamin E is an antioxidant and its deficiency could con-
tribute to inflammation and lung disease in CF. Vitamin E
deficiency leads to delayed stretch reflexes, cerebellar ataxia,
and peripheral neuropathy. Some studies have shown a corre-
lation between vitamin E status, polyunsaturated fatty acid
(PUFA) status, and inflammation in CF. Eight different forms
of vitamin E exist, the most common form being alpha-
tocopherol acetate. The recommended intake for CF patients
is 20 times the general recommended intake. Serum vitamin E
is influenced by serum lipid levels.
343
344 Cystic Fibrosis, Nutrition in
Vitamin K deficiency is associated with coagulation abnor-
malities and bone disease. Vitamin K is found in green vege-
tables, and vitamin K status is monitored by a serum
prothrombin time. As intestinal bacteria are a source of vita-
min K, supplementation should be provided during courses of
antibiotic, a common occurrence in CF patients.
Essential fatty acids are long-chain PUFAs and include
omega-3 and omega-6 fatty acids. Their deficiency may con-
tribute to an inflammatory state including scaly dermatitis,
alopecia, and growth failure and tends to be more common
in infants. Routine supplementation is not recommended at
this time. However, a systematic review showed that supple-
mentation with omega-3 fatty acids improved several markers
of lung disease.
CF patients are prone to hyponatremic dehydration under
conditions of heat stress secondary to sodium losses through
sweat. Sodium chloride supplementation is used especially in
warm months or climates and in infants.
The CFF recommends a trial of zinc supplementation for
children < 2 years not growing well. Dermatitis can be a symp-
tom of zinc deficiency.
Iron deficiency has been noted in several studies of CF
patients and is monitored yearly by serum hemoglobin and
hematocrit.
Oral supplements are used in CF but are often less effec-
tive because they may displace ordinary food. A systematic
review showed that calorie-protein supplements are not supe-
rior to monitoring and dietary advice from a health profes-
sional and a nutritionist. Enteral nutrition is generally started
via gastrostomy tube for patients with growth failure.
Although there are no randomized control trials to support
this, enteral nutrition is thought to improve and maintain
lung function in patients with CF. For toddlers, a combina-
tion of daytime feeds, daytime meals, and nighttime contin-
uous feeds is used. Concentrated formula delivers more
calories in less volume for overnight feeds to help prevent
excessive nocturnal urination.
In conclusion, CF is a disease in which nutritional status
is highly important to overall disease outcomes. It is impor-
tant to monitor nutritional status regularly, looking at growth
and overall digestive issues including signs of malabsorption,
and to screen for nutritional deficiencies. Fortunately,
advances in these nutritional measures over the past several
decades have resulted in improved life expectancy for this
chronic disease.
See also: Calcium: Physiology; Calcium: Properties and
Determination; Fats: Classification and Analysis; Malnutrition: Concept,
Classification and Magnitude.
Further Reading
Adde FV, Rodrigues JC, and Cardoso AL (2004) Nutritional follow-up of cystic fibrosis
patients: the role of nutritional education. Jornal de Pediatria 80: 475–482.
Bell SC, et al. (1998) Nutrition in adults with cystic fibrosis. Clinical Nutrition
17: 211–215.
Brady MS, Richard K, Yu PL, and Eigan H (1992) Effectiveness of enteric coated
pancreatic enzymes given before meals in reducing steatorrhea in children with
cystic fibrosis. Journal of the American Dietetic Association 92: 813–817.
Ellis JA, Bond SA, and Wootton SA (1992) Energy and protein intakes of patients with
cystic fibrosis. Journal of Human Nutrition and Dietetics 5: 333–342.
Flegal KM, Graubard BI, Williamson DF, and Gail MH (2005) Excess deaths associated
with underweight, overweight, and obesity. JAMA 293: 1861–1867.
Gaskin KJ (2004) Exocrine pancreatic function. In: Walker WA, Goulet O, Kleinman RE,
Sherman PM, Schneider BL, and Sanderson IR (eds.) Pediatric gastrointestinal
disease, pp. 1607–1623. Hamilton, ON: B.C. Decker.
Hanning RM, Blimkie CJ, Bar-Or O, Lands LC, Moss LA, and Wilson WM (1993)
Relationships among nutritional status and skeletal and respiratory muscle function
in cystic fibrosis: does early dietary supplementation make a difference? American
Journal of Clinical Nutrition 57: 580–587.
Lloyd-still JD, Smith AE, and Wessel HU (1989) Fat intake is low in cystic fibrosis
despite unrestricted dietary practices. Journal of Parenteral and Enteral Nutrition
13: 296–298.
Luder E, et al. (1989) Efficacy of a nonrestricted fat diet in patients with cystic fibrosis.
American Journal of Diseases of Children 143: 458–464.
Orenstein DM (2004) Cystic fibrosis: a guide for patient and family. Philadelphia, PA:
Lippincott Williams.
Paccou J, Zeboulen N, Combescure C, Gossec L, and Cortet B (2010) The prevalence
of osteoporosis, osteopenia, and fractures among adults with cystic fibrosis:
a systematic literature review with meta-analysis. Calcified Tissue International
86(1): 1.
Peterson ML, Jacobs DR Jr, and Milla CE (2003) Longitudinal changes in growth
parameters are correlated with changes in pulmonary function in children with cystic
fibrosis. Pediatrics 112(3 Pt 1): 588–592.
Powers SW, Byers KC, Mitchell MJ, Patton SR, Schindler T, and Zeller MH (2003) A
randomized pilot study of behavioral treatment to increase caloric intake in toddlers
with cystic fibrosis. Children’s Health Care 32: 297–311.
Powers SW, Jone JS, Ferguson KS, Piazza-Wagoner C, Daines C, and Acton JD (2005)
Randomized clinical trial of behavioral and nutrition treatment to improve energy
intake and growth in toddler and preschoolers with cystic fibrosis. Pediatrics
116(6): 1442.
Rhodes B, Nash EF, Tullis E, Pencharz PB, Brotherwood M, Dupuis A, and
Stephenson A (2010) Prevalence of dyslipidemia in adults with cystic fibrosis.
Journal of Cystic Fibrosis 9(1): 24–28.
Stallings VA, et al. (2008) Evidence-based practice recommendations for nutrition-
related management of children and adults with cystic fibrosis and pancreatic
sufficiency insufficiency: result of a systematic review. Journal of the American
Dietetic Association 108: 832–839.
Stark LJ, Opipari LC, Jelalian E, et al. (2003) Contribution of behavioral therapy to
dietary treatment in cystic fibrosis: a randomized controlled study with 2-year
follow-up. Behavior Therapy 34: 237–258.
Stark LJ, Opipari-Arrigan L, Quittner AL, Bean J, and Powers SW (2011) The effects of
an intensive behavior and nutrition intervention compared to standard of care on
weight outcomes in CF. Pediatric Pulmonology 46(1): 31.
Steinkamp G and Wiedemann B (2002) Relationship between nutritional status and lung
function in cystic fibrosis: cross sectional and longitudinal analyses from the
German CF quality assurance (CFQA) project. Thorax 57: 596–601.
Zhang Z and Lai HJ (2004) Comparison of the use of body mass index percentiles and
percentage of ideal body weight to screen for malnutrition in children with cystic
fibrosis. American Journal of Clinical Nutrition 80: 982–991.
D
Dahi
CD Khedkar, College of Dairy Technology, Pusad, India
SD Kalyankar, Government College of Dairy Technology, Udgir, India
SS Deosarkar, College of Dairy Technology, Pusad, India
AM Patil, Shivaji College, Udgir, India
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Indian curd, known as dahi, is a well-known fermented milk
product consumed by large sections of the population
throughout the Indian subcontinent, either as a part of the
daily diet or as a refreshing beverage. This product finds its
name in the ancient Hindu scriptures and is used in several of
Ayurvedic formulations (traditional Indian medicinal system)
because of its potential therapeutic properties. The beneficial
aspects of dahi and its products have been often investigated.
Dahi prepared from buffalo milk is white in color and pos-
sesses a firm body and slightly granular texture, whereas dahi
prepared from cow milk is less firm and smooth in texture.
Dahi may be consumed directly, either sweetened or salted and
spiced. It is also consumed with other foods such as rice and
wheat loaf. Dahi has assumed a special place in the daily diet of
Indian populations, who prefer to take dahi once or twice a day
in morning or evening meals. In 2010–11, the production of
dahi was estimated at almost 8% of the total dairy production
in India. Since conversion of milk into dahi is an important
intermediate step in the manufacture of indigenous fat-rich
dairy products like butter and ghee, it can be said that over
40% of the total milk production in India is converted into
dahi. Dahi is also used as a vehicle to incorporate probiotics,
and several varieties of dahi are developed for the benefit of
consumers, like fruit dahi, sweetened dahi, whole-milk dahi,
and skim-milk dahi. Though nowadays, dahi is mostly sold
in polythene pouches and in set form in HDPE containers,
traditional dahi was being sold in earthen pots. Dahi sold in
pouches does not have the three-dimensional network struc-
tures that are typical of set dahi, whereas the products sold in
the rigid plastic containers possess a firm body and smooth
texture that appeals to consumers. Its typical flavor is the fore-
most quality attribute of dahi that determines its acceptance. The
pleasant flavor of dahi is attributed to its diacetyl content, which
is produced by citrate-fermenting lactic acid bacteria (LAB).
Definition
According to the Bureau of Indian Standards (BIS), dahi is a
product obtained by lactic fermentation of cow or buffalo milk
or mixed milk through the action of single or mixed strains of
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00212-9
LAB. This definition does not include milk coagulated by the
addition of acids and milk-coagulating enzymes. As per the
Prevention of Food Adulteration Act of India (PFA, 1988), dahi
or curd is a product obtained from pasteurized or boiled milk
by souring naturally or otherwise by adding harmless lactic
acid and/or other aroma-producing lactic acid bacterial cul-
tures. Dahi may contain additional cane sugar. It should have
the same percentage of fat and nonfat solids as the milk from
which it is prepared. Where dahi or curd, other than skimmed-
milk dahi, is sold or offered for sale without any indication of
the class of milk, the standards prescribed for dahi prepared
from buffalo milk should apply. This is an equivalent of yogurt
made from boiled milk after inoculation through back-
slopping, which consists of the leftover dahi from the previous
lot. However, it differs from yogurt in having less acidity.
Starter Cultures Used for the Preparation of Dahi
In the production process of dahi, inoculation with small
quantities of the desired fermenting microbiota, that is,
through back-slopping or via the starter culture, takes place.
The most important microorganisms in starter cultures are
the homofermentative and heterofermentative LAB. The dahi
starter cultures can be classified into two groups, according to
the characteristics of the microorganisms: mesophilic and ther-
mophilic cultures.
Mesophilic starter cultures are used at temperatures varying
between 16 and 30  C (Table 1). The characteristics of dahi
starter cultures and their metabolic products are given in
Table 2. The most typical representatives are the homofermen-
tative Lac. lactis subsp. lactis and Lac. lactis subsp. cremoris.
These are similar, though the former is able to produce free
amino acids, which are believed to stimulate the growth of Lac.
lactis subsp. cremoris strains. Another important species in the
mesophilic starter category is the homofermentative Lac. lactis
subsp. lactis biovar diacetylactis, which is able – besides lactic
acid production – of fermenting citric acid into a number of
other compounds such as diacetyl and carbon dioxide. How-
ever, at a pH below 5, carbon dioxide and a number of
compounds similar to diacetyl, but nonaromatic, are pro-
duced. Heterofermentative aroma-producing starter cultures,
like Leu. mesenteroides subsp. dextranicum and Leu. mesenteroides
subsp. plantarum, are also able to ferment citric acid, that is,
345
346 Dahi
Table 1 Designation of dahi based on types of starter cultures
Sr.
no. Designation Cultures used
1. Sweet dahi Lactococcus lactis subsp. lactis, single or in
combination with Lac. lactis subsp. cremoris,
2. Sour dahi Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus salivarius subsp. thermophilus
Table 2 Characteristics of starter cultures used in dahi making and their principal metabolic products
Starter organisms
Lactobacillus delbrueckii subsp. bulgaricus
Lactococcus lactis subsp. lactis
Str. salivarius subsp. thermophilus
Lactococcus lactis subsp. cremoris
Leuconostoc mesenteroides subsp. dextranicum
Lac. lactis subsp. lactis biovar diacetylactis
into carbon dioxide, diacetyl, and some other products. Unfor-
tunately, leuconostocs may reduce diacetyl into nonaromatic
compounds again. The intensity of reduction varies between
the various representatives of the strains.
Homofermentative thermophilic starter microorganisms, like
L. delbrueckii subsp. bulgaricus and Str. salivarius subsp. thermo-
philus, are used to obtain mildly sour dahi. These starters play an
important role in producing a good quality dahi having a firm
and uniform consistency with a sweet aroma and acidic taste.
Classification of Dahi
Broadly speaking, dahi may be classified into two types:
• Dahi for churning into desi (indigenous) butter
• Dahi for direct consumption
The dahi for direct consumption is further classified into the
following subtypes:
• (i) Whole milk dahi and (ii) skim-milk dahi
• (i) Sweet (or mildly sour) dahi, (ii) sour dahi, and (iii)
sweetened dahi
Technology of Sweet and Sour Dahi Manufacture
The flow diagrams showing the steps involved in manufacture
of sweet (or mildly sour) and sour dahi are given in Figure 1.
Preliminary Treatment of Milk
The preparation of the basic mix involves selection of milk,
preheating, filtration/clarification, and standardization of the
milk. The different methods for standardization indicate
the possibilities that exist for either increasing or decreasing
the various milk constituents, especially the fat percent. How-
ever, the choice of any particular system is primarily governed
by the following factors:
Remarks
With or without aroma producers like Leuconostoc mesenteroides subsp.
dextranicum, Leu. mesenteroides subsp. plantarum, Lac. lactis subsp.
lactis biovar diacetylactis
Single or in combination with or without aroma producers
Metabolic products Lactose fermentation
Lactate, diacetyl, acetaldehyde Homofermentative
Lactate Homofermentative
Lactate, diacetyl, acetaldehyde Homofermentative
Lactate Homofermentative
Lactate, diacetyl Heterofermentative
Lactate, diacetyl, CO2 Heterofermentative
(i) Cost and availability of the raw materials
(ii) Scale of production
The composition of dahi varies considerably. The average com-
position of dahi made from buffalo milk is delineated in
Table 3. Commercial dahi tends to contain around 14% of
milk solids.
The use of ultrafiltration to concentrate the solids in cow
milk and skim milk is being considered as a feasible alterna-
tive. The importance of total solids stems from the improved
consistency imparted to the dahi coagulum, an improvement
that is carried further in the homogenization stage by employ-
ing a pressure of 2500 psi. Homogenization of milk imparts a
smooth texture to the coagulum. It also reduces the chances of
whey separation. Homogenization is followed by a final heat
treatment of 80–90  C for a prolonged period of 20 min. The
effect of this drastic heat treatment can be summarized as
follows:
• The bacterial load in the milk is reduced, and hence, the
starter culture has less competition from the contaminating
microorganisms.
• There is a denaturation of whey proteins (albumins and
globulins) and an aggregation of the casein micelles into a
three-dimensional network. The network traps the whey
proteins, and the dahi coagulum produced subsequently is
rendered more viscous.
• There is a reduction in the amount of oxygen in the milk,
and as normal dahi cultures are microaerophilic, the low-
ered oxygen tension encourages their growth.
• Some limited damage to the milk proteins may occur dur-
ing heating, and the breakdown products can stimulate
starter activity.
Fermentation of Milk
The acidification of milk during the manufacture of dahi is an
important biochemical process, which must be carried out
under controlled conditions in special incubators and/or
 Dahi 347

Selection of milk

Preheating (40 °C)

Filtration/clarification

Standardization (3% fat + 10% nonfat milk solids)

Heat treatment (60 °C)

Homogenization (2500 psi)

Final heat treatment (80–90 °C for 20 min)

Cooling to 22–25 °C

Inoculation with active starter culture @ 1–1.5%

(For sweet dahi Lac. lactis subsp. lactis + Lac. lactis subsp. cremoris + Leu.
mesenteroides subsp. dextranicum)

(For sour dahi L. delbrueckii subsp. bulgaricus + Str. salivarius subsp. thermophilus)

Distribution in the packages of desired size

Incubation at 27–30 °C/18 h (for preparation of sweet dahi )

and 37–38 °C/10–12 h (for preparation of sour dahi )

Cooling to 5 °C or below

Storage and distribution

Figure 1 Flow diagram for the preparation of dahi.

fermentation tanks. Fermentation tanks are used as incubators
only, and they are usually insulated in order to maintain
appropriate temperature. The processing of the milk and the
cooling of dahi is carried out in other equipment on the pro-
duction line. Cabinets are also used for this purpose. These
comprise a small insulated room, which is divided into com-
partments, and most incubators of this type are multipurpose
chambers capable of circulating hot or cold air. Hot air is
circulated during the fermentation period followed by cold
air during the cooling stage. Sometimes, these cabinets are
used as incubators only, and the dahi can be cooled in refrig-
erated cold storage. In the case of mildly sour dahi manufac-
ture, the temperature is maintained at 27–30  C, while for sour
dahi, it is 37–38  C.
348 Dahi
Table 3 Average composition of buffalo milk dahi
Constituents % flat and textural defects like gassiness, weak body, wheying off,
Moisture 85–88
Fat 5–8
Protein 3.2–3.4
Lactose 4.6–5.2
Ash 0.70–0.75
Lactic acid 0.5–1.0
Calcium 0.12–0.14
Phosphorus 0.09–0.11
Cooling of Dahi
After the incubation period is over, dahi is cooled in order to
control the level of lactic acid in the product. The rate of cool-
ing can affect the structure of the coagulum. Thus, very rapid
cooling can lead to whey separation, and this is due to a too
rapid concentration of protein filaments, which, in turn, affects
their hydrophilic properties.
Microbiological Quality of Dahi
In general terms, dahi can be regarded as ‘hygienically safe.’ The
reason for this confidence stems from the level of acidity
present around 0.7–1.0% (lactic acid). Under this situation,
potential pathogens such as Salmonella spp. will be largely
inactive. Similarly, coliforms will be unable to survive the
low pH encountered. Inhibition of some pathogens may be
reinforced by the production of antimicrobial substances by
the dahi starter organisms. Several studies have confirmed that
certain LAB isolated from dahi can produce bacteriocins effec-
tive against several of the human Gram-positive pathogens. As
far as the microbiological standards for dahi are concerned, the
maximum acceptable number of yeasts and mold and coli-
forms of 100 and 10 cfu g 1, respectively, is accepted by the
BIS for both the sweet and sour types of dahi. In both types, it is
mandatory that a phosphatase test should be negative.
Technology of Sweetened Dahi (Misti Dahi)
Manufacture
Misti dahi (syn. sweetened dahi, red dahi, payodhi) is a tradi-
tional sweetened fermented milk product of the Eastern region
of India. It is prepared on cottage scale by halwais (sweetmeat
makers) every day to cater to local demands. Traditionally,
milk added with cane sugar is continuously heated in an
open pan at simmering temperature (68–70  C) for 6–7 h to
concentrate and develop an intense cooked flavor and brown
color, besides increasing the viscosity and inducing other
physicochemical changes. After cooling to about 40  C, the
mix is inoculated with a commercial misti dahi starter culture,
which generally comprises a mixed lactic culture like Lactoba-
cillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus,
and some aroma-producing organisms like Lac. lactis subsp.
cremoris, as mentioned in the case of mildly sour and sour dahi.
Wide variations in total solids content (27–43%), nonfat milk
solids (11–16%), and sucrose (13–19%) in the market samples
of misti dahi sold in Kolkata city have been reported. Many
flavor defects such as overly fruity, alcoholic, highly acidic, and
and thick crust on top surface were observed in most of the
market samples. In view of the increasing nationwide demand
for this product and the growing interest of the organized dairy
plants for large-scale manufacture, the technology was devel-
oped to suit industrial-scale production. This technology is
accepted and invariably adopted for dahi manufacture on
large scale. Manufacturing technology for industrial produc-
tion of misti dahi is given in Figure 2.
To prepare Misti dahi, fresh buffalo milk is standardized to
3.5% fat and 9% nonfat milk solids, and it is then heated to
65  C in a plate heat exchanger and homogenized at a pressure
of 56 kg cm 2 (one stage). Milk is concentrated to 1.44-fold
in a vacuum evaporator. After adding cane sugar, the milk is
heated at 85  C for 10 min to generate cooked flavor. The mix
is cooled to 40  C before inoculation with a commercially avail-
able lactic culture (e.g., LF-40). In some cases, sugar, caramel,
jaggery, and artificial colors are added to impart brown color.
The inoculated mix is aseptically distributed into presterilized
polystyrene containers of desired size and mechanically trans-
ferred to an incubation chamber at 40  C. During the curd
formation, milk must remain stationary. In all postfermentation
activities, the gel should be subjected to a minimum amount of
external influences. Diacetyl is the predominant flavor compo-
nent, but microquantities of acetoin, acetic acid, and carbonyls
are also present. Off-flavors in this product can be traced back to
poor-quality raw milk, the use of contaminated cultures, high
incubation temperature, and contaminated packages.
Rheological Properties
Consistency, firmness and viscosity of dahi are important tex-
tural parameters that govern the quality of the product. These
attributes can be measured objectively by a texture analyzer or
cone penetrometer. The texture analyzer works on the principle
that a cylindrical steel probe penetrates into the product sam-
ples and experiences resistance during the penetration. The
resistance offered by the dahi sample (5  C) during the pene-
tration of the probe up to a specified distance is recorded as
firmness (newtons) of dahi. A cone penetrometer is a simple
and useful instrument for determining the firmness or hard-
ness of food materials. In this instrument, a conical metal
probe is allowed to freely fall into the sample for a specified
time, for example, 5 s. Then, its fall is arrested by an electro-
magnetic mechanism, and the depth of penetration is mea-
sured on a dial scale and expressed as (0.1) mm. The higher
the depth, the softer the dahi sample must be. Since dahi is
generally a soft material, a metal probe would be too heavy for
the purpose, so a rubber conical probe can be used.
Factors Affecting Rheological Properties of Dahi
Rheological parameters of dahi are significantly influenced by
the heat treatment of the milk used for dahi making. It has been
reported from the texture analyzer parameters that by the heat
treatment of milk, the firmness of dahi increases from 0.039 N
in no heat treatment (control) to 0.110 N in dahi prepared
 Dahi 349

Receiving of buffalo milk

Standardization (fat, 3.5% + SNF, 9.0%)

Preheating (65 °C–70 °C)

Homogenization (56 kg cm2 at 65 °C)

Concentration to 1.44 fold by heating

Addition of cane sugar (approx. 15%)

Heating at 85–90 °C for 10 min

Cooling to 37–40 °C

Inoculation with active starter culture @ 2%

(LF-40 or L. delbrueckii subsp. bulgaricus + Streptococcus salivarius subsp. thermophilus)

Aseptic packaging in the pre-sanitized retail packs

Incubation at 37–40 °C for 12–13 h

Storage at 5 °C or below

Distribution
Figure 2 Flow diagram for preparation of Misti dahi.

from boiled milk. The low-temperature, long-time (LTLT)
treated milk dahi has a 0.070 N firmness. Similar observations
were recorded with consistency value, but the LTLT treated
(0.33 N s) and boiled-milk (0.31 N s) dahi display almost the
same consistency, yet much higher than that of the control
(0.071 N s). Index of viscosity values are lower for control dahi
(0.0012 N s), whereas those for LTLT treated milk dahi and
boiled-milk dahi are 0.007 and 0.012 N s, respectively. This
trend is also reflected in parameters measured by cone
penetrometer.
The effect of a heat treatment of milk on the rheological
parameters can be attributed mainly to the denaturation of
proteins. In milk, casein exists in a colloidal state, which is
sustained by calcium bridges between the casein micelles. The
whey proteins exist in a soluble state and have no interaction
with casein micelles. But by heat treatment of milk, the whey
proteins get denatured and involved in interactions with the
casein micelles without affecting the calcium bridges. As inten-
sity of heat treatment given to milk increases, casein–whey
protein interactions take place. However, during the
350 Dahi
fermentation process, the calcium bridges between the protein
particles dissolve because the calcium goes from the colloidal
state to solution state, as the acidity rises and pH decreases. It
also results in the slow precipitation of the casein–whey protein
particles. The precipitated casein–whey protein particles
arrange themselves in specific manner forming three-
dimensional networks, which impart firmness to dahi. This
network can easily be broken by subtle forces of agitation, but
once broken, the network cannot be built back. However, part
of the original viscosity may be recovered during storage, which
is known as thixotropic character.
The sticky character acquired by the curd during fermenta-
tion is responsible for the thixotropic character and can be
attributed to mucilaginous substances produced by starter bac-
teria. This is the reason why it is widely believed in the sub-
continent that the milk should not be disturbed while setting
of milk is in progress otherwise firm curd would not form.
Disturbing the milk during setting means disturbing the net-
work being formed during acid production. A firm, well-set
curd is most preferred than stirred curd in India. However,
most of the curd sold is stirred curd because of convenient
processing steps, but manufacturers are realizing the people’s
preference for set curd, so set curd with a thick, firm consis-
tency is now gaining market. In this regard, the extent of heat
treatment of milk and use of starter cultures, which produce
not only lactic acid and diacetyl but also texturizing com-
pounds (heteropolysaccharides), are important. Some of the
workers in this field have advocated application of inulin and
texturizing substances for improving the consistency of dahi,
whereas others used xylooligosaccharides for the purpose. The
observations of these workers show that apart from heat treat-
ment, external polysaccharides could also be employed to
enhance the consistency of products like dahi.
Effect of Heat Treatment on the Syneresis and Acid
Development in Dahi
Syneresis is the separation of whey from the curd. The extent of
syneresis is indicative of the quality of dahi. Consumers prefer
dahi with no visible whey separation. The presence of separated
whey indicates excessive fermentation and stored dahi. During
fermentation, changes take place with regard to the milk pro-
teins, affecting water retention. Consumers tend to think that
the curd showing whey separation is not fresh and hence not
good. Dahi prepared from raw milk shows visible whey sepa-
ration measured as 7.7 ml after centrifugation, whereas dahi
prepared from milk pasteurized by LTLT shows a syneresis
value of 7.2 ml, which can also be visualized. However,
boiled-milk dahi exhibits no visible whey separation and
shows firm consistency.
When well-set curd is disturbed, the whey flows through the
channels in the network and finally separates out. The partially
denatured proteins hold more moisture or whey, so syneresis is
less in the curds prepared with high heat-treated milk (boil-
ing). In the case of raw milk, the whey proteins are in native
state so they possess low water-binding ability; hence, more
syneresis is expected to take place in that curd. In LTLT treated
milk, the whey protein denaturation has just begun so water
binding is slightly larger than in raw milk, leading to less
syneresis. In boiled milk, the proteins hold more moisture,
giving low syneresis values. Thus, the firmer the curd, the
lower the syneresis.
Effect of Heat Treatment on Sensory Quality and Acceptance
of Dahi
The acceptability of dahi samples is generally measured on a
9-point hedonic scale. It is observed that the boiled-milk dahi
scores highest in all the sensory attributes. Milk that does not
receive any heat treatment results in dahi with low sensory
scores that fall in the range of around 5–6, that is, ‘liked
slightly.’ LTLT heat-treated dahi scores better than raw-milk
dahi indicating that flavor, body, and texture improve by heat
treatment. By heat treatment, undesirable microorganisms are
killed and LAB grow with less competitive inhibition. When
milk is boiled, the same happens, but because of protein–
protein interaction, the body and texture improve further,
which will synergistically enhance the flavor attribute. For
these reasons, the overall acceptance scores are highest for the
boiled-milk dahi.
The intensity of heat treatment given to milk influences the
extent of denaturation of whey proteins. It is stated that during
dahi manufacturing, heating of milk (90–95  C/5 min) dena-
tures whey proteins, which consist of a-lactalbumin and
b-lactoglobulin. The denatured whey proteins interact with
casein micelles by coating on their surface. The denatured
whey proteins have a good water-binding ability, so they
enhance the thickness of the curd. They also help in sustaining
the three-dimensional structure of dahi. It is known that whey
proteins denaturation begins at 65  C and would almost
completely be denatured (93–95%) by heat treatment of milk
at 80  C for 45 min. The water-binding ability of proteins is
maximal only when they are partially denatured, that is, when
the protein structure is not completely damaged. This is
because of exposure of hydrophilic groups, which were other-
wise remaining embedded inside the tertiary or quaternary
structure. But when milk is intensely heat-treated, as in heat
sterilization, that is, more than 10 psi, then whey proteins are
completely denatured, their secondary structure is fully
straightened out, and the hydrophilic groups are lost; as a
result, the proteins lose water-binding ability. So, sterilization
of milk is detrimental to the curd firmness. It is now clear that
boiling the milk without holding will not lead to complete
damage to the secondary structure of whey proteins, but results
in only partial uncoiling leading to more number of hydro-
philic groups being exposed. Thus, water-binding ability is
increased.
Role of Dahi in Complementary Feeding
Conversion of milk into dahi helps to extend the shelf life of
milk, thereby allowing storage and transport. In fact, the gen-
eration of lactic and short-chain fatty acids from lactose and
the consequent fall in pH inhibit the growth of many patho-
genic microorganisms. This technology has been widely practi-
ced throughout India for a long time.
Dahi is nutritionally similar to unfermented milk, except
that some of the lactose is broken down to glucose and
galactose, which are further fermented, some vitamins are
 Dahi 351

Table 4 Mineral and vitamin content of dahia iron from unmodified cow’s milk is poorly absorbed, as a
result of the high content of protein and the low content of
Sr. No. Constituents Milk Dahi vitamin C compared with commercial infant formula. Further-
1. Mineral matter (g) 0.8 0.8 more, the early introduction of unmodified cow’s milk can
2. Calcium (mg) 118 149 cause blood loss from the intestinal tract, thereby having a
3. Phosphorous (mg) 96 102 negative influence on iron status, especially during the first 6
4. Vitamin A (I.U.) 4.6–5.2 96.0 months of life and also during the second half of infancy.
5. Thiamine (mg) 55 49 A number of health benefits have traditionally been attrib-
6. Riboflavin (mg) 167 157 uted to dahi and products made from it, which have been used
7. Nicotinic acid (mg) 96 86 to prevent a wide range of diseases including atherosclerosis,
8. Biotin (mg) 29.0 3.2 allergies, and gastrointestinal disorders. Although empirical
9. Pantothenic acid (mg) 202 183
findings are yet to be supported by controlled studies, initial
10. Folic acid (mg) 161 178
results from investigations into the antibacterial, immunologic,
11. Vitamin B12 (mg) 0.15 –
12. Ascorbic acid (mg) 1.4 1.3 antitumor, and hypocholesterolemic effects of dahi consump-
tion suggest potential benefits.
a
Values given per 100 g of the product.

synthesized, and the bioavailability of Ca and P is enhanced
due to increased acidity and release of some amino acids. This
makes dahi like other fermented milks an important food in
cases of lactose tolerance. In very young children, lactose
intolerance is often due to an impaired activity of intestinal
lactase following intestinal pathologies. Children with lactose
intolerance could receive dahi and other fermented milks
without showing the classical clinical signs of impaired
absorption, such as intestinal bloating and gas production.
In addition to that, lactase activity of live microorganisms
present in dahi also remains after ingestion in the gastrointes-
tinal tract, improving lactose digestion. The use of special
milk formulation for lactose-intolerant subjects could not be
affordable in poor regions in which the use of dahi can be a
valid alternative. Although dahi is primarily regarded as a
useful source of protein and minerals, vitamins from the
original milk or secreted by the starter culture bacteria are
usually present in detectable quantities (Table 4). The nutri-
tional value of milk protein is self-evident, but it is important
that the level in dahi is due to the concentration of milk solids,
higher than in liquid milk, as is the concentration of minerals
such as calcium and phosphorous. Furthermore, the low pH
of dahi tends to make the latter more accessible for absorption
through the intestinal wall.
Dahi is indeed able to enhance the absorption of some
minerals, such as calcium and nonheme iron, as a result of its
lower pH. The increased calcium absorption is related to both a
direct effect of the acidification of intestinal lumen and the
reduction of the mineral complex formation with other dietary
components. If dahi is consumed at mealtimes, the lactic acid is
likely to have a positive effect on the absorption of iron from
other foods. The bioavailability of iron is more important than
the total amount of iron in the diet. In contrast to breast milk,
See also: Acidophilus Milk; Fermented Foods: Composition and
Health Effects; Fermented Foods: Origins and Applications; Fermented
Foods: Use of Starter Cultures; Lactic Acid Bacteria; Malnutrition:
Prevention and Management; Probiotics; Protein: Digestion,
Absorption and Metabolism; Single Cell Proteins; Yogurt: Dietary
Importance; Yogurt: The Product and its Manufacture.
Further Reading
De S (1977) Outlines of dairy technology. Oxford: Oxford University Press.
Department of Health (1991) In: Dietary reference values for food, energy and
nutrients for United Kingdom. Report on health and social subjects Vol. 41. London:
HMSO.
Ghosh J and Rajorhia GS (1987) Chemical, microbiological and sensory properties of
Misti Dahi sold in Calcutta. Asian Journal of Dairy Research 6: 11–15.
Ghosh J and Rajorhia GS (1990) Technology for production of Misti Dahi – a traditional
fermented milk product. Indian Journal of Dairy Science 43: 239–244.
Indian Standards Institution (1973). ISI Document No. 7035, New Delhi: Manak Bhavan.
International Dairy Federation (1983) Cultured foods in human nutrition. Brussels:
International Dairy Federation, Doc. No. 159, pp. 18–28.
Khedkar CD and Ouwehand AC (2006) Modifying the gastrointestinal microbiota with
probiotics. In Gastrointestinal Microbiology. pp. 315–333. New York: Taylor &
Francis.
Laxminarayana H, Nambudripad VKN, Laxmi NV, Anantaramiah SN, and
Sreenivasamurty V (1952) Composition of Dahi. Indian Journal of Veterinary
Science and Animal Husbandry 22: 13–16.
Mathur BN (1998) In: Advances in cheese and fermented milk products. A compendium
of short term course notespp. 210–217. Karnal (Haryana), India: National Dairy
Research Institute.
Shahani KM and Chandan RC (1979) Nutritional and healthful aspects of cultured and
culture-containing dairy foods. Journal of Dairy Science 62: 1685–1694.
Yadav JS, Grover S, and Batish VK (1993) A comprehensive dairy microbiology. New
Delhi, India: Metropolitan.
Yamauchi K (1992) Biologically functional proteins of milk and peptides derived from
milk proteins. Bulletin of the International Dairy Federation 272: 51–58.
 Dairy Products: Dietary and Medical Importance
F Visioli, University of Padova, Padova, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Milk is a widely disseminated beverage worldwide and is essen-
tial to the diet of several millions worldwide because it pro-
vides important macro- and micronutrients. Dairy products are
also important food staples that provide protein and micronu-
trients in a rather concentrated form.
Milk and dairy products are perceived as nutritionally rele-
vant foods, mostly because their calcium, protein, phosphorus,
etc., content promotes skeletal, muscular, and neurological
development. However, their relatively high saturated fat pro-
portion (milk fat contains about 70% of saturated fatty acid
(SFA); myristic and palmitic acids combined account for
50%, while the rest is mostly short- and medium-chain fatty
acids, including oleic acid) often raises issues in terms of adverse
effects on cardiometabolism. However, the vast majority of
recent epidemiological and intervention studies indicate
that dairy products do not adversely affect surrogate markers
of cardiovascular disease and cardiovascular prognosis. Con-
versely, short-chain SFA from milk and its derivatives are –
apparently – benign or even beneficial with regard to
inflammation. Extensive calcium intake is often frowned upon
because of potentially noxious actions on coronary calcification.
However, accumulated epidemiological evidence suggests that
calcium does not play major roles in coronary calcification and
its intake is inversely associated with blood pressure. Therefore,
the hypothesized association between calcium intake and car-
diovascular risk is not supported by current data and, actually,
the converse might be true. In addition, some milk components
such as trans-fatty acids, tripeptides, calcium, phosphorus,
lactoferrin, and oligosaccharides might exert useful – though
as yet unproven – physiological actions. Finally, milk is an
efficient vehicle for fat- and lipid-soluble nutrient absorption
because milk fat is highly dispersed in very small micelles; this
makes milk an excellent matrix for functional foods.
In this article, the latest published evidence on milk, dairy
products, and human health is summarized.
Milk, Dairy Products, and Cardiometabolism
Cardiovascular disease is the leading cause of death in the
Western world and its incidence can be modulated by diet. As
mentioned, milk is often perceived as a double-edged sword
chiefly because of its SFA content. In addition, milk and dairy
products contain cholesterol (approximately 80 mg/100 g).
Therefore, several people worry that the intake of either one
might theoretically have detrimental effects on their choles-
terol concentrations. The net result is that cardiovascular
patients or individual at risk (real or perceived) often stops
using milk and its derivatives precisely to improve their prog-
nosis. In this respect, it is noteworthy that the real contribution
of dietary cholesterol to cardiovascular risk is being debated;
352 Encyclopedia of Food and Health
the current consensus is that it depends on individual, inher-
ited ability to synthesize versus absorb cholesterol. Also, the
extent and the exact nature of the role of SFA in cardiovascular
disease onset and development are being reexamined; more-
over, genetic background is a predictor of the response to SFA
intake.
The first review on the association between dairy products
and circulating cholesterol concentrations has been published
by St-Onge et al. In that paper, the authors mention an old study
carried out in the African Maasai ethnic group. Maasais consume
large amounts of milk, which, in that study, was inversely corre-
lated with cholesterolemia. The same authors proposed milk as
an inhibitor of cholesterol synthesis. Other publications
expanded on the cholesterol-lowering effects of both milk and
skimmed milk (which were previously suggested by Hepner
et al.). In their review, St-Onge et al. hypothesized that the
cholesterol-lowering effects of milk are due to the intestinal
microbial fermentation of indigestible carbohydrates (see
succeeding text), which would positively alter the microbiota,
inhibit cholesterol synthesis, and interfere with cholesterol
enterohepatic circulation, in turn lowering cholesterolemia.
A recent study performed by Høstmark et al. in 18 770 sub-
jects examined the association between cheese consumption and
circulating concentrations of high-density lipoprotein (HDL)
cholesterol (which was positive) and triacylglycerols (which
was negative), in different age groups. The authors attributed
this effect to the fatty acid composition of cheese and its bacterial
content, although this hypothesis needs confirmation.
One of the questions arising from these studies is whether
various dairy products have different effects on cardiovascular
risk markers. In this respect, one study equicalorically (20% of
total calories, normalized for lactose and casein) compared the
effects of milk, cheese, and butter, for 3 weeks. Cheese slightly
increased low-density lipoprotein (LDL) cholesterol, whereas
the effects of whole milk and butter were similar. These data
have been replicated by Biong et al., who reported that con-
sumption of cheese induced a lower increase of cholesterol
concentrations than that of an identical amount of fat from
butter. Along these lines, Nestel et al. administered 40 g day 1
from either cheese or butter to mildly hypercholesterolemic
subjects. Total and LDL cholesterol increased significantly –
after 4 weeks – in the butter group as compared with the cheese
one. Some authors suggest the inclusion of modest amounts of
cheese in the diets of mildly hypercholesterolemic subjects,
even though current guidelines recommend otherwise. Indeed,
in a study by Hjerpsted et al., 13% of total daily calories were
replaced with either 143 g of cheese or 47 g of butter (with the
same lipidic content), for 6 weeks, in a randomized crossover
trial on healthy subjects. The result indicates that cheese does
not increase LDL cholesterol compared with the run-in period;
rather – as compared with butter – cheese induces a signifi-
cantly lower increase in total (5.7%) cholesterol and LDL
(6.9%) cholesterol.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00214-2

Mechanistically, several hypotheses have been to explain
the differential effects of cheese and butter on cholesterolemia.
One such hypothesis is that calcium – whose concentrations
are higher in cheese than in butter – binds to fatty acids in the
intestine and forms insoluble soaps, thereby decreasing fat
absorption. This hypothesis is corroborated by the higher-fat
fecal excretion recorded in the cheese group with respect to the
butter one. Also, the higher protein and probiotic content of
cheese might theoretically contribute to its almost neutral
effect on plasma cholesterol. However, it should be noted
that the study by Tholstrup et al. reported a lack of difference
in cholesterolemic effect of the diets containing whole milk
and butter. In summary, whole milk likely raises LDL-
cholesterol concentrations similar to butter, and its use by
hypercholesterolemic patients or by individuals with elevated
cardiovascular risk should be considered with caution. Trans-
fatty acids are also a matter of concern in cardiometabolism,
and laws are being approved to eliminate them from the
diet. However, dairy trans-fatty acids might have different phys-
iological actions than the industrial ones. As an example, con-
jugated linoleic acid is being promoted as healthful even
though solid scientific evidence of its putative health effects is
lacking. Yet, there are efforts to increase its concentrations in
bovine milk.
Two of the most researched issues in cardiometabolism
are oxidative stress and inflammation, because they play
major roles in the onset and development of the metabolic
syndrome (MetS).
Two large studies, that is, the Nurses’ Health Study and the
Multi-Ethnic Study of Atherosclerosis, reported an inverse asso-
ciation between a prudent diet that included low-fat dairy
products and biomarkers of inflammation. In addition,
Australian study reported an inverse association between
MetS and type II diabetes incidence and dairy consumption
in 1807 and 1824 patients, respectively. In particular, those
who had the highest consumption level of dairy products
witnessed a MetS risk reduction of 59%. A similar association
was recorded when the incidence of diabetes was computed.
These publications triggered a series of investigations aimed at
clarifying mechanisms of actions and whether this association
was casual or causal. Zemel et al. supplemented obese patients
with skimmed milk for 28 days and recorded a significantly
lower oxidative stress and inflammation. In addition, a 12-
week diet administered to MetS patients that included 3.5
servings/day of milk and/or yogurt reduced oxidative stress
markers after 7 days, as compared with an isocaloric diet that
provided 0.5 servings/day of dairy products. After 12 weeks,
decreased systemic inflammation was also recorded in the
dairy group though not in the control arm.
It is noteworthy that the observed decrease in oxidative
stress and inflammation markers was independent of body
weight changes and became apparent shortly after the admin-
istration of dairy products. This suggests causation and an
active role of milk and dairy products in reducing two hall-
marks of the MetS.
A recent meta-analysis of eight randomized and controlled
studies in overweight or obese subjects concluded that dairy
product consumption does not negatively impact on bio-
markers of inflammation. Yet, further ad hoc studies on
Dairy Products: Dietary and Medical Importance 353
the effects of milk and dairy products on inflammation are
warranted before solid conclusions can be drawn.
Cancer
Cancer is the second cause of death in industrialized countries
(and in some of them, it is actually number one). Milk and
dairy products contain compounds that have suggested, in
in vitro studies, as chemopreventive. Some epidemiological
studies have been carried to ascertain whether milk and dairy
product use is associated with cancer incidence. For example,
Kampman et al. published an observational study in which
331 men and 350 women have been followed for 4 and
8 years, respectively, and no association between milk and
fermented milk consumption and colorectal cancer was
found. Other such studies yielded mixed outcomes: some of
them found positive associations between dairy use and
prostate and ovary cancers, whereas others found the converse
when the incidence of colorectal, lung, or breast cancers was
evaluated. A very large prospective study by Park et al. analyzed
the diets and cancer incidence of 293 907 men and 198 903
women, with a follow-up of 7 years. In this study, calcium
and dairy product use was inversely associated with gastro-
intestinal (GI) (especially colorectal) cancer incidence. In par-
ticular, the reduction of GI cancer risk was of 16% for men and
of 23% for women. Other cancers were also inversely associ-
ated with milk and cheese consumption, namely, those of the
head and neck, esophagus, stomach, colon, and bladder. As
mentioned, milk contains putatively chemopreventive com-
pounds such as calcium, vitamin D, and conjugated linoleic
acid that might explain these observations. In vitro, calcium
inhibits cell proliferation and stimulates differentiation and
apoptosis in the GI tract and in the mammalian gland. As
mentioned earlier, calcium also binds to fatty acids and biliary
salts in the intestine, therefore lessening their potentially nox-
ious effects on carcinogens the mucosa. Accordingly, two Euro-
pean studies reported an inverse association between milk use
and colorectal cancer. In the first one, 45 241 subjects have
been followed for 12 years, and their use of yogurt was mon-
itored and plotted against colorectal cancer incidence, showing
an inverse association that was stronger in men. A systematic
review by Aune et al. took into account 19 cohort studies and
reported a significantly inverse association between milk, total
dairy products yet not cheese consumption, and colorectal
cancer incidence. On the other hand, calcium also interacts
with vitamin D and with insulin-like growth factor-1, which
would increase prostate cancer risk, as shown by Park et al.
Concerning breast cancer incidence, one report by Knekt
et al. found a significantly inverse association between milk use
and breast cancer incidence in 4697 Finnish women. Shin et al.
also published a cohort study, in which they did not find any
association between dairy, calcium, or vitamin D consumption
and breast cancer incidence in postmenopausal women. Con-
versely, in premenopausal women, a significant reduction of
breast cancer risk as associated with low-fat dairy products was
reported, suggesting that hormonal status might modulate the
effects of milk and its derivatives on breast cancer, via
unknown mechanisms. A large meta-analysis of more than
354 Dairy Products: Dietary and Medical Importance
20 studies did not find any association between breast cancer
incidence and dairy product use. These results have been sub-
sequently confirmed. Conversely, Dong et al. performed a
meta-analysis on 1 063 471 participants in 18 prospective
cohort studies and reported an inverse association between
dairy – though not milk – use and breast cancer, notably in
premenopausal women and when low-fat products were sepa-
rately computed. In summary, based on accumulated data,
there is currently no indication that milk and dairy use favors
or contrasts the incidence of breast cancer.
The association between milk use and bladder cancer inci-
dence has also been analyzed. One meta-analysis by Mao et al.
reported that high milk consumption was associated with a
16% reduction of bladder cancer risk; this inverse association
was stronger in Asian subjects than in North American and was
not found in Europeans. Also, the association depended upon
the kind of dairy product that was analyzed. Another meta-
analysis did not find any correlation among variables, after
computing 14 studies on milk (4879 cases of bladder cancer)
and six studies on dairy products (3087 cases), for a total of
324 241 subjects.
When other cancers, including lung, ovary, esophagus and
stomach, and oropharyngeal cancers, are taken into account,
no clear association between milk consumption and incidence
emerges. Conversely, positive associations between dairy use
and prostate cancers have been published and several mecha-
nisms of action have been proposed to explain this hypothet-
ical effect.
Milk as a Vehicle for Bioactive Molecules
Fat is found in milk in extremely small micelles: the average
diameter of the fat globules in homogenized bovine milk is
1–3 mm; estimations indicate that 1 g of fat in milk is dispersed
in 1010–12 micelles, that is, a very wide surface of about one
square meter. Therefore, it is conceivable that lipid-soluble
compounds, such as added fatty acids, liposoluble bioactive
molecules, and vitamin E, are highly bioavailable when
ingested with milk (Figure 1). In addition to exogenous com-
pounds (which render some milks functional foods), milk
naturally contains biologically active molecules. One example
is that of oligosaccharides, which are not metabolized along
human GI tract, so they reach the colon intact where they serve
as specific nutrients for probiotic strains. Even though bovine
milk contains only trace amounts of these potentially healthful
molecules, some researchers are working toward introducing
human milk oligosaccharides in that of transgenic animals.
One current issue is the lack of suitable commercial standards
for bovine oligosaccharides; consequently, we can only iden-
tify approximately 70 fully annotated oligosaccharides in
human milk and approximately 40 in bovine milk. It is note-
worthy that human milk is rich in oligosaccharides, which
have been proposed as important contributors to child
development, largely because they are prebiotics and help in
creating a healthy microbiota. Indeed, neutral oligosaccharides
– namely, the monomer N-acetylglucosamine and fucose – are
essential to the development of the correct microbiota of
breast-fed neonates, because of their immunomodulating
actions. Also, acidic oligosaccharides (where the monomer is
6
5
4
µMols
3
b b
2
1 a a
0
TC TG HDL-c
Figure 1 Milk is an efficient vehicle for omega 3 fatty acid
administration. In addition to high bioavailability of added fatty acids
(and, in this case, of vitamin E), this functional food improves lipid
surrogate markers of cardiovascular disease. Values are the means  SD.
T0, initial values; T3, after 3 weeks of treatment; T6, after 6 weeks of
treatment. Values marked with letters are statistically different from those
at T0 as follows: ap < 0.05; bp < 0.005. Data are from Visioli et al. (2000),
where full details of the study are provided.
sialic acid) help in preventing the adhesion of pathogens to the
intestinal mucosa. Interestingly, bovine milk also contains
these oligosaccharides, which are abundant in colostrum.
Fruits and vegetables also contain oligosaccharides and some
of them have been synthesized; yet, those from milk are pecu-
liar in that they exhibit a branched rather than linear structure.
Moreover, they contain fucose and sialic acid, which are almost
absent in other oligosaccharides. This structural difference
might confirm milk oligosaccharide activities that are different
than those of synthetic or vegetal origin. It must be under-
scored that the concentration of oligosaccharides in bovine
milk decreases in a time-dependent fashion: ad hoc investiga-
tions are being carried out to stabilize oligosaccharides in milk
or to formulate them as nutraceuticals or as probiotic compo-
nents of functional foods.
In terms of dairy products, it is noteworthy that some kinds of
cheeses, namely, those infected with Penicillium such as Roque-
fort, Stilton, and Gorgonzola, contain high amounts of andrastins
A–D, which – in vitro – are potent inhibitors of farnesyltransferase,
a key enzyme in cholesterol synthesis. Other peptides formed
during ripening-induced proteolysis might further contribute
healthful – albeit as yet unexplored – properties that would
partially explain the paradoxically relatively low incidence of
cardiovascular disease (CVD) in high-cheese-use countries.
The rapidly growing market of functional foods indicates
milk as a suitable vehicle for bioactive compounds: as men-
tioned, milk is an excellent vehicle for fat-soluble molecules.
Indeed, milk and some dairy products such as yogurt provide
an excellent matrix for the administration of bioactive mole-
cules, for example, phytosterols, and are being marketed to
target population groups, pending appropriate regulatory
evaluation. Nowadays, any supermarket exhibits a large num-
ber of fortified milks and dairy products, some of which do
indeed contribute to well-being.
In summary, either fortified or ‘natural’ milk and dairy
products contain several compounds – even though often
present in low concentrations – that might in the future be
exploited for pharmanutrition applications.

Conclusions
While milk and dairy products are often regarded as double-
edged sword because of their high saturated fat content and
abundance of important micro- and macronutrients, accumu-
lated evidence suggests that their use within the frame of a
balanced diet might be advantageous from a health viewpoint.
It is also worth reminding that milk and dairy products account
for a substantial proportion of calories and macro- and micro-
nutrients in several populations worldwide. Therefore, their
nutritional value is quantitatively relevant on a global scale.
Of course, subjects with cardiometabolic disturbances
should consume dairy products in moderation and should
discuss their use with their physician/nutritionist. Even though
it is difficult to single out the health contribution of a particular
food item, future studies might further elucidate the role of
milk and dairy products in human health and disease.
See also: Acidophilus Milk; Bioactive Peptides in Foods; Butter:
Properties and Analysis; Cheese: Composition and Health Effects; Fatty
Acids: Trans Fatty Acids; Fermented Foods: Fermented Milks; Food
Intolerance: Lactose Intolerance; Lactose; Milk: Role in the Diet;
Obesity: The Role of Diet; Yogurt: Dietary Importance.
Further Reading
Aune D, Lau R, Chan DS, et al. (2012) Dairy products and colorectal cancer risk: a
systematic review and meta-analysis of cohort studies. Annals of Oncology
23: 37–45.
Dairy Products: Dietary and Medical Importance 355
Chowdhury R, Warnakula S, Kunutsor S, et al. (2014) Association of dietary, circulating,
and supplement fatty acids with coronary risk: a systematic review and meta-
analysis. Annals of Internal Medicine 160: 398–406.
Fonolla J, Lopez-Huertas E, Machado FJ, et al. (2009) Milk enriched with ‘healthy fatty
acids’ improves cardiovascular risk markers and nutritional status in human
volunteers. Nutrition 25: 408–414.
Lawrence GD (2013) Dietary fats and health: dietary recommendations in the context of
scientific evidence. Advances in Nutrition 4: 294–302.
Lopez-Huertas E (2010) Health effects of oleic acid and long chain omega-3 fatty acids
(EPA and DHA) enriched milks. A review of intervention studies. Pharmacological
Research 61: 200–207.
Nettleton JA, Steffen LM, Mayer-Davis EJ, et al. (2006) Dietary patterns are associated
with biochemical markers of inflammation and endothelial activation in the Multi-
Ethnic Study of Atherosclerosis (MESA). American Journal of Clinical Nutrition
83: 1369–1379.
Samelson EJ, Booth SL, Fox CS, et al. (2012) Calcium intake is not associated with
increased coronary artery calcification: the Framingham Study. American Journal of
Clinical Nutrition 96: 1274–1280.
St-Onge MP, Farnworth ER, and Jones PJ (2000) Consumption of fermented and
nonfermented dairy products: effects on cholesterol concentrations and metabolism.
American Journal of Clinical Nutrition 71: 674–681.
Visioli F (2013) Pharma and nutrition: crossing the Rubicon. PharmaNutrition 1: 9.
Visioli F and Strata A (2014) Milk, dairy products, and their functional effects in
humans: a narrative review of recent evidence. Advances in Nutrition 5: 131–143.
Visioli F, Rise P, Plasmati E, Pazzucconi F, Sirtori CR, and Galli C (2000) Very low
intakes of N-3 fatty acids incorporated into bovine milk reduce plasma
triacylglycerol and increase HDL-cholesterol concentrations in healthy subjects.
Pharmacological Research 41: 571–576.
Willett W and Mozaffarian D (2008) Ruminant or industrial sources of trans fatty acids:
public health issue or food label skirmish? American Journal of Clinical Nutrition
87: 515–516.
Relevant Websites
http://www.journals.elsevier.com/pharmanutrition – Elsevier Journal: PharmaNutrition.
 Date Palm: A Wealth of Healthy Food
KM Farag, Damanhour University, Damanhour, Egypt
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Date is one of the healthiest foods in the world. It has been part
of the people’s life in the desert and a way of surviving arid
conditions. Although about three-fourths or more of the fruit
consists of sugars, there are many other nutritious components
such as minerals, vitamins, antioxidants, and dietary fibers.
The diversity of date palm cultivars, species, and clones adds
to the wealth of this tree. The different date fruit types, whether
soft, semidry, or dry, provide consumers with many choices
and the ability to grow palm trees in a variety of climates. Even
the edibility of the fruit varies from those that could be con-
sumed at full coloration, as a mature fruit, called the bisr or
khalal stage, to others that must be converted to the rutab stage
or the ripe fruits. The mature fruit could be yellow or red (rarely
orange) but not necessarily free of soluble tannins while the
ripe fruit is full of phenols and tannins in a polymerized form
with a consumable taste. However, the initiation of ripening
means the initiation of rutab development, and the fruit
becomes soft and edible. Firm texture of the rutab fruits
could be maintained by applying the polyamine putrescine.
The predominant sugar in dry dates is sucrose because the
invertase enzymatic activity is low due to low water or high
temperature stress that inhibits such activity. It was found that
when a reduced rate of gain in fresh weight and volume occurs,
a decrease in the rate of gain in reducing sugars and a rapid
increase in accumulation of sucrose and total sugars take place.
Thus, sucrose accumulation starts late in the season when the
weather is usually hot, and the rate of sucrose inversion to
reducing sugars at this stage varies in different date cultivars.
The predominant sugars in soft dates are glucose and fructose
as in Zaghloul and Barhee dates.
Description of Dates
Date palm tree is a dioecious plant where male and female
flowers are born on separate palms. The tree has only one point
of vegetative growth represented in the apical meristem. Date
palm cultivars have specific leaf morphology whether in the
distribution of leaflets (pinnae) or their angle in the three
dimensions. The fruit is a berry that develops from one of
three carpels found in each of the pistillate flowers. Fruit
shape is usually more or less oblong or ellipsoidal. The seed
is bony called the pit and about three times as long as it is wide,
and it contains mainly cellulose. Female trees need artificial
pollination to obtain a fair fruit set. Variations in pollen source
could affect some fruit characteristics outside the embryo and
the endosperm such as fruit length and size, the pulp to pit
ratio, color intensity, and even the time of ripening in
marginal-production areas. This has been known as the
metaxenic effect of pollen grains. When pollination is pre-
vented or when fertilization fails, all or one of the three carpels
356 Encyclopedia of Food and Health
of the pistillate flowers will develop into a parthenocarpic fruit.
These are called parthenocarpic single or triplets, respectively.
The parthenocarpic triplets are hollow, whereas the partheno-
carpic singles contain quasi-degenerated seeds. Both types of
parthenocarpic fruits are found in different cultivars at various
ratios, especially when failure occurs in pollination and fertil-
ization. Data on parthenocarpic fruits showed the same pattern
of growth, though at a different rate. They change their color at
the same time as seed-bearing fruits but do not reach matura-
tion, if at all, at the same time.
It was reported that the date fruit has five phases that can be
distinguished. The Arabic terms for these different stages have
been used in the literature. The hababouk stage of slow growth
rate begins from fruit set and continues 4–5 weeks. In this time,
the content of moisture in the fruit is 85–90%. This stage is
followed by a rapid increase in fruit weight ranging from 6 to
10 weeks (the kimri stage), then a declining rate of growth (the
depression phase of the kimri stage) until reaching to the bisr
or khalal stage, which is fruit coloration. Fruit skin is relatively
thin at the early stages of hababouk and kimri, which could not
resist the exerted water pressure leading to cracking. Similarly,
the fruit tip at the initiation of the khalal stage could crack due
to the increase in water absorption while there is a reduced rate
of water loss because the relative humidity is high. That is one
of the factors that creates a driving force for water loss.
During the khalal stage, the fruit starts to turn from green to
yellow (or red), weight gain is slow, but sucrose content
increases while moisture goes down to 50–55%. In some cul-
tivars such as Barhee and Zaghloul, tannins start to precipitate
and lose their astringency. The khalal stage could be extended
by applying a calcium formulation after harvest and modifying
the method of packaging. During the fourth stage, namely the
rutab stage, dates become half ripe and soft and turn to light
brown color while the sucrose turns to invert sugars. Fruit
moisture is about 35–40%, whereas the astringent taste disap-
pear. Finally, during the tamr stage, dates become semidry or
dry contain about 50% sucrose (or higher if temperature is
high and water content is low). Usually the fruit moisture
ranges between 20% and 25%.
Types of Cultivars
Dates can be marketed in three types, namely, soft dates,
semidry, or dry. The division is based on the texture, water
content, appearance, and their heat summation units. Soft
dates are distinguished by their soft flesh, high moisture con-
tent (more than 30% reaching to 70%), and high sugar
content, especially glucose and fructose. Examples of soft
dates are Barhee, Halawy, Hayany, Zaghloul, and Samany.
Semidry dates have a relatively firm flesh, fairly low moisture
(20–30%), and high sugar content especially reducing and
sucrose in similar amounts ranging from 35% to 37%. This
http://dx.doi.org/10.1016/B978-0-12-384947-2.00215-4

type includes Sewi, Al-Amry, Al-Ajlani, Dayri, and Khadrawi.
Furthermore, dry dates have a hard texture, low water content
(<20%), and great sugar content, especially sucrose. Examples
are Zahdi, Sayer, and Sackouttti. Moreover, soft dates could be
edible at the khalal (fully colored) or at the initiation of the
rutab stage depending on their soluble tannins content at this
stage because they have to lose their astringent taste. It is
suggested that the rate at which soluble tannins are converted
to insoluble tannins varies within cultivars, making some of
them edible as fresh juicy dates. Brownish color at the rutab
stage is ascribed to date phenolase that works on substances
such as catechin or the dactylifric acids. Furthermore, heat
requirements vary among date cultivars. Soft dates require
relatively lower summation of heat units as compared with
semidry and dry heat. These requirements are calculated from
full bloom to maturity and estimated to be 2500, above
2500–3150, and above 3150–6000 heat units for soft,
semidry, and dry dates, respectively.
Production of Different Areas
In history date palm cultivation has been extending along
desert areas and around the rivers. The tree favors high tem-
perature and sunshine. Thus, date palm trees are confined to
arid and semiarid regions. No wonder that date palm figures
were part of ancient history and civilizations. It is reported that
date palm is currently grown for fruit production used for local
consumption or trade in 37 countries around the world, in
addition to few other countries growing the crop on small
areas. Almost all parts of the tree have been beneficial whether
as an energy source or providing shade and shelter against the
harsh conditions in the desert regions. Many traditional by-
products have been made ranging from hand fans to furniture.
According to FAO (2011), Table 1 illustrates the production
areas of date palm in the leading countries.
The data in Table 1 indicated that the greatest production of
dates is in United Arab Emirates even though there are much
larger areas grown with date palm in other major producing
countries such Saudi Arabia, Iraq, and Iran.
Date palm germplasms may exceed 5000 cultivars and
clones. Most clones are less productive and susceptible to
Table 1 Cultivated area and the total production of date palm in the
leading countries
2011 Data
Countries Cultivated area (ha) Total production (tons)
Egypt 41 652 1373 570
Iran 154 274 1 016 608
Iraq 123 230 619 182
Saudi Arabia 172 297 1122 822
Oman 31 348 268 011
Morocco 70 433 117 867
Tunisia 51 000 (Feddans) 180 000
The United States 3399 30 209
United Arab Emirates 41 159 2 391 643
Source: FAO statistics handbook (2011). Date Palm Statistics.
Date Palm: A Wealth of Healthy Food 357
diseases than known-commercial cultivars. Many threatened
clones and cultivars are subjected to extinction due to the
spread of Fusarium fungi in Morocco, Tunisia, and Algeria.
This is why there has been a narrow genetic diversity in these
regions such as Deglet Noor in Tunisia and Algeria, although it
represents over 65% of the total population, and Boufeggous-
O-Moussa and Jihal in Morocco.
The cultivated area has increased by 0.3 million hectares
over the previous decade. The largest date palm areas are
located in the Asian continent, which include the Middle East-
ern producing countries, followed by Africa, North whereas in
the Americas and Europe, the production of date palm covers
only a few thousands hectares. The date crop has been also
established in California, Arizona, and Mexico. The common
required temperature is 35  C necessary for an optimum devel-
opment of pollen and low relative humidity for fruit setting
and ripening. Date palm trees grow in nearly rainless regions at
9–39 O North latitude, which are represented by the Saharan
and Southern fringe of the Near East.
The increase in relative humidity around the bunches
causes some physiological disorders such as checking at the
early kimri stage or black nose by reaching to initiation of
maturity (the khalal stage). In both disorders, the rate of
water absorption is greater than its loss from the fruit, which
creates a pressure on fruit skin, resulting in surface cracking.
With regard to the distribution of date palm, this crop
cultivation is spreading in both the old world (Near East,
North Africa, Spain) and the new world (Australia and the
American continent), where dates are grown and consumed
in large quantities.
The distribution of date palm according to the latitude for
both Northern and Southern Hemispheres is between 10 N
(Somalia) and 39 N (Elche/Spain or Turkmenistan). Favor-
able areas are located between 24 and 34 (Morocco, Algeria,
Tunisia, Libya, Egypt, Iraq, and Iran). In the United States, date
palms are found between 33 and 35 N. Because of the climatic
factors, the date palm will grow but will not fruit properly
outside these defined geographical limits. Altitude is very
important because it imposes the availability of water and the
temperature limits that largely determine the distribution of
date palm in the world. Date palm grows well from 392 m
below sea level to 1500 m above with an altitude range of
1892 m.
Consumption of Dates as a Food
Dates have been the essential food in many regions around the
world, especially arid zones of the old world and part of the
diet all year-round. Even in the new world and Europe, date
fruits have attracted the attention of the public and sport
dieticians due to the high glucose and fructose content in
semidry and soft dates. It was reported that one pound of
dates (453 g) supplies the human body with 5.33 kJ of physi-
ological energy.
A comparison was made to show the relative value of four
types of food (cow’s milk, meat, honey, and orange juice)
against date fruits. The comparison showed that protein con-
tent in dates was very close to that of cow’s milk, but greater
than that found in honey or fresh orange juice. Meanwhile, the
358 Date Palm: A Wealth of Healthy Food
date fruits were superior to all the other four types of food in
potassium. Furthermore, calcium in dates was three times
greater than that found in cow’s meat and 15 times greater
than that in honey while about half of the calcium content in
the cow’s milk. Moreover, phosphorus content in 100 g of
dates was about two-thirds of that found in cow’s milk but
three times more than that found in honey. Iron, on the other
hand, is much more in date fruits when compared with cow’s
milk, honey, or orange juice. Iodine content, as an essential
element for the gland activity, in dates is equal to that found in
honey or fresh orange juice. Moreover, sodium, as an impor-
tant element required for water balance in the human body
and that affects muscles and the nervous system, is contained
in dates in smaller amounts than that found in cow’s milk or
meat but greater than that found in honey or fresh orange
juice. Even carotenes in dates are much greater than that
found in cow’s milk or orange juice. These carotenes are impor-
tant antioxidants for providing protection against cancer.
Dates are also an excellent source for carbohydrates, pro-
teins, fats, dietary fibers, essential minerals, and vitamins.
Edible dates have been classified into four stages termed
kimri, khalal (bisr), rutab, and tamr.
These represent the astringent green, mature full-colored,
soft brown, and hard raisin-like stages of development, respec-
tively. For human consumption, in general, dates become
edible at the khalal stage where the fruit color changes to a
yellow or red tone, depending on the cultivar, over a period of
3–5 weeks. Fruits are rarely orange in color at the khalal stage.
Few date cultivars could be consumed at the khalal stage such
as Zaghloul, Samany, and Barhee because they are not astrin-
gent at this stage, and soluble tannins do not represent a
problem. However, many date cultivars and clones, especially
in the Arabian Peninsula need to initiate the rutab stage to get
rid of the astringent taste. At the rutab stage, dates begin to
soften in about a 2–4 week period due to an increased loss of
moisture content and an increase in enzymatic activity of some
cell wall hydrolyses such as pectinase and polygalacturonase.
The conversion to the rutab stage in some cultivars is required
to obtain the insoluble form of tannins. During ripening
(rutab development) there is a sharp reduction in both the
amount and concentration of soluble tannins indicating the
possibility of ceasing their accumulation together with break-
down and/or a conversion into insoluble form during devel-
opment and ripening. It was also reported that soluble tannins
are polymerized when converted to the insoluble form and
lose their ability to compete or bind to the binding sites of
Table 2 The macro- and microelements content of two commercial date cultivars
Cultivars
Macroelements Hallawi
Calcium 184
Phosphorus 16
Potassium 854
Sodium 14
Chlorine 260
Magnesium 56
Source: Yousef, A. and Kado, A. (1982). Chemical composition of four Iraqi date cultivars. Baghdad: Agriculture and Water Resources Research Center.
ethylene. Date fruits are also heavily consumed at the tamr
stage whether semidry or dry. Dates are drier and rather firm
in texture at this stage, and their color turns to a darker tone.
The lower water content of dates at the tamr stage enhances
their storability and shelf life. Dates, however, continue to
ripen after harvest, exhibiting a characteristic respiratory rise
before ripening preceded by a spike in ethylene production,
which accelerates the process. The most important category of
accumulated proteins involved in membrane and cell wall
degradation are polygalacturonase and pectinesterases that
ensure the softening of the fruit. There are also some other
proteins that are responsible for the pigment degradation and
color change during various stages from kimri to the tamr
stages. Thus, physiological changes that occur in dates during
ripening begin with the recognition of external signals, includ-
ing ethylene spike that occurs before the respiratory rise.
Nutritional Value of Dates
The high content of carbohydrates in date fruit, especially
sugars that reach to 88%, may lead to the impression that little
is left to contribute to the nutritional value of dates. However,
the fruit is full of essential nutrients such as potassium, phos-
phorus, sodium, zinc, manganese, magnesium, copper, iron,
fluorine, and selenium. Dates are a great source of potassium
that helps in the maintenance of a healthy nervous system and
in balancing the body’s nervous system. Phosphorus functions
with calcium to help with bone strength and growth. More-
over, selenium is important for cell growth and repair. Iron is
essential to production of red blood cells, which carry all the
nutrients to cells throughout the body. As an example, the
macro- and microelements of the two well-known cultivars,
Hallawi and Zahdi, were reported (on a dry weight basis) in
Table 2.
The amount of sodium in dates is low but ideal when
looking at the recommended amount per day for an individual
(more than 2.4–2.0 mg day 1). Consuming too much sodium
may increase the risk of heart disease and hypertension. Thus,
dates are perfect for a healthy lifestyle because they contain
reducing sugars, low sodium, no fats or cholesterol, great
potassium, and calcium contents in addition to dietary fibers.
The dietary fiber content of widely retailed dates was estimated
at 4.4% with 3.2% insoluble and 1.2% soluble. Others
reported a greater percentage of dietary fibers ranging from
6.4% to 11.5%. Variations were reported to be dependent on
Cultivars
Zahdi Microelements Hallawi Zahdi
207 Iron 5.26 10.37
14 Manganese 5.86 5.16
887 Copper 2.77 2.75
5 Zinc 1.39 0.74
342 Cobalt 0.76 0.95
59 Fluorine 0.20 0.12

Table 3 Variations in sugar percentage within different date types
Sugars (%)
Total Reducing Nonreducing
Date cultivar sugars sugar (sucrose)
Zaghloul (soft) 80.70 79.50 1.20
Ajlany 78.80 35.40 43.40
(semidry)
Sakoutti (dry) 78.70 14.40 63.30
Source: Ibrahim, A. M. and Kholaif, M, N. (1998). Date palm production and hasbandry
(in Arabic). Egypt: Al-Maaref Press.
the stage of ripeness. Six to seven dates, approximately 100 g,
consumed daily by an adult would provide 50–100% of the
recommended daily fibers uptake. Thus, dates proved to be a
better snack than others containing artificial preservatives.
Fluorine in dates proved to be essential to protect against
tooth decay. It is even added to toothpaste, whereas selenium
plays an important role in protecting against cancer and stim-
ulating the immune system.
Dates are very rich in carbohydrates, especially sugars such
as fructose, glucose, mannose, maltose, and other nonreducing
sugars such as sucrose. The percentage of the reducing sugars,
namely glucose and fructose, depend on the type of dates (dry,
semidry, or soft) and on the cultivar. In Deglet Noor, for
example, the predominant sugar was sucrose, whereas in Allig
it was reduced sugars, the more abundant sugar. This trend was
early reported because it was shown that rutab dates had much
less sucrose than reduced sugars. This difference was ascribed
to the potential presence of high invertase activity in Allig
dates. A similar trend of sugars was reported for Zaghloul,
Ajlany, and Sakoutti as soft, semidry, and dry dates, respec-
tively (Table 3).
The high increase in sugars reflects on the energy produced
by 1 kg of apricot, banana, orange, and dates to be 520, 970,
480, and more than 3000 calories, respectively. Meanwhile,
1 kg of cooked rice, wheat flour, or meat (without fats) pro-
duces 1800, 2295, and 2245 calories, respectively.
Dates are also rich in many vitamins such as B-carotene
(vitamin A), thiamine (B1) and riboflavin (B2). The contents,
however, vary with the cultivar and the stage of ripeness. For
example, vitamin C content (ascorbic acid) in rutab dates is
greater than dry dates. Ripe fruits were reported to contain a
substantial amount of carotenoids including lutin and various
forms of b-carotene with the total content of carotenoids
decreasing toward the final ripening stages and in storage.
Furthermore, dates contain a moderate amount of folic acid.
Table 4 reports vitamin contents of two date cultivars on a dry
weight basis, namely Hallawi and Zahdi.
With regard to phenolic compounds in dates, it was found
that dates contain a number of phenolics such as phenolic acids,
hydroxycinnamates and flavonoids including tannins. These
compounds are beneficial for human health as antioxidants
and maintaining a specific cellular homeostasis. Phenolics have
also been considered a defense mechanism against pathogens.
Concerning dietary fibers in dates, it was reported that six to
seven dates (about 100 g) consumed daily by an adult would
provide 50–100% of the recommended daily need.
Date Palm: A Wealth of Healthy Food 359
Table 4 Vitamin content of two commercial dry date cultivars
(mg per 100 g)
Date cultivars
Vitamins Hallawi Zahdi
Thiamine (B1) 99 80
Riboflavin (B2) 173 167
Biotin (H) 4.63 5.74
Folic acid (folacin) 57 63
Ascorbic acid (C) 3.56 2.41
Source: Yousef, A. and Kado, A. (1982). Chemical composition of four Iraqi date
cultivars. Baghdad: Agriculture and Water Resources Research Center.
Seeds of date also contain beneficial constituents that could
be utilized for animal feeding. They contain carbohydrates
(62.5%), fibers (16.2%), oils (8.5%), proteins (5.2%), ash
(1.1%), and moisture (6.5%). Date seeds also represent a
source of sterols and alkali-soluble polysaccharide. The seeds
yield a yellow green moisturizing oil rich in essential fatty acids
such as lauric (8%), myristic (4%), palmitic (25%), stearic
(10%), oleic (45%), and linoleic (10%) as well as traces of
caprylic and capric acids, suitable for use in soap and cosmetic
products.
The health benefit of dates is a relatively new important
aspect of date palm. It was found that alterations induced by
cerebral ischemia were significantly attenuated by 15 days of
pretreatment with methanolic extract of date fruits (100 and
300 mg kg 1), whereas a 30 mg kg 1 dose was insignificant in
this regard. These results suggested the possible use of dates
against bilateral common carotid artery occlusion induced
oxidative stress and neuronal damage. In another recent study
they investigated the role of date Palm fruit extract in protection
against oxidative damage and hepatotoxicity induced by sub-
chronic exposure to dimethoate (20 mg kg 1 day 1) in rat liver.
Such chemical induces an excessive production of free radicals
that are responsible for several cell alterations in the organism.
The study provided evidence that pretreatment with date palm
extract restored the liver damage induced by dimethoate as
revealed by inhibition of hepatic lipid peroxidation and preven-
tion of oxidative stress induced hepatotoxicity. Moreover,
another important beneficial health aspect of date palm extract
was found with rat experiments where such extract was able to
significantly reduce the frequency of defecation and decreased
gastrointestinal motility. It was concluded that date palm extract
may contain some pharmacologically active substances with
antidiarrheal properties.
See also: Adolescent Nutrition; Anemia: Prevention and Dietary
Strategies; Antioxidants: Characterization and Analysis; Antioxidants:
Role on Health and Prevention; Ascorbic Acid: Properties, Determination
and Uses; Berries and Related Fruits; Browning: Enzymatic Browning;
Diarrheal Diseases; Dietary Fiber: Determination; Fatty Acids: Essential
Fatty Acids; Food and Agriculture Organization of the United Nations;
Fructose: Sources, Metabolism, and Health; Fruit Juices; Fruits of
Tropical Climates: Biodiversity and Dietary Importance; Functional Foods;
Lauric Oils; Malnutrition: Prevention and Management; Meat: Structure;
Mediterranean Diet; Milk: Sources and Composition; Phenolic
360 Date Palm: A Wealth of Healthy Food
Compounds: Occurrence, Classes, and Analysis; Phospholipids:
Properties and Occurrence; Protein: Food Sources; Snack Foods: Types
and Composition; Sucrose: Dietary Importance; Sugar Alcohols; Tannins;
Traditional Foods; Vitamins: Overview.
Further Reading
Agbon AN, Kwaneshie HO, and Hamman WO (2013) Antidiarrheal activity of aqueous
fruit extract of Phoenix dactylifera (date palm) in Wistar rats. British Journal of
Pharmacology and Toxicology 4: 121–127.
Al-Gboory B and Krepl V (2010) Importance of date palms as a source of nutrition.
Agricultura Tropica Et Subtropica 43: 341–347.
Al-Hooti S, Jiuan S, and Quabazard H (1995a) Studies on the physico-chemical
characteristics of date fruits of five UAE cultivars at different stages of maturity. Arab
Gulf Journal of Scientific Research 13: 553–569.
Al-Qurashi AD (2010) Physico-chemical changes during development and ripening of
"Helali" date palm fruit. Journal of Food, Agriculture and Environment 8: 404–408.
Al-Shahib W and Marshall RJ (2003a) The fruit of the date palm: it’s possible use as the
best food for the future. International Journal of Food Science and Nutrition
54: 247–259.
Al-Shahib W and Marshall RJ (2003b) Fatty acid content of seeds from 14 varieties of
date palm Phoenix dactylifera L. International Journal of Food Science and
Technology 38: 709–712.
El Hadrami A and Al-Khayri JM (2010) Socioeconomic and traditional importance of
date palm. Emirates Journal of Food and Agriculture 24: 371–385.
El Hadrami I and El Hadramin A (2009) Breeding date palm. In: Jain SM and
Priyadarshan PM (eds.) Breeding plantation tree crops, pp. 99–216. New York: Springer.
Elleuch M, Beshes S, Roiseux O, Becker C, Deroanne ND, and Attia H (2008) Date flesh:
chemical composition and characteristics of the dietary fiber. Food Chemistry
111: 676–682.
El-Sohaimy SA and Hafez EE (2010) Biochemical and nutritional characteristics of date
palm fruits (Phoenix dactylifera L.). Journal of Applied Sciences Research
6: 1060–1067.
El-Zoghbi M (1994) Biochemical changes in some tropical fruits during ripening. Food
Chemistry 49: 33–37.
FAO Stat and FAO Statistical Yearbook (2011) Statistics Division, Food and Agriculture
Organization of the United Nations. Italy: Rome.
Farag KM (1998) Development to the rutab stage without accompanied fruit softening of
Zaghloul dates by some postharvest treatments. Proceedings of the first
International conference on date palm, 1 pp. 417–425. Al-Ain, UAE: United Arab
Emirates University.
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 Diarrheal Diseases
Z Bhutta, Centre for Global Child Health, Toronto, ON, Canada
S Syed, Emory University, Atlanta, GA, USA
ã 2016 Published by Elsevier Ltd.
Epidemiology and Global Burden of Diarrhea
The WHO defines diarrhea as the passage of three or more
loose or liquid stools per day, or more frequently than is
normal for the individual. Diarrhea is the leading cause of
mortality among children under 5 years of age accounting for
one in ten child deaths worldwide annually. It disproportion-
ally affects young children in low- and middle-income coun-
tries (Figure 1). In 2010, there were 1.731 billion episodes of
diarrhea (36 million of which progressed to severe episodes)
and in 2011, an estimated 700 000 deaths were due to diarrhea.
Incidence and mortality from diarrhea and pneumonia vary by
age (Figure 2) with the burden of disease mainly concentrated
in the younger age groups. The epidemiology of childhood
diarrhea and pneumonia overlap, likely because of shared
risk factors, such as undernutrition, suboptimum breastfeed-
ing, and zinc deficiency. Children are at the highest risk during
the first 2 years of life, with 72% of deaths being secondary to
diarrhea. Diarrheal mortality rates are declining by about 4%
annually with disease incidence decreasing from an estimated
3.4 episodes/child-year in 1990 to 2.9 episodes/child-year in
2010. The highest burden of disease has remained consistent
with respect to age in the 6–11-month-old age group having
the highest incidence. The burden of mortality due to diarrhea
in children less than 5 years of age in 2010 was highest in the
WHO regions of Africa and Southeast Asia. It is estimated that a
third of severe diarrhea episodes is preventable by vaccination
(i.e., against rotavirus and cholera). Furthermore, under nutri-
tion is a key underlying risk factor for morbidity and mortality
associated with both diarrhea and pneumonia. Diarrhea dis-
ease severity in children under-five is mostly mild (
65% cases
for which no care was sought/no dehydration) lasting about 4
days or moderate (
35% cases for which care was sought/any
dehydration on presentation) with only 0.5% of cases being
severe (cases for which care was sought, with severe dehydra-
tion on presentation) and lasting 8 days. Older children and
adults have predominantly mild episodes (95%) with 4.95%
being moderate and 0.05% being severe. Among individuals
16 years or older, severe episodes typically last
3 days and
cause dehydration in
93% of cases.
Morbidity and Risk Factors for Diarrhea
Despite reduction of diarrheal mortality by use of oral rehy-
dration therapy, persistent global burden of enteric infections
results in high morbidity of up to 43% of stunted growth,
affecting one-fifth of children worldwide and one-third of
children in developing countries. Children suffering from diar-
rhea during the first 2 years of life might have on average an
8 cm growth shortfall and 10 IQ point decrement by the time
they are 7–9 years old. The proposed link between enteric
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00223-3
infections and child growth and development is a double
burden of enteric infections and malnutrition, and the poten-
tial link of both of these factors to obesity later in life is an
interrelated ‘triple burden’ (Figure 3). Important risk factors
for diarrhea include lack of exclusive breastfeeding in infants
younger than 6 months, undernutrition and zinc deficiency.
While evidence suggests that vitamin A deficiency increases the
risk of severe diarrhea, it is not an important risk factor for the
incidence of diarrhea. Associations of specific water and sani-
tation risk factors (i.e., unwashed hands and poor water qual-
ity) with diarrhea morbidity and mortality have been shown.
However, for some outcomes such as inappropriate excreta
disposal, poor availability of evidence permits only rough
estimates of risk. In a stratified subgroup of infants aged less
than 6 months, acute lower-respiratory-tract infections are a
risk factor for diarrhea. Measles is an established risk factor for
diarrhea. A study from the 1980s showed that with moderate
coverage of the measles vaccine (45–90%) in infants, the inci-
dence of diarrhea could be reduced by 0.6–3.8%, and diarrhea
mortality reduced by 6–26% in children younger than 5 years.
Table 1 shows the effect sizes for specific confirmed biological
risk factors for diarrhea.
Diarrhea Classification and Pathophysiology
Diarrhea can be classified based on duration as prolonged/
acute (1–13 days) and persistent (14 days or more). Prolonged
diarrhea can further be classified as prolonged/acute watery
diarrhea which is loose or watery stools at least three times in
a 24 h period or prolonged/acute invasive diarrhea which is
gross blood (by history or inspection) in the stool of less than
14 days duration, typically accompanied by fever. Diarrhea can
be secondary to one or more of the following pathophysiolog-
ical processes: secretory, osmotic and inflammatory diarrhea.
Secretory diarrhea occurs when there is a significant increase in
the volume of intestinal fluid output which exceeds the reab-
sorptive ability of the gastrointestinal epithelium. Research
initially done in patients with cholera revealed that while the
secretory diarrhea in cholera does lead to significant fecal water
and electrolytes loss, the gut still retains the ability to reabsorb
water and electrolytes due to an intact Na-coupled solute
cotransport mechanism. Chloride-mediated secretion is stim-
ulated by second messengers in diarrheal infections secondary
to Vibrio cholerae 01 and 139, Shigella, Salmonella, Escherichia
coli strains and other bacterial pathogens who produce enter-
ocyte receptor binding pathogens, causing chloride-mediated
secretion stimulated by second messengers (e.g., cAMP, cGMP,
and calcium). It is important to recognize that there may be a
combination of osmotic and secretory diarrhea such as in the
case of Rotavirus where there enterotoxin mediated secretory
diarrhea along with osmotic diarrhea secondary to enterocyte
361
362 Diarrheal Diseases

Pneumonia

4%
14%
Preterm birth
complications 14%

Neonatal Intrapartum-related
death: 40% complications 9%
Other disorders 18%

Sepsis or meningitis 5%

Meningitis 2% Other neonatal disorders 2%

AIDS 2%
Congenital abnormalities 4%
Tetanus 1%
Malaria 7%
1%
Injury 5%
Measles 1% 10%

Diarrhoea

Figure 1 Global causes of childhood deaths in 2010, diarrhea kills 2195 children daily – more than malaria, measles, and AIDS combined. (Note – new
figures show that neonatal component may be 44%). Reproduced from Liu, L., Johnson, H.L., Cousens, S., et al. (2012). Global, regional, and
national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379(9832), 2151–2161.

vasoactive intestinal peptide or serotonin, or released by the

100 Diarrhoea incidence
Diarrhoea mortality
immune system, such as histamine, serotonin, prostaglandins,
90 Pneumonia incidence or interleukin-1. Lastly, increased intestinal permeability due
Pneumonia mortality to loss of cellular tight junctions can lead to increased intesti-
80 nal fluid loss (Figure 4).
Poorly absorbed osmotically active substances in the gut
70
lumen retain fluid, pull water and secondarily ions to maintain
osmotic equilibrium with body fluids – this is the key under-
Proportion (%)

60
lying process behind osmotic diarrhea. This can occur secondary
50 to an absorptive defect or enzyme deficiency from pancreas or
intestinal wall causing maldigestion and/or malabsorption.
40
An osmotic gradient is created that can only be neutralized
30 by maintaining excessive amounts of water within the intes-
tine. Examples include, malabsorption with congenital or
20 acquired lactase deficiency, gluten-sensitive enteropathy (celiac
10 disease) and maldigestion, as seen in pancreatic insufficiency
(Figure 5).
0 Gastrointestinal barrier disruption by bacteria (Salmonella,
0 12 24 36 48 60 E. coli, Campylobacter), viruses (rotaviruses, coronaviruses),
Age (months) and protozoa (Cryptosporium, Giardia) in addition to idio-
pathic inflammatory bowel diseases are common causes of
Figure 2 Distribution of cases of and deaths from diarrhea and inflammatory diarrhea. Immune cells after activation release
pneumonia in children aged 0–4 years. Reproduced from Walker, C.L.,
inflammatory mediators and cytokines which stimulate secre-
Rudan, I., Liu, L., et al. (2013). Global burden of childhood pneumonia
and diarrhoea. Lancet 381(9875), 1405–1416.
tion and activate enteric nerves. Subepithelial myofibroblasts
destroy the basement membrane by metalloproteinases, dam-
aged enterocytes are extruded, and villous atrophy develops
damage. Phosphorylation-induced activation (by increase in followed by regenerative crypt hyperplasia in the small intes-
cAMP, cGMP, or calcium) of these channels can be set off by tine and colon. These surfaces are covered with immature
exogenous or endogenous substances. Exogenous mediators enterocytes that typically are deficient in the brush border
include bacterial toxins such as cholera or shiga toxins, or enzymes and transporters necessary for absorption of nutrients
parasites. Endogenous mediators can be endocrine, such as and water.
 Diarrheal Diseases 363

Enteric
infections
+/- diarrhoea

Water Antimicrobials
Sanitation Vaccines
Worsened Environment
Intestinal
infection damage and
intensity and inflammation
damage
Repeated and Human
persisting genome
Impaired infections
innate Microbiome
and acquired Nutrient
mucosal malabsorption
defences and/or loss

Probiotics Repair
Microflora nutrients

Malnutrition

Impaired vaccine responses

+

Cognitive impairment
Obesity and
associated
comorbidities
Poverty and costs

Figure 3 The vicious cycles of diseases of poverty. Enteric infections, especially in the first 2–3 years of life, with or without overt diarrhea, can
predispose an individual to malnutrition and stunted growth through multiple mechanisms. Stunting by 2 years of age, in turn, is associated with
impaired cognitive development that extends into later childhood and even adulthood and adult productivity. In addition, malnourished children
experience both greater frequency and duration of diarrheal illnesses, and, documented in animal models, heavier infections. The latter is documented
with Cryptosporidium and with enteroaggregative E. coli. Finally, enteric infections or stunting can predispose to obesity and its comorbidities of
diabetes, hypertension, cardiovascular disease, metabolic syndrome and burgeoning healthcare expenditures, contributing to individual and societal
poverty in vicious cycles. Reproduced from Guerrant, R.L., DeBoer, M.D., Moore, S.R., Scharf, R.J., and Lima, A.A. (2013). The impoverished gut – a
triple burden of diarrhoea, stunting and chronic disease. Nature Reviews Gastroenterology & Hepatology 10(4), 220–229.

Etiology of Diarrhea parasitic etiologic agents of diarrhea in developing countries

Major etiologies for prolonged/acute and persistent diarrhea
are outlined in Table 2. The most predominant cause of
diarrhea in developing countries is infectious (viral, bacterial,
parasitic). A systematic review of articles published between
1990 and 2011 reporting etiological agents causing diarrhea
in hospitalized children less than 5 years of age showed that
rotavirus, calicivirus, enteropathogenic, and enterotoxigenic
E. coli cause more than half of all diarrheal deaths in this
age group globally. A recent 3-year prospective study in four
sites in Africa (the Gambia, Mali, Mozambique, Kenya) and
three in Asia (India, Bangladesh, Pakistan) showed the most
attributable cases of moderate to severe diarrhea in children
aged 0–59 months was secondary to infection with rotavirus,
Shigella, ST-ETEC, cryptosporidium, and enteropathogenic
E. coli. Table 3 shows a comparison of viral, bacterial, and
and the United States. There is a seasonality in the transmis-
sion patterns of infectious agents in developing countries
with bacterial diarrheas peaking in the hot months and viral
diarrheas typically occurring throughout the year with some
peaking in the cooler months. More than one pathogenic
agent can exist with 11% (in community studies) and 12%
(in hospital based studies) of diarrhea cases documented as
having two or more enteric pathogens. Transmission of infec-
tious agents causing diarrhea is primarily ‘fecal–oral’ in the
setting of poor hand washing hygiene, unsanitary water and
dirty food preparation. The dose of infectious agent required
to cause diarrhea also matters with low infectious dose (e.g.,
shigella, giardia, rotavirus, cryptosporidium) can be transmit-
ted by person-to-person contact and high infectious dose
(e.g., salmonella, E. coli, vibrios) usually transmitted by
water or food.
364 Diarrheal Diseases

Table 1 Risk factors with direct biological links to diarrhea

Morbidity Mortality

Not exclusively breastfeeding (0–5 months; vs. exclusive breastfeeding)

Partially breastfed RR 1.7 (95% CI 1.0–2.8)a RR 4.6 (95% CI 1.8–11.8)a
Not breastfed RR 2.7 (95% CI 1.7–4.1)a RR 10.5 (95% CI 2.8–39.6)a
No breastfeeding (6–23 months; vs. receives any breast milk) RR 1.3 (95% CI 1.1–1.6)b RR 2.2 (95% CI 1.1–4.2)a
Underweight (vs. >–2 WAZ for morbidity and >–1 WAZ for mortality)
–2 to <–1 WAZ – OR 2.1 (95% CI 1.6–2.7)c
–3 to <–2 WAZ RR 1.2 (95% CI 1.1–1.4)d OR 3.4 (95% CI 2.7–4.4)c
Less than <–3 WAZ – OR 9.5 (95% CI 5.5–16.5)c
Stunting (vs. >–1 HAZ)
–2 to <–1 HAZ – OR 1.2 (95% CI 0.9–1.7)c
–3 to <–2 HAZ – OR 1.6 (95% CI 1.1–2.5)c
<–3 HAZ – OR 4.6 (95% CI 2.7–8.1)c
Wastingc (vs. >–WHZ)
–2 to <–1 WHZ – OR 1.2 (95% CI 0.7–1.9)c
–3 to <–2 WHZ – OR 2.9 (95% CI 1.8–4.5)c
<–3 WHZ – OR 6.3 (95% CI 2.7–14.7)c
Vitamin A deficiency (vs. not deficient) RR 1.5 (95% CI 1.3–1.8)e
Zinc deficiency (vs. not deficient) RR 1.2 (95% CI 1.1–1.2)f RR 1.2 (95% CI 1.0–1.6)f

Abbreviations: RR, relative risk; WAZ, weight-for age z-score; OR, odds ratio; HAZ, height-for-age z-score; WHZ, weight-for-height z-score.
a
Mix of cohort, observational, and case–control studies. Adjusted risk relations from individual studies used when available.
b
Mix of cohort, observational, and case–control studies. Adjusted risk relations from individual studies used when available. Data only available for ages 6–11 months for this risk
relation.
c
Prospective datasets adjusted for socioeconomic and non-nutritional determinants of mortality.
d
Mix of cohort, observational and case–control studies; analysis done with unadjusted risk estimates.
e
Meta-analysis of observed risk reductions from supplementation trials.
f
Meta-analysis of observed risk reductions from randomized controlled trials.
Source: Walker, C.L., Rudan, I., Liu, L., et al. (2013). Global burden of childhood pneumonia and diarrhoea. Lancet 381(9875), 1405–1416.

Diagnosis and Management of Acute/Prolonged
and Persistent Diarrhea
Despite clinical clues, determining the causative agent of diar-
rhea in an individual patient on the basis of clinical grounds
alone can be difficult. Both acute/prolonged and persistent
diarrheas have similar initial evaluation and management.
This chapter focuses on assessing the severity of illness and
the need for rehydration. A brief history and directed clinical
exam can help determine this and further guide the
management.
History
Specific points in history include, time of onset of diarrhea,
frequency, amount, consistency, and contents (blood, mucous,
pus) of stool, associated signs and symptoms (fever, abdomi-
nal pain, nausea, vomiting, bloating, flatus, tenesmus), recent
travel, dietary history, exposure to pets or cattle, drug history
(specifically antibiotics and laxatives), pertinent past medical
and surgical history, family history and day care attendance.
For persistent diarrhea, the age of symptom onset and a
detailed dietary history with relation between onset of symp-
toms and introduction to new foods is relevant.
Physical Examination
The initial physical exam focuses on signs and symptoms of
dehydration (discussed below) and sepsis (fever and toxic
look) which are major complications of acute and chronic
diarrhea and require urgent management. Nutritional assess-
ment is necessary and includes height, weight, vital signs, and
global nutritional appearance.
Assessing Dehydration and IMCI Guidelines
Early recognition of dehydration, which is the main cause of
mortality, is the most critical part of diarrhea management. Loss
of water and electrolytes (sodium, chloride, potassium, and
bicarbonate) via liquid stools, emesis, sweat, urine, and breath-
ing leads to dehydration if left unreplaced. The WHO suggests
assessment of dehydration on a scale of three (Figure 6).
1. Early dehydration with no signs or symptoms,
2. Moderate dehydration notable for thirst, restless, or irrita-
ble behavior, decreased skin elasticity, sunken eyes,
3. Severe dehydration when symptoms become more severe
with diminished consciousness, lack of urine output, cool,
moist extremities, a rapid and feeble pulse, low or unde-
tectable blood pressure, and pale skin in the setting of
clinical shock.
Lab Evaluation
For acute diarrhea, maintaining adequate intravascular volume
and correcting fluid and electrolyte disturbances take priority over
the identification of the causative agent. Stool cultures are usually
unnecessary for immune-competent patients who present within
24 h after the onset of acute, watery diarrhea. Lab testing including
 Diarrheal Diseases 365

Figure 4 Pathophysiology of secretory diarrhea: diarrhea secondary to cholera toxin cholera toxin induces secretion, in part, through activation of an
intramural neural reflex. Toxin attaches to enterochromaffin cells in the epithelium, causing an increase in (cAMP) there. In response to the latter,
serotonin (5-HT), neurotensin (NT), and possibly additional peptides are released. These activate afferent neurons (I), whose axons course from close to
the epithelium to the myenteric neural plexus, where they connect with interneurons (II) that, in turn, through release of acetylcholine (ACh) and/or
substance P (SP), activate secreto-motor neurons (III) in the submucosal neural plexus. Axons from these secreto-motor neurons reach the epithelial
surface, in both the villus and the crypt regions, releasing VIP and thereby stimulating secretion in crypts and inhibiting nutrient-independent salt
absorption in villi. Reproduced from Field, M. (2003). Intestinal ion transport and the pathophysiology of diarrhea. The Journal of Clinical Investigation
111(7), 931–943.

stool studies, blood tests, intestinal imaging, and immunologic
and serologic evaluation are indicated in bloody diarrhea, high
fever (>104 F), systemic illness, severe, or prolonged diarrhea,
history suggestive of food poisoning, recent history of travel
overseas or possibility of immune dysfunction. Supplementary
laboratory studies, including serum electrolytes, to assess patients
with acute diarrhea usually are unnecessary.
Treatment Approach
Fluids Resuscitation
Dehydration is the primary cause of death in patients with
diarrhea and effectively addressing this is the cornerstone of
diarrhea treatment. Oral rehydration solution (ORS), devel-
oped in the 1960s and 1970s by researchers in South Asia, is
a simple cost-effective solution containing glucose and
electrolytes that can be used to prevent and treat dehydration
due to diarrhea of any cause across all ages. Development of
the ORS took advantage of the glucose-coupled intestinal
sodium transport mechanism discovered in the 1960s by
Robert Crane. Absorption of sodium and glucose molecules
across the luminal membrane is facilitated by the protein
sodium glucose co-transporter 1 (SGLT1). Although nutrient-
independent sodium absorption across the intestinal epithelial
brush border membrane is impaired in diarrhea, cotransport of
sodium and glucose is preserved. This allows absorption of
sodium and thus water as provided by ORSs (Figure 7).
Once in the enterocyte, the transport of glucose into the
blood is facilitated by GLUT2 (glucose transporter type 2) in
the basolateral membrane with the Na þ Kþ ATPase provides
the gradient that drives the process. Key in the success of use of
ORS for rehydration is the fact that these physiological pro-
cesses stay intact, even in the setting of severe diarrhea.
366 Diarrheal Diseases

(a) Carbohydrate malabsorption (b) H2O

Osmotic laxatives H2O H2O H2O
Malabsorption syndromes H 2O H 2O H2O
-short bowel H2O
-bacterial overgrowth H2O
H 2O H2O
-mucosal destruction
-pancreatic insufficiency H2 O
H2O H 2O
H2O H 2O H2O H2O
Absorption H2O H2O

Secretion

Villus

Crypt

Figure 5 Pathophysiology of osmotic diarrhea. The figure demonstrates the pro-secretory effects of osmotic agents on intestinal fluid movement. (a)
Represents the normal luminal environment and (b) represents the effects of osmotic agents such as non-absorbed carbohydrates. Image reproduced
from the Internet http://cancerlink.ru/?page_id=1612 – free image source.

The original ORS recommended by the WHO in the 1970s be administered (20 ml kg1 body weight) until pulse, perfu-
had a formulation with a total osmolarity of 311 mmol l1 sion, and mental status return to normal.
(Table 4) but subsequent investigations lead to the current
WHO recommendations from 2004 of a low osmolarity ORS Zinc Supplementation
(total osmolarity of 245 mmol l1, decreased concentrations
Zinc deficiency is widespread among young children in low-
of glucose and sodium). The current formulation when com-
and middle-income countries. It contributes to mortality and
pared to the original formulation is associated with decrease in
morbidity related to diarrhea, malaria, pneumonia, and
stool volume by about 25% when compared to the original
restricts growth. Children with diarrhea suffer due to poor
World Health Organization/United Nations Children’s Fund
baseline dietary zinc intake with aggravated net losses of zinc
(WHO/UNICEF) ORS solution, reduced vomiting by almost
during the period of illness. The WHO and UNICEF has recom-
30%, and reduced need for unscheduled IV therapy by more
mended zinc for the treatment of diarrhea since 2004. Evi-
than 30%. A 2010 review by Munos and colleagues assessed
dence suggests that zinc administration (20 mg per day until
205 studies and showed that universal coverage with ORS
the diarrhea ceases) reduces the duration, severity and hospital
would reduce diarrhea-related deaths by 93%. There are two
admissions associated with diarrheal episodes in children in
phases to treatment: rehydration and maintenance. In the
developing countries. Zinc for the treatment of diarrhea is
rehydration phase, there is quick replacement of the fluid
estimated to decrease diarrhea mortality by 23% as well as
deficit over 3–4 h to achieve clinical hydration. In the mainte-
significantly reducing the severity and duration of diarrheal
nance phase, maintenance calories and fluids are administered
episodes in children as well as a reduction in treatment failure
with reintroductions of enteral feeding with an age-appropriate
or deaths in diarrheal episodes of longer duration (>14 days).
unrestricted diet, including solids. In children with mild to
moderate dehydration, 50–100 ml of ORS/kg body weight
during 2–4 h should be administered to replace the estimated
Diet
fluid deficit, with additional ORS administered to replace
ongoing losses. In the setting of severe dehydration, there is Current WHO guidelines for the management and treatment of
an emergent immediate need for IV rehydration. Lactated diarrhea in children strongly recommend continued feeding
Ringer’s solution, normal saline, or a similar solution should alongside appropriate management. An extensive review on
 Diarrheal Diseases 367

Table 2 Differential diagnosis for prolonged/acute and persistent diarrhea

Prolonged/acute diarrhea (1–13d)

Infectious Viral (rotavirus, Norwalk, enterovirus, calcivirus); bacterial (E. coli, Shigella, Salmonella, Yersinia,
Campylobacter, C. difficile, V. cholera, Aeromonas); Protozoa (Giardia, cryptosporidium, E. histolytica)
Allergy Short exposure to allergen, challenge to known allergen
Toxic Drug side effects, acute abdomen with diarrhea as presenting symptom, intussusception
Extra-intestinal infections Respiratory, urinary, sepsis
Persistent diarrhea (>¼14 days)
Infantile protracted diarrhea with Post infectious, food allergy, malnutrition congenital histological dysmorphism (e.g., tufting enteropathy,
villous atrophy microvillus inclusion disease)
Infectious Bacterial, parasitic
Inborn errors of metabolism Familial chloride diarrhea, sodium–hydrogen exchange defect, abetalipoproteinemia and
hypobetalipoproteinemia, Galactosemia, Tyrosinemia, Acrodermatitis enteropathica
Carbohydrate malabsorption Congenital (lactase deficiency, glucose–galactose malabsorption, sucrase–isomaltase deficiency, fructose
malabsorption) secondary (acquired mono and disaccharidase deficiencies)
GI organ pathology Small intestine (celiac disease, tropical sprue, Whipple’s disease, intestinal lymphangiectasia, eosinophilic
gastroenteropathy, enterokinase deficiency); short bowel syndrome; ischemia; lymphoma; motility disorders
(for e.g., small bowel overgrowth); pancreas (all conditions with exocrine insufficiency for e.g., cystic
fibrosis); liver (all conditions leading to cholestasis, bile salt deficiency)
Immune defects Isolated IgA deficiency, SCIDS, AIDS, autoimmune enteropathy
IBD Crohn’s disease, ulcerative colitis
Fecal impaction with overflow Hirschsprung’s disease, anorectal malfunction, functional constipation
Dietary Overfeeding, nondigestible carbohydrates
Others Toddler’s diarrhea, Munchausen by proxy, factitious diarrhea, non-GI causes (tumors – APUDoma,
ganglioneuroma, neuroblastoma)

Source: Robert Wyllie, J.S.H. (2011). Pediatric gastrointestinal and liver disease (4th ed). Diarrhea.

Table 3 Comparison of viral, bacterial, and parasitic etiologic agents of diarrhea in developing countries and the United States

Etiologic agent Developing country United States

Viral
Rotavirus Important Very important
Noroviruses Probably important Important
Enteric adenoviruses Minor Probably important
Bacteria
Enterotoxigenic E. coli Very important Minor
Campylobacter Important Important
Shigella Important Minor
Salmonella Variable Important
Enterohemorrhagic E. coli Minor Important
Parasites
Cryptosporidium Important Minor
Giardia Minor Minor
Strongyloides Minor Minor
Entamoeba histolytica Minor Minor

Source: Table modified from Lanata, C.F., Fischer-Walker, C.L., Olascoaga, A.C., Torres, C.X., Aryee, M.J., Black, R.E., and Child Health Epidemiology Reference Group of the World
Health Organization and UNICEF (2013). Global causes of diarrheal disease mortality in children <5 years of age: a systematic review. PLoS One 8(9), e72788.

feeding strategies and food-based interventions in children
younger than 5 years with diarrhea in low- and middle-income
countries showed that although illness duration was shorter
and risk of treatment failure 47% lower in children with acute
diarrhea who consumed lactose-free diet, there was no effect of
lactose avoidance on stool output or weight gain. WHO rec-
ommends the following:
• An age-appropriate diet regardless of the fluid used for ORT
(oral rehydration therapy)/maintenance
• More frequent breastfeeding or bottle feeding – special
formulas or dilutions are unnecessary
• Age-appropriate fluid resuscitation
• Frequent, small meals throughout the day (six meals/day)
• Energy and micronutrient-rich foods (grains, meats, fruits,
and vegetables)
• Increasing energy intake as tolerated following the diarrheal
episode
• Avoid: canned fruit juices and other high-sugar food and
drinks – these are hyperosmolar and can aggravate diarrhea.
368 Diarrheal Diseases
Does the child have diarrhoea?
If yes, ask: Look and feel:
For how long? Look at the child’s general
Is there blood in the stool? condition. Is the child:
Lethargic or
unconscious?
Restless and irritable?
Look for sunken eyes.
Offer the child fluid. Is the
child:
Not able to drink or
drinking poorly?
Drinking eagerly,
thirsty?
Pinch the skin of the
abdomen. Does it go back:
Very slowly (longer
than 2 seconds)?
Slowly?
Figure 6 IMCI flow chart for diarrhea in children under 5 years of age. Reproduced from WHO (2014). Maternal, newborn, child and adolescent health.
IMCI Chart Booklet. Page 3/76.
Figure 7 Coupled transport of sodium and glucose in intestinal epithelial cells. Reproduced from Duggan, C., Fontaine, O., Pierce, N.F., et al. (2004).
Scientific rationale for a change in the composition of oral rehydration solution. JAMA 291(21), 2628–2631.
Antibiotics for Cholera, Shigella, and Cryptosporidium
Antibiotics should not be used for relatively mild, self-limited
infectious diarrhea to minimize development of drug resis-
tance. When the diarrhea is hemorrhagic, seriously dehydrat-
ing, associated with serious systemic signs and symptoms, or of
Two of the following signs: Pink: If child has no other severe classification:
Lethargic or unconscious SEVERE Give fluid for severe dehydration (Plan C)
for DEHYDRATION Sunken eyes DEHYDRATION OR
Not able to drink or drinking If child also has another severe
poorly classification:
Classify DIARRHOEA
Skin pinch goes back very Refer URGENTLY to hospital with mother
giving frequent sips of ORS on the way
slowly.
Advise the mother to continue
breastfeeding
If child is 2 years or older and there is
cholera in your area, give antibiotic for
cholera
Two of the following signs: Yellow: Give fluid, zinc supplements, and food for some
Restless, irritable SOME dehydration (Plan B)
Sunken eyes If child also has a severe classification:
DEHYDRATION
Drinks eagerly, thirsty Refer URGENTLY to hospital with mother
Skin pinch goes back giving frequent sips of ORS on the way
slowly. Advise the mother to continue
breastfeeding
Advise the mother to return immediately
Follow-up in 5 days if not improving
Not enough signs to classify Green: Give fluid, zinc supplements, and food to treat
as some or severe NO DEHYDRATION diarrhoea at home (Plan A)
dehydration. Advise mother when to return immediately
Follow-up in 5 days if not improving
Dehydration present. Pink: Treat dehydration before referral unless the
and if diarrhoea 14 SEVERE child has another severe classification
days or more PERSISTENT Refer to hospital
DIARRHOEA
No dehydration. Yellow: Advise the mother to feeding a child who has
PERSISTENT PERSISTENT DIARRHOEA
DIARRHOEA Give multivitamins and
minerals (including zinc) for 14 days
Follow-up in 5 days
Blood in the stool. Yellow: Give ciprofloxacin for 3 days
and if blood in stool
DYSENTERY Follow-up in 3 days
longer than 5 days’ duration without improvement, antibiotic
use is appropriate. A review by Traa and colleagues assessed the
effectiveness of WHO-recommended antibiotics – ciprofloxa-
cin, ceftriaxone, and pivmecillinam – for the treatment of
dysentery, and concluded that antibiotics can be expected to

Table 4 Composition of standard and reduced-osmolarity WHO ORS
Standard WHO Reduced-osmolarity WHO
(1975) (2002)
Glucose 111 75
(mmol l1)
Sodium 90 75
(mequiv. l1)
Potassium 20 20
(mequiv. l1)
Chloride 80 65
(mequiv. l1)
Citrate (mmol l1) 10 10
Osmolarity 311 245
(mOsml l1)
Abbreviations: ORS, oral rehydration solution; WHO, World Health Organization.
Source: Duggan, C., Fontaine, O., Pierce, N.F. et al. (2004). Scientific rationale for a
change in the composition of oral rehydration solution. JAMA 291(21), 2628–2631.
decrease diarrhea mortality attributable to dysentery by more
than 99%. There is evidence to recommend antibiotic use for
reduction of morbidity and mortality due to cholera, Shigella
spp. and cryptosporidium – Das et al. showed that in the
developing country setting antibiotic management of cholera
resulted in a 63% reduction in rates of clinical failure and a
75% reduction in rates of bacteriological failure. They also
showed that antibiotic management of Shigella resulted in an
82% reduction in rates of clinical failure and a 96% reduction
in rates of bacteriological failure. Antibiotics for treatment of
cryptosporidiosis reduced mortality by 76%, rates of clinical
failure by 52%, and rates of parasitological failure by 38%. The
empiric antibiotic regimen should be determined by host fac-
tors, severity of illness, local resistance patterns, and history of
travel to an area with increased resistance.
Probiotics
Probiotics are defined by the joint Food and Agriculture Orga-
nization/WHO Working Group as ‘live microorganisms that
when administered in adequate amount concern a health ben-
efit on the host.’ Recent Cochrane systematic reviews on the
use of probiotics as an adjunct therapy to ORS have shown that
probiotics shorten the duration of diarrhea and reduce stool
frequency but the observation remains inconclusive due to few
trials with small numbers of participants. Most commonly
used probiotics in trials include Bifidobacterium, two genera
of lactic acid bacteria (Lactobacillus and Streptococcus) and
the yeast Saccharomyces. There is insufficient evidence to rec-
ommend their widespread use at this time.
Antiemetics
Vomiting associated with diarrhea causes significant additional
physical discomfort and is of concern for both parents and
physicians. Additionally, vomiting can limit the success of ORS
and can result in increased use of intravenous (IV) rehydration,
need for prolonged emergency department stays and hospitali-
zations. A recent systematic review evaluated seven RCTs which
used ondansetron, metoclopramide, dimenhydrinate, and
Diarrheal Diseases 369
dexamethasone in children aged 0–12 years. They found that
use of antiemetics significantly reduced the incidence of vomit-
ing and hospitalization by 54% and reduced IV fluid require-
ments by 60%. A 2011 Cochrane Review on the use of
antiemetics for reducing vomiting related to acute gastroenteritis
in children and adolescents concluded that oral ondansetron
increased the proportion of patients who had ceased vomiting
and reduced the number needing IV rehydration and immediate
hospital admission. IV ondansetron and metoclopramide
reduced the number of episodes of vomiting and hospital admis-
sion, and dimenhydrinate as a suppository reduced the duration
of vomiting. Antiemetics are effective for the management of
gastroenteritis in children and have the potential to decrease
morbidity and mortality burden due to diarrhea.
Breast Feeding
Breast milk provides various immunological, psychological,
social, economic, and environmental benefits, and is therefore
recommended as the best feeding option for newborn babies and
young infants in developing countries, including HIV-infected
populations. Lamberti and colleagues reviewed 18 studies from
developing countries reporting the effect of breastfeeding on
diarrhea morbidity and mortality. The investigators estimated
that not breastfeeding was associated with a 165% increase in
diarrhea incidence and mortality in 0–23-month-olds.
Vaccines for Rotavirus
Rotavirus is the most common cause of severe dehydrating
diarrhea in infants worldwide. Rotavirus vaccines represent
an important preventive approach to reducing the burden of
diarrheal disease due to rotavirus. Since 2006 two rotavirus
vaccines are currently licensed for vaccinating infants in
North and South American, European, and Eastern Mediterra-
nean countries: RotaTeq® (Merck) and Rotarix® (GlaxoS-
mithKline). RotaTeq®/RV5 – is a live, oral vaccine that
contains five reassortant rotaviruses developed from human
and bovine parent rotavirus strains and is given orally in
three doses at ages 2, 4, and 6 months. Rotarix®/RV1 is a live,
oral vaccine licensed in 2008 for use in the United States that
contains a human rotavirus strain, this is given orally in two
doses at ages 2 and 4 months. This first dose of either vaccine is
most effective if it is given before a child is 15 weeks of age and
children should receive all doses of rotavirus vaccine before
they turn 8 months old.
In the global health setting, there is evidence suggesting that
rotavirus vaccine efficacy may vary by setting due to regional
differences in circulating rotavirus vaccine strains, and reduced
efficacy of oral vaccines in countries with high burden of malnu-
trition and enteric infections. A meta-analyses by Munos et al.
assessed the effectiveness of two currently marketed vaccines the
pentavalent human-bovine reassortant rotavirus vaccine and
monovalent attenuated human rotavirus vaccine in Phase III tri-
als in European and Latin American sites for both vaccines, as well
as the United States (including Navajo and Apache populations)
and Taiwanese sites for the pentavalent vaccine. The effectiveness
studies of the monovalent and pentavalent vaccines were con-
ducted in northern Australia and Nicaragua, respectively. The
authors estimated that these rotavirus vaccines could prevent
370 Diarrheal Diseases
74% of rotavirus deaths and 47–57% of rotavirus hospitaliza-
tions. Data published from a randomized, placebo-controlled,
multicenter trial in South Africa and Malawi evaluating the effi-
cacy of a live, oral rotavirus vaccine in preventing severe rotavirus
gastroenteritis showed that the rotavirus vaccine significantly
reduced the incidence of severe rotavirus gastroenteritis among
African infants during the first year of life. The ROTAVAC rotavi-
rus vaccine study group recently published results from a Phase III
clinical trial using ROTAVAC which is a new rotavirus vaccine that
consists of a strain of the virus that was isolated, manufactured,
and tested in India. Infants aged 6–7 weeks received three doses of
oral human-bovine natural reassortant vaccine (116E) with effi-
cacy results from the study showing the vaccine to be effective and
well tolerated in Indian infants.
Vaccines for Cholera
Although case management with oral rehydration therapy has
substantially improved case-fatality rates for cholera, the infec-
tion can still kill rapidly, especially in outbreak settings. Old-
generation injectable cholera vaccines, abandoned since the
1970s because of their restricted effectiveness and local side
effects. However, studies have shown vaccines to be effective in
reducing risk of cholera infection in children younger than 5
years by 52%, increase in vibriocidal antibodies by 124% and a
relative risk of one or more adverse events of 1.4. Such evidence
for the effectiveness of oral cholera vaccines makes them good
candidates for cholera control in endemic areas.
Improved Water Provision, Use, Sanitation, and Hygiene
Promotion
Maintaining water quality (protection or treatment of water at
source or point of use) is more effective than improving water
supply (improved source of water or improved distribution,
or both). Cairncross and colleagues estimated the effect of
water, sanitation, and hygiene strategies and estimated risk
reductions for diarrhea of 48% for hand washing with soap,
17% with improved water quality, and 36% with proper
excreta disposal.
Community-Based Approaches
Although evidence confirms the efficacy and effectiveness of
many interventions, many interventions are not accessible to
people in need; hence, the focus on delivery strategies has
increased. One method of community-based case manage-
ment is to provide these amenities through community
health workers with home visitation and community-based
sessions for education and promotion of care seeking. These
approaches have been assessed and concluded that that
community-based interventions are associated with a 160%
significant increase in the use of ORS and an 80% increase in
the use of zinc in diarrhea. Furthermore, findings showed a
9% increase in care seeking for diarrhea and noted a 75%
significant decline in inappropriate use of antibiotics for
diarrhea.
Global Action Plan for Pneumonia and Diarrhea
The 2013 Lancet Childhood Pneumonia and Diarrhea series
reviewed existing preventive and therapeutic interventions
that could have a role in reducing the morbidity and mor-
tality burden in children younger than 5 years due to diar-
rhea and pneumonia. The interventions with the maximum
effect include breastfeeding, ORS, and community case man-
agement. The series concluded that despite a large global
burden childhood diarrhea and pneumonia deaths are avoid-
able and 15 interventions delivered at scale can prevent most
of these deaths (Figures 8 and 9). Estimates modeled with
the Lives Saved Tool showed that if the interventions are
scaled up by 80% in the 75 countdown countries, 95% of
diarrheal, and 67% of pneumonia deaths in children youn-
ger than 5 years can be prevented by 2025. Scaling up of
diarrheal and pneumonia interventions would cost US$
6.715 billion, only $2.9 billion more than present levels of
spending. Assessment is needed of the cost-effectiveness of
these interventions in national health delivery frameworks.
We have a clear idea of effective interventions for both
diarrhea and pneumonia and with community health-worker
programs increasingly being employed to reach underserved
populations, real opportunities exist to scale-up community
advocacy/education programs, early case detection and man-
agement strategies.
The series also included summary recommendations after
a series of consultations with several hundred front line pub-
lic health practitioners in target countries were held to iden-
tify key barriers to scaling up of evidence-based interventions
to reduce pneumonia and diarrhea mortality. Critical barriers
included: absence of national coordination within ministries
and other stakeholders to deliver interventions, insufficient
financial resources, inadequate training and support for
health workers, poor systems for monitoring and assessment
of key programmatic indicators and sporadic availability of
key commodities. The final list of recommendations
included: (1) Improve coordination between various groups
working on preventive and treatment interventions to control
pneumonia and diarrhea. (2) Substantially increase resources
for child survival programs, with an emphasis on pneumonia
and diarrhea control efforts. (3) Enhance efforts to attract,
train, and retain a competent work force of caregivers. (4)
Invest in better systems for harmonization of the collection of
essential programmatic indicators, and ensure that this infor-
mation is shared throughout the system. (5) Strengthen sup-
ply systems that deliver essential commodities. In summary,
the cost to achieve the end of preventable deaths from pneu-
monia and diarrhea by 2025 is estimated to be around US$
6.715 billion. Despite large reductions in child mortality
between 2000 and 2010 (both in all-cause mortality, and
specifically diarrhea and pneumonia associated), these dis-
eases remain causes for preventable deaths. The achievement
of the fourth Millennium Development Goal and the longer-
term target of reduction of child mortality to 20 deaths or
fewer per 1000 live births in all countries by 2035 requires
significant decreases in mortality from both diarrhea and
 Diarrheal Diseases 371

Environmental
WASH,* reduce overcrowding
and household air pollution Increased
susceptibility Delivery platforms
Promotion of community-based
health and behavioral change
Nutrition
Breastfeeding promotion,*
preventive vitamin A or zinc
supplementation*
Financial incentives to promote
care seeking
Vaccines Exposure
Measles, Haemophilus influenzae
type b, pneumococcal infection, Integrated community case
rotavirus, cholera management

Treatment
Oral rehydration solution,
continued feeding after Facility-based IMCI
diarrhoea, zinc for diarrhoea Pneumonia
treatment, probiotic use,
antibiotics and oxygen therapy Diarrhoea
for pneumonia, antibiotics for
dysentery

Survival Death

Figure 8 Conceptual framework of the effect of interventions for diarrhea and pneumonia – WASH, water, sanitation, and hygiene; IMCI, Integrated
Management of Childhood Illness. Asterisk (*), interventions common to both diarrhoea and pneumonia. Reproduced from Bhutta, Z.A., Das, J.K.,
Walker, N., et al. (2013). Interventions to address deaths from childhood pneumonia and diarrhoea equitably: what works and at what cost? Lancet 381
(9875), 1417–1429.

Pneumococcal vaccine
Case management of neonatal infections
Breastfeeding promotion
Case management of pneumonia infections
Improved water source
Zinc supplementation
Hib vaccine
Hand washing with soap
Improved sanitation
Oral rehydration solution
Rotavirus vaccine
Hygienic disposal of children’s stools
Vitamin A supplementation
Zinc for treatment of diarrhoea
Antibiotics for dysentery

0 50 000 100 000 150 000 200 000 250 000 300 000 350 000
Number of deaths averted

Figure 9 Sequential effect of 15 individual interventions on deaths due to diarrhea and pneumonia Haemophilus influenzae type b – Hib. Reproduced
from Bhutta, Z.A., Das, J.K., Walker, N., et al. (2013). Interventions to address deaths from childhood pneumonia and diarrhoea equitably: what works
and at what cost? Lancet 381(9875), 1417–1429.
372 Diarrheal Diseases
pneumonia. Our knowledge on diarrhea has drastically
evolved over the past five decades and the present challenge
is to implement these evidence-based strategies for reducing
morbidity and mortality due to diarrhea.
See also: Clostridium botulinum; Clostridium: Food Poisoning by
Clostridium perfringens; Clostridium: Occurrence and Detection of
Clostridium botulinum and Botulinum Neurotoxin; Clostridium:
Occurrence and Detection of Clostridium perfringens; Colon: Structure
and Function; Escherichia coli and Other Enterobacteriaceae: Food
Poisoning and Health Effects; Escherichia coli and Other
Enterobacteriaceae: Occurrence and Detection; Malnutrition: Concept,
Classification and Magnitude; Malnutrition: Prevention and
Management; Nutrition and Infection; Probiotics; Salmonella:
Detection; Salmonella: Properties and Occurrence; Salmonella:
Salmonellosis; Sanitization; Staphylococcus: Food Poisoning; World
Health Organization; Zinc: Physiology and Health Effects; Zinc:
Properties and Determination.
Further Reading
Baqui AH, Black RE, El Arifeen S, Yunus M, Chakraborty J, Ahmed S, and Vaughan JP
(2002) Effect of zinc supplementation started during diarrhoea on morbidity and
mortality in Bangladeshi children: community randomised trial. BMJ 325: 1059.
Bernaola Aponte G, Bada Mancilla CA, Carreazo Pariasca NY, and Rojas Galarza RA
(2010) Probiotics for treating persistent diarrhoea in children. Cochrane Database of
Systematic Reviews. CD007401.
Bhandari N, Rongsen-Chandola T, Bavdekar A, et al. (2014) Efficacy of a monovalent
human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-
blind, placebo-controlled trial. Lancet 383: 2136–2143.
Bhutta ZA, Das JK, Walker N, Rizvi A, Campbell H, Rudan I, and Black RE (2013)
Interventions to address deaths from childhood pneumonia and diarrhoea equitably:
what works and at what cost? Lancet 381: 1417–1429.
Cash RA, Nalin DR, Rochat R, Reller LB, Haque ZA, and Rahman AS (1970) A clinical
trial of oral therapy in a rural cholera-treatment center. The American Journal of
Tropical Medicine and Hygiene 19: 653–656.
Crane RK, Miller D, and Bihler I (1961) The restrictions on possible mechanisms of
intestinal transport of sugars. In: Kleinzeller A and Kotyk A (eds.) Membrane
transport and metabolism, Proceedings of a Symposium held in Prague, 22–27
August, 1960, pp. 439–449. Prague: Czech Academy of Sciences, Model of
cotransport on page 448.
Das JK, Ali A, Salam RA, and Bhutta ZA (2013a) Antibiotics for the treatment of Cholera,
Shigella and Cryptosporidium in children. BMC Public Health 13(Suppl. 3): S10.
Das JK, Kumar R, Salam RA, Freedman S, and Bhutta ZA (2013b) The effect of
antiemetics in childhood gastroenteritis. BMC Public Health 13(Suppl. 3): S9.
Das JK, Tripathi A, Ali A, Hassan A, Dojosoeandy C, and Bhutta ZA (2013c) Vaccines for
the prevention of diarrhea due to cholera, shigella, ETEC and rotavirus. BMC Public
Health 13(Suppl 3): S11.
Das JK, Lassi ZS, Salam RA, and Bhutta ZA (2013d) Effect of community based
interventions on childhood diarrhea and pneumonia: uptake of treatment modalities
and impact on mortality. BMC Public Health 13(Suppl 3): S29.
Duggan C, Fontaine O, Pierce NF, et al. (2004) Scientific rationale for a change in the
composition of oral rehydration solution. JAMA, the Journal of the American
Medical Association 291: 2628–2631.
Faure C (2013) Role of antidiarrhoeal drugs as adjunctive therapies for acute diarrhoea
in children. International Journal of Pediatrics 2013: 612403.
Field M (2003) Intestinal ion transport and the pathophysiology of diarrhea. The Journal
of Clinical Investigation 111: 931–943.
Gill CJ, Young M, Schroder K, et al. (2013) Bottlenecks, barriers, and solutions: results
from multicountry consultations focused on reduction of childhood pneumonia and
diarrhoea deaths. Lancet 381: 1487–1498.
Glass RI, Guttmacher AE, and Black RE (2012) Ending preventable child death in a
generation. JAMA 308: 141–142.
Guerrant RL, DeBoer MD, Moore SR, Scharf RJ, and Lima AA (2013) The impoverished
gut—A triple burden of diarrhoea, stunting and chronic disease. Nature Reviews.
Gastroenterology & Hepatology 10: 220–229.
King CK, Glass R, Bresee JS, and Duggan C (2003) Managing acute gastroenteritis
among children – oral rehydration, maintenance, and nutritional therapy. MMWR
52(RR16): 1–16.
Kotloff KL, Nataro JP, Blackwelder WC, et al. (2013) Burden and aetiology of diarrhoeal
disease in infants and young children in developing countries (the Global Enteric
Multicenter Study, GEMS): a prospective, case–control study. Lancet
382: 209–222.
Lamberti LM, Fischer Walker CL, Noiman A, Victora C, and Black RE (2011)
Breastfeeding and the risk for diarrhea morbidity and mortality. BMC Public Health
11(Suppl. 3): S15.
Lanata CF, Fischer-Walker CL, Olascoaga AC, Torres CX, Aryee MJ, and Black RE
(2013) Global causes of diarrheal disease mortality in children <5 years of age: a
systematic review. PLoS One 8: e72788.
Liu L, Johnson HL, Cousens S, et al. (2012) Global, regional, and national causes of
child mortality: an updated systematic analysis for 2010 with time trends since
2000. Lancet 379: 2151–2161.
Madhi SA, Cunliffe NA, Steele D, et al. (2010) Effect of human rotavirus vaccine on
severe diarrhea in African infants. The New England Journal of Medicine
362: 289–298.
Pierce NF, Banwell JG, Rupak DM, et al. (1968) Effect of intragastric glucose-electrolyte
infusion upon water and electrolyte balance in Asiatic cholera. Gastroenterology
55: 333–343.
Traa BS, Walker CL, Munos M, and Black RE (2010) Antibiotics for the treatment of
dysentery in children. International Journal of Epidemiology 39(Suppl 1): i70–i74.
Walker CL and Black RE (2010) Zinc for the treatment of diarrhoea: effect on diarrhoea
morbidity, mortality and incidence of future episodes. International Journal of
Epidemiology 39(Suppl. 1): i63–i69.
WHO (2014) Diarrhoea treatment guidelines: including new recommendations for the
use of ORS and zinc supplementation for clinic-based healthcare workers. Geneva:
WHO.
Wyllie R, Hyams J, and Kay M (2011) Diarrhea. Pediatric gastrointestinal and liver
disease, 4th ed. Philadelphia, PA: Elsevier Saunders.
Relevant Websites
http://www.cdc.gov/healthywater/global/diarrhea-burden.html – Centers for Disease
Control and Prevention.
http://www.cdc.gov/rotavirus/index.html?s_cid¼cs_281 – Centers for Disease Control
and Prevention.
http://www.who.int/topics/diarrhoea/en/ – World Health Organization.
http://www.who.int/maternal_child_adolescent/documents/fch_cah_06_1/en/ – World
Health Organization.
http://www.who.int/maternal_child_adolescent/topics/child/imci/en/ – World Health
Organization.
 Dietary Exposure Assessment
D Arcella, F Héraud, and M Gilsenan, European Food Safety Authority (EFSA), Parma, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Food contains, naturally, intentionally, or unintentionally, a
wide range of substances that may constitute a health hazard.
In this context, risk assessment provides the scientific foundation
upon which the risk analysis process is built. Risk assessments are
performed in a four-step process: hazard identification, hazard
characterization, exposure assessment, and risk characterization.
The first stage of a risk assessment, hazard identification, is
primarily a question of identifying the effects that are considered
as adverse, irrespective of the dose needed or the specific mech-
anism involved to elicit this effect. The next step, hazard
characterization, is centered on the quantification of these effects,
whereas dietary exposure assessment is the qualitative and/or
quantitative evaluation of the likely intake of biological,
chemical, or physical agents via food. Risk characterization is
the final stage of risk assessment that integrates information
from exposure assessment and hazard characterization into
advice suitable for use in decision-making.
Dietary exposure assessments are often performed accord-
ing to the fit-for-purpose principle with the consequence that
there is little harmonization across disciplines with each hav-
ing their own agreed international guidelines. In order to
calculate reliable estimates of the amounts ingested through
the diet of a specific chemical substance, three elements have to
be taken into account: (1) levels and fate of the chemical in
food, (2) food consumption patterns, and (3) integration of
these elements to determine exposure. In all three areas, the
limitations of the approaches currently used lead to uncer-
tainties that can cause either an over- or underestimation of
real intakes and thus of risk.
Presence and Levels of Chemicals in Food
A central aspect in the assessment of exposure is the concentra-
tion of the hazardous substance in food as consumed. Factors
influencing the variability of the chemical concentration levels
in ready-to-eat food products are of environmental origin or
related to agricultural and storage practices, food processing,
and cooking habits. Quantitative information on the level of a
chemical in food can be obtained through analytic determina-
tion. Inaccurate measurements can lead to results differing sig-
nificantly from the real concentration of the chemical in food
when the amount is determined analytically. Above all, food
sampling procedures for subsequent analysis can critically deter-
mine how close the measured value is to the real value. Aspects
such as climate, ripeness, and soil conditions are likely to influ-
ence the absence or presence of an environmental or agricultural
contaminant chemical, and this should be considered when
food products are sampled for analysis and methods are chosen.
For example, the presence of mycotoxin or fungicide residues in
crops and derived foods is related to climatic differences and
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00226-9
year-to-year variation. A further source of uncertainty is there-
fore linked to the possible effects of food processing, like wash-
ing, peeling, or cooking, on the chemical of interest, due to both
industrial and household preparations.
Most of the analytic determinations of a chemical in a food
that are used in dietary exposure assessments are carried out
within monitoring and surveillance programs. In these cases,
the concentration of a defined chemical in a food is commonly
assessed by means of random stratified sampling plans or
targeted sampling. The first tries to obtain a representative
picture of chemical levels present in food, whereas the second
is aimed at sampling those products expected to contain higher
levels in a cost-effective way. Targeted data are often collected
for enforcement purposes in response to specific problems.
They should be used with caution in dietary exposure
assessments, as they may not be representative of all foods
available for sale. However, clearly representative data are
often not available, and targeted data have to be used as an
alternative resulting in more conservative estimates of expo-
sure. A major limitation of using monitoring data is that often,
not all commodities entering the food chain are monitored.
This information might therefore not be sufficient to estimate
exposure from all possible dietary sources.
After representative samples are taken, the accuracy,
specificity, and sensitivity of the analytic methods used are
also important. Generally, analytic methods used for screening
purposes (e.g., for enforcement of maximum residue levels
(MRLs) or maximum levels) tend to be less precise and are
not suitable to detect residues at low levels. This is to a large
extent due to the fact that the objective of analytic methods
used for screening purposes is to separate the compliant from
the noncompliant results. For accurate dietary exposure assess-
ments, the limits of detection (LOD) and quantification
(LOQ) should be as low as technically possible. Different
recommendations exist for the handling of nondetected or
nonquantified values when assessing exposure. In the substi-
tution method, the most commonly used approach, three
different scenarios are considered: the lower bound obtained
by assigning a value of zero to all the samples reported as less
than the LOD or LOQ, the middle bound by assigning half of
the LOD or LOQ, and the upper bound by assigning the LOD
or LOQ as the sample result. Other factors influencing the final
result are the accuracy of the method, laboratory-to-laboratory
variation, repeatability, and recovery.
A complementary tool to the monitoring and surveillance
programs relies on the total diet studies (TDS), which are
designed to collect representative information on the chemical
occurrence in food as consumed by the population. In the
framework of the TDS, both sampling frame and analytic
techniques and performances are adapted in order to fit for
the purpose of dietary exposure assessment.
Besides these two main sources of data, chemical concen-
tration data can also originate from experimental trials,
373
374 Dietary Exposure Assessment
legislated maximum permitted levels (MPLs), MRLs, and usage
levels as reported by manufacturers. In the framework of pre-
regulatory exposure assessment (e.g., pesticides), the primary
sources of residue data in food are the supervised trials. These
data must be submitted by the applicants in support of the
registration of a pesticide. The trials are usually performed by a
manufacturer and simulate a maximum registered use sce-
nario. The trials are designed to determine the maximum res-
idue concentration that may be present in food or feed at the
earliest point at which these food commodities could enter
into commerce and are used to establish legally enforceable
residue limits.
When evaluating the presence and levels of chemicals in
food, it is valuable to distinguish between substances intention-
ally added, like additives, flavorings, or enzymes, from others,
such as contaminants. In the case of chemical substances inten-
tionally added to food at various processing steps, the amounts
that may be added are usually regulated by MPLs or MRLs. They
are defined in the food legislation for each food category to
ensure product safety. MPLs or MRLs can be used for a prelim-
inary assessment of exposure in order to identify those sub-
stances for which acceptable or tolerable intake limits may be
exceeded in a worst-case scenario. Data on the use patterns of
chemical substances intentionally added to food are difficult to
obtain. Information on the concentrations in brand foods is
considered to be commercially sensitive and is therefore not
widely available. Additives, like all other ingredients, must be
included on the product label in descending order by weight at
the time of manufacture. This may provide information on the
simple presence and in some cases on the amounts used, but the
collection of such data from labels requires major efforts.
Occurrence levels can also be obtained from the producers.
These levels range from the MPL to nothing at all. Manufac-
turers use the chemicals, for example, an antioxidant, at the
lowest effective levels in a given formulation; unnecessarily
high addition is usually avoided for cost reasons and prohib-
ited by the requirement to work according to ‘good
manufacturing practice.’ Proposed usage levels provided by
the industry are often the only available information in pre-
marketing situations.
Flavoring substances do not follow the same regulation as
food additives. Individual flavor components are usually not
specifically indicated on the label beyond the statement that
flavorings are used and maximum levels for these substances
were not established. Another category of substances that are
intentionally added during food processing is that of food
enzymes, which are primarily used as processing aids.
Food Consumption Patterns
When assessing exposure, three different sources of informa-
tion regarding food consumption can be used: food supply
data, data from household consumption surveys and from
individual dietary surveys, and data from duplicate diet and
biomarker studies from other sources of exposure data,
whereby these measures reflect both the consumption of
food and the concentration of the chemical in these foods.
Disappearance data provide gross annual estimates of the
national availability of food commodities. Food supply data are
calculated in food balance sheets (FBSs), which are accounts, on
a national level, of the annual production of food, changes in
stocks, imports and exports, agricultural use, and industrial use.
The result is an estimate of the average supply per head of the
population, irrespective of, for instance, age or gender. Food
supply data refer to food availability, which gives only a crude
(usually overestimated) impression of the potential average
consumption. Food losses prior to consumption, due to proces-
sing, spoilage, trimming, and waste, may not be adequately
accounted for. As a result of their long history, FBSs are espe-
cially used for assessing trends over time. International FBSs are
prepared and published by Eurostat and the FAO. They are
compiled in the so-called GEMS/Food cluster diets, which are
used by joint FAO/WHO expert meetings while performing
international risk assessment.
Food available at the household level may be estimated
from budget surveys and consumption surveys. The first type
of survey gives information on the purchases of food in terms
of expenditure and is used for economic policy. In household
consumption surveys, the amounts of food and drinks brought
into the household are also recorded, but not usually the meals
consumed out of home. In general, household surveys do not
provide information on how food is handled within the
household or on actual consumption by its members. Data
on the quantity of and/or expenditure on food maybe collected
by record keeping, by interviews, or by both methods.
The data from dietary surveys among individuals more
closely reflect actual consumption. To collect dietary intake
data at an individual level, several methods can be used. The
methods can be divided into two categories: recall and record
methods. Recall methods reflect past consumption, varying
from intake over the previous day (24 h recall) to usual food
intake (dietary history or food frequency). Record methods
collect information on current intake over one or more days.
The food frequency method consists of a questionnaire
containing a given list of foods, for which subjects are asked
to estimate the habitual frequency of consumption during a
specified period of time. The 24 h dietary recall is the most
common recall method used. During the interview, an indi-
vidual recalls actual food intake during the preceding day.
Food quantities are usually assessed by the use of household
measures, food models, or picture books. The interviewer
obtains information during a face-to-face interview or by tele-
phone. In the weighed record technique, the subject is taught
to weigh and record all foods and beverages immediately
before eating and to weigh and describe any leftovers. The
weighed method differs from the estimated record where sub-
jects do not use a scale but estimate the portion sizes using
picture books or household measures. The record is usually
done over 3–7 days.
The duplicate diet method differs from the other methods
in that the intake estimation does not rely on chemical data
from other data sources. The concentration is measured by the
chemical analysis of the duplicate diet collected.
The quality of the results of a food chemical exposure
assessment will depend largely on the quality of the food
consumption data used in the assessment. Collecting food
consumption data in surveys and in duplicate diet samples
for exposure assessment can alter the diet of study participants,
resulting in a bias in intake estimate. Intake may also be altered

if participants change their diet due to fatigue or loss of inter-
est. It is important to highlight that many dietary surveys
reported energy intakes consistently lower than would be
expected from the estimated basal metabolic expenditure and
physical activity. This problem of underreporting of energy
intakes is widespread and is one of the major biases in expo-
sure assessments based on dietary surveys.
Another important issue is the food coding system. In
dietary assessment, food products have to be identified and
thereafter categorized in line with the purposes of the study.
For this, national and international systems exist, but with
regard to exposure assessment, unconventional categorization
systems are often needed. An important limitation is that
analyses of residues in cereals, fruits, and vegetables are mainly
conducted in raw agricultural commodities (RACs) including
peel and nonedible parts. Either processed or prepared foods
are not monitored or the number of samples is very small. This
is due to the fact that in the legislation, the limits of residues are
mainly set for RACs, and the purpose of monitoring is usually
to assess compliance, rather than exposure.
In order to estimate acute exposure, it is sufficient to record
food intake for only one day, including details of the foods
consumed per meal and details on the time of each eating
occasion. However, food consumption data for several days
per subject are required in order to assess chronic exposure.
Information on food consumption for a few days may not
actually represent usual intake. However, statistical techniques
can be used to estimate usual intake, provided that two or more
repeated and independent days are available, although consec-
utive days cannot be considered as independent days.
Methods Used in Dietary Exposure Assessment
Up to 15 years ago, assessment of exposure was mainly based
on the estimates obtained by combining MPLs with mean food
consumption data for all chemicals. However, mean consump-
tion is not informative in relation to subjects consuming high
levels of one or more food categories (‘high consumers’) who
are more likely to ingest hazardous chemicals in excessive
quantities. The methodologies now adopted to assess exposure
from diet take into special consideration nonaverage
individuals and in particular those who consume relatively
large quantities of foods containing higher concentrations of
substances that may potentially lead to a health risk. The
definition of high-level consumers varies, it is normally the
90th, 95th, 97.5th, or 99th percentile of the distribution of
individual intake values. A high percentile, rather than the
maximum value, is usually chosen because maximum intakes
are unlikely to be maintained over long periods of time and
hence are not representative of high-level intakes in relation to
chronic exposure. The reliability of estimates depends on the
sample size available and should be considered when choosing
the high percentile value to retain for the exposure assessment.
It is also important to highlight that the definition of high-level
consumers is a major issue since, in practice, it defines the
proportion of the population that would have to exceed the
‘at-risk dose’ before risk mitigation measures are to be consid-
ered to reduce exposure levels. This is therefore both a scientific
and a political/ethical question.
Dietary Exposure Assessment 375
Ideally, dietary exposure to chemical substances should be
assessed by combining data on the concentration in all food
products with data on their consumption. Unfortunately, with
the exception of duplicate diet studies, exposure assessments
are not performed on the basis of consumption, occurrence,
and concentration data related to the same individuals within a
population. Therefore, assessments of exposure to dietary com-
ponents will usually require some degree of modeling to
attempt to create a representation of the real-life exposure
situation. There are a number of different models for combin-
ing or integrating the consumption data with the residue/con-
centration data. The method chosen usually depends on a
number of factors, including the purpose of the assessment
(target chemical, population group, and degree of accuracy
required) and the availability of data. In general, it is consid-
ered neither cost-effective nor necessary to collect detailed food
consumption and chemical concentration data for every haz-
ardous substance. A stepwise procedure is commonly used to
minimize estimation costs and focus resources on the most
important issues. The stepwise approach to the dietary exposure
assessment of food chemicals is such that as the accuracy of
dietary exposure assessments increases, the cost of collecting
adequate data and resources needed to undertake the assess-
ments also increases. The aim of the stepwise procedure is to
target dietary exposure estimates to chemicals that might be of
health concern for ‘average consumers’ or individuals belonging
to certain at-risk groups. If the estimated exposure to a given
pesticide residue, food additive, veterinary drug residue, and
contaminant exceeds its safety limit (e.g., acceptable daily intake
(ADI)), a more accurate method of dietary exposure assessment
should be applied. Exposure is therefore first assessed by using
methods following a deterministic approach based on conserva-
tive assumptions. Especially at this stage, the methodologies
adopted to assess exposure from the diet take into special con-
sideration ‘high consumers.’ However, for high consumers in
particular, food consumption data are not commonly available
at the individual level. Consequently, highly conservative
methods are used. It is important to remind that these methods
are not suitable for predicting actual exposure since they are
designed to cover the worst-case scenario. Nevertheless, they
are useful and inexpensive screening tools for identifying those
substances for which safety limits of intake may be exceeded.
The so-called budget method is the most known screening
method; it is above all used in the field of food additives. This
method uses theoretical food consumption data and MPLs for
additives. The budget method is based on the physiological
upper limit to the amount of food and drinks that can be
consumed each day and thus of food additives. For beverages,
this is assumed to be 100 ml, and for solids, 25 g kg 1 b.w. per
day. A further assumption is that only a certain proportion of
the diet is likely to contain food additives. The default propor-
tion of beverages and solid food that could contain the additive
is typically assumed to be 25% but varies depending on the
additive in question and the population under consideration.
A model diet is, for example, used for the assessment of expo-
sure to feed additives and veterinary drug residues. It assumes
the daily consumption of 300 g of muscle (from mammals,
birds, and/or fish), 100 g of liver (from mammals and/or
birds), 50 or 90 g of fat (from mammals or birds, respectively),
50 or 10 g of kidney (from mammals or birds, respectively),
376 Dietary Exposure Assessment
1500 g of milk, and 100 g of eggs. Screening methods are also
used for assessing acute exposure; an example is the
international estimated short-term intake, which is used in
the case of pesticide residues. This method requires data on
the consumption of large portions (usually the 97.5th percen-
tile from the single-day consumption data among consumers)
together with typical unit weights of the edible part of the
commodities and the body weights of the population associ-
ated with the food consumption data. The baseline assump-
tion is that a consumer may eat a large portion (high-level
consumer at the top end of the distribution curve among
consumers only) of a food that may contain a high residue
level, as derived from supervised field trials. The acute assess-
ment is conducted for each commodity separately as it is
considered unlikely that a consumer will eat two or more
different commodities with a large portion size within a short
period of time and that those commodities have the highest
level of the same pesticide.
When screening methods cannot rule out a safety concern,
more refined methods should be applied. With access to food
consumption survey information, actual mean and high per-
centile consumption can be used as inputs into an exposure
model. Such calculations can be further refined by splitting the
population into age groups and by using the average body
weight within each group to estimate the mean and high
percentile consumption per kg body weight. A further refine-
ment is possible when food consumption is available at the
individual level. In such cases, chemical concentrations and
food consumption can be directly matched for each subject
and the respective food or food category and a distribution of
total exposure estimates produced, where any percentile of
interest can be calculated. Dietary exposure estimates are in
the majority of cases produced by considering the consump-
tion of all foods and all subjects involved in the dietary surveys,
but estimates for ‘consumers only’ are also calculated under
specific circumstances.
The method applied in any dietary exposure assessment
should be clearly stated and reproducible. Information about
the model and data sources used, assumptions, limitations,
and uncertainties should also be documented. When assessing
the exposure, the contribution of variability (heterogeneity)
and true uncertainty (lack of knowledge) should be as far as
possible distinguished. Although it may be challenging to
identify all the uncertainties and to distinguish them from
the real variability, each scientific output should describe the
types of uncertainties encountered and considered during the
different risk assessment steps and indicate their relative
importance and influence on the assessment outcome.
Over the past years, to get a more realistic view of exposure to
hazardous substances, risk managers are becoming more inter-
ested in probabilistic modeling. In contrast, probabilistic
approaches are typically more resource-intensive than determin-
istic approaches, but they permit the characterization of the
variability and uncertainty that may exist in such exposure esti-
mates and thus facilitate more meaningful and realistic assess-
ments. The results, however, are only as good as the input data,
algorithms, and assumptions. The critical aspects of the food
consumption and chemical concentration data used to assess
deterministic estimates must therefore be taken into account
also when using a probabilistic approach. Moreover, the impact
of the assumptions should always be tested carefully and the
results should be fully documented. A modeling tool must be
structured so that all algorithms and assumptions inherent to
the model can be identified and validated. Sensitivity analyses
should be used to set priorities for risk reduction measures and
to define the main sources of uncertainty in order to plan further
studies and improve the exposure estimates.
In addition, more and more concerns stand in the risk
resulting from the combined exposure to multiple chemicals.
One challenge is to identify which chemical mixtures the pop-
ulation are exposed. Developments in the research field are
under way to address this challenge. Once the mixtures of
interest are defined, methodologies to assess combined expo-
sure in a risk assessment perspective do not differ greatly from
the ones previously described for a single chemical. The main
issue, especially when assessing acute exposure, is to address
the co-occurrence of the chemicals composing the mixture in
foods. Future developments in this direction mainly require
adaptation of the occurrence data collection for multiple che-
micals in same food samples.
Finally, there is also an increasing interest for the potential
use of biomarkers for assessing exposure to food chemicals. It
needs to be recognized that this approach takes account of
exposure from all sources, including nondietary sources. It is
thus important to understand to what extent such other
sources exist and are likely to be relevant, especially if attempt-
ing to compare the results of such measurements with dietary
exposure estimates assessed by means of conservative models.
A number of methods for specific individual food chemicals
are currently being developed, and it seems that this trend will
continue.
See also: Dietary Surveys: National Food Intake; Food Classification
and Description; Food Composition Databases; Risk Assessment of
Foods and Chemicals in Foods.
Further Reading
Dodd KW, Guenther PM, Freedman LS, et al. (2006) Statistical methods for estimating
usual intake of nutrients and foods: a review of the theory. Journal of the American
Dietetic Association 106: 1640–1650.
EFSA (European Food Safety Authority) (2008). Scientific Colloquium Report -
European Food Consumption Database – Current and medium to long term
strategies. Brussels, Belgium, 28–29 April 2005. Available at http://www.efsa.
europa.eu/en/events/event/colloque081204-m.pdf.
EFSA (European Food Safety Authority) (2009) General principles for the collection of
national food consumption data in the view of a pan-European dietary survey. EFSA
Journal 7(12): 1435.
EFSA (European Food Safety Authority) (2011) Use of the EFSA comprehensive
European food consumption database in exposure assessment. EFSA Journal 9(3):
2097.
EFSA (European Food Safety Authority) (2013) International framework dealing with
human risk assessment of combined exposure to multiple chemicals. EFSA Journal
11(7): 3313. http://dx.doi.org/10.2903/j.efsa.2013.3313. [69 pp.]. Available online:
www.efsa.europa.eu/efsajournal.
EFSA (European Food Safety Authority), FAO (Food and Agriculture Organization of the
United Nations), and WHO (World Health Organization) (2011) Towards a
harmonised Total Diet Study approach: a guidance document. EFSA Journal 9(11):
2450.
Kroes R, Muller D, Lambe J, et al. (2002) Assessment of intake from the diet. Food and
Chemical Toxicology 40(2–3): 327–385.

Lawrie CA and Rees NMA (1996) The approach adopted in the UK for the
estimation of the intake of food additives. Food Additives and Contaminants 13(4):
411–416.
Lowik MRH (1996) Possible use of food consumption surveys to estimate exposure to
additives. Food Additives and Contaminants 13(4): 427–442.
Moy G and Vannoort R (2013) Total diet studies. New York: Springer, 548 pp.
WHO (World Health Organization) (1997). Guidelines for predicting dietary intake of
pesticide residues (revised). Prepared by the Global Environment Monitoring
System – food contamination monitoring and assessment programme (GEMS/
Food) in collaboration with the Codex Committee on Pesticide Residues, WHO/FSF/
FOS/97.7. Geneva: WHO.
Dietary Exposure Assessment 377
WHO (World Health Organization) (2000) Methodology for exposure assessment of
contaminants and toxins in food. Geneva: WHO. Available at http://www.who.int/
foodsafety/publications/chem/en/exposure_june2000.pdf.
WHO (World Health Organization) (2009) Dietary exposure assessment of chemicals in
food. (Chapter 6)Principles and methods for the risk assessment of chemicals in
food. Environmental Health Criteria240: Geneva: WHO. FAO/WHO. International
Programme on Chemical Safety (IPCS), http://www.who.int/ipcs/food/principles/
en/index.html.
WHO/FAO (1997). Food consumption and exposure assessment of chemicals. Report of
a FAO/WHO Consultation, Geneva, Switzerland, 10–14 February, WHO/FSF/FOS/
97.5, 69 pp.
 Dietary Fiber: Bran
A Kamal-Eldin, United Arab Emirates University (UAEU), Al-Ain, United Arab Emirates
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Anatomically, a cereal grain is a one-seeded fruit, called caryop-
sis, in which the fruit coat (or pericarp) is fused with the seed coat
(cuticle or testa) to form what we refer to as the cereal bran. The
outermost layer of the cereal kernel is thus the pericarp or the
ripened ovary wall, which lacks cytoplasm and has lignified walls
and is dead at harvest ripeness. The pericarp is composed of the
outer epidermis, hypodermis, parenchyma, intermediate cells,
cross cells, and tube cells. The seed coat (arising from the integ-
uments) and an underneath pigment strand cover the seed
completely and control water migrations between the enclosed
seed and the pericarp cells. Beneath the seed coat is the aleurone
layer, or the outermost layer of the endosperm tissue, which is
the only endosperm tissue that will be alive at maturity. Unlike
the starchy endosperm cells, the aleurone layer cells have large
prominent nuclei that are not crushed during milling. The bran
(Figure 1) represents these outer parts of the grains including the
pericarp, the testa, and the aleurone layer. These are all fibrous
layers containing cellulose, hemicellulose, lignified materials,
and a wide range of bioactive compounds. Commercial bran
preparations contain variable amounts of the starchy endosperm
and germ depending on the variety of cereal and the milling
process (Figure 2). The amount of endosperm in the commercial
bran will depend on the shape of the kernel and the extraction
rate with, for example, rye being more irregular in shape than
wheat and, therefore, more difficult to mill to prepare ‘pure’ bran
fractions. The relative distribution of bran, endosperm, and germ
in selected cereal kernels is given in Table 1.
Composition and Properties
Brans from different cereals are different in the shape and
hardness of their particles, chemical composition, color, flavor,
and other sensory properties. The color of the cereal bran may
vary from white, yellow, green, blue, black, and purple red
depending on the variety and the type and amounts of pig-
ments as well as the inclusion of starchy endosperm. For
example, oat bran is lighter in color than rye, wheat, rice, and
barley brans. Wheat bran is more yellow in color, barley bran
followed by oat bran has more redness, while rye bran is
greener in color. All cereal brans have a pleasant flavor with
the exception of rye bran, which imparts an unpleasant feeling
of grittiness, roughness, and lack of smoothness and
creaminess.
Table 2 presents the major and most important constitu-
ents of cereal brans. The major chemical component in cereal
brans is dietary fiber, which is defined by the American Asso-
ciation of Cereal Chemists as consisting of “the remnants of
edible plant cells, polysaccharides, lignin and associated sub-
stances resistant to (hydrolysis) digestion by the alimentary
378 Encyclopedia of Food and Health
enzymes of humans.” Dietary fibers in cereal brans include
water-insoluble and water-soluble types, where wheat and rye
brans contain 1–5% soluble fiber, rice bran contains about
10% soluble fiber, while oats and barley contain about
30–40% soluble fibers. The insoluble dietary fiber is composed
of cellulose, hemicelluloses, and small amounts of klason
lignin. The hemicellulosic fraction is mainly composed of
arabinoxylan (or pentosan) structured as b-(1 ! 4)-
xylopyranosyl backbone with a-L-arabinofuranosyl residues
linked to the main chain as (1 ! 2) or (1 ! 3) side units. This
hemicellulose fraction also contains side units of D-glucuronic
acid or oligosaccharide side chains of galactose or xylose, and
some arabinofuranosyl side units contain ferulic acid. Because
of the complex structure of this fraction, it is often referred to as
a heteroxylan. It is believed that the heteroxylan fraction acts as
a fill in the spaces between the cellulosic microfibrils and forms
a cross-linked structure with other cell wall components, via di-
and triferulate bridges, that act as a cement solidifying the cell
wall structure and enhancing its insolubility. Klason lignin is a
complex three-dimensional hydrophobic polymeric network
of aromatic p-hydroxyphenyl, guaiacyl, and syringyl units
resistant to chemical and enzymatic degradation. Soluble die-
tary fiber is composed partly of soluble arabinoxylan, fructan,
and mainly highly soluble and viscous b-glucan, which is a
linear polysaccharide that consists only of b-D-glucopyranosyl
units joined by either (1 ! 3)- or (1 ! 4)-b-D-linkages, hence
the name mixed-linkage (1 ! 3), (1 ! 4)-b-D-glucan.
Cereal brans contain considerable amounts of minerals and
vitamins and contribute considerably to the recommended
daily intake (RDI) of some of these nutrients (Table 3). For
example, wheat bran is a good source of the B vitamins,
including niacin (19% of RDI/100 g), vitamin B6 (18% of
RDI/100 g), riboflavin and thiamine (10% of RDI/100 g),
and folates (B9) and pantothenic acid (B5) (6% of RDI/
100 g), and it provides small amounts of the lipid-soluble
vitamins E and K. Wheat bran is also a rich source of eight
important minerals being exceptionally high in manganese
(161% of RDI/100 g), and very high in magnesium (43% of
RDI/100 g), selenium (31% of RDI/100 g), and phosphorus
(28% of RDI/100 g), and still a good source of iron (16% of
RDI/100 g), zinc and copper (14% of RDI/100 g), and potas-
sium (9% of RDI/100 g). Rice bran has higher values and oat
bran significantly less values compared to wheat bran.
Besides fiber, cereal brans are rich sources of a wide range of
bioactive secondary metabolites including phytic acid, tocoph-
erols and tocotrienols, carotenoids, g-aminobutyric acid, octa-
cosanol, squalene, unsaturated fatty acids, phytosterols, and
phenolic compounds. The phenolic components of cereal
brans have received special interest because of their antioxidant
properties. These phenolic compounds (including free
aglycones, soluble conjugates, and polymeric and insoluble-
bound forms) differ widely in their structures and water solu-
bility. The free phenolic compounds are mainly present within
http://dx.doi.org/10.1016/B978-0-12-384947-2.00227-0
 Dietary Fiber: Bran 379

Wheat Rye
Pericarp

Pigment strand
Nucellar epidermis and seed coat
Aleurone layer

Subaleurone region of endosperm

100 µm
Outer Epidermis

100 µm
Pericarp Hypodermis

Bran
Inner Cross cells Oats Barley
Pericarp Tube cells
Testa (Seed coat)
Pigmented layer
Aleurone layer

Sub-aleurone layer

100 µm
100 µm

Starchy endosperm

Figure 1 Scanning electron micrographs showing the outer parts of wheat, rye, oats, and barley grains (modified from Dornez, E., Holopainen, U.,
Cuyvers, S., et al. (2011). Study of grain cell wall structures by microscopic analysis with four different staining techniques. Journal of Cereal Science
54, 363–373, with permission from Elsevier) and a schematic demonstration of the component cells of a cereal bran composed of the fruit coat
(the pericarp) and the seed coat (testa). Technical bran fractions obtained by milling usually contain a great part of the aleurone layer and some amount
of the subaleurone layer and the starchy endosperm depending on the cereal grain and the milling process.

Figure 2 Microstructures of unstained wheat bran (left) and rye bran (right) showing that the rye bran contained higher amount of the starchy
endosperm due to the difficulty in separating this anatomical fraction during milling. Reproduced from Kamal-Eldin, A., Laerke, H. N., Bach Knudsen, K.
E., et al. (2009). Physical, microscopic and chemical characterisation of industrial rye and wheat brans from the Nordic countries. Food & Nutrition
Research 53, 1–11.

the plant cell vacuoles, the soluble esters or conjugates are
esterified to sugars and other low-molecular-weight compo-
nents, while the polymeric proanthocyanidins and the
insoluble-bound forms are covalently bound to cell wall struc-
tural components such as cellulose, hemicellulose, lignin,
pectin, and rod-shaped structural proteins. Analysis of 51
Canadian wheat cultivars revealed a variation in the contents
of phenolic compounds (expressed in mg gallic acid equiva-
lents/g of bran) with the contents of free phenolic compounds
extractable with 80% ethanol ranging 850–1750 and the
content of bound phenols ranging 2300–5400 with the total
phenolic content ranging from 3400 to 6700 and averaging
c.5200. The main phenolic compounds in nonpigmented
cereal brans are the phenolic acids ferulic and p-coumaric
acids, which are found mainly linked to dietary fiber. An
indicative detailed high-performance liquid chromatography
analysis is available for rice bran (Table 4), and it shows that
pigmented brans of cereals contain much higher levels of
phenolic compounds imparted by flavonoids, anthocyanins,
and proanthocyanidin pigments. Consequently, brans from
380 Dietary Fiber: Bran

pigmented cereal cultivars have higher antioxidant activities
compared with brans from nonpigmented cereals.
A group of important bioactive compounds in cereal brans
include sterols, steryl esters, and steryl ferulates (the steryl fer-
ulates in rice bran are referred to as oryzanols), which are
particularly interesting because of their cholesterol-lowering
properties. Cereal brans are also characterized by their contents
of tocotrienols and policosanol waxes (mixture of long chain
alcohols) believed to have anticarcinogenic effects. Cereal brans
also contain considerable amounts of phytic acid, which is not
only known for its antioxidant properties but also considered
an antinutritional compound as will be discussed later. Wheat
and rye kernels are characterized by their contents of alkylre-
sorcinols, which can be used as specific intake biomarkers for
whole-grain and bran-rich diets including these cereals.
could come from cereal products and the other half from fruits
and vegetables. Soluble and insoluble dietary fibers are appre-
ciated for several health benefits including weight and diabetes
management, lowering of blood cholesterol, regulation of
blood glucose levels, modulation of the immune system, and
anti-inflammatory effects leading to lower risk of heart disease
and cancer. Insoluble dietary fibers (cellulose, some arabinox-
ylans, and lignins) are effective in enhancing the feeling of
fullness, increasing stool size, and helping reduce constipation
and hemorrhoids. Soluble fibers (some arabinoxylan and
b-glucan) form a gel in the intestine and increase the water
content of the stool. Cereal dietary fibers also improve micro-
bial fermentation, promote the growth of beneficial bacteria,
Table 3 Minerals and vitamins in the three commercially available
cereal brans

Component Wheat bran Oat bran Rice bran

Health Effects
Minerals (mg/100 g FW)
Cereal brans provide an invaluable source of soluble and Calcium 73 58 57
insoluble dietary fiber for human nutrition. It is believed that Iron 10.6 5.4 18.5
fiber intake of 30–40 g day1 is desirable and that half of it Magnesium 611 235 781
Phosphorus 1013 734 1677
Potassium 1182 566 1485
Table 1 The relative proportions of bran, endosperm, and germ in Sodium 2 4 5
selected cereal grains Zinc 7.3 3.1 6.0
Vitamins (mg/100 g FW)
Relative percentage (wt%)
Thiamine 523 1170 2753
Cereal grain Pericarp Endosperm Germ Riboflavin 577 220 284
Niacin 13 578 934 33 995
Wheat (Triticum aestivum) 4–6 91–93 2–4 Vitamin B6 1303 165 4070
Rice (Oryza sativa) 2–3 95–96 2–3 Folate (vitamin B9) 79 52 63
Maize (Zea maize) 5–6 80–85 10–15 Vitamin E 1490 1000 4920
Sorghum (Sorghum bicolor) 6–7 83–85 9–10 Vitamin K 1.9 3.2 1.9
Pearl millet (Pennisetum glaucutn) 7–9 75 15–18
Source: USDA National Nutrients Database (http://ndb.nal.usda.gov/).

Table 2 Important chemical components in samples of cereal brans (g/100 g DM)

Cereal bran Wheat Rye Oats Barley Rice

Proximate composition
Crude protein 16.9 16.5 17.4 13.8 13.7
Ash 5.5 4.6 1.7 4.9 7.8
Fat 5.6 4.3 12.3 4.9 22.4
Dietary fiber 40.0 42.1 17.3 26.3 45.2
Starch, soluble carbohydrates, and other minor components 32.0 32.5 46.6 50.1 10.8
Dietary fiber components
Arabinoxylan 22.4 21.5 6.4 10.3 9.9
b-Glucan 2.2 4.3 9.0 7.8 trace
Cellulose 9.3 5.6 2.2 Unknown 19.0
Klason lignin 3.3 4.1 3.5 Unknown Unknown
Fructan 2.8 6.6 – Unknown Unknown
Antinutritional factors
Phytic acid 4.2 2.7 2.7 4.0 4.3
Polyphenols 0.32 Unknown 0.05 0.65 0.58
Tannins 0.30 Unknown 0.62 0.34 0.07
Oxalates 0.04 Unknown 0.03 0.03 0.04
Saponins 0.26 Unknown 0.20 0.32 0.30

Source: Kamal-Eldin, A., Laerke, H. N., Bach Knudsen, K. E., et al. (2009). Physical, microscopic and chemical characterisation of industrial rye and wheat brans from the Nordic
countries. Food & Nutrition Research 53, 1–11; Kaur, S., Sharma, S., and Nagi, H.P.S. (2011). Functional properties and anti-nutritional factors in cereal bran. Australian Journal of
Food and Agro-Industry 4, 122–131.
 Dietary Fiber: Bran 381

Table 4 Classification and contents of phenolic compound in rice bran (mg/100 g DM)

Pigmented rice bran Nonpigmented rice bran

Phenolic compound Soluble Insoluble Total Soluble Insoluble Total

Phenolic acids
Gallic acid 1.18–8.3 1.69–4.06 5.4 0.05–0.48 0.20–2.1 1.35
Protocatechuic acid 1.98–9.7 6.04–24.5 16.2 0.33–1.07 0.04–0.72 0.94
p-Hydroxybenzoic acid 1.25–26.4 4.0–52.5 31.2 0.25–1.39 0.06–6.73 3.71
Vanillic acid 1.09–16.6 7.2 54.8 0.28–1.64 0.02–0.34 0.86
Syringic acid 0.07–5.59 – – 0.07–0.58 0.08–0.21 0.36
Chlorogenic acid 9.79–15.4 – – 0.11–1.55 0.21 1.05
Caffeic acid 2.78–25.3 11.1 25.6 0.14–0.79 0.04–0.15 0.45
p-Coumaric acid 1.64–6.2 16.8–144 46.2 0.10–1.57 8.1–74.2 42.2
Sinapic acid 0.65–5.0 15.0–25.9 22.8 0.04–0.60 0.20–3.47 2.06
Ferulic acid 2.95–19.1 15.3–94.8 96.6 1.37–8.69 11.9–225 120.3
Cinnamic acid – – – 0.02 0.07 0.09
Ellagic acid 5.5–12.2 – – 4.2 – 4.2
Total 298.8 177.5
Flavonoids
Luteolin 18.4 – 18.4 – – –
Apigenin 7.8 – 7.8 0.04 0.32 0.4
Tricin 11.9–193 – 101 0.50–4.86 0.65–2.85 3.3
Quercetin 1.4–6.3 – 3.4
Isorhamnetin 0.04–0.67 – 0.2
Total 130.8 3.7
Anthocyanins
Peonidin-3-O-glucoside 11.4–534 – 124 0.04–2.6 – 0.96
Cyanidin-3-O-glucoside 9.1–2640 – 700 7.36 – 7.36
Cyanidin-3-O-galactoside 2.93–50 – 28.3 – – –
Cyanidin-3-O-rutinoside 3.17–96.6 – 55.5 6.19 – 6.2
Total 908 14.5
Proanthocyanidin residues
Catechin 5.8–48.7 – 20.9 0.55–2.35 – 1.4
Epicatechin 46.5 – 46.5 – – –
Total 67.4 1.4
Grand total phenolics 1405 197

Source: Goufo, P., and Trindade, H. (2014). Rice antioxidants: phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, g-oryzanol, and phytic acid.
Food Science and Nutrition 2, 75–104.

and increase the production of short-chain fatty acids in the
large intestine. Scientists are still in search for the putative
mechanisms behind the beneficial health benefits of cereal
fibers. In a metabolomic study, the intake of a high-fiber rye
diet was found to increase the concentrations of the ribose
metabolites ribitol and ribonic acid and the tryptophan metab-
olite indole acetic acid, and it was speculated that high-fiber
diets increase the synthesis of serotonin and reduced hunger. A
high intake of bread of whole-grain rye with added rye bran by
males in the early stage of prostate cancer was found to elevate
plasma betaine and dimethylglycine and 3-hydroxybutyric
acid and acetone, which is explained by a shift in energy
metabolism from anabolic to catabolic state. The increase in
betaine explains the reduction of homocysteine and the
increase of dimethylglycine and methionine.
The intake of high levels of cereal brans exposes individuals
to higher intake of some antinutritional factors. The antinutri-
ent phytic acid is known to act as a powerful chelating agent
that forms insoluble complexes with divalent cations such as
calcium, magnesium, zinc, and iron, thereby reducing their
bioavailability. Phenolic compounds also chelate metals and
reduce their bioavailability. As shown in Table 4, the content
of polyphenols varies widely in wheat, rice, barley, and oat
brans. Rice bran contains higher levels of phenolic compounds
and oxalates followed by wheat, barley, and oat bran.
Food Applications
Brans are major side streams generated in the milling of cereal
grains by the industry. Brans of wheat, oats, and rice are widely
commercially available and are well characterized with respect
to their composition and properties, while brans of other
cereal grains are much less known. Cereal brans can be added
to a number of foods to increase their fiber content and to
improve their value as functional foods (the US Institute of
Medicine, 2002 recommended that the intake of total dietary
fiber for children and adults should be above 25 g day1).
Cereal brans can be used as ingredients in a number of healthy
food products including breads, cookies, crackers, snack foods,
beverages, sauces, dairy products, imitation cheeses, meats and
meat analogues, and many other products. One application
382 Dietary Fiber: Bran
area for wheat bran is its incorporation in pasta manufacturing
as it was shown that the addition of 25–30% of common
wheat bran to a commercial pasta preparation increased its
dietary fiber to a level similar to that obtained by using
whole-grain durum flour and provided pasta with very good
sensory quality and lower losses of dry matter and higher
resistance to overcooking compared with whole-grain durum
pasta. Arabinoxylans, from wheat and rye brans, may be added
as thickening agents in gluten-free bread, and b-glucan may be
added to contribute viscosity to, for example, yogurts and other
dairy and nondairy products. The sensory and nutritional
value of cereal brans may be improved by fermentation,
which adds taste and flavor and removes phytic acid and
some of the antinutritional factors. An important product
from rice bran is rice bran oil, which is high in g-oryzanols
and tocotrienols and well known for its cholesterol-lowering
properties.
In this article, the main characteristics and properties of
cereal brans have been discussed. Cereal brans can be exploited
as ingredients in a number of functional foods aiming for
control of energy intake and health benefits against diabetes,
coronary heart disease, and some cancers. The possibilities for
applications in food products are wide and unlimited and offer
the industry new opportunities for the production of innova-
tive and healthy food products having high contents of fiber
and associated bioactive compounds.
See also: Barley; Carbohydrate: Digestion, Absorption and
Metabolism; Cellulose; Cereals: Dietary Importance; Cereals: Types
and Composition; Dietary Fiber: Determination; Oats; Rice: Types and
Composition; Wheat: Grain Structure of Wheat and Wheat-based
Products.
Further Reading
Brij Verma PH and Chibbar RN (2008) Phenolic content and antioxidant properties of
bran in 51 wheat cultivars. Cereal Chemistry 85: 544–549.
Cordain L (1999) Cereal grains: humanity’s double-edged sword. In: Simopoulos AP
(ed.) Evolutionary aspects of nutrition and health: diet, exercise, genetics and
chronic disease. World Review of Nutrition and Dietetics, vol. 84, pp. 19–73. Basel:
Karger.
Delcour JA and Hoseney RC (2010) Structure of cereals. Chapter 1, In: Principles
of cereal science and technology, 3rd ed., pp. 1–22. St. Paul, MN: AACC
International.
Devries JW, Prosky L, Li B, and Cho S (1999) A Historical perspective on defining
dietary fiber. Cereal Foods World 44: 367–369.
Dornez E, Holopainen U, Cuyvers S, et al. (2011) Study of grain cell wall structures by
microscopic analysis with four different staining techniques. Journal of Cereal
Science 54: 363–373.
Friedman M (2013) Rice brans, rice bran oils, and rice hulls: composition, food and
industrial uses, and bioactivities in humans, animals, and cells. Journal of
Agricultural and Food Chemistry 61: 10626–10641.
Goufo P and Trindade H (2014) Rice antioxidants: phenolic acids, flavonoids,
anthocyanins, proanthocyanidins, tocopherols, tocotrienols, g-oryzanol, and phytic
acid. Food Science and Nutrition 2: 75–104.
Harris PJ, Chavan RR, and Ferguson LR (2005) Production and characterization of two
wheat-bran fractions: an aleurone-rich and a pericarp-rich fraction. Molecular
Nutrition & Food Research 49: 536–545.
Howarth NC, Saltzman E, and Roberts SB (2001) Dietary fiber and weight regulation.
Nutrition Reviews 59: 129–139.
Institute of Medicine (2002) Dietary, functional, and total fiber. In: Dietary reference
intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and
amino acids, pp. 265–334. Washington, DC: National Academies Press.
Kamal-Eldin A, Laerke HN, Bach Knudsen KE, et al. (2009) Physical, microscopic and
chemical characterisation of industrial rye and wheat brans from the Nordic
countries. Food & Nutrition Research 53: 1–11.
Kaur S, Sharma S, and Nagi HPS (2011) Functional properties and anti-nutritional
factors in cereal bran. Australian Journal of Food and Agro-Industry 4: 122–131.
Maffei HVL (2014) Dietary fiber and wheat bran in childhood constipation and health.
In: Watson RR, Preedy V, and Zibadi S (eds.) Wheat and rice in disease prevention
and health, 239–277.
Saunders RM (1978) Wheat bran: composition and digestibility. Chapter 2,
In: Spiller GA (ed.) Topics in dietary fiber research, pp. 43–58. New York: Plenum
Press.
Sharif MK, Butt MS, Anjum FM, and Khan SH (2014) Rice bran: a
novel functional ingredient. Critical Reviews in Food Science and Nutrition
54: 807–816.
Smith CE and Tucker KL (2011) Health benefits of cereal fibre: a review of clinical trials.
Nutrition Research Reviews 24: 118–131.
Waniska RD and Rooney LW (2000) Structure and chemistry of the sorghum caryopsis.
In: Smith CW and Frederiksen RA (eds.) Sorghum: origin, history, technology, and
production, pp. 649–688. New York: Wiley.
Relevant Websites
http://www.aaccnet.org/initiatives/definitions/Pages/OatBran.aspx – Association of
Analytical Cereal Chemists.
http://www.aaccnet.org/initiatives/definitions/Pages/DietaryFiber.aspx – Association of
Analytical Cereal Chemists.
 Dietary Fiber: Determination
R Mongeau and SPJ Brooks, 3W Banting Research Centre, Ottawa, QC, Canada
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from the Encyclopedia of Food Sciences and Nutrition, volume 3, pp. 1823–1832, ã 2003, Elsevier Science Ltd.,
with an updated Bibliography section supplied by the Editor.
Introduction
This article presents a brief overview and comparison of
commonly utilized methods for quantifying dietary fiber in
food. There are, fundamentally, only two types of methods.
Both rely on the removal of nonfiber components by a combi-
nation of physical, chemical, and enzymatic procedures prior to
measurement of dietary fiber. The gravimetric methods mea-
sure dietary fiber by weighing the residue, and the gas–liquid
chromatographic (GLC) methods measure dietary fiber by
hydrolyzing a treated sample with acid to sugar monomers
prior to quantification by GLC. Although, on the surface, both
types of methods appear similar, there are fundamental practi-
cal differences that have come about because of variations in
the individual method steps and differences in the choice of
reagents and enzymes. These practical differences have signifi-
cant consequences since they determine the final composition
of dietary fiber as measured by each method.
Procedures have been described for isolating and analyzing
dietary fiber in specific foods as well as analyzing carbohydrate
polymers that are not digested in the small intestine and are
not identified as dietary fiber by the current Association of
Official Analytical Chemists (AOAC) methods. This article,
however, will focus on practical methods that are applicable
to the routine analysis of a wide range of foods. Most total
dietary fiber (TDF) methods also allow for the quantification
of different fiber fractions such as soluble fiber, insoluble fiber,
hemicelluloses, cellulose, and lignin. In this article, only the
methods that measure TDF are reviewed. This is partly because
of space limitations and partly because the individual fiber
components have not yet been positively associated with
human physiological effects. For example, soluble and insolu-
ble fiber can be readily measured, but these designations have
little physiological meaning since the digestive properties and
physiological benefits associated with some soluble fiber can
be found in some insoluble fibers and the physiological ben-
efits associated with insoluble fibers are also found with some
soluble fibers. In addition, some processing techniques can
also partly depolymerize insoluble fiber that may then be
found in the soluble fiber fraction.
The early work of Van Soest focussed on measuring rumi-
nant fiber (mainly hemicellulose, cellulose, and lignin), but
these procedures could not be used to measure TDF because
they would have underestimated the TDF content of human
foods by missing a significant proportion of the soluble dietary
fiber. All the modern methods incorporate steps to measure
soluble fiber. This is important since it can represent a signif-
icant proportion of the TDF of a food. The chemical complexity
and physical variability of the dietary fiber fraction in various
foodstuffs as well as the various food-processing techniques
have contributed to the difficulties encountered in devising
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00228-2
adequate dietary fiber methods. Many of the present-day TDF
procedures are based on the pioneering work of Southgate.
Ideal reference standards do not exist for dietary fiber anal-
ysis. Purified fiber components and unpurified standardized
‘reference samples’ are both used as standards of analysis, but
the former group rarely represents the complex dietary fiber
matrix found in foods and the latter group remains variable in
composition, structure, and properties.
Historical Perspectives on Dietary Fiber Values
Several different measures of dietary fiber and dietary fiber
fractions have been reported for foods over the last 150 years.
It is important to put these values into some sort of context to
understand the differences between these measurements and
the implications for human dietary fiber research.
The crude fiber method was standardized in the late
nineteenth century in Germany. It was initially developed to
analyze ruminant feeds and silage. It is a highly empirical
method that uses sequential extractions with hot dilute acid
and alkali. The crude fiber residue contains no soluble fiber and
retains only about 15% of the total hemicelluloses, 50–100%
cellulose, and 10–50% lignin. The variability in the method
and the variable losses of various dietary fiber fractions mean
that crude fiber values are of little value in human nutrition.
Acid detergent fiber (ADF) represented the first method that
reproducibly maintained specific dietary fiber fractions. ADF is
obtained by refluxing in 1 N mol l
1 sulfuric acid containing
cetylmethylammonium bromide detergent followed by filter-
ing and washing. This fraction contains mostly cellulose
and lignin.
Neutral detergent fiber (NDF) represents the insoluble frac-
tion of TDF. It is obtained by refluxing in a solution of sodium
lauryl sulfate followed by filtration and washing. NDF includes
most of the cell wall material but excludes the soluble b-glucans
and soluble hemicelluloses. It includes the ADF fraction as well
as the insoluble hemicelluloses. It thus underestimates TDF
because it does not measure soluble dietary fiber.
In some older tables, TDF values are reported as the sum of
NDF and pectin (estimated TDF) but, as shown in Figure 1,
this value is not always accurate. Figure 1 also presents the
relationship between insoluble dietary fiber (essentially NDF
but using porcine pancreatic a-amylase to remove starch; see
succeeding text) and TDF (as determined by the rapid health
protection branch (HPB) method; see succeeding text) deter-
mined for the same foods. The insoluble dietary fiber underes-
timates the TDF value by  40%, but this is not constant. The
differences between TDF and insoluble dietary fiber represent
actual differences measured on the same food sample, but the
differences between the estimated TDF and the actual TDF
383
384 Dietary Fiber: Determination
10
8
Estimated TDF, IDF, or CRF
6 glass crucibles of defined porosities. It is, therefore, important
(g g–1 'as is')
4 minimizes particle loss during filtration. Many different gravi-
2
0 Lee method. Before presenting these methods, we will first
0 2 4 6 8 10 12
TDF (rapid HPB method)
(g g–1 'as is')
Figure 1 Relationship between crude fiber (CRF; white triangles),
insoluble fiber (IDF; gray circles), estimated total dietary fiber (TDF, total
of pectin þ neutral detergent fiber (NDF); black circles), and TDF values
for different types of foods. The TDF and CRF values are from the
Canadian Nutrient File (Health Canada 1997; Mongeau et al., 1989). TDF
and IDF values were determined by the rapid HPB method (AOAC
992.16, see text). The solid line represents a 1:1 correspondence. The
dashed line (IDF) and the dotted line (CRF) are regression lines
with the intercept fixed at the origin. Insoluble dietary fiber is essentially
NDF but includes a treatment with porcine pancreatic a-amylase.
The NDF þ pectin values are ultimately from the USDA Nutrient
Database (1999).
values were performed at different times with different proce-
dures using different food samples so that procedural variations
may account for some of the differences. For example, it is
known that some previously determined NDF values were mea-
sured after incubation at lower (suboptimal) temperatures that
may have given higher and more variable values due to incom-
plete starch digestion.
In addition to the relationships described earlier, Figure 1
shows the relationship between crude fiber (CRF) and TDF (as
determined by the rapid HPB method) for foods from the
Canadian Nutrient File. Like the insoluble dietary fiber values,
the CRF values significantly underestimate the TDF values;
however, in this case, the variability is largely due to the CRF
method. Figure 1 shows no consistent relationship between
TDF and CRF or insoluble dietary fiber (and thus NDF). Thus,
it is not possible to predict TDF values from either CRF or
insoluble dietary fiber: a complete TDF measure is required.
Gravimetric Methods
Gravimetric methods use enzyme and/or chemical treatments
to remove material that is digestible in the small intestine.
Ideally, these treatments should mimic the digestive processes
to arrive at a sample that is representative of the material
entering the large intestine of humans. Because dietary fiber
represents the structural components of plant cell wall mate-
rial, the weight of the residue is corrected for the presence of
noncombustible materials (such as minerals) and corrections
may also be applied for components that are incompletely
digested by the procedure (such as protein). The remaining
weight is considered to be dietary fiber. In order to measure the
remaining material gravimetrically, it must be filtered through
when preparing the sample to ensure that it is not too finely
ground. The use of a Wiley mill avoids these problems and
metric methods have been published with many variations in
reagents, temperatures, and incubation times. However, rela-
tively few of these methods have been approved by the AOAC
after rigorous testing and interlaboratory verification. Three
AOAC-approved methods will be discussed in detail in this
section: the rapid HPB method, the Prosky method, and the
discuss the detergent system.
The Detergent System
When introduced in 1963, the detergent system represented a
great improvement over the CRF method, and it is still in use
today for measuring insoluble dietary fiber. It has been widely
used to measure silage and ruminant feed fiber. The system
includes the NDF and the ADF procedures, which use hot
detergent to remove digestible material and soluble fiber. The
detergent system discriminates among the insoluble fibers –
hemicelluloses, cellulose, and lignin. A chelating agent in the
neutral detergent solution permits the solubilization of insolu-
ble pectin. When NDF and ADF procedures are performed
sequentially, the difference in weight between the NDF and
the ADF extracts is an estimation of the insoluble hemicellulose
fraction. The ADF residue contains lignin, cellulose, and (if
present) cutin. Lignin can be measured by gently oxidizing the
ADF residue with potassium permanganate. A 72% sulfuric acid
treatment hydrolyzes cellulose leaving cutin, phlobaphenes,
and the Maillard products.
NDF and Porcine a-Amylase
Since water-insoluble dietary fibers represent a major fraction of
TDF, the NDF procedure represents a simple method that can
form part of an overall procedure for measuring TDF in human
foods. Nonfiber materials such as starch, most protein, and any
fiber-like artifacts remaining in the NDF residue can be effi-
ciently removed by rapid treatment with a-amylase from porcine
pancreas (A-3176, Sigma Chemical Co., St Louis, MO); the rapid
treatment consists of 5 and 60 min incubations at 55  C and is
at least as effective as the 18 h treatment at 37  C originally
proposed by Schaller. The source of the enzyme is specified
since pancreatin and other preparations of porcine pancreas
a-amylase are much less efficient. Unpurified porcine a-amylase
has unique amylolytic and proteolytic activities when applied
after the neutral detergent extraction step, making it an ideal
enzyme to use for measuring insoluble dietary fiber.
The heat-stable a-amylase from Bacillus subtilis has also
been used to digest starch after the neutral detergent extraction
 Dietary Fiber: Determination 385

step, but several potential problems arise with this enzyme. Rapid HPB Method
It may contain fiber-digesting enzymes that could lead to an
The rapid HPB method described here is the AOAC No. 992.16
underestimation of the cellulose and hemicellulose fractions.
method. In order to obtain a measure of the TDF in a sample,
On the other hand, it does not completely remove the nonfiber
one needs only to combine the insoluble dietary fiber from
material (such as starch), which will lead to an overestimation
the NDF þ porcine pancreas a-amylase treatment with a specif-
of the lignin and possibly the hemicellulose fractions. The final
ically tailored method for measuring soluble fiber. In the rapid
value, therefore, represents a compromise between these two
HPB method, the soluble fiber procedure was devised to max-
competing factors. Table 1 shows that, for shredded wheat, the
imize the recovery of soluble fiber while excluding protein and
NDF residue obtained after treatment with B. subtilis a-amylase
other precipitable, nonfiber materials. This latter characteristic
contained 30% more material than that obtained after treat-
is important if one wishes to avoid time-consuming protein
ment with porcine pancreas a-amylase. This residue apparently
determinations that are part of other procedures. The soluble
contained three times more lignin and four times more pro-
fiber procedure includes a gelatinization step in acetate buffer
tein. Both the NDF residue and the apparent lignin content
at pH 4.5 (121  C) followed by a treatment with heat-stable
were also higher in samples of bran flakes. Other problems are
a-amylase (100  C) to remove any starch. Starch gelatinization
apparent with the B. subtilis a-amylase. For example, toasting
is important to ensure complete starch hydrolysis. The soluble
whole wheat bread increased the B. subtilis a-amylase-
material is separated from insoluble fiber by filtration and is
measured NDF and lignin values by two- and fivefold, respec-
treated at 60  C with amyloglucosidase (to finish starch diges-
tively (Table 1), but no such differences were noted with
tion by hydrolyzing branched residues) and then with protease
the porcine pancreas a-amylase. The efficacy of the porcine
(to hydrolyze the peptide bonds of protein). The soluble fiber
pancreas a-amylase treatment has been verified in various
is then precipitated in 80% ethanol at room temperature; the
foods and food products and confirmed by other researchers.
sugar and amino acid residues remain soluble in ethanol.
In addition to its demonstrated capabilities in avoiding
Acetate buffer at pH 4.5 favors the exclusion of protein and
artifactual increases in the estimation of various dietary fiber
appears to prevent the degradation of fiber components that
fractions, porcine pancreas a-amylase enzyme also facilitates
may occur at high temperatures. This does not occur as readily
rapid filtration in subsequent steps (starch and protein tend to
at the relatively higher pH values used by other TDF procedures
clog the crucibles). The neutral detergent–a-amylase combina-
(e.g., phosphate buffer at pH 7). The heat-processed reference
tion is advantageous for research analyses since the neutral
standards extracted from apple and carrot, and used in some
detergent solution discriminates well between plant and bac-
collaborative studies, may show lower TDF values due to the
terial cell walls. This is important because bacterial cell walls
loss of pectin during analysis. The differences are less apparent
interfere with the fiber analysis of fecal samples.
when expressed on a ‘per gram fresh weight’ basis. Without a
Other modifications from the original Van Soest NDF
method for accurately measuring all cell wall components, it is
method include the deletion of decalin, 2-ethoxyethanol, and
difficult to provide an absolute compositional measure of the
sulfite (to maximize the recovery of lignin).
constituents of dietary fiber as measured by different methods.
However, the use of differential procedures to obtain fiber
fractions (ADF and NDF), coupled with sulfuric acid digestion,
Table 1 Effect of two sources of a-amylase on the apparent neutral permanganate oxidation, and total nitrogen analysis, allows
detergent fiber (NDF) and lignin content of breakfast cereals and bread an estimate of the relative contribution of several plant compo-
(g 100 g
1, dry weight basis) nents to the final TDF value.
Figure 2 shows a comparison between several methods for
Porcine pancreas
measuring dietary fiber and the relative amounts of the com-
a-amylasea Bacillus subtilis a-amylase
ponents retained by each method. The rapid HPB method
NDF Lignin Protein b
NDF Lignin Proteinb retains lignin (a structural polyphenol), the cell wall-associated
protein, and a small amount of starch. The significant amount
Shredded 10.2 0.8 0.4 13.4c 2.7c 1.7c of starch retained by the Prosky method (see succeeding text)
wheat is readily apparent when TDF values are measured in legumes.
Bran flakes 10.1 0.9 15.8 2.4
Table 2 shows a comparison of four different methods for
Whole 5.2 0.3 5.9 0.4
wheat
measuring TDF in green peas. The starch in the residue was
breadd estimated by subtracting the values from the total nonstarch
Whole 5.4 0.4 10.1 2.2 polysaccharides (NSPs) þ lignin measured by the Englyst pro-
wheat cedure. The table also shows the effect of adding porcine
bread, pancreas a-amylase.
toastedd The method is considered ‘rapid’ when compared to other
a
fiber methods, particularly when the Fibertec E (for soluble
a-Amylase from porcine pancreas (Sigma Chemical Co., catalog no. A-3176).
fiber) and the Fibertec I (for insoluble fiber) are used. For
b
Protein measured in NDF residue (N  6.25).
c
From Van Soest, P. J. (1978). Fiber analysis tables. American Journal of Clinical
example, the maximum number of duplicate determinations
Nutrition 31, S284. using the Prosky TDF method is 20 per week, while 44 duplicate
d
From Mongeau, R and Brassard, R. (1980). Rapid digestion of starch and artifact TDF determinations per week are possible using the rapid
fibre in the measurement of neutral detergent fibre of cereal products. Getreide Mehl und HPB method. This evaluation takes into account the time
Brot 34, 125–127. required for preparing the reagents and performing the
386 Dietary Fiber: Determination

Structural protein Nonstructural

Lignin NSP
Structural NSP

Starch
Maillard
Englyst products?

Uppsala no. 994.13

Prosky no. 985.29

Lee no. 991.43

Rapid HPB no. 992.16

Figure 2 Material measured as dietary fiber by unmodified methods for all food categories. The surface area of the box is proportional to its total
dietary fiber (TDF) value. It shows whether the method includes lignin and structural fiber protein or if it excludes starch and the Maillard products.
The cell wall material corresponds to the structural nonstarch polysaccharides (NSP), lignin, and structural protein. The larger amounts of starch
retained by the Theander, Prosky, and Lee methods are most evident when measuring TDF in legumes. AOAC numbers for each procedure are indicated.
Adapted from Mongeau, R. (1995). HPB, Health Protection Branch, Health Canada.

Table 2 Total dietary fiber (TDF) in boiled dried green peas as measure TDF gives a more accurate and reliable measurement
measured by four different methods with or without added porcine because it eliminates potential cross contamination that could
pancreas a-amylase give rise to falsely high TDF readings. For example, if the insol-
uble fiber fraction contains some small amount of soluble fiber,
Estimated it will be counted twice in the final value (once in the insoluble
TDF on a ‘per g dry starch content fraction and once in the soluble fraction). The far right column
Method weight basis’ (%) (%)
of Table 3 shows that the TDF values are comparable when
Rapid HPB method, 9.9  0.1 2.7 measured by either procedure and demonstrates the variability
separate in the procedure.
Prosky TDF method 19.0  0.2 11.8 In a comparison of 28 foods from various categories, the
Prosky TDF 12.3  0.3 5.1 rapid sequential HPB method agreed well with the Prosky TDF
method þ porcine method (slope ¼ 0.99, intercept ¼
0.11, r2 ¼ 0.985) and with
pancreas a-amylase the Englyst GLC method (slope ¼ 1.03, intercept ¼ 0.15,
Lee TDF method 18.8  0.3 11.6 r2 ¼ 0.989). For comparison with the Englyst method, it was
Lee TDF method þ porcine 12.3  0.3 5.1
necessary to measure lignin in each food and add its weight to
pancreas a-amylase
the total NSP (obtained from the Englyst analysis). This gave a
Englyst method 7.2  0.2
(NSPs þ lignin) TDF value that was comparable to that measured gravimetri-
Englyst method (omit 10.4  0.1 3.2 cally. The regression parameters indicate that the rapid HPB
DMSO) method and the Prosky TDF method retain similar material in
the final fraction (Figure 2). However, the negative intercept
NSPs, nonstarch polysaccharides; DMSO, dimethyl sulfoxide. shows that the Prosky TDF method gives a higher value than
Starch content estimated by subtracting the NSP þ lignin value from the TDF estimate. the rapid HPB method when measuring foods containing low
Source: Mongeau, R. and Brassard, R. (1994). Comparison and assessment of the
amounts of fiber.
difference in total dietary fiber in cooked dried legumes as determined by five methods.
Journal of AOAC International 77, 1197–1202.

The Prosky, Lee, and Asp Methods

analysis, but not the time required for preparing the samples
(freeze-drying and grinding).
Both separate and sequential rapid HPB methods are avail-
able and Table 3 presents a comparison between them. In the
sequential method, the insoluble residue generated during the
soluble fiber determination is treated with neutral detergent
and porcine a-amylase yielding the insoluble fiber residue.
Thus, a single sample generates both insoluble and soluble
fiber measurements to give the TDF value. The separate method
requires two samples for analysis. Using a single sample to
The Prosky TDF method has also been referred to as the AOAC
method since 1985. The Prosky, Lee, and Asp methods are
similar but differ in the sources and types of enzymes as well
as in the type and pH value of the buffers. The TDF values from
all three methods are comparable (see Table 2 for a compari-
son between the Prosky and the Lee methods for green peas).
In the Prosky and Lee TDF methods, duplicate samples are
treated with a-amylase, protease, and amyloglucosidase to
remove digestible material. Four volumes of 95% ethanol are
 Dietary Fiber: Determination 387

Table 3 Comparison of total dietary fiber (TDF) values by gas–liquid chromatography (GLC: nonstarch polysaccharides (NSPs) þ lignin) or
gravimetric methods (on a g 100 g
1 dry weight basis)

Gravimetric TDF valuesc

Food NSPa NSPb Permanganate lignin Prosky Rapid separated Rapid sequential

Apple 9.9–14.7 13.4 0.8 14.6 14.1 14.6

Beans, green 27.8–32.2 30.6 2.1 33.7 31.3 29.1
Bread, white wheat 2.6–3.3 3.0 0.6 3.5 3.0 3.9
Carrot 22.8–28.9 23.5 1.8 25.4 23.4 23.4
Corn kernels, canned 6.7–8.9 5.9 1.0 7.7 7.8 7.6
Cornflakes 0.7–2.4 1.1 <0.1 2.5 1.1 2.6
Flour, white wheat 3.0–3.7 2.6 <0.1 3.0 3.9 3.6
Oats, rolled 7.1–9.5 8.2 0.9 10.5 11.0 9.6
Peas, canned 20.4–22.3 – – – 23.3 20.9
a
Range of values reported by Anderson and Bridges (1988), Englyst and Cummings (1989), Englyst et al. (1995), and Marlett (1992).
b
NSP as determined by the Englyst GLC method.
c
TDF as determined by the Prosky method and the rapid separate and sequential methods.
d
The separate method required analyzing two separate samples to obtain the TDF value.

then added and the precipitate filtered through Celite C-211 Table 4 Polysaccharide and unidentified material in the Prosky total
(Thermo Fisher Scientific, Mississauga, Ontario, Canada). The dietary fiber (TDF) method for selected food groups
residue contains a substantial amount of nitrogen requiring a
Prosky TDF method residue (g 100 g
1 dry matter)
total nitrogen analysis (N) and subsequent back-calculation to
correct for protein content (N content  6.25). One of the Total Unidentified
duplicates is also analyzed for ash as in other gravimetric Food Starch polysaccharides material
analyses, and this, too, is subtracted from the residue weight.
It is proposed that the use of this protein conversion factor Bread 1.0 5.1 0.8
leads to overestimation of dietary fiber (Table 4). The addi- Other cereals 0.3 3.8 1.0
tional protein determination step prevents this method from Green 1.5 25.6 4.6
vegetables
being considered rapid. As pointed out earlier and in Tables 3
Other vegetables 1.8 10.2 7.5
and 4, the final insoluble dietary fiber residue can retain sig-
Canned 2.3 12.9 4.0
nificant amounts of starch. In a simplified version of the vegetables
Prosky method, the protease step is skipped since protein is Fresh fruit 0.2 8.6 3.5
measured in the final residue. This means that a larger amount Fruit products 0.1 2.5 1.5
of protein is subtracted as compared with the original Prosky Nuts 0.4 6.7 2.5
procedure and this may contribute to some variability. The
nature of the Celite used in different laboratories has also Values represent TDF measurements after correction for ash and ‘protein’ (N  6.25).
Part of the unidentified material represents lignin but the values are too large to be
been considered as a source of variability. Other modifications
accounted for as lignin only.
have also been proposed to improve the precision of the
Source: Englyst, H. N., Quigley, M. E., Englyst, K. N., Bravo, L. and Hudson, G. J.
Prosky method. The method comparisons in Tables 2 and 3 (1995). Dietary fibre. Measurement by the Englyst NSP Procedure. Measurement by the
were confined to the results obtained with the original version AOAC Procedure. Explanation of Differences Medical Research Council & University of
of the Prosky method. Cambridge. Report of a Study Commissioned by MAFF, UK.

Complete Polysaccharidic Components (GLC Methods)
Southgate published the original TDF method that required
the hydrolysis of fiber into its individual polysaccharidic com-
ponents. In Southgate’s method, TDF represented NSPs plus
lignin, but the method also provided values for hemicellulose
and cellulose content. The method used 85% (v/v) alcohol to
precipitate soluble starch residues followed by gelatinization
and starch digestion with an appropriate enzyme. The original
enzyme used by Southgate (takadiastase) had both amylolytic
and proteolytic activities. However, in the 1970s, this enzyme
became unavailable and other enzymes were used as replace-
ments. It is apparent that some of these replacement enzymes
are often less effective (Tables 2 and 3) but the a-amylase from
porcine pancreas appears to be adequate. The original
Southgate method measured the NSPs colorimetrically, but
now, more specific methods are available such as GLC.
The more modern chemical methods use a combination of
enzyme and/or chemical treatments to remove digestible mate-
rial. The residue is then hydrolyzed by acid prior to the mea-
surement of the sugar content. The acid treatment needs to be
severe enough to assure complete hydrolysis but mild enough
to prevent monomer degradation. The neutral fiber polysac-
charidic constituents are measured chemically, usually by GLC
after derivatization to a measurable form (e.g., by forming
alditol acetates). Chemical derivatization is not required when
high-performance liquid chromatography is used to measure
the residue concentration. Acidic sugars (uronic acids) are mea-
sured colorimetrically. The total NSP content is obtained by
appropriate calculations and TDF is calculated as the sum of
388 Dietary Fiber: Determination
NSPs þ lignin. The main GLC methods have been published by
Anderson, Englyst, and Theander and their coworkers. Marlett
used a modified Uppsala method. The GLC methods are differ-
ent in several aspects, including the starch gelatinization step,
enzyme treatments, and conditions of acid hydrolysis. This
explains why the methods may be in agreement for some
foods, but not for others.
The Englyst Method
Like all GLC methods, the Englyst method measures NSPs (neu-
tral sugarsþ uronic acids). However, unlike other methods, the
Englyst method is completely applicable to processed foods
because it makes use of dimethyl sulfoxide (DMSO) during the
gelatinization step to disperse the starch completely. This is fol-
lowed by treatment with a heat-stable a-amylase and by a mixture
of pullulanase–pancreatin in acetate buffer at pH 5.2. This treat-
ment completely removes all the starch, leaving only the non-
starch polypeptides (Figure 2). The total insoluble and soluble
NSPs are then precipitated in 80% ethanol and collected by
centrifugation prior to hydrolysis with sulfuric acid. Neutral
sugar residues are measured by GLC as alditol acetates and uronic
acids are measured colorimetrically. According to Englyst and
Cummings, the method can be performed in approximately the
same amount of time as the Prosky TDF method. However, the
GLC instrument must be equipped with an autosampler for the
comparison to be valid. A rapid version of the Englyst method
also exists with the sugar components measured colorimetrically,
but this does not represent a substantial reduction in assay time
when compared to the GLC method with an autosampler.
The Englyst method is the only dietary fiber method that
does not include lignin. The role of noncarbohydrate compo-
nents (which include lignin) in in vivo fiber digestion may be
significant. There is some debate over whether some of the
starch and structural protein should be considered as dietary
fiber. These components would have to be measured by other
methods if they were included in the TDF value.
The Uppsala Method
In the Uppsala method, there is a provision for an initial
extraction in 80% ethanol if the sample contains very large
amounts of mono-, di-, and oligosaccharides. This extraction
appears important in reducing any coprecipitation of nonpo-
lymeric sugars and fiber. Starch is digested in acetate buffer at
pH 5.0 by incubating with a thermostable a-amylase and then
with amyloglucosidase. DMSO is not used and starch may be
less efficiently dispersed and, therefore, less efficiently removed
(Figure 2). After reprecipitating in ethanol and centrifuging,
the residue is hydrolyzed with sulfuric acid. As in the Englyst
method, neutral polysaccharides are analyzed as alditol ace-
tates by GLC and uronic acids by decarboxylation with special
equipment or by colorimetry.
Unlike the Englyst method, the Klason lignin is measured
gravimetrically. Potential errors can arise when lignin is measured
independently and added back to NSPs. For example, the lignin
fraction may include other nondigested fiber materials (such as
cellulose), which would then overestimate insoluble fiber. This
was apparently the reason behind an apparent lignin value of
0.9–3.5% (dry weight basis) measured in apples, peas, or a food
composite. This value is suspect because low permanganate lignin
values (obtained after the treatment of the insoluble fiber residue
with porcine pancreas a-amylase and subsequent workup to
obtain an ADF residue) were obtained, which were independent
of any food-processing method (similar to the values in Table 1).
Marlett had discussed the necessity of measuring lignin and the
problems related to its measurement.
The Anderson Method
As in the Englyst method, the Anderson GLC method uses
DMSO to disperse starch prior to hydrolysis. The starch is
then digested with porcine pancreas a-amylase in acetate buffer
at pH 5.2. After precipitation with four volumes of ethanol, the
residue is extracted with water at 100  C to obtain soluble and
insoluble fiber fractions. Following centrifugation, the soluble
fiber from the supernatant and the insoluble fiber from the
pellet are hydrolyzed with sulfuric acid. The Klason lignin is
measured as the loss in weight of the nonhydrolyzed insoluble
residue after ashing.
Comparison of GLC and Gravimetric Methods
for Measuring TDF
As already indicated, there is general agreement between the
rapid HPB method and the Prosky and Englyst methods. The
regression coefficients were 0.985 and 0.989 for the Prosky
and Englyst methods, respectively. Other comparisons have
also shown good agreement between values. For example, in
a comparison of 25 different foods, TDF values as measured by
the rapid HPB method agreed with those reported by Anderson
and Bridges (modified Englyst method) and with those
reported by Marlett (modified Uppsala method). However,
more direct comparisons are needed to determine the potential
problems with each method.
Table 3 presents a comparison of the TDF values expressed
on a ‘per gram dry weight’ basis to magnify differences that
may be partly hidden when the values are expressed on a ‘per
gram fresh weight’ basis. In order to compare TDF values, the
permanganate lignin values need to be added to the NSP
values. Three different levels of variability are apparent from
the data shown in Table 3. The range of NSP values obtained
from three different laboratories (using three slightly different
methods and different food samples) illustrates, in part, the
intermethod variability. In addition, there is some interlabora-
tory variability, as shown by a comparison of four laboratories
as well as an inherent variability in the samples (including
stage of maturation of the product). Direct comparisons are
only possible for the results in the five right-hand columns.
These samples were composites based on winter and summer
collections of up to 20 individual foods over 30 months
in different locations. The results show that, when lignin is
included in the final TDF value, there is good agreement
between GLC and gravimetric methods for the indicated foods.
Nutritional Labeling and Analysis
It is difficult to decide on a single method for measuring the
dietary fiber content of all foods. The challenge is to find a

single method that works adequately for all food groups and is
independent of sample processing. Several candidates are
possible, but to a large extent, the final decision will reflect
the definition of dietary fiber. The dietary fiber definition must
not be driven by available methodologies but must be arrived
at independently of methodological considerations. As shown
in Figure 2, it is possible to make a case for including structural
protein, lignin, structural, and nonstructural NSPs, as well as
some starch. Each method includes variable amounts of all
these components. In addition to these complications, it is
also possible to separate TDF into insoluble and soluble com-
ponents. Once again, each method uses different procedures to
measure these fractions so that the measured content of insol-
uble and soluble fiber will differ from laboratory to laboratory.
Consumption of these different fiber fractions has not yet been
related to definite physiological effects, although there appears
to be a role for viscous soluble dietary fiber in reducing the risk
factors for cardiovascular disease. The role of starch and other
nonstarch polymers not traditionally associated with dietary
fiber has yet to be determined as well. Therefore, the following
discussion deals only with practical considerations in choosing
a TDF method for food labeling.
Equipment
A method requiring simple, inexpensive equipment would be
the most widely applicable because it would be more accept-
able to nations unwilling or unable to spend considerable
sums of money to monitor labeling. In general, the gravimetric
methods require simpler equipment and the rapid HPB
method is less costly than other gravimetric procedures
because it does not require elemental nitrogen analysis. The
Englyst colorimetric method can be completed with a simple
spectrophotometer, although this method is less robust than
the GLC method. Except for the Englyst method, all methods
require a muffle furnace for ashing the samples. Although the
gravimetric systems require simpler equipment, a hot-water
supply is needed and analyses are greatly aided by purchasing
semiautomated filtering/refluxing equipment that would
increase the cost. However, these pieces of equipment are
usually less expensive to maintain than GLCs.
Operator Time
Because it does not include a protein determination, the rapid
HPB method requires the least amount of operator time
per duplicate determination. Methods that require sugar deriv-
atization and analysis by GLC tend to be longer than the
gravimetric methods, especially when lignin measurement is
included. The time required for these methods is greatly reduced
if the GLC system includes an autosampler. Semiautomatic
equipment is available for the gravimetric determinations, but
filtering time can be slow, especially for samples that contain a
considerable amount of starch.
Lignin
The Englyst method is the only one that does not include lignin
in the final TDF value. Most researchers consider lignin to be
an important dietary fiber constituent, and, consequently,
Dietary Fiber: Determination 389
most methods include a measure of the lignin content. In the
GLC methods, the adoption of the Klason method (residue
after sulfuric acid digestion) for measuring lignin means that
other nonfiber materials such as the Maillard products may be
included in the lignin fraction. This could lead to overestima-
tion of the lignin content and, consequently, overestimation of
the insoluble dietary fiber and TDF values. Lignin may be more
accurately measured by a combination of NDF þ porcine pan-
creas a-amylase and ADF treatments prior to permanganate
oxidation. This would, however, increase the time required
for analysis as well as increase the amount of sample needed
per assay.
Independence from Food Processing
Different methods include different amounts of starch in the
final TDF estimate (Figure 2). The only method that does not
include any starch is the Englyst method because it completely
solubilizes the starch with DMSO prior to enzymatic hydroly-
sis. There are several different types of starch, some of which
arise from food preparation such as retrograde starch, and so
the inclusion of starch in a method could be problematic. An
example of the potential for problems comes from measuring
the TDF values in cooked and uncooked rice and potatoes
(high-starch foods) using the Prosky method. The Prosky
method gave higher TDF values in the cooked foods. Problems
were also noted with roasted peanuts where nonfiber material
was included with TDF as well as toasted whole wheat bread
(Table 1). Lower cooking temperatures (i.e., 95  C compared
to 100–120  C) artificially increased the TDF content of white
navy beans when the Prosky and Lee methods were used. Some
of the problems may have been related to the choice of enzyme
used to degrade starch, as was observed for toasted whole
wheat bread (Table 1).
In general, the Englyst method is independent of the effects
of food processing because it uses DMSO to aid in removing
starch and nonpolymeric sugars. This helps exclude food prep-
aration artifacts, such as retrograde starch, as well as the Mail-
lard products, from the final analysis, but it also means that
lignin is not included. It also means that lignin is excluded
from the final analysis. Other methods that rely on GLC iden-
tification of NSPs include the Klason lignin, but this analysis
has other problems (see preceding text). Of the gravimetric
methods, the rapid HPB method appears to be independent
from food processing and, at the same time, includes lignin in
the final value.
Precision
Method variability pooled over all foods has been measured in
a US Department of Agriculture study using 25 duplicate food
samples. Different laboratories were asked to measure TDF in
the samples without knowing their composition or source. The
coefficient of variation for a single observation was 3.0% with
the rapid HPB method, 4.7% with the Prosky TDF method,
and 8.4% for the Uppsala method. This represents an estima-
tion of the intralaboratory precision for selected unprocessed
and processed foods. More interlaboratory work comparing
precision of several methods is needed.
390 Dietary Fiber: Determination
Remaining Problems
The general agreement between the different methods for mea-
suring fiber is encouraging because it opens the door to uni-
versal acceptance of a dietary fiber method. However, many
problems remain. First, an alcohol precipitation step is used to
separate fiber material from nonfiber material. This step usu-
ally follows starch digestion and is designed to precipitate
sugar polymers. It is possible that some of the fiber compo-
nents are not completely precipitated and that some sugars
released by starch digestion are coprecipitated. This may vary
with the method and may be the source of some of the inter-
method variability. In addition, the selectivity of this step in
separating sugar polymers from nonpolymeric compounds has
been questioned. In particular, polymers such as oligofructose
(actually a mixture of oligomers and polymers) do not precip-
itate during the subsequent centrifugation. The ‘dietary fiber’
nature of these polymers is currently being debated.
Second, structural fiber protein may represent up to 10% of
dietary fiber in immature plant tissues. It is impossible, at the
present time, to quantify this protein (although the rapid HPB
gravimetric method presumably includes it). Except for the
rapid HPB method, this fraction is excluded from the reported
TDF values.
Third, the colorimetric method for the determination of
uronic acid values (included in most GLC methods) needs to
be reexamined. This is because the uronic acid content of
vegetables was shown to be higher when measured by a decar-
boxylation method.
Fourthly, there are a small number of foods where significant
method disagreement occurs. For example, for soya bean fiber
isolate, the NSP value using the Englyst GLC method is lower
than the TDF value measured gravimetrically. The reasons for
these discrepancies need to be examined in order to determine
the exact TDF composition measured by each method. These
comparisons will greatly improve fiber methodology.
Finally, although both acetone and ether have been used to
remove fat from the samples prior to analysis, their efficacy is
questionable and only the defatting procedure of Bligh and
Dyer is efficient enough to defat all food samples completely.
Caution is warranted when substituting defatting procedures
since TDF methods have been validated with their respective
defatting methods and substitutions may produce unexpected
results. For example, the degree of lignin removal with the
Bligh and Dyer procedure is unknown. There is some question
as to the necessity for complete defatting since the methods
were validated with a wide variety of food samples, all with
different fat contents. It may be necessary to reexamine the
importance of fat removal prior to analysis in samples contain-
ing high amounts of fat.
Conclusion
Comparable TDF values can be obtained for many foods using
any of the methodologies discussed earlier. Some foods are
more problematic and require a careful choice of methodol-
ogy. Significant causes of error have been identified, including
the potential precipitation of incompletely digested starch and
the measurement of the Klason lignin, particularly in processed
foods. The inclusion of the Maillard products in the Klason
lignin may significantly increase the final TDF value. The fol-
lowing treatment is recommended: NDF, porcine pancreas a-
amylase, ADF, and permanganate treatment. This procedure
gives permanganate lignin values that are independent of food
processing and, when added to NSPs determined by GLC,
provides a detailed and known measurement of dietary fiber.
The rapid HPB method, including the neutral detergent and
porcine a-amylase treatments, has several advantages and
would serve as a good method for food labeling.
See also: Cellulose; Chromatography: Focus on Multidimensional
GC; Chromatography: High-Performance Liquid Chromatography;
Starch: Sources and Processing; Starch: Structure, Property, and
Determination.
Further Reading
Anderson JW and Bridges SR (1988) Dietary fiber content of selected foods. American
Journal of Clinical Nutrition 47: 440–447.
Buttriss JL and Stokes CS (2008) Dietary fibre and health: an overview. Nutrition
Bulletin 33(3): 186–200.
DeVries JW and Rader JI (2005) Historical perspective as a guide for identifying and
developing applicable methods for dietary fiber. Journal of AOAC International
88(5): 1349–1366.
Elleuch M, Bedigian D, Roiseux O, Besbes S, Blecker C, and Attia H (2011) Dietary fibre
and fibre-rich by-products of food processing: characterisation, technological
functionality and commercial applications: a review. Food Chemistry 124(2):
411–421.
Englyst H and Cummings J (1989) Dietary fibre and starch: definition, classification and
measurement. In: Leeds AR (ed.) Dietary fibre perspectives. Reviews and
bibliography, pp. 3–26. London: John Libbey.
Englyst, H. N., Quigley, M. E., Englyst, K. N., Bravo, L. and Hudson, G. J. (1995).
Dietary fibre. Measurement by the Englyst NSP Procedure. Measurement by the
AOAC Procedure. Explanation of Differences Medical Research Council & University
of Cambridge. Report of a Study Commissioned by MAFF, UK.
Huang H, Haiyan Y, Huirong X, and Ying Y (2008) Near infrared spectroscopy for on/in-
line monitoring of quality in foods and beverages: a review. Journal of Food
Engineering 87(3): 303–313.
Jenkins DA, Kendall CWC, and Vuksan V (2000) Viscous fibers, health claims, and
strategies to reduce cardiovascular disease risk. American Journal of Clinical
Nutrition 71: 401–402.
Knudsen Bach KE and Mongeau R (1995) Analytical methods for the determination of
components conventionally considered as dietary fibre. In: Sørensen A, Knudsen
Bach KE, Englyst HN, Gudmand-Høyer E, and Nyman M (eds.) Metabolic and
physiological aspects of dietary fibre in food. Recent Progress in the Analysis of
Dietary Fibre COST 92, pp. 205–206. Luxembourg: Commission of the European
Communitites.
Lupton JR, Betteridge VA, and Pijls LTJ (2009) Codex final definition of dietary fibre:
issues of implementation. Quality Assurance and Safety of Crops & Foods 1(4):
206–212, Special issue: Dietary Fibre.
McCleary B and Prosky L (2008) Measurement of dietary fibre and fibre components.
In: McCleary B and Prosk L (eds.) Advanced dietary fibre technology, pp. 61–117.
Oxford: Wiley.
McCleary BV, Vries D, Jonathan W, et al. (2010) Determination of total dietary fiber
(CODEX definition) by enzymatic-gravimetric method and liquid chromatography:
collaborative study. Journal of AOAC International 93(1): 221–233.
McCleary BV, DeVries JW, Rader JI, et al. (2011) Collaborative study report:
determination of insoluble, soluble, and total dietary fiber (codex definition) by an
enzymatic-gravimetric method and liquid chromatography. AACC International
Report 56(6).
Mongeau R (1995) Experiences with different official analytical methods in Canada.
In: Sørensen A, Bach Knudsen KE, Englyst HN, Gudmand-Høyer E, and Nyman M
(eds.) Metabolic and physiological aspects of dietary fibre in food. Recent progress

in the analysis of dietary fibre COST 92, pp. 143–151. Luxembourg: European
Commission.
Mongeau R, Brassard R, and Verdier P (1989) Measurement of dietary fiber in a total
diet study. Journal of Food Composition and Analysis 2: 317–326.
Mongeau R and Brooks SPJ (2001) Chemistry and analysis of lignin. In: Cho SS and
Dreher ML (eds.) Handbook of dietary fiber, pp. 321–373. New York, Basel: Marcel
Dekker.
Dietary Fiber: Determination 391
Theander O, Åman P, Westerlund E, Andersson R, and Pettersson D (1995) Total dietary
fiber determined as neutral sugar residues, uronic acid residues, and Klason lignin
(the Uppsala method): collaborative study. Journal of AOAC International
78: 1030–1044.
Wehling P and DeVries JW (2012) On the use of the Horwitz Ratio (HorRat) as an
acceptance criterion for dietary fiber collaborative studies. Journal of AOAC
International 95(5): 1541–1546.
 Dietary Fiber: Energy Value
R Mongeau and SPJ Brooks, 3W Banting Research Centre, Ottawa, QC, Canada
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from the Encyclopedia of Food Sciences and Nutrition, volume 3, pp. 1850–1858, ã 2003, Elsevier Science Ltd., with an
updated Bibliography section supplied by the editor.
Caloric Value in Human Nutrition
All food contains energy, which is needed by the body to fuel
metabolic processes. This energy is obtained through the
oxidation of macronutrients (carbohydrate, fat, and protein)
and is partly captured as chemical energy in compounds
such as adenosine triphosphate (ATP). These ‘high-energy’
compounds are used to drive many processes, including the
biosynthesis of cellular components, the maintenance of ion
concentrations across membranes, the initial phases of cata-
bolic pathways, cell replication, mechanical function, etc. The
large amount of energy released by the hydrolysis of ATP
(
7 kcal mol1 under cellular conditions) is captured by
coupling its hydrolysis to other chemical reactions to drive
them to completion.
The energy captured as ATP equivalents is one end of a long
chain of digestive and metabolic processes that starts as the
gross energy of the macronutrients in ingested food (Figure 1).
Significant losses in energy occur during the digestive process.
These can be identified and quantified as losses due to incom-
plete digestion and absorption (loss in fecal energy to give
apparent digestible energy; DEapp), losses to fermentation
gases and urinary nitrogen energy (to give apparent metabo-
lizable energy), and losses associated with the digestive process
itself and with the absorption and storage of food (to give net
energy). Calculating the metabolizable energy from a food
requires the collection of all excreta (including methane and
hydrogen gases). For substances that are fermented, extra infor-
mation is also required as discussed later in this article. Net
energy values are more difficult to estimate and require some
assumptions about digestive processes (see section on factorial
models of fermentation, eqn [19]).
Metabolizable energy values are the commonly reported
food energy values found on product labels. These were initially
determined in the 1900s by a German scientist who was a
pioneer in this area. To honor his name, these factors are com-
monly referred to as ‘Atwater factors.’ Both general and specific
Atwater factors exist. The specific values were measured in indi-
vidual foods. The general values (4 for carbohydrate, 9 for fat, 4
for protein, and 7 for alcohol) were calculated from the specific
Atwater factors using average intakes of mixed diets. The more
modern food energy values, as contained in tables such as the
US Department of Agriculture Handbook #8, represent specific
Atwater factors as modified by Merrill and Watt. Although we
refer to these factors as metabolizable energy values for macro-
nutrients, the values for carbohydrate and fat are, in reality,
DEapp values. This is because urinary and gaseous energy losses
were considered as insignificant for these macronutrients. In the
case of the protein Atwater factors, urinary energy losses were
taken into account, but gaseous energy losses were, again,
assumed to be negligible. The Atwater factors for fat, protein,
392 Encyclopedia of Food and Health
and carbohydrate represent the total energy available for metab-
olism, and this heat should be equivalent to the energy mea-
sured with a whole-body calorimeter.
Digestible sugars, fats, and proteins are readily absorbed in
the small intestine by physiological and biochemical processes
that capture the maximum energy from these food compo-
nents. These macronutrients are virtually completely absorbed
by the small intestine with little energy finding its way into the
large intestine. In fact, with diets high in digestible sugars, fats,
and proteins, most of the energy entering the large intestine
comes from small intestine secretions and from the sloughing
off of dead intestinal cells. It is the relative simplicity of the
digestive process coupled with the high degree of absorption
that allows the comparatively easy metabolic energy calcula-
tion for these macronutrients.
Not all food components are readily digested and absorbed
in the small intestine. Nonstarch long-chain polysaccharides
such as those associated with plant cell walls are not digested
by human enzymatic secretions. Many other carbohydrates are
not well absorbed by the small intestine. These pass into
the large intestine where they are anaerobically fermented by
the bacterial population that resides there. For the sake of
clarity, the terms ‘undigested’ and ‘resistant’ will be used here
to refer to the degradation processes occurring in the upper
gastrointestinal tract (mostly by host digestive enzymes), and
the term ‘fermentation’ and related words will be used to refer
to the bacterial degradation processes occurring in the lower
gastrointestinal tract. The energy losses associated with fermen-
tation are not as readily measured, and so calculation of the
metabolizable energy for fermentable food components is not
as straightforward as it is for sugars, fats, and proteins. The
present chapter outlines different methods for obtaining the
metabolizable energy of food components not digested and
absorbed in the small intestine.
A short note about the methods used to determine macro-
nutrient content of foods is in order. In some countries like the
United States and Canada, the carbohydrate content is deter-
mined by subtracting the fat, protein, water, and ash content of
the food from the total weight (difference method). This mea-
surement includes both ‘available carbohydrate’ (i.e., carbohy-
drate that can be digested in the small intestine) and dietary
fiber. Other countries, such as Great Britain and Australia,
measure ‘available carbohydrate’ directly using chromato-
graphic methods. The carbohydrate values from this latter
method do not include dietary fiber. Specific Atwater factors
used in the United States and Canada take into account some
(but not all) of the energy losses associated with dietary fiber
because they rely on the apparent digestibility of carbohydrate
to calculate food energy. This approach overestimates energy
availability from mixed diets. In Europe, the general Atwater
factor for carbohydrate (4 kcal g1, 16.7 kJ) is used to calculate
http://dx.doi.org/10.1016/B978-0-12-384947-2.00782-0

Figure 1 Diagrammatic representation of the energy losses associated
with various stages of digestion and the resulting determined energy
values.
carbohydrate energy after subtracting the dietary fiber content.
This method underestimates the energy of mixed diets. When
‘available carbohydrate’ is measured directly, one must also
measure dietary fiber and use a dietary fiber-specific energy
value to calculate the energy contribution from dietary fiber.
Digestible Energy and Metabolizable Energy:
Calculations
Digestible Energy
As noted earlier, the digestible energy of a substance is the
difference between the ingested energy and the energy excreted
in the feces. This is, in principle, a simple definition but gives
rise to two different digestible energy values depending on how
the measurement is made: the apparent digestible energy and
the partial digestible energy. We begin by defining IMs as the
ingested mass of a measured substance FMs as the amount of
the substance of interest. This substance can represent any
dietary material, but for the purposes of this article, we will
assume that it escapes digestion in the small intestine and
reaches the large intestine where it may (or may not) be fer-
mented. If we further define recovered in the feces, the appar-
ent digestibility (Dapp) can be calculated as
Dapp ¼ ðIMs  FMs Þ=IMs [1]
The apparent digestible energy value (DEapp) is simply the
apparent digestibility multiplied by the heat of combustion
(DHc) of the material:
Dietary Fiber: Energy Value 393
DEapp ¼ Dapp  DHc [2]
The DEapp takes into account only the energy loss associated
with incomplete digestion of a single food component and not
any potential interaction of the component with macronutri-
ents. If the food component is fermented, part of its energy
value will be captured and utilized by the host, part of the
remaining energy will appear in the feces, and part will be lost
to other processes (discussed in the succeeding text). Thus, the
DEapp overestimates the true digestible energy.
Partial digestible energy values (DEpart) describe the
influence of a dietary substance on the energy digestibility
of the whole diet. As such, they represent a more complete
picture of the energy intake and excretion associated with
ingestion of a particular food component. Calculating
DEpart values requires measurement of overall energy bal-
ance. The first step is to define the energy digestibility
(Denergy) of the overall diet as
Denergy ¼ ðIET  FEÞ=IET [3]
where IET is the total ingested energy and FE is the fecal energy.
Partial digestible energy values are determined by adding a
varying amount of a food component (as a supplement) to
an unchanging basal diet. The energy digestibilities of the
unsupplemented and supplemented diets are then determined
(eqn [3]) and are plotted as a function of the fraction of
ingested energy that is derived from the supplement (IES/IET).
If the supplement has a lower digestibility than that of the
overall diet, the graph should be a straight line with a negative
slope – the energy digestibility of the overall diet will decrease
with increasing amounts of the supplement. The partial indi-
gestibility of the supplement (SD) is given by


SD ¼ DDenergy =DðIES =IET Þ þ 1  Denergy, 0 [4]
where Denergy,0 is the energy digestibility of the basal (unsup-
plemented) diet. The DEpart is obtained by multiplying the
partial digestibility (1  the partial indigestibility) of the
supplement by its heat of combustion:
DEpart ¼ DHc  ð1  SD Þ [5]
Partial and apparent digestibilities can differ by a consider-
able amount. In general, DEpart < DEapp because DEpart
includes energy losses that are not accounted for by DEapp
measurements. This is illustrated by an experiment that com-
pared the DEapp and DEpart values for the fiber component of
hard red wheat bran (bran is
40% fiber). Measurement of the
diet and fecal fiber content and the diet and fecal energy
content gave a DEapp ¼ 1.82  0.17 kcal g1 (7.6 kJ g1) and a
DEpart ¼ 0.45  0.10 kcal g1 (1.9 kJ g1). The DEapp value
shows that, for every gram of wheat bran fiber ingested,
4.15  1.82 ¼ 2.33 kcal (9.7 kJ) of energy was lost to feces
in the form of fiber. The DEpart value shows that
1.82  0.45 ¼ 1.37 kcal (5.8 kJ) of extra energy was lost to the
feces in addition to that lost from fecal fiber excretion. Thus,
only 0.45/1.82  100% ¼ 25% of the energy of the fermented
wheat fiber was potentially retained by the rats (see later in text
for other calculations on the metabolizable energy value of
wheat fiber). These calculations show the importance of deter-
mining the total energy losses in a digestion experiment involv-
ing fermentable substances.
394 Dietary Fiber: Energy Value

Metabolizable Energy metabolizable energy must be employed for diets containing

Like digestible energy values, it is possible to define two differ-
ent metabolizable energy values. Livesey (1993) called the
Atwater metabolizable energy values ‘apparent metabolizable
energy’ values (MEapp) because they do not take into account
all the energy losses normally associated with metabolizable
energy measurements:
ME ¼ IE  ðFE þ UE þ GEÞ [6]
where UE is the urinary energy and GE is the gaseous energy.
If a strict parallel between digestible energy and metabolizable
energy is to be made, one could also define MEapp as
DEapp  (UE þ GE). In essence, this is the definition used by
Atwater with some assumptions. For fat and carbohydrate, it
was assumed that GE and UE were negligible. For protein, it
was assumed that GE was negligible but UE was not.
Partial metabolizable energy values (MEpart) can also be
calculated in a fashion analogous to DEpart values. In this
case, we define the total energy loss as
X researchers have called it a net metabolizable energy (NME).
Elosses ¼ FE þ GE þ UE [7]
Once again, we define the energy metabolizability of the
total diet as
 X  many more energy losses. The NME is a more practical measure
Menergy ¼ IET  Elosses =IET [8]
and use this value to define a partial energy ‘nonmetaboliz-
ability’ (SM):


SM ¼ DMenergy =DðIES =IET Þ þ 1  Menergy, 0 [9]
appreciable amounts of undigested and fermented carbohy-
drates as the greatest differences occur with these food compo-
nents. Many methods can be used to obtain the ME values of
food components that are not digested by the small intestine.
Whole-body calorimetric measurements (in a chamber where
an individual can be placed for experimentation) can be used
to determine the energy value directly, estimates of the meta-
bolizability can be obtained from mixed diets, body energy
measurements can be made in laboratory animals and related
back to the metabolizable energy of the food, and theoretical
calculations can provide a general model for estimating metab-
olizable energy.
Large differences exist between the energy losses associated
with the digestion of macronutrients (fat, carbohydrate,
and protein) and undigested and fermented carbohydrates.
Figure 2 shows an example of the various processes that
occur in the gastrointestinal tract during the consumption of
a partially digested and partially fermented food. Because the
final energy value is net of all major losses of energy, many
This should not be confused with the net energy values used for
livestock, which represent different energy measurements.
Also, it is not the net energy of Figure 1, which incorporates
of food energy for undigested and fermented substrates
because it takes into account energy losses that do not occur
for fat, carbohydrate, and protein. Use of this value would
allow comparisons between the energy values of different
food components.

where Menergy,0 is the energy metabolizability of the basal

(unsupplemented) diet. The MEpart is then given by
Mathematical Models for Undigested and Fermented
MEpart ¼ DHc  ð1  SM Þ [10] Compounds: Sugar Alcohols
Equations that minimize the error structure for experimen-
It is technically difficult and expensive to measure all stages of
tally determining DEpart and MEpart are also available.
the fermentation process and determine the energy losses
Like DEpart values, MEpart values describe the influence of a
part of the diet on the energy metabolizability of the whole
diet. For protein and fat, it is thought that MEpart > MEapp
because fecal excretion of bacterial nitrogen and fat will
decrease the MEapp value. Note that energy losses to bacterial
fat and nitrogen are not simply due to the loss of macronutri-
ent mass. Energy is required for the de novo bacterial synthesis
of fat- and protein-containing macromolecular structures,
which are then excreted in the feces. This energy is also lost.
For carbohydrates, it is thought that MEpart and MEapp are
equivalent since bacteria do not contain appreciable amounts
of carbohydrate. For carbohydrates that escape digestion in the
small intestine and are partially or completely fermented, like
those in dietary fiber, MEapp > MEpart because of the extra
energy losses that occur during fermentation. This energy loss
is the subject of this article.

Metabolizable or Net Energy? Figure 2 Scheme showing various stages of digestion of a

macronutrient that is partially digested in the small intestine and partially
The discrepancies between MEapp and MEpart energy values fermented in the large intestine. The letters represent fractions of the
mean that a more accurate system for measuring food initial amount that proceed down various pathways (see text).

associated with each step. This is true of human whole-body
calorimetric measurements since analysis requires a complete
energy balance profile for each subject (see succeeding text). A
more simplistic approach involves the construction of a math-
ematical model of the digestive events in the large intestine.
This approach has most commonly been applied to the energy
value of sugar alcohols but can also be extended to other
partially or completely fermentable carbohydrates.
Common dietary sugars (such as glucose, galactose, and
fructose) have a ketone or aldehyde group that allows cycliza-
tion to a hexose or pentose ring structure. Reduction of this
carbonyl group to an alcohol produces a sugar alcohol that
cannot cyclize. The resulting sugar alcohols are not well
absorbed in the small intestine so that a significant percentage
can pass intact into the large intestine. Isomalt (Palatinit®),
an equimolar mixture of two disaccharide alcohols (a-D-
glucopyranosyl-1,1-D-mannitol and a-D-glucopyranosyl-1,6-D-
sorbitol), is an example of a sugar alcohol that is virtually
unabsorbed in the small intestine. It passes intact into the
large intestine, where it is rapidly and completely fermented
by the bacterial population that resides there. As such, it
represents a model for a theoretical dietary fiber that is
completely fermented in the large intestine and can be used
as a basis for constructing a mathematical model.
A good mathematical model seeks to define all losses that
occur during fermentation of ingested material and assign rea-
sonable factors to these losses. Figure 2 presents the scheme for
such a model. Fermentable substances provide carbon units for
bacterial growth and reproduction and serve as metabolizable
substrates to meet the colonic bacterial population’s energy
requirements for maintenance and growth. Four major products
result from anaerobic colonic fermentation: increased bacterial
mass (factor a, Figure 2), heat energy loss due to fermentation
(factor b, Figure 2), methane and hydrogen gas (factor c,
Figure 2), and short-chain fatty acids (SCFAs). Humans can
only use SCFAs as an energy source. Thus, the metabolic energy
yield of the sugar alcohol has been reduced by the losses to
bacterial mass, as well as the heat and gas produced during
fermentation. It has also been reported that SCFAs may produce
significantly less ATP per gram of absorbed SCFA than glucose,
so an additional energy loss to this difference in metabolic
efficiency should also be included (factor d, Figure 2).
The fermentation model of Figure 2 can be expressed as an
equation that incorporates the four major processes by which
energy is lost during fermentation. The equation describing the
scheme of Figure 2, as it applies to sugar alcohols, was first
developed for the Nutrition Council of Holland and has been
recognized by several authors as an alternative method
for calculating the energy value of sugar alcohols. For partly
fermentable substances, it can be written as
NME ¼ DHc  ½ðA  BÞ þ a  ð1  A  CÞ  b [11]
where DHc is the heat of combustion of the substance in
question, A represents the fraction of the ingested substance
absorbed in the small intestine, B represents the fraction of
absorbed substance that is metabolized, and C represents the
fraction of the ingested substance that is excreted intact in feces
(Figure 2). The factor a represents the proportion of energy
derived from fermentation of the substance. It can be calcu-
lated from eqn [12]:
Dietary Fiber: Energy Value 395
a ¼ ð1  a  b  cÞ  d [12]
The factor b was not present in the original equation and
represents the extra energy lost due to the presence of the
fermentable carbohydrate. This can come about through bind-
ing to macronutrients, changes in osmotic balance, alterations
in transit time, changes in intestinal viscosity, inhibition of
digestive enzymes, inhibition of macronutrient uptake, or
increased sloughing of intestinal cells. As discussed earlier
(see section on DEpart and MEpart), measuring the appearance
of an undigested food component in the feces is not sufficient
to account for all of the energy losses that can occur when a diet
is supplemented with a fermentable carbohydrate. In a practi-
cal sense, it is impossible to differentiate between any ‘extra’
energy lost to the feces (b) and the energy lost due to increased
bacterial mass that results from fermentation (factor a). The
solution is to set b ¼ 0 in eqn [11] and measure total fecal
output.
Using the mathematical model of eqns [11] and [12]
requires a knowledge of factors A, B, and C, which are obtained
from experimental evidence. For sugar alcohols, values for
factor A have been obtained from ileostomy patients or by
direct sampling using multiple lumen tubes. Factor B (the
amount of sugar alcohol metabolized) has been estimated
indirectly by measuring urinary sugar alcohol content (the
amount of sugar alcohol not metabolized).
In the original factorial model paper, the Nutrition Council
of Holland held a to be constant for all sugar alcohols and
estimated it at 0.5. This value included fermentation energy
losses resulting from increased microbial mass excreted in the
feces (a  0.2), loss of H2 and CH4 (c ¼ 0.03–0.08), heat
produced by microbes (b ¼ 0.02–0.05), and higher heat loss
during utilization of SCFAs as compared with glucose
(d ¼ 0.80–0.85). Summing these factors gives a value of
0.40–0.53. The value of 0.5 was adopted because the Nutrition
Council of Holland did not want to suggest that the value was
known with high precision (they could have used 0.46–0.47 as
the average) and because they wanted to include some energy
lost to increased cell proliferation in the large intestine caused
by increased fermentation. Thus, the slightly higher value of
0.5 was chosen as an estimate of the total losses during fer-
mentation. In a later review of the value of a, it was determined
that the individual factors that contribute to a should give a
value closer to 0.6. However, in the same review, the author
noted that the factorial procedure overestimates energy values
to the extent that it takes no account of osmotic effects of the
sugar alcohol. Thus, the results of energy values calculated by
the Dutch method are probably fairly accurate. Many
researchers agree that the value of 0.5 represents a reasonable
estimate of the fermentation process.
For completely fermentable substrates that are not
absorbed by the small intestine, like sugar alcohols, the MNE
value is 0.5  DHc because A ¼ 0 and C ¼ 0. As indicated earlier,
the value of b is set to 0, and any ‘extra’ energy lost due to
ingestion is incorporated into the factor a, which has been
estimated at 0.5. Thus, for the sugar alcohol isomalt (which is
completely fermented and not absorbed by the small intes-
tine), the energy value is
1.9 kcal g1 (8 kJ g1, approxi-
mately half the heat of combustion). For completely
fermentable dietary fibers, the 0.5  DHc rule of thumb
396 Dietary Fiber: Energy Value

represents an upper limit of the NME since dietary fiber is
known to bind fats and cholesterol as well as inhibit carbohy-
drate absorption. Thus, dietary fiber may interact with other
macronutrients to decrease their metabolizable energy values.
This will give a lower NME value for dietary fiber since there
will be a higher excretion of fecal energy.
When all the energy losses are included, the energy value of
dietary fiber can be small compared with the ingested value.
For example, measurements with hard red spring wheat fiber
give NME values of
0.3 kcal g1, even though the total
ingested energy is
4 kcal g1. In the case of hard red spring
wheat bran, much of the energy is lost to excretion; wheat bran
dietary fiber is incompletely fermented so that
60% is lost
directly to the feces. In addition, a significant loss of energy
comes from dietary fiber interactions with macronutrients plus
other losses due to the presence of wheat bran fiber (b). Finally,
energy is lost during the fermentation process itself. The energy
value of hard red spring wheat fiber is not typical of other
dietary fibers, as shall be shown in the following sections
(Table 1).
Whole-body Calorimetric Measurements: Lactitol as
a Model
is estimated from nitrogen excretion, fat and carbohydrate
oxidation can be calculated as follows:
Nonprotein RQ ¼ 1:00  carbohydrate oxidation
þ 0:707  fat oxidation [14]
However, caution is needed when estimating fuel oxidation
from nonprotein RQ values, since the RQ itself can vary con-
siderably depending on the fat and protein mixture being
oxidized. In addition to the problem of obtaining an accurate
picture of fat and carbohydrate oxidation from nonprotein RQ
values, it is also not possible to distinguish between the oxida-
tion of carbohydrate þ fat and the oxidation of SCFAs pro-
duced by carbohydrate fermentation. When measuring the
apparent metabolizable energy value of a macronutrient that
represents a small proportion of the total diet, there is a high
potential for error when macronutrient oxidation profiles are
measured. However, theoretical calculations show that there is
much less error associated with measuring energy loss.
An example of the use of indirect calorimetry for measuring
the metabolic energy of completely fermentable substances is
the determination of the net energy value for lactitol, a disac-
charide sugar alcohol (4-O-(b-D-galactopyranosyl)-D-sorbitol).
The total energy balance was measured during the experimen-
tal period in the calorimeter. The retained energy of each
subject was then calculated as

Indirect calorimetry (a type of whole-body calorimetry where REðretained energy Þ ¼ IET

all gas consumption and gas exhalation are continuously mon-
itored) can be used to determine energy expenditure through
the use of equations relating total O2 consumption, CO2 expi-
ration, and urinary nitrogen excretion to energy utilization.
One such equation is
Heat production ¼ 16:175 O2 þ 5:021 CO2
 5:987 urea N  4:5 H2
In addition to total energy production, indirect calorimetry
provides information on macronutrient utilization through
the nonprotein respiratory quotient (RQ). The RQ is the ratio
of CO2 expired to O2 consumed during a defined period. The
theoretical RQ values are known for fat, carbohydrate, and
protein oxidation, making it possible to determine total fat,
carbohydrate, and protein oxidation. When protein oxidation
 ðfecal E þ urine E þ gaseous E þ expended EÞ
[15]
The energy needed to maintain energy balance (metaboliz-
able energy for maintenance; MEm) could then be determined
as
[13] MEm ¼ metabolizable energy intake þ negative RE
 1:11  positive RE [16]
The factor 1.11 represents the cost of depositing body
energy and was measured in an earlier experiment. The differ-
ence in MEm on a diet supplemented with sucrose versus one
supplemented with lactitol is a measure of the energy lost from
lactitol and can be expressed as the change in MEm adjusted for
the metabolic weight of the individuals (weight0.75):

Table 1 Potential error associated with oxidation of 50 g of a carbohydrate versus fermentation and oxidation of the released short-chain fatty acidsa
c
Substrate RQ Oxidation substrates (mol)b O2 consumed (mol) Heq kcal per 50 g

50 g of sucrose 1.00 0.146 CHO 1.753 5.012 250.6

50 g of lactitol 0.943 0.409 (CHO) þ 0.099 (fat) 1.113 (CHO) þ 0.269 (fat) 4.981 (CHO) þ 4.682 (fat) 153.1
50 g of lactitol 0.943 0.508 SCFA 1.382 4.663 144.5
a
The table illustrates the potential error involved in measuring the apparent metabolic energy value of a completely fermentable supplement (lactitol) by indirect calorimetry. This was
done by calculating the apparent metabolic energy value using two different methods. It was assumed that lactitol was fermented to a mixture of SCFAs followed by complete oxidation
to CO2 and water plus fermentation gases. In an indirect calorimeter, oxidation of SCFAs can be mistaken as oxidation of fat and carbohydrate in a 0.195:0.808 ratio. Oxidation of these
substrates would give 153.1 kcal per 50 g. Oxidation of the SCFA directly would give 144.5 kcal per 50 g of original lactitol. Thus, an error of
6% can occur when indirect
calorimetry is used to measure the metabolic energy value of lactitol. The yield of SCFAs from lactitol and the proportion of acetate, propionate, and butyrate (115.1:35.38:24.5 per
100 mol of glucose) represent weighted averages of literature values for ruminants. As such, these calculations approximate the actual error associated with indirect calorimetry, which
varies from individual to individual.
b
Equivalent mol of sucrose or SCFAs obtained from 50 g of substrate.
c eq
H values from Livesey, G. and Elia, M. (1988). Estimation of energy expenditure, net carbohydrate utilization, and net oxidation and synthesis by indirect calorimetry: evaluation of
errors with special reference to the detailed composition of fuels. American Journal of Clinical Nutrition 47: 608–628. The Heq value for glucose was used for carbohydrate and the Heq
value for the fat mixture of an omnivore diet was used for fat. The Heq value for SCFAs represents a weighted average.




DMEm ¼ MEm =weight0:75 lactitol  MEm =weight0:75 sucrose [17]
and the NME can then be calculated as


NME ¼ MEpart  DMEm = IS=weight0:75 lactitol [18]
where MEpart is obtained from eqn [10] and IS is the ingested
mass of the supplement (lactitol). The MEm values are divided
by the metabolic weight to account for differences in metabolic
mass. Using this procedure, a value of 2.4 kcal g1 (10 kJ g1)
for lactitol was obtained. Note that some measure of the energy
expenditure is required in this experiment (eqn [15]). The
authors relied on diaries and ankle actometers and corrected
to equal actometer counts. They acknowledged that this was
not very satisfactory; there is a potential 15% error in the final
net energy value for lactitol.
Metabolizable Energy Value of Dietary Fiber in
Mixed Diets
Up to this point, this article has focussed on undigested and
fermented food components as a general group of substances.
This section will deal specifically with dietary fiber, a partly
fermented food component that passes through the small
intestine unacted upon by human digestive enzymes. Dietary
fiber is a mixture of nonstarch polysaccharides and lignin that
are structural components of plant cell walls.
As indicated earlier, it is not appropriate to use metaboliz-
able energy values for dietary fiber since many more energy
losses are associated with fiber digestion as compared with the
digestion of fat, carbohydrate, and protein. Equations have been
developed to take account of these extra losses and provide a
method to calculate the NME value of a fermentable dietary
component when the fecal energy losses are known. These
equations show that approximately one-half of the potential
energy is available to the host when undigested carbohydrates
are completely fermented. This value is widely accepted.
Knowledge of the energy yield from the fermented portion of
dietary fiber is not sufficient in and of itself to allow the calcu-
lation of dietary fiber NME since a significant proportion of the
‘fermentable’ dietary components are not fermented but appear
in the feces. In addition, some ‘extra’ energy (factor b, eqn [13])
may be lost during digestion because of the intimate physical
interaction between dietary fiber and the food matrix. The aim
of these studies is to determine a general NME value for dietary
fiber that can be used to calculate the metabolizable energy of
mixed human diets. To overcome these problems, two solutions
have been employed. The first solution involved determining
individual digestibility factors for specific foods and using these
to calculate metabolizable energy intake. This method has the
advantage that macronutrient digestibility is measured directly
for specific foods and multiplied by the specific heats of com-
bustion when available. In this method, the calculated digestible
energies of the diets are close to the measured values for mixed
diets since the final value reflects the sum of the individual
components. This method has some problems. In the system
of Merrill and Watt, the digestibility of carbohydrates was deter-
mined by difference, so accurate values for carbohydrate digest-
ibility are lacking. This also means that a digestibility value for
the fermentable food component portion of the carbohydrate
Dietary Fiber: Energy Value 397
fraction (as determined by difference) is lacking. It is, therefore,
not possible to calculate the metabolic energy of a food of
known composition since values for undigested and fermented
components are not available. This limits the usefulness of this
system to foods that have been measured. In addition to this
problem, the NME value is appropriate for undigested and
fermented food components, whereas the digestible energy
value is not. Thus, this system does not allow any correction
for energy lost when food components are resistant to digestion
but fermented in the lower gut.
The second solution is to use an average digestibility value
determined by examining typical mixed diets. This can be
calculated by two separate methods: using a factorial approach
and using an empirical model. Factorial models relate total
protein, fat, carbohydrate, and dietary fiber intake to digestible
energy through an equation of the type:
MEðkcaÞ ¼ 4:0  proteinðgÞ þ 9:0  fatðgÞ þ 3:75
 carbohydrateðgÞ þ NMEDF
 dietary fiberðgÞ [19]
In the development of eqn [19], carbohydrate was deter-
mined directly (not determined by difference) and is expressed
as monomeric units. Dietary fiber must also be measured
directly through an approved, reproducible method that is
not affected by food processing. The factors 4 (protein) and 9
(fat) are the general Atwater factors derived from mixed diets.
The value of 3.75 for carbohydrate is slightly lower than the
general Atwater factor of 4. This reflects the fact that the carbo-
hydrate is expressed as the total monomeric weight.
The value of NMEDF (the NME for dietary fiber) comes from
an analysis of the dietary fiber digestibility of mixed diets and
the loss of energy to feces in mixed Western diets. This value is
determined as follows. Firstly, the DEpart value for dietary fiber
is obtained by comparing the digestible energies of mixed diets
to the metabolizable energy calculated by eqn [19]. The appar-
ent digestible and metabolizable energies for carbohydrate and
fat are identical since the macronutrient digestibilities were
accounted for, and urine and gaseous losses of these compo-
nents were negligible. Protein losses are corrected for urine
nitrogen and protein digestibilities. The digestible energy for
dietary fiber (DE dietary fiber) was estimated by
DE dietary fiber ¼ DHc  Dapp  ð1  aÞ
¼ 4:1 kcal g1  0:7  0:7


¼ 2:0 kcal g1 8:4 kJ g1 [20]
The apparent digestibility of dietary fiber is around 0.7. The
value of factor a in eqn [20] needs to be defined for dietary fiber.
For sugar alcohols, the value is
0.2 (see preceding text). A value
of 0.3 has been adopted for dietary fibers in humans. This value
reflects the fact that the majority of energy loss estimates
lie between 0.2 and 0.4 kcal kcal1 of fermented energy. As
already mentioned, a value of 0.2 was obtained when measuring
lactitol energy utilization in human subjects. It has also been
suggested that between 20% and 30% of the energy in undi-
gested and fermented carbohydrates is mikuutilized by bacteria
in humans. A value of 0.2 kcal kcal1 of fermented material has
been measured in rats fed with an a-amylase-resistant pea starch,
and a value of 0.4 kcal kcal1 of fermented material has been
measured in rats fed with guar gum. In human diets with high
intakes of dietary fiber, an increased fecal energy output of 0.3
398 Dietary Fiber: Energy Value
and 0.39 kcal kcal1 of energy fermented, respectively, has been
measured. Overall, these data suggest that about 0.3 kcal of
energy is lost to the feces per kcal of energy fermented. Thus,
1.0  0.3 ¼ 0.7 kcal kcal1 is retained.
As indicated earlier, the digestible energy value does not
give an accurate picture of the energy losses that occur during
fermentation of undigested food components. Correction fac-
tors must be included for energy losses to the heat of fermen-
tation (factor b), losses to fermentation gases (factor c), and the
relative efficiency of utilization of SCFAs (factor d). Correcting
the digestible energy for these values gives
NMEDF ¼ DHc  Dapp  0:54
¼ 4:1 kcal g1  0:7  0:54 ¼ 1:5 kcal g1 [21]
Equation [21] shows that the NME value for dietary fiber is
approximately 1.5 kcal g1. Equation [21] applies strictly to
dietary fiber because the DHc value and digestibilities were
determined for dietary fiber. It is useful for calculating the
metabolic energy values of mixed diets because the digestibil-
ities were determined for mixed diets. It is comparable,
therefore, with the general Atwater factors of 4, 9, and 4 for
carbohydrate, fat, and protein.
Empirical models represent another method for determin-
ing total energy intake. Empirical models are, in general, supe-
rior to the factorial models for a number of reasons, the most
important being that the empirical models are much more
robust to errors in carbohydrate, fat, and protein determina-
tions as well as to the use of general Atwater factors. An exam-
ple of an empirical equation is as follows:
ME ¼ 0:96  total energy ðkcalÞ  2:5 kcal g1
 dietary fiberðgÞ  12 kcal g1
 ingested nitrogenðgÞ [22]
Equation [22] relates the metabolizable energy to total
energy, protein, and dietary fiber only. The factor 0.96 repre-
sents the metabolizability of mixed diets in the absence of
dietary fiber. The value of 2.5 kcal g1 represents the NME
energy loss associated with ingestion of dietary fiber. This
value is therefore the difference between the heat of combus-
tion of dietary fiber and its NMEDF value (determined in the
preceding):
2:5 kcal g1 ¼ DHc  NMEDF
¼ 4:0 kcal g1  1:5 kcal g1 [23]
A version of eqn [22] was tested for bias and accuracy of
prediction by comparison with 43 human diets containing
varied intakes and sources of dietary fiber. The equation
showed no bias over a wide range of dietary fiber intake
(2–93 g daily). By necessity, this test was performed by com-
paring DEpart values because MEpart values cannot be measured
directly for fermentable food components. This analysis pro-
vides further evidence for a general NME for dietary fiber of
1.5 kcal g1.
Overall Conclusion
Eating food components that are not digestible but that are
fermentable lowers the amount of metabolizable and
digestible energy available from food. This occurs because of
two different processes. Fermentable food components, like
dietary fiber, are not 100% fermentable, and so the NME for
these components is lower than that of protein, carbohydrate,
and fat. In addition, fermentation itself is associated with many
different energy losses, including losses to increased bacterial
mass, the heat of fermentation, fermentation gases, and a lower
efficiency of host utilization of SCFAs. Because the digestion of
fermentable food components is so different from that of
macronutrients, special equations are needed to determine
the NME yield.
It is possible to utilize data from several sources to obtain
an estimate of the relative losses that occur during fermentative
digestion and back-correct to obtain an unbiased estimate of
the energy value of dietary fiber. Taking into account all these
energy losses, an NME value for dietary fiber of 1.5 kcal g1
(6.3 kJ g1) has been calculated. This appears to accurately
predict energy losses associated with mixed Western diets
when analyzed using either factorial or empirical models.
Thus, the NME of 1.5 kcal g1 (6.3 kJ g1) can be used in a
fashion similar to the general Atwater factors of 4, 9, and 4 for
protein, fat, and carbohydrate to predict the metabolizable
energy of mixed Western diets. Individually measured NME
values for fiber in whole foods may differ from this value
since dietary fiber is fermented to different extents and is
intimately associated with the food matrix, which may limit
macronutrient digestibility. Thus, individual measurements
are needed for direct comparison and may be required for
specific types of foods.
See also: Dietary Fiber: Bran; Dietary Fiber: Determination; Dietary
Fiber: Physiological Effects.
Further Reading
Bär A (1990) Factorial calculation model for the estimation of the physiological caloric
value of polyols. In: Hosoya N (ed.) Proceedings of the international symposium on
caloric evaluation of carbohydrates, research foundation for sugar metabolism,
Tokyo 209–257.
Bernier JJ and Pascal G (1990) Valeur énergétique des polyols (sucres-alcools).
Médicine et Nutrition XXVI: 221–238.
British Nutrition Foundation (1990) Complex carbohydrates in foods. The Report of the
British Nutrition Foundation’s Task Force. London: Chapman & Hall, pp. 56–66.
Burton-Freeman B (2000) Dietary fiber and energy regulation. Journal of Nutrition
130(2): 272S–275S.
Buttriss JL and Stokes CS (2008) Dietary fibre and health: an overview. Nutrition
Bulletin 33(3): 186–200. http://dx.doi.org/10.1111/j.1467-3010.2008.00705.x.
Calloway DH and Kretsch MJ (1978) Protein and energy utilization in men given a rural
Guatemalan diet and egg formulas with and without added oat bran. American
Journal of Clinical Nutrition 31: 1118–1126.
Cummings JH (1983) Fermentation in the human large intestine: evidence and
implications for health. Lancet 1: 1206–1208.
FASEB (1994) The evaluation of the energy of certain sugar alcohols used as food
ingredients life sciences research office. Bethesda, MD: Federation of American
Societies for Experimental Biology (FASEB).
Goranzon H, Forsum E, and Thilen M (1983) Calculation and determination of
metabolizable energy in mixed diets to humans. American Journal of Clinical
Nutrition 38: 954–963.
Johnson L, Mander AP, Jones LR, Emmett PM, and Jebb SA (2008) A prospective
analysis of dietary energy density at age 5 and 7 years and fatness at 9 years among
UK children. International Journal of Obesity 32: 586–593. http://dx.doi.org/
10.1038/sj.ijo.0803746, published online 2 October 2007.

Kristensen M and Jensen MG (2011) Dietary fibres in the regulation of appetite and food
intake. Importance of viscosity. Appetite 56(1): 65–70.
Livesey G (1989) Procedure for calculating the digestible and metabolizable energy
values of food components making a small contribution to dietary intake. Journal of
the Science of Food and Agriculture 48: 475–481.
Livesey G (1990) Energy values of unavailable carbohydrate and diets: an inquiry and
analysis. American Journal of Clinical Nutrition 51: 617–637.
Livesey G (1991) Calculating the energy values of foods: towards new empirical
formulae based on diets with varied intakes of unavailable complex carbohydrates.
European Journal of Clinical Nutrition 45: 1–12.
Livesey G (1992) The energy values of dietary fibre and sugar alcohols for man.
Nutrition Research Reviews 5: 61–84.
Livesey G (1993) Comments on the methods used to determine the energy values of
carbohydrates: dietary fibre, sugar alcohols and other bulking agents. International
Journal of Food Sciences and Nutrition 44: 221–241.
Livesey G (1995) Metabolizable energy of macronutrients. American Journal of Clinical
Nutrition 62(Suppl.): 1135S–1142S.
Livesey G and Elia M (1988) Estimation of energy expenditure, net carbohydrate
utilization, and net oxidation and synthesis by indirect calorimetry: evaluation of
errors with special reference to the detailed composition of fuels. American Journal
of Clinical Nutrition 47: 608–628.
Dietary Fiber: Energy Value 399
Merrill AL and Watt BK (1973) Energy value of foods. Basis and derivation. Agriculture
handbook #74. Washington, DC: US Department of Agriculture and US Department
of Health and Human Services.
Mongeau R and Brassard R (1993) Enzymatic–gravimetric determination in foods of
dietary fibre as sum of insoluble and soluble fibre fractions: summary of
collaborative study. Journal of AOAC International 76: 923–925.
Mongeau R, Brooks SPJ, Lampi BJ, and Brassard R (1997) Hard wheat bran and hard
wheat bran fiber energy values measured in rats after 6 and 16 weeks.
In: Kritchevsky D and Bonfield C (eds.) Dietary fiber in health and disease,
pp. 267–289. New York: Plenum Press.
Nutrition Council of Holland (1987) The energy value of sugar alcohols. The Hague,
Netherlands: Recommendations of the Committee on Polyalcohols.
Pérez-Escamilla R, Obbagy JE, Altman JM, et al. (2012) Dietary energy density and
body weight in adults and children: a systematic review. Journal of the Academy of
Nutrition and Dietetics 112(5): 671–684.
van Es AJH, de Groot L, and Vogt JE (1986) Energy balances of eight volunteers fed on
diets supplemented with either lactitol or saccharose. British Journal of Nutrition
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subjective appetite, energy intake and body weight: a systematic review of
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 Dietary Fiber: Physiological Effects
IT Johnson, Institute of Food Research, Norwich, UK
ã 2016 Elsevier Ltd. All rights reserved.
This article is a revised version of an article originally published from the Encyclopedia of Food Sciences and Nutrition, volume 3, pp. 1833–1837,
ã 2003, Elsevier Science Ltd., with an updated Bibliography section supplied by the editor.
Introduction
The term ‘crude fiber’ dates from the nineteenth century, when
it was used to describe the material that remained after rigorous
nonenzymatic hydrolysis of animal feedstuffs. The importance
of similar undigestible residues in human foods was recognized
in the early 1970s by the physician and epidemiologist Hugh
Trowell, but he also argued that the crude fiber figures then
available for foods had little physiological significance and
were of no value in the context of human diets. He therefore
coined the term ‘dietary fiber’ for what he and others came to
define as “The sum of lignin and the plant polysaccharides that
are not digested by the endogenous secretions of the mamma-
lian digestive tract.” In practice, these residues contain resistant
starch molecules, but for many years, a narrower definition,
which included only ‘nonstarch polysaccharides’ (NSP), found
favor, particularly in the United Kingdom. However, the most
recent CODEX definition (2009) states that “Dietary fiber
means carbohydrate polymers with 10 or more monomeric
units, which are not hydrolyzed by the endogenous enzymes
in the small intestine of humans.” This definition, which has
been widely adopted internationally, includes both resistant
starch molecules and cell wall polysaccharides, together with
lignin and a few other minor components. Certain isolated or
synthetic polysaccharides can also be included in the defini-
tion, so long as they have been shown to exert beneficial phys-
iological effects. Dietary fiber thus includes a large and complex
mixture of polysaccharides that share few properties other than
resistance to digestive enzymes. The presence of undigested cell
wall fragments and dispersed polysaccharides in the intestinal
lumen can alter physiological processes throughout the gut, but
the effects of different fiber components depend upon their
varied physical and chemical properties during digestion and
their varying susceptibilities to degradation by bacterial
enzymes in the colon. It is important to realize that a single
analytic value for the fiber content of a food is a poor guide to its
physiological effects. This article will review the main mecha-
nisms of action of resistant polysaccharides in the alimentary
tract and their implications for human health.
Fiber in the Alimentary Tract
Food is conveyed through the alimentary tract by the rhythmic
muscular activity of peristalsis. Digestive enzymes are released
into the lumen at intervals to facilitate the breakdown of
macromolecules into fragments that can then be absorbed
and utilized by the body. The earliest stages of digestion
begin in the mouth, where food particles are reduced in size
and lubricated with saliva, which also contains the digestive
enzyme salivary amylase. In the stomach, any large particles
that remain are broken down by rigorous muscular activity in
400 Encyclopedia of Food and Health
the presence of hydrochloric acid and proteolytic enzymes. The
need to disrupt and disperse intractable food particles and cell
walls delays gastric emptying, and since cell wall polysaccha-
rides help to determine the texture of many plant foods, and
hard foods tend to be chewed more thoroughly than soft
foods, the presence of dietary fiber in unrefined foods may
begin to regulate digestion at a very early stage. Apples, for
example, tend to be consumed more slowly than apple juice,
and the absorption of sugar from apples is also slower. Simi-
larly, the rate at which the products of starch digestion are
absorbed from cubes of cooked potato has been shown to be
much slower when they are swallowed whole than when they
are chewed normally. The nutritional significance of such
effects is not entirely clear, but they may limit the rate at
which glucose from some foods enters the circulation.
Small Intestine
The small intestine is the largest of the digestive organs, with
the greatest surface area, as befits its role as the main site of
nutrient absorption. The semiliquid products of gastric diges-
tion are released at intervals into the small intestine and pro-
pelled away from the gastroduodenal junction by peristaltic
movements at about 1 cm min 1. The hydrolysis of proteins,
triglycerides, and starch continues within the duodenum and
upper jejunum, under the influence of pancreatic enzymes.
The final stages of digestion occur at the mucosal surface,
where the products are absorbed into the circulation, along
with water and electrolytes, via the specialized epithelial cells
of the intestinal villi. Muscular activity in the small intestinal
wall, together with rhythmic contractions of the villi, ensures
that the partially digested chyme is well stirred. In adults, the
first fermentable residues from a meal containing complex
carbohydrates enter the colon  4.5 h after ingestion. When a
solution containing indigestible sugar is swallowed without
food, it reaches the colon about 1.5 h earlier than when the
same material is added to a solid meal containing dietary fiber.
The effects of dietary fiber on transit through the small intes-
tine are complex and difficult to study. Experiments with
human subjects suggest that though coarse bran added to
experimental meals delays gastric emptying, it may accelerate
small intestinal transit. However, there is also some evidence
that phase separation occurs, so that bran may accumulate in
the distal ileum without any delay in transit of the liquid phase
to the colon. Viscous polysaccharides such as guar gum appear
to have little effect on small intestinal transit in humans.
Carbohydrate Metabolism
In his early work, Trowell proposed that dietary fiber was a
major factor in the prevention of type 2 diabetes. Indeed, he
http://dx.doi.org/10.1016/B978-0-12-384947-2.00783-2

argued that this condition was largely unknown in Western
Europe prior to the introduction of mechanized flour milling
because until then, the consumption of unrefined carbohydrate
foods had favored slow absorption of glucose, which placed less
strain upon the ability of the pancreas to maintain glucose
homeostasis. Whatever its merits, this hypothesis stimulated a
great deal of research on the effects of cell wall polysaccharides
on the digestion and absorption of carbohydrates, and this has
undoubtedly shed light on the role of dietary fiber in the man-
agement of diabetes. The ‘glycemic index’ (GI) quantifies the
appearance of glucose in the bloodstream after ingestion of a
carbohydrate-rich food. To determine the GI for a specific food,
fasted subjects are given a test meal containing a standardized
quantity of carbohydrate. The area under the blood-glucose
curve in response to the test meal, divided by that produced by
an equal quantity of a reference carbohydrate such as glucose,
gives a ratio that, expressed as a percentage, is the GI. When
glucose is used as the standard, unrefined foods containing
dietary fiber often have a GI substantially lower than 100%.
The resistance of plant cell walls to digestion during passage
through the gut varies considerably from one food to another,
and any cell walls that remain intact in the small intestine will
impede the access of pancreatic amylase to starch. Even when
enzymes and their substrates do come into contact, the presence
of cell wall polysaccharides may slow the diffusion of hydrolytic
products through the partially digested matrix in the gut lumen.
Pulses tend to give particularly low GI values, probably because
legume seeds have relatively thick cell walls that resist destruction
during processing and cooking. This particular effect of fiber on
GI cannot be predicted from simple analytic values for total fiber
because it reflects the structure, as opposed to the absolute quan-
tity, of cell wall polysaccharides within the food.
Many studies on postprandial glycemia have been con-
ducted using isolated fiber supplements added to glucose test
meals or to low-fiber sources of starch. They demonstrate that,
contrary to Trowell’s original hypothesis, wheat bran and other
insoluble cell wall materials have little effect on glucose metab-
olism. However, certain polysaccharides, such as guar gum,
pectin, and oat beta-glucan, which form viscous solutions in
the stomach and small intestine, do reduce the GI of foods to
which they are added. Highly viscous food components may
delay gastric emptying but the primary mechanism of action
appears to be suppression of convective stirring in the fluid
layer adjacent to the mucosal surface. The rapid uptake of
monosaccharides by the epithelial cells tends to reduce the
concentration of glucose in this boundary layer, so that absorp-
tion from the gut lumen becomes rate-limited by the relatively
slow process of diffusion. The overall effect is to delay the
assimilation of glucose and hence suppress the glycemic
response to glucose or starchy foods. A similar mechanism
probably inhibits the reabsorption of cholesterol and bile
salts in the distal ileum, and this may account for another
important physiological effect of viscous polysaccharides
such as oat beta-glucan, which is to reduce plasma cholesterol
levels in humans.
Mineral Metabolism
Polysaccharides and phenolic components of cell walls often
contain polar groups that can interact with ionized solutes in
the gastrointestinal contents. Such molecular binding effects
Dietary Fiber: Physiological Effects 401
do occur in vitro and might, in principle, restrict the availability
of nutrients and other substances for absorption in the small
intestine. Iron, zinc, and calcium are absorbed relatively poorly
from the human diet, and there have been some concerns that
high intakes of dietary fiber might exert an adverse effect on
human mineral nutrition. However, studies with subjects who,
for clinical reasons, have had the colon removed and the small
bowel brought to the abdominal surface (ileostomists) suggest
that neutral polysaccharides do not bind minerals to any sig-
nificant extent. Charged polysaccharides such as pectin can
bind mineral cations, but fermentation of cell walls in the
colon probably releases iron and calcium into the lumen,
whence they may be salvaged by colonic absorption. There is
little objective evidence that dietary fiber per se has an adverse
effect on mineral metabolism. Furthermore, consumption of
highly fermentable oligosaccharides such as inulin has been
shown to improve calcium absorption in humans and animal
models, apparently because the production of short-chain fatty
acids stimulates active mineral transport in the colon.
The effect of phytate (myoinositol hexaphosphate) is gen-
erally considered to be more significant than that of fiber.
Phytate is often present in close association with cell wall
polysaccharides in unprocessed legume seeds, oats, and other
cereals. Phytate does exert a potent binding effect on minerals
and has been shown to reduce the availability of magnesium,
zinc, and calcium for absorption in trials with human volun-
teers. Phytate levels in foods can be reduced by the activity of
endogenous phytase, by hydrolysis with exogenous enzymes,
or by fermentation. Dephytinized products may be of benefit
to individuals at risk of suboptimal mineral status.
Large Intestine
The large intestine, which is located distal to the small bowel,
salvages energy from food residues and from endogenous pro-
teins and carbohydrates that have escaped digestion and
absorption. Its other main role is to form and store feces.
Fecal microorganisms degrade undigested starch and many of
the polysaccharides that comprise dietary fiber, to yield the
volatile fatty acids butyrate, propionate, and acetate. Whereas
the foregut of ruminants is adapted to provide a watery,
nutrient-rich environment for bacterial fermentation, it is the
colon that provides the main site of permanent bacterial colo-
nization in the human alimentary tract. The development of
rapid and inexpensive gene sequencing technology has revolu-
tionized the study of this gastrointestinal microbiota in recent
years, and the gut microbiome, as it is now known, derives
much of its nutrition from dietary fiber. The metabolic activi-
ties of the gut bacteria are now thought to exert a major
influence on human metabolic health, and the effects of diet
on the composition of the microbiome are a major focus of
interest.
Absorption and metabolism of short-chain fatty acids
derived from carbohydrate fermentation provides an impor-
tant route for the recovery of energy from undigested poly-
saccharides. Butyrate functions as a major fuel for the colonic
mucosal cells, whereas propionate and acetate are absorbed
and metabolized systemically. The other major breakdown
products of carbohydrate fermentation are hydrogen, meth-
ane, and carbon dioxide, which together comprise flatus gas.
402 Dietary Fiber: Physiological Effects
Excess gas production is said to cause distention and pain in
some individuals, especially those attempting to increase their
fiber consumption, but this is probably caused more by fer-
mentation of oligosaccharides such as stachyose and verbas-
cose, found, for example, in legume seeds, rather than the cell
wall polysaccharides themselves. The proximal colon contains
about 200 g of dilute fecal material, portions of which are
transferred at regular intervals from the right colon into the
transverse and distal segments for partial dehydration and
storage. The pattern of motility in the large intestine is similar
in principle to that of the rest of the alimentary tract, but the
rate of transit is slower. In healthy individuals, stools are
passed with a frequency varying from once or twice a day to
once every 2–3 days.
Fecal Bulk and Composition
The ability of dietary fiber to prevent cancer and various degen-
erative diseases of the alimentary tract was proposed by Denis
Burkitt, who based his hypothesis largely on the concept of
fecal bulk. His field observations in Africa, where cancer and
other chronic bowel diseases were rare, suggested that popula-
tions consuming traditional rural diets rich in vegetables and
cereal foods produced bulkier, more frequent stools than per-
sons living in the industrialized West. Burkitt argued that con-
sumption of highly processed cereals in industrialized societies
led to chronic constipation and that this caused prolonged
high pressures both within the colonic lumen and also within
the lower abdomen as a result of straining to pass hard stools.
This in turn was thought to increase the risk of various diseases
of muscular degeneration including varicose veins, hemor-
rhoids, hiatus hernia, and colonic diverticula. Furthermore,
infrequent defecation was thought to cause prolonged expo-
sure of the colonic epithelial cells to mutagenic chemicals that
could initiate cancer. Although Burkitt’s overall hypothesis for
the beneficial effects of fecal bulk is undoubtedly an oversim-
plification, it has never been comprehensively refuted. There is
relatively strong epidemiological evidence for a protective
effect of dietary fiber against colorectal cancer, and although
there is no conclusive evidence from randomized controlled
trials, this association provides one of the main reasons for
recommendations to maintain relatively high levels of fiber
consumption in the United Kingdom (30 g day 1 in adults)
and elsewhere.
It is certainly true that the consumption of dietary fiber is a
major determinant of both fecal bulk and bowel habit, but the
magnitude of the effect depends upon the type of fiber con-
sumed. Soluble cell wall polysaccharides such as pectin are
readily fermented by the microflora, whereas lignified tissues
such as wheat bran tend to remain partially intact in the feces.
Both classes of dietary fiber can contribute to fecal bulk but by
different mechanisms. The increment in stool mass caused by
wheat bran depends to some extent on particle size, but in
healthy Western populations, it has been shown that for every
1 g of wheat bran consumed per day, the output of stool is
increased by between 3 and 5 g. Other sources of dietary fiber
also favor water retention. For example, ispaghula, which is a
mucilaginous material derived from Psyllium, is used pharma-
ceutically as a bulk laxative. Soluble polysaccharides such as
guar and oat b-glucan are readily fermented by anaerobic
bacteria, but solubility is no guarantee of fermentability, as is
illustrated by modified cellulose gums such as methylcellulose,
which is highly resistant to degradation in the human gut.
Fermentation reduces the mass and water-holding capacity of
soluble polysaccharides considerably, but the bacterial cells
derived from them do make some contribution to total fecal
output. Thus, although all forms of dietary fiber exert some
fecal bulking effects, their magnitude cannot be deduced from
a single analytic measurement of total fiber content.
Apart from increasing the availability of energy from dietary
fiber, the major effect of bacterial fermentation is to regulate
the physical and chemical properties of the intraluminal envi-
ronment. The production of metabolites by the fecal micro-
biome is strongly influenced by both the level and type of
dietary fiber consumed by the host, and it is conjectured that
a high consumption of fiber favors a less carcinogenic chemical
environment in the fecal stream. Most colorectal carcinomas
develop progressively from precancerous lesions called adeno-
matous polyps. The gradual transition from a normal crypt via
a precancerous lesion to a malignant tumor is associated with a
progressive loss of differentiation, deregulation of cell growth,
and an accumulation of mutations in genes associated with the
control of cell proliferation and death. These somatic muta-
tions are assumed to be caused primarily by mutagenic chemi-
cals in the feces that have been shown to be carcinogenic in
animals. These include heterocyclic aromatic amines created
during the cooking of meat at high temperatures and
N-nitrosamines derived by bacterial metabolism from dietary
proteins. It has not been established conclusively that these
substances cause colorectal carcinogenesis in humans, but
there is strong circumstantial evidence that they do and it is
prudent to adopt nutritional strategies to reduce their concen-
tration in the colon.
The realization that colorectal carcinogenesis is a prolonged
multistage process involving changes to a complex array of
genes raises new questions about interactions between the
colonic epithelial cells, dietary fiber, and other constituents of
the fecal stream. Bile acids, which are produced by the liver and
secreted into the gut lumen, where they facilitate the digestion
and absorption of fat, are known to increase the rate at which
colonic mucosal cells divide. This is particularly true of second-
ary bile acids (e.g., deoxycholic acid and lithocholic acid) pro-
duced by bacterial metabolism of primary bile acids in the
colon. It has been proposed that bile salts act as tumor pro-
moters in the human colon, accelerating the carcinogenic pro-
cess initiated by fecal mutagens. High-fat diets tend to stimulate
the release of bile acids and increase their concentration in the
feces, and this may partially explain the adverse effects of
Western diets on the risk of bowel cancer in human popula-
tions. However, increased fecal bulk will tend to dilute the
concentration of bile salts and reduce their residence time.
Furthermore, recent studies suggest that fermentation of dietary
fiber shifts the composition and metabolic activities of the fecal
microbiome so as to inhibit the production of secondary bile
acids. The nonfermentable particulate components of plant cell
walls may also provide a finely dispersed solid phase on to
which bile acids can bind, so that their concentration in the
aqueous phase of the feces is reduced still further.

Cellular Physiology
Fermentation of carbohydrates lowers the pH of the feces and
increases the concentration of short-chain fatty acids in contact
with the mucosal cells. There is evidence that these changes
modify the intraluminal environment in such a way as to
regulate the birth and death of the mucosal cells. Programmed
cell death (apoptosis) is a mechanism whereby damaged cells
are removed selectively from a tissue in an orderly manner that
does not cause inflammation or general tissue destruction. This
may well provide a defense against the survival of cells carrying
tumorigenic mutations. Apart from the role of butyrate as an
essential source of energy for the colonic mucosa, it also sup-
presses proliferation, increases differentiation, and induces
apoptosis in many types of tumor cell grown in vitro. Tumor
cell lines established from adenomatous polyps and carcino-
mas from human colon undergo increased apoptosis in the
presence of butyrate, at concentrations close to those that occur
in vivo. Unabsorbed starch and fermentable components of
cereal fiber may provide a source of increased butyrate, which
helps to suppress the proliferation of tumor cells and increase
the likelihood of their deletion from the tissue by apoptosis. If
so, much may depend on the rate of fermentation. Sugars and
small oligosaccharides probably disappear too rapidly after
entering the colon to provide a supply of butyrate to the distal
colorectum, whereas lignified plant cell walls and some types
of resistant starch that are slowly fermented may deliver buty-
rate efficiently to more distal regions of the colonic mucosa.
Adverse Physiological Effects
There are few well-authenticated adverse effects of dietary fiber.
Some cases of pediatric malnutrition caused by very high fiber
intakes have been reported, but this problem appears to be
confined to grossly unbalanced diets. Similarly, the few cases of
gastrointestinal obstruction in the literature appear to stem
from very abnormal intakes of wheat bran or from swallowing
nonhydrated supplements of viscous polysaccharides intended
for consumption as a drink. There is some evidence that
patients with a history of gastrointestinal surgery are at
increased risk of obstruction due to abnormally high intakes
of cereal bran.
Sources of Fiber
As should now be obvious, although all plant foods provide
some form of dietary fiber, not all components of fiber have
Dietary Fiber: Physiological Effects 403
the same physiological effects. In the United Kingdom, about
47% of total fiber intake is obtained from bread of various
types and from breakfast cereals. The level of fiber in bread
depends upon the extraction rate, which is the proportion of
the original grain used to make flour. White flour has an
extraction rate of about 70% and contains about 3% NSP,
whereas ‘wholemeal’ (100% extraction) flour contains about
10% NSP. Wheat bran, which contains about 40% NSP, can be
added to the diet as a supplement, but it usually requires
processing to increase its palatability. One of the lasting effects
of the dietary fiber hypothesis has been to increase the variety
of high extraction cereal products in the market place. Such
products tend to contain primarily insoluble, poorly ferment-
able polysaccharides, and they provide an effective way of
increasing fiber intake to increase stool bulk. Oats, rye, and
barley contain higher quantities of soluble fiber than wheat.
Oat bran in particular is an important source of b-glucan,
which has been shown to reduce plasma low-density lipopro-
tein cholesterol levels in humans. A further 45% of total fiber
intake in the United Kingdom comes from fruits and vegeta-
bles. Typically, the levels of NSP in fruits and vegetables are
between 1% and 5% of fresh weight, and the polysaccharides
are mostly soluble pectins and arabinogalactans, which are
readily fermentable in the large bowel. For reasons described
earlier, soluble fiber from vegetables and fruit does not have
the bulk laxative effect of cereal bran, but it does provide
fermentable carbohydrate for fermentation in the colon.
See also: Cancer: Diet in Cancer Prevention; Carbohydrate: Digestion,
Absorption and Metabolism; Dietary Fiber: Determination; Phytic Acid:
Properties, Uses, and Determination.
Further Reading
Burkitt DP and Trowell HC (eds.) (1975) Refined carbohydrate foods: some implications
of dietary fibre. London: Academic Press.
Flint HJ (2012) The impact of nutrition on the human microbiome. Nutrition Reviews
70(Suppl. 1): S10–S13. PMID:22861801, http://dx.doi.rg/10.1111/j.1753-
4887.2012.00499.x.
Johnson IT and Southgate DAT (1994) Dietary fibre and related substances. London:
Chapman & Hall.
Jones JM (2014) CODEX-aligned dietary fiber definitions help to bridge the ’fiber gap’.
Nutrition Journal 13: 34. http://dx.doi.org/10.1186/1475-2891-13-34.
Kumar V, Sinha AK, Makkar HP, de Boeck G, and Becker K (2012) Dietary roles of non-
starch polysaccharides in human nutrition: a review. Critical Reviews in Food
Science and Nutrition 52(10): 899–935. http://dx.doi.org/10.1080/
10408398.2010.512671.
Southgate DAT (1992) Determination of food carbohydrates, 2nd ed. London: Elsevier
Applied Science.
 Dietary Fiber: Properties and Sources
R Mongeau and SPJ Brooks, 3W Banting Research Centre, Ottawa, QC, Canada
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from the Encyclopedia of food sciences and nutrition, vol. 3, pp. 1813–1822, ã 2003, Elsevier Science Ltd., with an
updated Bibliography section supplied by the editor.
Background
Although dietary fiber has been known for more than 2000
years under various terms (e.g., bran and roughage), the term
‘dietary fiber’ first appeared in 1953 and referred to
hemicellulose, cellulose, and lignin. The term ‘fiber’ is some-
what misleading since only a fraction (cellulose) of dietary
fiber is fibrillar in nature. To correct this misnomer, other
terms (e.g., plantix) have been proposed, but despite these
efforts, the term ‘dietary fiber’ has survived.
This section deals with several different aspects of dietary
fiber, including its determination and many of its physiologi-
cal effects. Most of this information is relatively new and is the
result of progress in research brought about in the last 30 years.
The first part of this article will focus on the nature and com-
position of dietary fiber, its properties, and examples of sources
of dietary fiber. This is followed by a definition of dietary fiber.
Although this should be a relatively straightforward descrip-
tion, there are many different viewpoints concerning the
nature and physiological effects that dietary fiber should
have. These will be discussed in relationship to the physiolog-
ical effects of dietary fiber.
Chemical Structure
The composition and structure of dietary fiber differ from plant
to plant. It is also a function of the portion of the plant that is
edible and the stage of maturation and is largely composed of the
cell wall (structural) components that give the plant physical
stability. As such, it is made of highly interlinked sugar-based
and phenolic-based polymers (hemicelluloses, pectic substances,
phenolics, glycoproteins, and proteoglycans) in a matrix of amor-
phous structure with some enmeshed cellulose microfibrils. The
cell wall components are intimately linked together through
various linkages including protein–sugar bonds.
Polysaccharide Fiber Components
Dietary fiber comprises carbohydrate and noncarbohydrate
polymers; most of these are structural components. The carbo-
hydrate polymers are often described as nonstarch polysaccha-
rides (NSPs) to differentiate them from the (relatively) easily
digested starch components of food. Figure 1 shows the pro-
portion of the monomeric constituents of fiber polysaccha-
rides from three food categories (11 cereals, 13 fruits, and 11
vegetables) and from canned mushrooms, peanuts, and baked
white beans. As can be seen, arabinose is a major NSP compo-
nent of cereals, peanuts, and beans. Xylose is present in large
amounts only in cereals. Pectic substances (uronic acids) rep-
resent a substantial part of fruit, vegetable, peanut, and bean
NSPs. Mushroom NSP is formed mostly of glucose residues.
404 Encyclopedia of Food and Health
These results were based on the analysis of composites made of
up to 20 individual foods purchased over a 30-month period
using the Englyst gas–liquid chromatography method. A more
recent version of this method would have given a higher pro-
portion of xylose relative to glucose.
The different monomeric residues of Figure 1 are the basic
units that form the major NSP macromolecular constituents
shown in the upper part of Table 1. Cellulose and b-glucans are
generally unbranched polymers containing mostly glucose res-
idues. The b-glucans have mixed b-1,3 and b-1,4 interresidue
linkages and are a constituent of dietary fiber from barley and
oats. The glucose residues of cellulose are joined by b-1,4
linkages. In addition to their resistance to the enzymes of the
human digestive tract, these linkages allow greater inter- and
intrapolymer hydrogen bonding than the a-1,4 linkages of
starch do. This allows the formation of tightly condensed
crystalline regions within the cellulose microfibrils. Cellulose
includes structurally amorphous regions where the presence of
other sugars can be found. The final cellulose structure is a
function of the glucose and nonglucose contents: the degree of
cellulose crystallization is inversely proportional to the non-
glucose residue content. Cellulose is a minor fraction of the
total cereal dietary fiber but is a major fraction of dietary fiber
in other foods. The proportion of cellulose in the dietary fiber
of a food is not reflected by the glucose content of the NSP
fraction, since glucose is also a major component of other
fractions such as b-glucans and hemicelluloses.
Xylose is a part of the primary chain of hemicelluloses and
gums and the secondary chain of gums and pectin. Arabinose
is in the primary chain of gums and the secondary chain of
hemicelluloses and pectin. Hemicelluloses constitute a major
fraction of dietary fiber; this fraction is largely made of arabi-
nose and xylose in cereals and of xylose and glucose in fruits
and vegetables (Table 1). Pectic substances are prevalent in
citrus fruits. They are also present in vegetables, legumes, and
cereals in small amounts (Table 1). Other polysaccharide con-
stituents are mainly represented by galactose or mannose in
bananas. Nonstructural NSPs represent a small portion of
dietary fiber. The monomeric NSP composition is of limited
usefulness in predicting the properties and functions of dietary
fiber. This is because the structural architecture of dietary fiber
plays a large role in many of its physiological properties, and
this structure cannot be predicted by its composition. Indeed,
two dietary fibers with a similar component profile (e.g., wheat
bran and corn bran) can have clearly different structures, even
under optic microscopy, and can show different behaviors in
the gastrointestinal tract.
Nonsaccharide Fiber Components
The minor components of dietary fiber that are not polysac-
charides and, hence, not NSPs also play an important
http://dx.doi.org/10.1016/B978-0-12-384947-2.00784-4
 Dietary Fiber: Properties and Sources 405

tissues. In most fruits and vegetables, lignin represents only a

100
small part of dietary fiber, although it can be as high as 4% of
dry matter in mature pears.
Cutin, waxes, or suberin may also be present in some
Distribution (%)

tissues. These compounds are mixtures of lipids, proteins,

and carbohydrates that form the waterproof covering and cuti-
50 cle on the outer cell wall of plants. They are especially resistant
to digestion and fermentation. As such, they impair the digest-
ibility of other cell wall components and are usually found in
the feces.

Mushrooms
Fruits

Peanuts

White Beans
Structure
Vegetables
Cereals

Models of primary cell walls of plants were often obtained from

Figure 1 Distribution of the main polysaccharide constituents of dietary
fiber from some foods and food categories. The values are means of
n ¼ 11 cereals, n ¼ 13 fruits, and n ¼ 11 vegetables. Other values are
for n ¼ 1. ▪: glucose; : arabinose; : xylose; : uronic acid; and □:
other sugars.
Table 1 Main macromolecular constituents of dietary fiber
Fruits and
Constituent vegetables
Polysaccharides
Hemicelluloses
Xyloglucans X X
Glucuronoxylans X
Arabinoxylans
Glucuronoarabinoxylans
Galactomannans
Cellulose X X
b-D-Glucans
Pectic substances (pectin) X X
Others
Lignin
Phenolic esters X X
Protein
Glycoproteins X X
structural role. Table 1 shows the distribution of the nonsac-
charidic polymers that are intimately associated with dietary
fiber structure. These polymers include glycoproteins (fruits,
vegetables, and legumes), proteins (cereals), phenolic esters
(cereals), and lignins (lignified tissues of fruits, vegetables,
and cereals). Lignin is of special interest because of its role in
slowing down the fermentation of dietary fiber. It is a complex
group of phenyl propane polymers formed by the condensa-
tion of aromatic alcohols and is especially important in con-
ferring structural stability. As such, highly lignified tissues are
found in the stems of plants such as trees and bushes and in the
stalks of cereals. Lignification occurs at the expense of water
and pectin as cells differentiate and mature. This increases cell
wall rigidity in critical areas. Since edible plant tissues are
consumed when relatively immature, their cells are largely
undifferentiated and, largely, unlignified. Wheat bran and the
small seeds covering strawberries are examples of lignified
nonfood materials such as wood and tobacco. Because the cell
wall material forms a large part of these plants, the models
provide a wealth of information on the interactions between
cell wall components such as carbohydrates and lignin and
protein and carbohydrates. More recent reports have provided
some structural information on the cell walls of plant foods.
These reports have shown that, in carrots, protein–protein or
protein–polysaccharide linkages may contribute to the forma-
tion of a rigid, inextensible cell wall. Other data have shown that
protein may form up to 10% of cell walls in immature plants
and appears to be important for structural cohesion. The major
Cereals Legumes part of the flesh of fruits and vegetables contains undifferen-
tiated types of cell wall that have significant amounts of highly
branched hemicellulosic types of polysaccharides. The cellulose
content is usually low, and cellulose is laid down in a more
oriented arrangement in the matrix. This may reflect stresses on
the plant. The cell walls are often rich in pectic substances and
X
X
contain
10% protein, as described earlier. These cell walls are
X thin and elastic with the cell form maintained by osmotic
X pressure.
X In contrast, the outer layers of many seeds and nuts contain
X thick lignified cell walls that are difficult to break. These serve
as mechanical protection to the seed within and as a vapor
X barrier to prevent desiccation.
X
Physical Properties
Soluble and Insoluble Dietary Fibers
It is possible to separate the total dietary fiber (TDF) into
soluble and insoluble components based on solubility in
water, but quantifying the amount of soluble fiber remains
problematic. This is because the conditions utilized to measure
the proportion of soluble fiber differ among laboratories (e.g.,
time, temperature, pH, and type of buffer; see the section
‘Definition (Based on Function and Structure)’). It should
be noted that the distinction between soluble and insoluble
fibers is somewhat artificial, since it is difficult to predict the
actual fiber solubility in the gastrointestinal tract.
For many years, it was thought that soluble fiber was not
commonly found in foods. Reports on pectin, guar, legume
fiber, and oat bran appeared in the early 1960s, and currently,
it is estimated that about a third of the daily TDF intake is
soluble fiber. Pectic polyuronides are the major soluble dietary
fiber components of vegetables and fruits, but the exact com-
position varies from plant to plant. When measuring dietary
406 Dietary Fiber: Properties and Sources
fiber, one must also take into account the method of food
preparation because the soluble fiber content of a fiber source
is influenced by the method of food preparation. For example,
some polyuronides are susceptible to depolymerization at the
high temperatures used to cook the foods. Similarly, some
insoluble fiber is susceptible to depolymerization at higher
temperatures and may be found in the soluble fiber fraction.
In addition to the distinction between soluble and insolu-
ble fibers, viscosity may also be an important fiber characteris-
tic. This is because viscous fibers modestly lower blood
cholesterol and triacylglycerol concentrations and attenuate
the postprandial glucose response. It is thought that the
increased viscosity of the intestinal lumen reduces the rate of
diffusion of glucose, cholesterol, and triacylglycerols to their
respective receptors along the intestinal wall, reducing absorp-
tion. The results of a recent meta-analysis using several differ-
ent clinical studies showed a statistically significant lowering of
plasma triacylglycerols and cholesterol in subjects fed for at
least 14 days with a diet supplemented with oats, psyllium,
pectin, or guar gum, but the effect was modest: a decrease of

1 mg dl1 g1 of soluble fiber was observed. If we consider
that an individual with moderately higher blood cholesterol
has a total serum cholesterol value of
300 mg dl1, the
change brought about from the consumption of viscous, solu-
ble fibers is on the order of 0.3%. Considering that oat bran
contains about 20% fiber by weight and only half of this is
soluble, one can calculate that 10 g of oat bran per day is
needed for each 0.3% change in total cholesterol.
Insoluble dietary fiber is, by definition, the fraction of the
TDF that is not soluble in hot buffer solution. However, the
measured proportion of insoluble fiber can vary depending on
the dietary fiber methodology. Insoluble dietary fiber consists
primarily of hemicellulose, cellulose, and lignin. It is often
incorrectly believed that insoluble fiber is not fermented to
any great extent by the bacteria present in the large bowel.
However, actual measurements of fiber fermentability have
shown that a significant proportion of insoluble dietary fiber
is fermented. This is true even for wheat bran, which has a
relatively lower insoluble fiber fermentability of between 30%
and 40% (
90% of the wheat bran TDF is insoluble dietary
fiber). The fermentability of oat bran is even greater – up to
80% of the insoluble fiber (approximately half of the TDF) is
fermented in the human large intestine. The degree of ferment-
ability is a property unique to each dietary fiber and depends
largely on the nature and the structural arrangement of the
fiber components and also on other physical characteristics
such as particle size. For example, the structure of coarse
wheat bran favors a high water-holding capacity (see later)
and a slow rate of fermentation. This permits the microtrap-
ping of slowly released fermentation gases, increasing fecal
bulk and stimulating defecation.
Water-Holding Capacity
Dietary fiber holds water by adsorption and absorption. Some
water is also retained outside the fiber matrix (free water). The
particle size, chemical composition, and structure of dietary
fiber influence the water-holding capacity. The in vitro water-
holding capacity cannot be used to predict the impact of highly
fermentable fiber on colonic function.
Table 2 Effect of grinding on the properties of wheat bran insoluble
fibera
Mesh Apertureb MPS Glycocholate
sieve (mm) (mm)c WHCd bindinge
As is 800 9.5  0.1 26.5  1.5
20 840 420 8.1  0.1 24.9  1.2
40 420 280 6.6  0.2 23.8  0.8
60 250 180 5.8  0.1 22.1  1.0
80 175 160 5.6  0.1 21.5  0.3
a
Residue after neutral detergent and porcine pancreatic a-amylase treatments.
b
Aperture size of sieve used with a Wiley mill, intermediate model.
c
MPS, geometric mean particle size.
d
WHC, water-holding capacity, grams of water per gram of insoluble fiber, using the
centrifugation method; mean  SEM of three measurements.
e
Grams of glycocholate bound per 0.2 g of insoluble fiber; mean  SEM of four
measurements.
Although lettuce fiber isolates retain much water, the water-
holding capacity expressed in grams of water held by fiber per
gram of edible portion of food has been found to be higher for
wheat bran fiber (4.5 g) and carrot fiber (2.1 g) than for that of
lettuce and various other foods (0.3–1.3 g). The water-holding
capacity of fiber from the bran of ready-to-eat breakfast cereals
is positively correlated (r  0.85 and P < 0.05) with its mean
particle size (MPS): the water-holding capacity of 160 mm MPS
wheat bran fiber is 59% of that of 800 mm MPS wheat bran
fiber (Table 2). A large portion of the water held by wheat bran
fiber appears to be free water.
The water-holding capacity has a significant effect on fecal
output and stool hardness, as discussed earlier. These factors
are important physiological effects of wheat bran and contrib-
ute significantly to its laxative effects.
Bile Salt Binding and Serum Cholesterol Reduction
There is considerable interest in the cholesterol-reducing prop-
erties of dietary fiber. This is because the influence of serum
cholesterol (low-density lipoprotein (LDL) and high-density
lipoprotein (HDL) cholesterol, in particular) on cardiovascular
disease is considerable. Three different mechanisms have been
proposed to account for the serum cholesterol-reducing prop-
erties of dietary fiber. As discussed earlier, a certain soluble
viscous fiber may modestly lower cholesterol by increasing
luminal viscosity and preventing cholesterol (re)absorption.
Acetic and propionic acids, produced by the fermentation of
dietary fibers in the gut, are thought to inhibit liver cholesterol
synthesis at the metabolic level. Dietary fibers also have the
ability to bind cholesterol directly: deconjugated bile salts are
bound to pectic substances by hydrogen bonding, and lignin
appears to bind bile salts through hydrophobic interactions.
The actual in vivo mechanism is unknown, and it is likely that
different fibers operate through different combinations of all
three mechanisms to influence blood cholesterol levels.
In vitro evidence suggests that the physical form of the fiber
may also play an important role in binding bile salts. In com-
monly ingested ready-to-eat cereals, glycocholate binding
(r ¼ 0.90, P < 0.001) and taurocholate binding (r ¼ 0.86,
P < 0.05) have been positively correlated with the MPS of the

neutral detergent fiber (NDF). Reducing the particle size of
wheat bran NDF from 800 to 160 mm reduces its glycocholate
binding by 19% (Table 2). The bile salt-binding capacity of
fiber isolates, although method-dependent, may reflect events
in the terminal small intestine. Purified fiber fractions may be
poor (cellulose) or strong (lignin) binders. Some rice bran
appear to have a high capacity to bind bile salts.
Human cholesterol-lowering studies have linked the con-
sumption of dietary fiber with lower total plasma cholesterol
and increased LDL/HDL ratios in addition to reductions in
serum triglycerides. Studies such as these have suggested that
cardiovascular risk may be reduced by consuming particular
types of foods high in dietary fiber, most notably whole cereal
grains. However, it should be noted that coronary heart disease
(CHD) is a complex disease and is affected by many factors. For
example, the reduction in CHD observed in a recent study was
larger than would be expected from the beneficial effects of
soluble dietary fiber on serum cholesterol levels only. Thus,
reductions in CHD are likely to be the result of many factors,
one of which is TDF intake.
Cation Exchange Capacity
Cation exchange is partly dependent on the presence of uronic
acid in the nonesterified form. The preparation of the fiber
material may decrease the number of nonesterified carboxyl
groups and the apparent cation exchange capacity. Wheat bran
contains little uronic acid, and its cation exchange capacity is
mainly due to diffusion within the dietary fiber network. The
cation exchange capacity of fiber from different sources is
difficult to compare, unless it is expressed per edible portion.
According to the affinity of minerals for carboxylic acid groups,
cabbage and coarse wheat bran show a high cation exchange
capacity compared with pectin.
There has been some concern that the ability of dietary fiber
to bind minerals (as measured in vitro) may lead to mineral
deficiencies in individuals consuming high-fiber diets. In
North American diets, calculations show that mineral intakes
far exceed the potential binding capacity of dietary fiber so that
no need for concern arises. In addition, minerals that are
bound to fibers or that are trapped in the (as yet undigested)
cell wall matrix may not be absorbed in the small intestine but
could be partially released and absorbed in the colon when the
fiber is degraded by bacteria. Absorption of minerals in the
colon has been suggested as a mechanism for accounting for
increased mineral absorption in rats fed with fructooligosac-
charides. This may explain why cation adsorption has not been
consistently related to mineral bioavailability. However, wheat
bran fibers can permanently bind heavy metal ions to decrease
their toxicity.
Viscosity and Gelling Properties
As mentioned earlier, certain soluble fibers, such as oat b-D-
glucans, are viscous when dissolved in water, while others,
such as pectins, show gelling properties. These fibers influence
gastric emptying and absorption rates in the small intestine.
Direct measurement of the viscosity produced by concentra-
tions of fiber likely to be used in diets has shown little effect of
low-methoxy pectin, a slight increase with wheat bran, and a
Dietary Fiber: Properties and Sources 407
significant increase with high-methoxy pectin. Some of the
nonstructural dietary fiber components can also increase
viscosity.
Microbial Degradation (Fermentability)
Most dietary fiber remains undegraded until it reaches the large
intestine, where the extent of fermentation depends on the
source and several other factors, including the physical struc-
ture of the fiber, presence of specific components in the fiber
matrix, nitrogen source, bacterial adaptation, and transit time.
It is generally accepted that soluble fiber is almost completely
fermented in the large intestine, but unfortunately, it is also
generally believed that insoluble fiber is not fermented. Actu-
ally, both insoluble and soluble fibers are extensively fermen-
ted. On average, 70–80% of the TDF from mixed diets (e.g.,
fruits, vegetables, legumes, and most cereals) is degraded by
colonic bacteria. Since insoluble dietary fiber is the major
dietary fiber fraction, representing approximately two-thirds
of the TDF in Western diets, this means that insoluble fiber is
highly fermented. Indeed, as indicated in the preceding text,
70–80% of the insoluble fiber from oat bran and up to 40% of
the insoluble dietary fiber from wheat bran are fermented.
There is some indication that fermentability depends on
particle size, with small particle sizes being more readily fer-
mented, but this has not been consistently reported, and the
effect appears modest. In rats fed with purified diets containing
15% hard red wheat bran, NDF fermentability was largely
unchanged (34–36%) when the MPS varied from 1275 to
394 mm. Similar trends were observed when American Associ-
ation of Cereal Chemists soft and hard wheat bran were fed at
various MPSs. In these experiments, 37.3–37.6% and
30.0–32.8% of the NDF were fermented, respectively.
Interaction between Structure and Physiology
Food Processing
The physicochemical properties of dietary fiber per se are
dependent on the chemical composition and the structural
characteristics of the fiber. However, as noted earlier, knowing
the former does not allow one to predict the latter. When
considering the potential in vivo effect of dietary fiber, one
must also take food processing practices into consideration.
Many different types of food processing practices are currently
employed. Foods can be boiled, canned, frozen, blanched,
parboiled, extruded, adiabatically extruded, and milled. All
processes can have an effect on dietary fiber content, as mea-
sured by current methodologies. For example, heat treatment
can break glycosidic linkages in dietary fiber polysaccharides.
This can solubilize some of the insoluble fiber and reduce the
TDF content if the polymers are broken down into small
molecular mass fragments. Heat treatment can also reduce
the overall length of the polysaccharide chains to lower viscos-
ity and the water-holding capacity of the fiber. The pentosans
in dietary fiber can react with amino acids such as lysine
(Maillard reaction) and form new polymers that do not behave
physiologically like dietary fiber. The boiling of vegetables
increases the apparent TDF content, but this seems to be due
to the loss of nonfiber components to the water.
408 Dietary Fiber: Properties and Sources
It is often difficult to predict beforehand the effect of a food
processing method on the TDF and the distribution between
insoluble and soluble fractions. For example, extrusion cook-
ing of some cereal bran apparently increases its water-holding
capacity in vitro, although this does not translate into a change
in fecal water-holding capacity in vivo. Extrusion cooking of
wheat bran not only causes small decreases in the TDF and
insoluble dietary fiber but also gives rise to small increases in
soluble dietary fiber. Processing of rice (abrasion or parboil-
ing) removes the seed coat and the majority of the dietary fiber.
Some collapsing of the fiber structure also occurs during food
preparation and/or mastication due to the reduction of particle
size, and the fiber structure of sources such as spinach may be
modified by cooking.
Milling is known to significantly affect TDF fermentation in
the human colon. Cereal grains have a tough outer seed coat
that would be completely resistant to digestion if the cereals
were not milled prior to consumption. After milling, the thin-
walled dietary fiber structures from the endosperm are partly
disrupted and, thus, easily broken during fermentation. Milling,
therefore, not only makes the endosperm accessible to the
digestive enzymes in the upper digestive tract but also makes
the dietary fiber more accessible to colonic bacteria. However,
milling can also negatively affect the physiological properties of
the dietary fiber in grains. While coarse grinding and adequate
mastication appear to be sufficient for the digestion of cereals,
fine grinding disrupts much of the cell wall architecture, and
further milling can even interfere with its determination by
gravimetric methods. Table 3 shows the effect of milling on
the dry bulk volume of wheat bran, as measured in a volumetric
cylinder. The volume of the coarse bran was reduced from
5 ml g1 to < 2 ml after grinding in an M5 Wiley mill using a
0.5 mm screen. Fiber from different sources ground under the
same conditions may behave differently during milling, so that
their resulting particle sizes may be different.
As discussed earlier, the particle size of unfermented wheat
bran fiber may be related to colonic function and fecal charac-
teristics in humans and laboratory animals. Finely ground
cellulose or even wheat bran, for example, may have a consti-
pating effect, whereas coarsely ground wheat bran prevents
constipation. The influence of particle size on fecal bulking
has been measured directly in rats. When rats were fed with
diets containing 12% hard wheat bran, the fecal wet density
was 0.796  0.010 (mean  SEM, n ¼ 12) in the coarse bran
group (geometric MPS: 850 mm) but increased to
0.888  0.013 in the fine bran group (geometric MPS:
308 mm). This increased density was not due to a change in
Table 3 Effect of grinding on dry bulk of wheat bran (ml g1):
duplicate measurements
Sieve aperture a
Bran A b
Bran B Bran C Bran D
As is 3.1 3.4 5.1 3.5
2.0 mm (10 mesh) 2.7 2.4 2.8 2.1
1.0 mm (18 mesh) 2.2 2.1 2.4 2.0
0.5 mm (35 mesh) 1.8 1.8 1.9 1.7
a
Using an M5 Wiley mill. Mesh sieve equivalence is indicated in parentheses.
b
Bran A was American Association of Cereal Chemists-certified soft white wheat bran;
bran B–D were soft or hard wheat bran from other sources.
fecal weight but was due to a decrease in fecal volume, parallel-
ing the effect demonstrated for bran in Table 3. In ready-to-eat
breakfast cereals, the fiber MPS has been found to vary between
350 and 2000 mm. Increased particle size normalizes gastroin-
testinal transit time, delays gastric emptying, and may also
affect the extent of insoluble fiber fermentation. Although
dietary fiber has many positive effects on the digestive tract,
excessive milling of dietary fiber to produce small particles in
the range of 7–70 mm may have negative effects. Smaller parti-
cles do not exhibit the transit time normalization associated
with larger bran particles, and the persorption of a small
amount of very fine fiber particles may occur in some regions
of the intestinal wall. Contrary to persorbed starch granules,
persorbed fiber particles are not degraded in the blood and
may impose an increased workload on the kidneys. Data on
persorption are scarce, and much work is needed on this
subject.
Studies with apples, apple puree, and apple juice have
highlighted the link between the physical integrity of the cell
wall and the beneficial effects of unprocessed dietary fiber, in
this case, a lowering of the glycemic index of a food (the rise in
blood glucose that accompanies the ingestion and digestion of
a carbohydrate-containing food). Only the unprocessed apple
had a reduced glycemic index; the apple puree, containing the
physically degraded dietary fiber, was similar to apple juice in
response. This demonstrates the importance of the physical
encapsulation of available macronutrients within the fiber
matrix. This effect has also been observed with rolled oats
where higher glycemic indexes were observed with more finely
cut oats. Studies such as these show that the integrity of the cell
wall is important in bringing about beneficial physiological
effects, even in the presence of viscous, soluble fiber types
(b-glucans in the cell wall; see the section ‘Bile Salt Binding
and Serum Cholesterol Reduction’).
In Vitro Measurements
In vitro properties of fiber isolates have been used to estimate
their potential in vivo effect. However, in vitro data should be
used with caution. The measurements are influenced by the
methods used to follow digestion in vitro and by the methods
used to prepare the fiber. Other confounding influences are
also present: the fiber residue obtained in vitro may have been
altered by heat or chemicals or may still contain residual
digestible materials. For these reasons, in vivo conditions gen-
erally result in a more extensive digestion than that measured
in vitro.
Digestive Events
An important consideration when examining the physiological
effect of dietary fiber is the fact that dietary fiber is concentrated
and denuded only in the terminal small intestine. This means
that the dietary fiber that is partially associated with digestible
materials in the jejunum and duodenum may have different
physical properties from those of an entirely denuded fiber
matrix at the terminal ileum. In the large intestine, most of
the soluble fiber and a large but variable portion of insoluble
dietary fiber are degraded by the colonic microflora. As already
 Dietary Fiber: Properties and Sources 409

discussed, fermentation plays an important role in mediating
many of the physiological effects associated with dietary fiber.
Definition (Based on Function and Structure)
In the early 1970s, it became evident that cellulose represents
only a small part of the TDF content of foods. Prior to this,
there was a tendency to equate dietary fiber with cellulose
because that is what was measured using the old crude fiber
method. While this helped reinforce the use of the term ‘dietary
fiber’ (cellulose has a fibrillar structure), cellulose represents
only a small part of the TDF content of foods. Thus, at that
time, new methodologies were emerging, and it was clear that
there was a need for a dietary fiber definition that was linked to
the actual physicochemical composition of dietary fiber.
It is in this context that the dietary fiber hypothesis appeared,
a few years after Cleave’s hypothesis in 1956, which related
refined foods and Western diseases. The dietary fiber hypothesis
thus appeared as a refocusing of Cleave’s original ideas, revers-
ing the original emphasis to become a diet high in unrefined
edible plant material that is protective against Western diseases
of the bowel. The dietary fiber hypothesis is important in under-
standing the ‘dietary fiber concept,’ since it links botanical struc-
ture to digestive processes and health outcomes – mainly
noninfective colonic diseases such as constipation, diverticular
disease, irritable colon, ulcerative colitis, appendicitis, hemor-
rhoids, polyps, and cancer of the large bowel. It is this link to
these and other health outcomes that has characterized much of
the work in the dietary fiber field over the last 30 years. The first
dietary fiber definition in 1972 sought to describe the plant
structures and components that were responsible for mediating
these health benefits: “that portion of food which is derived
from cellular walls of plants which is digested very poorly by
human beings. . . Fibre is composed largely of cellulose,. . .
hemicelluloses, pentosans, pectin and lignin.”
There are many different definitions of dietary fiber. How-
ever, they all include certain specific and critical aspects. First
and foremost, a definition must include the fact that dietary
fiber corresponds mostly to the structural material in the edible
part of plant foods in the usual human diet. This has been
consistently reflected throughout the decades by the inclusion
of the term ‘plant cell wall’ when referring to dietary fiber
(Table 4). The term ‘dietary fiber’ itself is something of a
misnomer, since dietary fiber is not fibrillar (except for cellu-
lose). To reflect this fact, many other terms have been proposed
over the years, including unavailable carbohydrate and plantix.
None of these terms have succeeded in replacing ‘dietary fiber.’
Another important aspect of any dietary fiber definition is
that it is resistant to human digestive secretions (including
enzymes) and not absorbed in the small intestine. The obser-
vation that dietary fiber increased fecal weight initially gave the
impression that dietary fiber was resistant to both human
digestive secretions and bacterial digestion in the lower gut
(fermentation). While it is true that dietary fiber is not digested
and absorbed in the small intestine, a large proportion of
dietary fiber is fermented in the colon. The extent of this
fermentation is a function of several different factors (see the
section ‘Microbial Degradation (Fermentability)’), meaning
that the extent of fermentation cannot be used as a criterion to
define dietary fiber.
Using ‘resistance to endogenous digestive secretions’ alone
as a criterion may be confusing. For example, some dietary
fiber may be partly degraded by acids in the stomach, while

Table 4 Use of the term ‘plant cell wall’ in defining dietary fiber over time

Year Text Reference

1885 The ‘cell membranes’ of Rubner
1929 The ‘skeletal framework of the plant’
1981 The ‘remnants of plant cells’
1985 ‘Derived from plant cell walls and not
digested by human alimentary
enzymes’
1990 The ‘structural plant cell wall
composed of polysaccharides and
lignin’
1995 ‘The “cell wall material of plant foods”
responsible for the physical form
and texture of unprocessed plant
foods’
1995 The ‘plant cell walls’ are the common
characteristic of plant foods that
constitute a high-fiber diet
associated with beneficial effects
1999 ‘The cell walls of edible plant tissues in
the traditional human diet’
Trowell, H. C. (1975). In: Burkitt, D. P. and Trowell, H. C. (eds.) Refined carbohydrate foods and
disease, p. 43. London: Academic Press
McCance, R. A. and Lawrence, R. D. (1929). The carbohydrate content of foods. Medical Research
Council special report series No. 135, pp. 1–173. London: HM Stationary Office
AOAC Fiber Consensus (1981). Association of Official Analytical Chemists 95th Annual Meeting,
October 1981, Washington, DC
Trowell, H., Burkitt, D. P. and Heaton, K. (eds.) (1985). Dietary fibre, fibre-depleted foods and
disease. London: Academic Press
Eastwood, M. (1990). Function of dietary fibre in the large intestine. In: Southgate, D. A. T., Waldron,
K., Johnson, I. T. and Fenwick, G. R. (eds.) Dietary fibre: chemical and biological aspects, pp.
211–219. Cambridge: Royal Society of Chemistry
Heaton, K. W. (1995). The dietary fibre concept – time for a re-evaluation? In: Sørensen, A., Bach
Knudsen, K. E., Englyst, H. N., Gudmand-Høyer, E. and Nyman, M. (eds.) Metabolic and
physiological aspects of dietary fibre in foods: recent progress in the analysis of dietary fibre, COST
92, p. 15. Luxembourg: Commission of the European Commission
Cummings, J. H., Hudson, G. J., Quigley, M. E. and Englyst, H. N. (1995). The classification and
measurement of dietary carbohydrates. In: Sørensen, A., Bach Knudsen, K. E., Englyst, H. N.,
Gudmand-Høyer, E. and Nyman, M. (eds.) Metabolic and physiological aspects of dietary fibre in
food. Recent progress in the analysis of dietary fibre in food, COST 92, pp. 17–36. Luxembourg:
European Commission
Mongeau, R., Scott, F. W. and Brassard, R. (1999). Definition and analysis of dietary fiber. In: Cho, S.
S., Prosky, L. and Dreher, M. (eds.) Complex carbohydrates in foods, pp. 305–316. New York &
Basel: Marcel Dekker
410 Dietary Fiber: Properties and Sources
some small intestine-digestible material can reach the colon
where bacterial fermentation occurs. In addition, a definition
based on the resistance to digestibility by endogenous digestive
secretions could be problematic if the material is not specified
as coming from the edible part of plant foods. Indeed, such a
definition could be erroneously interpreted as meaning that
dietary fiber is any material resistant to in vivo digestion or even
as any material measured as dietary fiber by a specific dietary
fiber method. Among the materials to be excluded from the
dietary fiber definition are ‘ruminant fiber’ and the products
formed during processing and/or cooking. There have been
requests to include some other plant components and manu-
factured polymers, and this is a subject of much debate. Among
the criteria that could be used to accept or reject a material as
dietary fiber, only ‘edible plant cell wall’ is specific enough to
allow an absolute definition. In addition, it links current die-
tary fiber definitions to the original ‘dietary fiber concept,’
which recognized that a diet high in minimally processed
edible plant foods provided many health benefits. Confusion
often arises because of the overlapping characteristics and
physiological effects of the different ‘candidate’ materials with
those of a dietary fiber ‘reference.’ Examples of ‘candidates’ are
polydextrose (a randomly bonded synthetic glucose polymer),
inulin and fructooligosaccharides (naturally occurring
glucose–fructose oligomers and polymers), and resistant starch
(formed by food processing methods and largely undigested in
the small intestine). This overlapping means that it becomes
virtually impossible to distinguish between fiber and nonfiber
material based on resistance to digestion alone. Debates such
as this started shortly after dietary fiber became popular in the
1970s. They are still ongoing, as demonstrated by the publica-
tion of a recent international survey of 147 professionals.
While a complete discussion of the merits of either side is
beyond the scope of this article, it is important to remember
the origins of the dietary fiber definition and the dietary fiber
hypothesis that firmly linked fiber to physiological outcome.
The definition of dietary fiber was first centered on the
‘original dietary fiber concept’ that linked the consumption of
‘plant cell walls’ in unrefined, traditional foods to positive
physiological outcomes. Table 5 shows two definitions that
were proposed after the turn of the millennium. These two
definitions have taken the original ideas of the dietary fiber
hypothesis into account and included a reference to some
specific physiological effects in humans.
While the newer definitions of dietary fiber specifically
mention the fact that dietary fiber has physiological benefits,
Table 5 Recent proposed definitions of dietary fiber
Year Text
2001 ‘. . .the edible parts of plants or analogous carbohydrates that are
resistant to digestion and absorption in the human small
intestine. . .promotes beneficial physiological effects including
laxation, and/or blood cholesterol attenuation, and/or glucose
attenuation’
2002 ‘Dietary Fiber consists of nondigestible carbohydrates and lignin that
are intrinsic and intact in plants. Functional Fiber consists of
isolated, nondigestible carbohydrates that have beneficial
physiological effects in humans’
the list is rather specific and deals primarily with effects
observed with viscous fibers (lowering postprandial serum
glucose and lowering serum cholesterol) or effects brought
about from partial fermentation and water-holding capacity
(laxation). These effects are well supported by clinical experi-
ments with laboratory animals and with humans. However,
the original dietary fiber concept related dietary fiber intake to
the diseases of Western man, including heart disease, cancer,
and obesity. As mentioned earlier, the link between viscous
dietary fiber and heart disease is supported in the literature
through its effect on serum cholesterol and triglycerides, but
the link with other Western diseases has been much harder to
demonstrate. It has been well established that eating patterns
that favor foods high in dietary fiber and low in red meat
significantly reduce the risk of cancer, heart disease, and obe-
sity. However, it has been relatively difficult to isolate this
effect to dietary fiber consumption per se. Part of the problem
comes from the almost exclusive reliance on epidemiological
studies to demonstrate any association. There are several rea-
sons why epidemiological studies are generally favored. First,
these studies provide data on free-living humans, rather than
inbred laboratory animals. Second, these studies follow the
long-term effect of dietary fiber consumption on health out-
comes. Third, the sample size can vary from a few hundred
(case control studies) to many thousands of individuals (pro-
spective cohort studies). This gives the studies good statistical
power and lends weight to the conclusions. However, epide-
miological studies also suffer from a number of shortcomings
including problems with assessing food intake for the past 2–3
years (case control studies) or with monitoring food intake 2–3
times over a period of 5 years and using this to predict disease
outcome 15 years later (prospective cohort studies), problems
that arise from the use of incomplete dietary tables to calculate
dietary fiber intake, and problems arising from the use of
inappropriate methods for correcting energy intake.
The most striking conclusion from epidemiological studies
is a failure to provide support for an inverse correlation
between dietary fiber intake and colon cancer. This concept
has long been entrenched in the literature largely because of
studies with laboratory animals that have provided much data
in favor of a protective effect of dietary fiber. The animal
studies are supported by plausible physiological and biochem-
ical mechanisms that can account for the observed results.
However, the epidemiological data are inconsistent in its sup-
port for an inverse relationship between colon cancer and
dietary fiber intake, and clinical trials that examined the effect
Reference
American Association of Cereal Chemists (2001). Report of the
Dietary Fiber Definition Committee to the Board of Directors of the
American Association of Cereal Chemists. 2001. The definition of
dietary fibre. Cereal Foods World 46, 112–129
National Academy of Sciences (2002). Food and Nutrition Board.
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Washington, DC: National Academy Press
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of diet on the reappearance of colon polyps (colon cell growths content expressed on an ‘as is’ basis. Since some methodolog-
that have a 1/20 chance of forming a cancerous tumor) found ical variability is inevitable, the letters ‘E,’ ‘M,’ and ‘P’ in the
no protective effect of dietary fiber. Even though the link right-hand column designate which methods would give TDF
between adenomatous polyps and cancer is not straightfor- values in the range shown. Other methods not included in the
ward, these studies show that more research is needed to comparison are likely to agree with these values. The letter ‘E’
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last two methods are gravimetric and include lignin in the final
Sources of Dietary Fiber determination. Table 6 shows a considerable agreement
between methodologies: 33 of 49 foods are designated ‘EMP,’
The TDF content of a food depends on many factors, including meaning that all three methods give similar values. In addition,
(1) the plant variety, (2) the stage of maturity when harvested, three foods are designated ‘MP’ and 13 foods ‘M’ only because
(3) the plant-growing conditions, and (4) the method of food sample size limitations precluded measurement with other
preparation. Although not all the methodological issues have methods. The TDF values are based on winter and summer
been completely resolved with respect to measuring dietary collections of up to 20 individual foods over 30 months in
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a majority of foods. Most fruits and vegetables contain low amounts of dietary
The names of 49 common dietary fiber sources are provided fiber (1.0–2.2% TDF) on an ‘as is’ basis. Legumes contain
in Table 6. The foods are classified according to their TDF intermediate amounts of dietary fiber (around 4% TDF), and
cereals contain between 1–2.2% TDF (corn kernels) and
Table 6 Sources of dietary fiber 15.5–15.8% TDF (oat bran). The dietary fiber content of
foods is greatly influenced by the moisture content. Thus,
TDFa Foods Methodsb cereal TDF values are high because these foods contain little
moisture (up to 10% at most), whereas fruits and vegetables
0.1–0.5 White rice, cooked M
contain 80–90% moisture on an ‘as is’ basis. The values of
Orange juice from concentrate M
0.6–0.9 Lettuce M
Table 6 are reported as a percentage of weight, but when
Cucumbers EMP calculating dietary fiber intake, it is important to factor in the
Pineapple, melons (cantaloupe) EMP serving size (30 g for breakfast cereals, 50 g for lettuce and
1.0–2.2 White bead EMP raisins, and 125 g for vegetables and fruits). For example, 1
Corn kernel (canned), potatoes (boiled), EMP serving of bran flakes will provide
4 g of dietary fiber, 1
green beans (boiled), cabbage (raw), celery serving of lettuce will provide 0.35 g of TDF, 1 serving of raisins
(raw), onion (cooked), green peppers (raw) will provide about 1.8 g of TDF, and 1 serving of corn will
Cauliflower (cooked), tomatoes M provide about 2 g of TDF.
Apple, bananas, orange, pears, strawberries, EMP
cherries, grapefruit, plums
Apple pie, blueberry pie, pasta (cooked) M
2.3–3.0 Rye bread M See also: Cellulose; Dietary Fiber: Bran; Dietary Fiber: Determination.
Mushroom (E ¼ 2.0) MP
Beets (canned), rutabaga (raw), carrots EMP
(raw), broccoli (raw)
Blueberries (E ¼ 1.9) MP Further Reading
3.1–4.3 Brown rice EMP
Raisins (seedless) M Anderson JW, Allgood LD, Lawrence A, et al. (2000) Cholesterol-lowering effects of
4.4–6.4 Bran muffins, wheat cereal, wholemeal bread EMP psyllium intake adjunctive to diet therapy in men and women with
hypercholesterolemia: meta-analysis of 8 controlled trials. American Journal of
White beans (baked) EMP
Clinical Nutrition 71: 472–479.
Kidney beans (baked), peanut butter M Baintner K (ed.) (1986) Intestinal absorption of macromolecules and immune
6.5–7.3 Peanuts EMP transmission from mother to young. Boca Raton, FL: CRC Press.
Green peas (boiled) M Bolton RP, Heaton KW, and Burroughs LF (1981) The role of dietary fibre in satiety,
8.0–9.3 Oatmeal cereal EMP glucose, and insulin: studies with fruit and fruit juice. American Journal of Clinical
9.4–10.3 Shredded wheat EMP Nutrition 34: 211–217.
12.3–13.1 Bran flakes EMP Brown L, Rosner B, Willett W, and Sacks FM (1999) Cholesterol-lowering effects of
15.5–15.8 Oat bran EMP dietary fiber: a meta-analysis. American Journal of Clinical Nutrition 69: 30–42.
40.6–40.9 AACC hard wheat bran (E ¼ 36.4) MP Brownlee IA (2011) The physiological roles of dietary fibre. Food Hydrocolloids 25(2):
238–250.
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TDF, total dietary fiber range in grams per 100 g on an ‘as is’ basis. Carpita NC (1990) The chemical structure of the cell walls of higher plants.
b In: Krichevsky D, Bonfield C, and Anderson JW (eds.) Dietary fiber: chemistry,
Methods: E, Englyst GLC method plus potassium permanganate lignin; M, Mongeau
physiology, and health effects, pp. 15–30. New York: Plenum Press.
rapid gravimetric method; and P, Prosky gravimetric method.
Cummings JH (1996) The effect of dietary fiber on fecal weight and composition.
Source: Mongeau, R. and Brassard, R. (1989). A comparison of three methods for In: Spiller GA (ed.) Handbook of dietary fiber in human nutrition, pp. 211–280. Boca
analyzing dietary fiber in 38 foods. Journal of Food Composition and Analysis 2, Raton, FL: CRC Press.
189–199; Mongeau, R., Brassard, R. and Verdier, P. (1989). Measurement of dietary Dikeman CL and Fahey Jr. GC Jr. (2006) Viscosity as related to dietary fiber: a review.
fiber in a total diet study. Journal of Food Composition and Analysis 2, 317–326. Critical Reviews in Food Science and Nutrition 46(8): 649–663.
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Dreher ML (ed.) (1987) Handbook of dietary fiber: an applied approach. New York:
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Waldron K, Johnson IT, and Fenwick GR (eds.) Dietary fibre: chemical and
biological aspects, pp. 211–219. Cambridge: Royal Society of Chemistry.
Heaton KW (1995) The dietary fibre concept – time for a re-evaluation? In: Recent
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and reference materials. Journal of AOAC International 78: 22–36.
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brans: bile salt binding and water-holding capacity in relation to particle size. Cereal
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Mongeau R, Scott FW, and Brassard R (1999) Definition and analysis of dietary fiber.
In: Cho SS, Prosky L, and Dreher M (eds.) Complex carbohydrates in foods,
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O’Shea N, Arendt EK, and Gallagher E (2012) Dietary fibre and phytochemical
characteristics of fruit and vegetable by-products and their recent applications as
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rheological and breadmaking properties of wheat doughs. Journal of Cereal Science
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fibre from vegetable products as source of functional ingredients. Trends in Food
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on inflammatory bowel disease and colon cancer: importance of fermentation
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of the cell wall matrix. In: Southgate DAT, Waldron K, Johnson IT, and Fenwick GA
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32–42.
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 Dietary Practices
AFG Cicero, University of Bologna, Bologna, Italy
T Stallone, National Welfare and Assistance Fund for Registered Biologists (ENPAB), Roma, Italy
ã 2016 Elsevier Ltd. All rights reserved.

How to Build the ‘Perfect Diet’?: A Methodological Clinical Data

Approach
From the era of the ‘fathers’ of medicine, like Aesculapius, a
large attention has been given to a correct diet as the best way
to maintain health. During the last century, we have sampled
many data on diets or specific diet components that could have
positive or deleterious effects on health. Nevertheless, what
kinds of data are available?
Preclinical Data
Preclinical data are those derived from test performed in cellu-
lar models or in animals. They are usually carried out on single
components of the diet or of a nutrient and furnish informa-
tion on the pharmacological activities of foods or food com-
ponents. Even if the obtained results are usually of scientific
interest, the proposed models do not adequately represent the
complexity of human diets and human metabolism, so they
can only constitute a basis for further research on humans.
Epidemiological Data
Epidemiological data relate the assumption of specific dietary
components or specific dietary patterns with the risk of devel-
oping a disease. They are actually the milestones on which we
built dietary guidelines and furnish dietary advices. They are
particularly relevant when obtained from large population
samples followed up for years with a standardized methodol-
ogy. The main limitations of epidemiological data are the
following:
• They are population-specific: It is not immediately true that
a protective habit for a population is also protective for a
different one because other factors could be involved, such
as other dietary components, physical activity, environ-
ment, and genetics/genomics. Moreover, the dietary ques-
tionnaires used in various studies are frequently different,
and they are not always validated in different populations.
• They are usually ‘old’: During the time in which the study is
carried out (from years to decades) and the data are ana-
lyzed, the dietary and behavioral habits of the population
could have changed. A typical example is the one of the
Mediterranean diet, codified on the basis of the southern
Italian habits in the 1950s, which are totally different from
the current ones.
• They are usually related to a lifelong exposition to a specific
diet or diet components. Nonetheless, observing that the
lifetime exposition to a diet component seems to be pro-
tective against the development of a given disease does not
immediately mean that it will be protective when intro-
duced/increased in the diet starting from the middle or
old age.
Data derived from clinical trials represent the best source of
evidence. They clearly show how changing a diet pattern or
increasing/reducing a specific diet component is associated
with a modification of biological parameters (usually a labo-
ratory or instrumental biomarker) in humans. The main limi-
tations of clinical trials are the large number of concomitant
parameters to be controlled (both in the diet and outside the
diet) and the lack of long-term large trials with hard outcomes
(morbidity or mortality).
Systematic Reviews/Meta-analyses/Metaregressions
Finally, we could ‘cumulate’ the evidence derived from epide-
miological studies or clinical trials in order to obtain a general
conclusion on a specific dietary exposition or intervention on
specific parameters of human health. These analyses, when
available, usually constitute the basis for new guidelines, even
if their value could not be absolute for the single subject or a
specific subpopulation. Then, their conclusion does not always
reflect a final answer to a scientific question, especially when
the available trials are strongly heterogeneous as it regards
design, number, and kind of subjects enrolled and other
study characteristics. Thus, from all these data, we still have
no definitive idea about the ‘perfect diet’ to be adopted by the
general population in order to conduct a healthy and pro-
longed life, but we can highlight some milestones.
General Rules for the General Population
Large-scale unfocused interventions on lifestyle habits have
been proved to be needed but hardly cost-effective. The best
way to plan dietary suggestions in order to improve the global
health of a whole population, apart from the individual needs,
is to attentively detect, between the most recent scientific liter-
ature, the most evidence-based milestones of a healthy diet and
healthy lifestyle, avoiding in particular to focus on intermedi-
ate outcomes, but rather concentrating on morbidity and mor-
tality. Moreover, the suggestions have to be as short, clear, and
simple as possible, feasible by the subjects, and possibly trans-
versally useful to prevent the most common chronic diseases in
a population. To do this, a possible way consists on focusing
our attention on the results of the largest available systematic
reviews and meta-analyses.
Overall, the main recent meta-analysis of clinical data
suggests the following:
• Water is a milestone of a healthy diet.
• Any excessive energy intake has to be avoided: Since pre-
clinical data show that a mildly reduced energy intake is per
se associated with an increased life span, an adequate

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414 Dietary Practices
energy intake is probably overall more relevant than any
other dietary indications.
• Increased sodium intake was associated with an increased
risk of stroke (
25%), stroke mortality (
60%), and cor-
onary heart disease mortality (
30%).
• A diet with a low glycemic index (GI) and glycemic load
(GL) is associated with a reduced risk of developing type 2
diabetes (T2D) and coronary heart disease (CHD). In par-
ticular, the risk of developing T2D is 33% higher in subjects
in the highest quintile of energy-adjusted GI than those in
the lowest one. Participants who consume a combination
diet high in GI or GL and low in cereal fiber have an
50%
higher risk of T2D. GL appears to be more relevant than GI
in increasing the risk of CHD (
50%) and stroke (
20%)
in a dose-dependent manner.
• Overall, nonrigid vegetarian diets (neither vegan nor frui-
tarian) are associated with a lower risk of developing car-
diovascular diseases, cancers, and autoimmune diseases.
• A diet rich in cereal and vegetable fiber is associated with a dose-
dependent reduction of developing cardiovascular disease
(
9% per 7 g per day) and various gastrointestinal
(
10–40% per 10 g per day) and some nongastrointestinal
cancers. The total intake of fruit and vegetables had a negative
association with risk of asthma in adults and children (
45%).
• Red meat is per se not significantly associated with total or
cause-specific mortality; conversely, the intake of processed
meat was associated with a 30% higher rate of cardiovascu-
lar disease (per 50 g per day) and higher cancer mortality.
Preservatives are the notable difference; the calculated
blood-pressure effects of sodium differences (
400%
higher in processed meats) explain most of the observed
higher risk.
3% of deaths could be prevented if all partic-
ipants had a processed meat consumption of less than 20 g
per day. Moreover, increasing the intake of red meat of
more than 0.50 servings per day is associated with an
increased risk of developing T2D of
55% while reducing
the intake of the same amount leads to a decreased risk
of
15%.
• The highest category of fish consumption (i.e.,  4 times
per week) is associated with the greatest risk reduction in
CHD (
20%). In dose-response analysis, each additional
100 g serving of fish per week was associated with an
5%
reduced risk.
• There is a linear association of consumption of total dairy
products, low-fat dairy products, cheese, and yogurt and the
risk of T2D. The relative risk reduction is
5% and
10%
for 200 g per day total and low-fat dairy consumption,
respectively. The relative risk reduction is
20% and

10% for 30 gper day cheese and 50 g per day yogurt
consumption, respectively.
• Dietary consumption of saturated fatty acids and choles-
terol is not significantly associated with an increase in
cardiovascular disease risk, if not because usually assumed
with high-energy diet.
• Extreme quintiles of total trans fatty acid intake (3–10 g per
day) are associated with an increased risk of
20% of CHD
and
25% of fatal CHD.
• The consumption of nuts is inversely associated with fatal
CHD (
25% less risk), nonfatal CHD (
20% less risk),
and T2D (
10% less risk).
• Coffee intake is associated with a lower risk of mortality for
every cause (high vs low category of coffee consumption:
RR ¼ 0.86). In particular, this seems to be related to a reduc-
tion in the risk of developing cardiovascular disease, T2D,
and some cancers too. Moreover, coffee consumption
seems associated with a lower risk of developing seroposi-
tive rheumatoid arthritis, urolithiasis, and Parkinson’s
disease.
• High-quality chocolate intake is associated with a reduced
risk of developing stroke of
20%.
• Low alcohol intake (from every source, not necessarily from
red wine) is associated with a reduced risk of CHD
(
20%), total stroke (
15%), ischemic stroke (
20%),
and stroke mortality (
30%), but heavy alcohol intake is
associated with an increased risk of total stroke (
20%).
Moreover, compared with lifetime abstainers, the RR for
T2D among men is most protective when consuming 22 g
per day alcohol and becomes deleterious at just over 60 g
per day alcohol. Among women, consumption of 24 g per
day alcohol is most protective and becomes deleterious at
about 50 g per day alcohol. Furthermore, low alcohol
intake also seems to be associated with a lower risk of
developing rheumatoid arthritis, urolithiasis, and
Parkinson’s disease. However, even a low alcohol intake is
a risk factor for (among others) gout, atrial fibrillation,
colorectal adenoma development, and upper aerodigestive
tract cancer mortality.
• Potassium intake is associated with a reduced risk of devel-
oping stroke (
20%).
• Dietary magnesium (per 200 mg per day increment) is
associated with a 22% lower risk of CHD. The association
of dietary magnesium with fatal CHD is nonlinear, with an
inverse association observed up to a threshold of
250 mg
per day, compared with lower intakes.
• For total calcium intake, each 300 mg per day increase is
associated with an approximately 8% reduced risk of colon
cancer. For supplementary calcium, each 300 mg per day
increase is associated with a 9% reduced risk of colon
cancer.
Overall, the available evidence suggests that a complete bal-
anced ‘Mediterranean-like’ antioxidant and anti-inflammatory
diet, mainly based on fresh vegetables and whole-grain
carbohydrates (nearly vegetarian, not necessarily vegan),
should be advised to most of the people in order to preserve
and promote their health (Box 1). Most of the successful diets
(Mediterranean, Asian, Atkins, and zone/dissociated) are low-
Box 1 Milestones of a Healthy Diet
n Total energy intake proportional to physical activity
n Low salt intake
n Carbohydrates with low glycemic index as the main source of energy
n Very low intake of simple sugars
n Large intake of water, fresh vegetables, legumes, and berries
n Fish and nuts and moderate amount of nonprocessed meat
n Moderate doses of dairy products (preferably fermented, rich in probio-
tics, and low-fat)
n Coffee, high-quality dark chocolate, and low quantity of alcohol (from
either wine or beer) not forbidden

energy diets fundamentally based on these principles. Low-
carb/high-protein diet could be considered for a short period
in selected overall healthy subjects to reduce body weight and
related cardiovascular risk factors, but then, a standard bal-
anced diet has to be recovered as soon as possible. Extreme
diets (high protein, vegan, etc.) have to be avoided, especially
on the long term for the risk of developing deficit of intakes
(for instance, of essential amino acids and some vitamins) or
excessive intakes (for instance, of nonessential amino acids) of
specific micro- and macronutrients.
Finally, all the potential suggestions previously reported
increase their potential effect on global health if associated
with a strong message on dramatic reduction of active and
passive exposition to cigarette smoking and on an improve-
ment of everyday physical activity amount.
The main advantage of this approach is that the same life-
style suggestions seem to be useful to reduce the risk of cardio-
vascular disease, obesity, type 2 diabetes, nonalcoholic liver
fatty acids, age-related degenerative maculopathy, the most
frequent cancers, some degenerative neurological diseases,
and rheumatologic diseases.
A factor that has to be considered is that a healthy diet is
often slightly but significantly more expensive than an
unhealthy diet, so, when formulating suggestions, the avail-
ability of the foods and their cost always have to be kept in
mind. For instance, even if Asian goji berries have a very high
antioxidant power, it is nonsense to recommend their
consumption in Europe, where various much less expensive
berries with high antioxidant power are available.
It is also relevant to remember that some of the previously
reported suggestions do not count for every subject. For
instance, obese, diabetics, hypertriglyceridemics, and patients
affected by hyperuricemia have to consume fruits in modera-
tion since they are a relevant source of energy and induce the
synthesis of uric acid.
Another factor to be considered is the modality of cooking.
Some foods (in particular, vegetables) have not to be cooked in
order not to lose vitamins and minerals. The best ways to cook
are with steam or on hot stones, less salting, boiling, or baking.
Pasta and rice have to be boiled just the time to make them
chewable (slightly underdone), but not overcooked, in order
not to increase their glycemic index. Salt has to be limited at
minimum and possibly substituted with spices (e.g., pepper,
chili, and aromatic herbs), lemon juice, or vinegar. Vegetable
oils (preferably extra virgin olive oil, less sunflower oil, corn oil,
or rice oil), rich in n-6 fatty acids, which can be useful in the
prevention of cardiovascular disease, have to be used without
reaching their smoking point, that is, before they are peroxidized.
Anyway, even though they are ‘healthy,’ vegetable oils constitute
a relevant source of energy and thus have to be consumed in
moderation. Animal fats have to be avoided; however, they are
always preferable than modified trans-hydrogenated fats.
The Individual Approach
Mind over Food and Eating Disorders
If we only had our core systems such as the brain stem and
hypothalamus, we would not probably have many of the
problems linked to food, such as obesity and related diseases
Dietary Practices 415
or eating disorders. The communication between the internal
system of fuel availability and the hypothalamus, where hun-
ger and the satiety centers are located, is regulated by a homeo-
static control system through hormones capable of crossing the
blood–brain barrier. In this way hunger, satiety, metabolic
functions, and fat reserves are adjusted in the short and long
terms.
The main molecules involved in homeostatic control are
leptin and adiponectin (produced by the adipose tissue), ghrelin
(produced by the stomach), peptide YY (produced by the intes-
tinal cells), and neuropeptide Y and agouti-related protein (pro-
duced by the hypothalamus), where ghrelin and neuropeptide
Y stimulate the appetite, while leptin and peptide YY inhibit
the appetite.
With respect to cardiovascular disease risk, adiponectin is a
‘good’ adipokine due to its anti-inflammatory, antiatherogenic,
antidiabetic, and cardioprotective effects and the promotion of
good endothelial function. Leptin, on the contrary, could be
considered a ‘bad’ adipokine given that it promotes insulin
resistance, high blood pressure, atherosclerosis, MI risk, vascular
inflammation, VSMC hypertrophy and proliferation, oxidative
stress, and endothelial dysfunction.
Our modern lifestyle puts pressure on the homeostatic
system. Many factors such as stress, anxiety, depression, and
nervousness could influence our body weight. Palatable food
high in fat, sugar, and salt can increase dopamine in the sub-
cortical mesolimbic system (the ventral tegmental area,
nucleus accumbens, amygdala, and hippocampus). This gen-
erates an intense pleasure and can corrupt neural functions of
the brain system involved in hedonic, motivational, and cog-
nitive processing. For this reason, it has been suggested that
there is a possibility for the development of a food addiction.
Further studies are needed to better describe these mechanisms
and to understand how to utilize the modern neuromarketing
to promote the consumption of healthy food.
An abnormal relationship with food can result in an eating
disorder (ED). The main consequences of EDs are malnutrition,
obesity, a worse quality of life due to the obsessive thoughts
related to food, and increased mortality. They are commonly
rare in the general population; the prevalence of all EDs is
about 5%. Patients who suffer from an ED tend to deny the
disease and avoid treatment. The derivation of EDs is psycho-
logical. The causes could be genetic or they could derive from
other pathologies such as anxiety, depression, and alcoholism.
The Diagnostic and Statistical Manual of Mental Disorders (5th
edition) distinguishes EDs as anorexia nervosa and bulimia
nervosa, which are common in adolescent women; binge eat-
ing disorders typical in men and adults; and not otherwise
specified EDs. The therapy for EDs consists in a team treatment
carried out by different professionals: psychiatrists, psycholo-
gists, nutritionists, nurses, and other medical professionals.
The nutritional rehabilitation of an ED should provide a grad-
ual modification of eating habits. The expectation is often not a
complete cure, rather the improvement of the nutritional status
and the quality of life of the individual.
Clinical Nutrition and Morbidities
Many diseases require a personalized nutrition for their care.
The diet thus becomes an integral part of the therapy. If you
416 Dietary Practices
think you are suffering from one of these conditions, you
should contact medical professionals and a nutritionist.
Overweight and obesity
The key strong recommendations for overweight and obesity
recently published in the Journal of the American College of
Cardiology are as follows:
• Advising overweight and obese individuals who would
benefit from weight loss to participate for  6 months in a
comprehensive lifestyle program that assists participants in
adhering to a lower calorie diet and increasing physical
activity through the use of behavioral strategies
• Prescribing on-site, high-intensity (i.e., 14 sessions in 6
months) comprehensive weight loss intervention
• Using a very-low-calorie diet (800 kcal day1) only in lim-
ited circumstances where a medical supervisor is required
• Advising overweight and obese individuals who have lost
weight to participate in a long-term ( 1 year) comprehen-
sive weight loss maintenance program
Cardiovascular disease and dyslipidemia
In order to lower the levels of total cholesterol and LDL choles-
terol in heart diseases, the American College of Cardiology and the
American Heart Association recommend normalizing body weight
and reducing the consumption of saturated fats, cholesterol, and
trans fats. Saturated fats are found primarily in dairy products,
meats, and certain oils (coconut and palm oils). Trans fats are
found in many snacks and fried foods and are listed on labels as
‘partially hydrogenated oils.’ The intake of soy protein and func-
tional foods rich in phytosterols could play a positive role in
reducing heart diseases. In order to lower triglycerides, it is useful
to reduce the consumption of alcohol, simple sugars, and
carbohydrates. Finally, a rise in unsaturated fats and constant
physical activity could help increase our HDL cholesterol levels.
However, in our attempts to cut ‘bad fats,’ we should be careful
not to increase the intake of other potentially dangerous nutrients,
such as sugars. Heart health is also linked to the adequate intro-
duction of protective foods, such as fruits and vegetables, recom-
mended by the World Health Organization in an amount of 400 g
per person per day. Cholesterolemia being strongly genetically
determined, recent guidelines also suggest to include in the every-
day diet some specific nutraceuticals and functional foods in order
to further reduce the LDL cholesterol plasma levels.
Diabetes
The American Diabetes Association recognizes the integral role of
nutrition therapy in overall diabetes management. For individ-
uals with type 1 diabetes, participation in an intensive flexible
insulin therapy education program, using the carbohydrate
counting meal planning approach, can result in improved
glycemic control. A simple diabetes meal planning approach,
such as portion control or healthy food choices, may be better
suited for individuals with type 2 diabetes. In summary, people
with diabetes should
• limit or avoid the intake of sugar-sweetened beverages;
• reduce sodium to less than 2300 mg per day, with addi-
tional reductions individualized for those who have a high
blood pressure;
• eat fatty fish at least 2 times (two servings) per week (they
do not benefit from the use of omega-3 (EPA/DHA) sup-
plements for the prevention or treatment of cardiovascular
diseases);
• not use vitamin or mineral supplements without proved
deficiencies (additionally, there is no evidence supporting
the use of cinnamon or other supplements).
Food allergies and food intolerances
Whereas a food allergy causes an immune system reaction, a
food intolerance does not. In some cases, an allergic reaction to
food can be serious, even deadly. Food intolerance symptoms
generally appear gradually and are limited to gastrointestinal
effects, whereas food allergy symptoms are more severe. If you
have a food allergy, you will need to avoid the allergenic food
completely. If you have a food intolerance, you may generally
be able to eat a small amount of the food in question. Lactose
intolerance is a common example of an enzymatic intolerance,
which is due to a lack of lactase needed to digest lactose. Celiac
disease is a permanent intolerance to gluten, a protein found in
oats, wheat, spelled, Kamut®, barley, and rye. Indeed, the
consumption of gluten, also in small quantities, can cause
several more or less serious consequences. The nonceliac gluten
sensitivity is today a still poorly defined condition. The aim of
the scientific community is to test and find a definition of the
specific markers and precise diagnostic criteria.
Inflammatory bowel diseases
Patients with inflammatory bowel diseases (IBDs) should
generally avoid foods that worsen symptoms, eat smaller
meals in frequent intervals, drink adequate fluids, and avoid
caffeine and alcohol. IBD patients should eliminate dairy
if they are lactose-intolerant, limit excess fat, reduce simple
carbohydrates, and reduce high-fiber foods during the inflam-
matory phase.
Chronic kidney disease
People with a chronic kidney disease (CKD) need to make
changes to their diet such as limiting fluids; eating a low-
protein diet; limiting sodium, potassium, phosphorous, and
other electrolytes; and paying attention to malnutrition. The
diet can be changed if the kidney disease worsens and during
dialysis.
Neurodegenerative diseases
Neurodegenerative diseases (NDs) are hereditary and sporadic
conditions, characterized by a progressive nervous system dys-
function. They include Alzheimer’s disease and other demen-
tias, such as Parkinson’s disease. The following guidelines
suggest how to prevent and delay the symptoms:
• The intake of saturated fats and trans fats should be reduced
to the lowest amount possible.
• Vegetables, legumes, fruits, and whole grains should replace
meats and dairy products.
• Vitamin E should come from foods, rather than from
supplements.
• A reliable source of vitamin B12 should be part of one’s
daily diet.

• If using multiple vitamins, choose those without iron and
copper and consume iron supplements only when directed
by your physician.
• Those who desire to minimize exposure to aluminum
should avoid the use of cookware, baking powder, or
other products that contain aluminum.
• Aerobic exercise should be included in an individual’s
routine.
Conclusion
A global healthy diet associated with a healthy lifestyle is
advisable for the general population and has to be founded
on some basic suggestions. The assumption of fresh foods,
without the addition of salt, in amounts proportional to the
energy expenditure, would be sufficient to prevent the largest
part of chronic diseases in Western countries. Only subjects
with specific diseases or characteristics should have a struc-
tured individual diet support.
See also: Alcohol: Metabolism and Health Effects; Aluminum:
Properties, Presence in Food and Beverages, Fate in Humans, and
Determination; Appetite Control in Humans: A Psychobiological
Approach; Bacillus cereus and Other Bacillus sp. Causing Foodborne
Poisonings, Detection of; Barley; Berries and Related Fruits; Bioactive
Peptides in Foods; Cereals: Types and Composition; Cheese:
Composition and Health Effects; Cholesterol: Factors Determining
Blood Cholesterol Levels; Cocoa: Composition and Health Effects;
Coffee: Health Effects; Colon: Diseases and Disorders; Dairy Products:
Dietary and Medical Importance; Eating Disorders; Energy: Intake and
Energy Requirements; Fatty Acids: Essential Fatty Acids; Fatty Acids:
Fatty Acids; Fatty Acids: Trans Fatty Acids; Fish: Dietary Importance
and Health Effects; Food Allergies; Fructose and High-Fructose Corn
Syrup; Fructose: Sources, Metabolism, and Health; Glucose: Glucose
Intolerance; Hypertension and Diet; Legumes in the Diet; Magnesium;
Meat: Role in the Diet; Nutritional Epidemiology; Nuts: Health Effects;
Obesity: The Role of Diet; Olive Oil: Its Role in the Diet; Potassium:
Properties and Determination; Protein Quality and Amino Acids in
Maternal and Child Nutrition and Health; Retinol: Properties and
Dietary Practices 417
Determination; Satiety; Spices and Flavoring Crops: Uses and Health
Effects; Starch; Vegetable Oils: Dietary Importance; Vegetarian Diets;
Vinegar.
Further Reading
Afshin A, Micha R, Khatibzadeh S, and Mozaffarian D (2014) Consumption of nuts and
legumes and risk of incident ischemic heart disease, stroke, and diabetes: a systematic
review and meta-analysis. American Journal of Clinical Nutrition 100(1): 278–288.
Barnard ND, Bush AI, Ceccarelli A, et al. (2014) Dietary and lifestyle guidelines for the
prevention of Alzheimer’s disease. Neurobiology of Aging 35: S74–S78.
Barry VW, Baruth M, Beets MW, et al. (2014) Fitness vs. fatness on all-cause mortality:
a meta-analysis. Progress in Cardiovascular Diseases 56(4): 382–390.
Berthoud HR (2012) The neurobiology of food intake in an obesogenic, environment.
Proceedings of the Nutrition Society 71(4): 478–487.
Bhattarai N, Prevost AT, Wright AJ, et al. (2013) Effectiveness of interventions to
promote healthy diet in primary care: systematic review and meta-analysis of
randomised controlled trials. BMC Public Health 13: 1203.
Castro-Quezada I, Román-Viñas B, and Serra-Majem L (2014) The Mediterranean diet
and nutritional adequacy: a review. Nutrients 6(1): 231–248.
Evert AB, Boucher JL, Cypress M, et al. (2013) Position statement. Nutrition therapy
recommendations for the management of adults with diabetes. Diabetes Care
36: 3821–3842.
Fan J, Song Y, Wang Y, Hui R, and Zhang W (2012) Dietary glycemic index, glycemic
load, and risk of coronary heart disease, stroke, and stroke mortality: a systematic
review with meta-analysis. PloS ONE 7(12): e52182.
Freedman LS, Schatzkin A, Midthune D, and Kipnis V (2011) Dealing with dietary
measurement error in nutritional cohort studies. Journal of the National Cancer
Institute 103: 1086–1092.
Gulliford MC, Bhattarai N, Charlton J, and Rudisill C (2014) Cost-effectiveness of a
universal strategy of brief dietary intervention for primary prevention in primary care:
population-based cohort study and Markov model. Cost Effectiveness and Resource
Allocation Journal 12(1): 4.
Kushner RF and Sorensen KW (2013) Lifestyle medicine: the future of chronic disease
management. Current Opinion in Endocrinology, Diabetes and Obesity 20(5): 389–395.
Matheson GO, Klügl M, Engebretsen L, et al. (2013) Prevention and management of
non-communicable disease: the IOC consensus statement, Lausanne 2013. British
Journal of Sports Medicine 47(16): 1003–1011.
Pallauf K, Giller K, Huebbe P, and Rimbach G (2013) Nutrition and healthy ageing:
calorie restriction or polyphenol-rich “MediterrAsian” diet? Oxidative Medicine and
Cellular Longevity 2013: 707421.
The American College of Cardiology, American Heart Association Task force on Practice
Guidelines and the Obesity society (2014) 2013 AHA/ACC/TOS Guideline for the
management of overweight and obesity in adults. Journal of the American College of
Cardiology 63(25): 2985–3023.
The Task Force for the management of dyslipidaemias of the European Society of Cardiology
(ESC) and the European Atherosclerosis Society (EAS) (2011) ESC/EAS Guidelines for
the management of dyslipidaemias. European Heart Journal 32(14): 1769–1818.
 Dietary References: US
J Dwyer, RD Tufts Medical Center, Boston, MA, USA
NJ Armstrong, Tufts University, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Dietary reference intakes (DRIs) are a set of nutrient-based
reference values that provide quantitative recommendations by
gender, age, life stage, or physiological condition (such as preg-
nancy or lactation) for nutrient intakes of individuals living in
the United States and Canada. The reference values include the
estimated average intake (EAR), the recommended dietary
allowance (RDA), the adequate intake (AI), the tolerable upper
intake level (UL), and, for energy providing nutrients, the
acceptable macronutrient distribution range (AMDR). DRIs pro-
vide nutrient standards that are used to shape various public
policies, such as nutrient standards for school lunch programs,
congregate and delivered elderly meal programs, and guidelines
for the women, infants, and children supplemental feeding
program. The DRIs are the basis for developing dietary advice
for patients requiring medical nutrition therapy and to establish
the daily values displayed on the nutrition fact panels of pack-
aged food and supplements. They are the foundation for formu-
lating the Dietary Guidelines for Americans.
History
The DRIs replaced the RDA, a nutrient standard used in the
United States from 1941 to 1989 and a similar Canadian
standard. These newer values were developed because of
changes in science, limitations of the existing RDA, and the
need to consider the role of nutrients in preventing dietary
deficiency diseases as well as their associations with diet-
related chronic degenerative diseases and toxicities. Starting
in 1994, expert committees examined the scientific data on
requirements of nutrients for these functions. The Standing
Committee on the Scientific Evaluation of DRIs coordinated
the effort under the direction of the Food and Nutrition Board,
Institute of Medicine (IOM) of the National Academy of
Sciences, and Health Canada.
Indicators Used to Establish DRIs
Many possible functional indicators and health outcomes exist
and are examined by the DRI committees, including chronic
disease indicators. The DRIs are based on the relationship
between a nutrient and a single functional indicator of the
health outcome of interest. These functional indicators may
involve intake of a nutrient such as vitamin D and a status
biomarker such as 25-OH-D of known sufficiency for bone
health outcomes. Alternatively, for other nutrients such as
ascorbic acid, the functional biomarker is a plasma ascorbic
acid level above the threshold, 0.2 mg dl
1, which is adequate
to prevent scurvy. For example, night blindness is associated
418 Encyclopedia of Food and Health
with vitamin A deficiency and its prevention is a desirable
health outcome. The ability of the retina to adapt to dim
light depends on AIs of vitamin A (retinal). It plays an impor-
tant role in rod function during dim light. Without an ade-
quate retinal supply, the functions of the rods become
compromised in dim light settings, and if chronic vitamin A
deficiency occurs, it can result in night blindness.
Definitions of DRIs
Table 1 summarizes definitions of the DRIs. After the choice of
a criterion for nutrient adequacy is established, the existing
experimental data are analyzed and summarized. The esti-
mated average requirement (EAR) is the amount of a nutrient
that meets the requirement of a specific criterion of adequacy
(as determined by the functional indicator) for half of the
healthy individuals within a defined age, gender, and life
stage. There must be enough scientific evidence available to
set an EAR for a nutrient and to calculate an RDA. The RDA is
the average daily dietary intake that meets the nutrient require-
ments of nearly all healthy individuals of a specific age, gender,
life stage, or physiological condition for the outcome of inter-
est. If the statistical distribution of the amounts of nutrient
intake needed to achieve the EAR, for example, the desired level
of the functional indicator for the specific population, is deter-
mined to be normal (Gaussian) (Figures 1 and 2), and the
standard deviation (SD) can be determined, then the following
calculation is used:
RDA ¼ EAR þ 2  SD
When there are insufficient data or variability in order to
calculate an SD for the nutrient, a symmetrical distribution is
assumed and then a coefficient of variation (CV) of 10% EAR is
usually used. The calculation then becomes
RDA ¼ EAR þ 2  CV or EAR þ 2ð0:1  EARÞ
Note: the CV is usually set at 10% unless variability is
known to be greater.
When nutrient requirements are not normally distributed,
as, for example, is the case for iron, then statistical transforma-
tion of the data is required before the EAR and RDA are set.
When insufficient evidence is available to determine an EAR
and RDA, then an AI is derived to provide an intake recom-
mendation. The AI is a recommended daily intake of a nutrient
that is based on observational and experimental data or obser-
vations of average intakes of a group or groups of presumably
healthy people. The AI represents an informed decision about
what seems to be an appropriate intake for an individual based
primarily on epidemiological rather than an experimentally
determined value and it should be used more cautiously than
the RDA.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00233-6
 Dietary References: US 419

Table 1 Terms used in US reference standards

Term Definition

Estimated average requirement (EAR)
Recommended dietary allowance
(RDA)
Adequate intake (AI)
Tolerable upper intake level (UL)
Acceptable macronutrient distribution
range (AMDR)
Estimated energy requirement (EER)
Total energy expenditure (TEE)
The amount of an individual nutrient that meets the requirement for a specific criterion of adequacy for half of
the healthy individuals within a defined age, gender, or life stage
The average daily dietary intake level that meets the nutrient requirement of nearly all healthy individuals of a
specific age, gender, life stage, or physiological condition
The nutrient intake amount that is set when decisive data on the requirement are lacking in order to set an EAR
and RDA
The highest level of daily nutrient intake that is likely to pose no risk of adverse health effect to almost all
individuals
A range of intakes for macronutrients expressed as the proportion of total energy intake that is associated
with chronic disease reduction and provides adequate amounts of the nutrient while maintaining energy
balance
The requirement for energy of healthy adults with a body mass index (BMI) in the normal range of 18.5 and 25
Set of equations that estimate the energy expenditure needed to maintain current body weight and activity
levels

EAR

50% 50%

34% 34%

RDA

13.5% 13.5%
2.5% 2.5%
–3 SD –2 SD –1 SD 0 +1 SD +2 SD +3 SD
Mean

Median
Percentile
rank 2.5 16 50 84 97.5
Figure 1 The normal distribution of a nutrient requirement and placement of the EAR and RDA. Reprinted with permission from Dietary reference
intakes: the essential guide to nutrient requirements, 2006 by the National Academy of Sciences, courtesy of the National Academies Press,
Washington, DC.

Table 2 presents the RDA and AI for vitamins, and Table 3
presents the same values for mineral elements. Table 4 presents
the RDA and AI for total water and macronutrients.
Little experimental data are available on nutrient require-
ments for children, and thus, the EAR, RDA, and UL are based
on extrapolations from adult values. For setting an EAR, the
extrapolations for infants include scaling down the require-
ments by a specified power to adjust for metabolic differences
or adjustments based upon reference body weights. The AIs are
estimated by observing the amounts of nutrients eaten by
healthy infants or children.
The AMDR provides acceptable high and low limits of
protein, carbohydrate, and fat (n
6 and n
3 polyunsaturated
fatty acids) expressed as percent of energy needs. It is based on
some intervention trials but primarily on epidemiological
studies indicating that an imbalance in macronutrients can
increase risk of several diet-related chronic degenerative dis-
eases including coronary heart disease, obesity, type 2 diabetes,
and some cancers. Table 5 presents the AMDRs. One of the key
features of the AMDR is that it gives an upper- and lower-level
boundary for the range since there is an increased risk resulting
from chronic intakes both above and below it. Note that the
Table 2 Dietary reference intakes (DRIs): recommended dietary allowances and adequate intakes, vitamins (Food and Nutrition Board, Institute of Medicine, National Academies)

Vitamin A Vitamin C Vitamin D Vitamin E Vitamin K Thiamin Riboflavin Niacin Vitamin B6 Folate Vitamin Pantothenic Biotin Choline
Life stage/ (mg (mg (mg (mg (mg (mg (mg (mg (mg (mg B12 (mg acid (mg (mg (mg
group day
1)a day
1) day
1)b,c day
1) d day
1) day
1) day
1) day
1)e day
1) day
1)f day
1) day
1) day
1) day
1)g

Infants
0–6 months 400* 40* 10 4* 2.0* 0.2* 0.3* 2* 0.1* 65* 0.4* 1.7* 5* 125*
6–12 500* 50* 10 5* 2.5* 0.3* 0.4* 4* 0.3* 80* 0.5* 1.8* 6* 150*
months
Children
1–3 years 300 15 15 6 30* 0.5 0.5 6 0.5 150 0.9 2* 8* 200*
4–8 years 400 25 15 7 55* 0.6 0.6 8 0.6 200 1.2 3* 12* 250*
Males
9–13 years 600 45 15 11 60* 0.9 0.9 12 1.0 300 1.8 4* 20* 375*
14–18 900 75 15 15 75* 1.2 1.3 16 1.3 400 2.4 5* 25* 550*
years
19–30 900 90 15 15 120* 1.2 1.3 16 1.3 400 2.4 5* 30* 550*
years
31–50 900 90 15 15 120* 1.2 1.3 16 1.3 400 2.4 5* 30* 550*
years
51–70 900 90 15 15 120* 1.2 1.3 16 1.7 400 2.4h 5* 30* 550*
years
>70 years 900 90 20 15 120* 1.2 1.3 16 1.7 400 2.4h 5* 30* 550*
Females
9–13 years 600 45 15 11 60* 0.9 0.9 12 1.0 300 1.8 4* 20* 375*
14–18 700 65 15 15 75* 1.0 1.0 14 1.2 400i 2.4 5* 25* 400*
years
19–30 700 75 15 15 90* 1.1 1.1 14 1.3 400i 2.4 5* 30* 425*
years
31–50 700 75 15 15 90* 1.1 1.1 14 1.3 400i 2.4 5* 30* 425*
years
51–70 700 75 15 15 90* 1.1 1.1 14 1.5 400 2.4h 5* 30* 425*
years
>70 years 700 75 20 15 90* 1.1 1.1 14 1.5 400 2.4h 5* 30* 425*
Pregnancy
14–18 750 80 15 15 75* 1.4 1.4 18 1.9 600j 2.6 6* 30* 450*
years
19–30 770 85 15 15 90* 1.4 1.4 18 1.9 600j 2.6 6* 30* 450*
years
31–50 770 85 15 15 90* 1.4 1.4 18 1.9 600j 2.6 6* 30* 450*
years
Lactation
14–18 1200 115 15 19 75* 1.4 1.6 17 2.0 500 2.8 7* 35* 550*
years
19–30 1300 120 15 19 90* 1.4 1.6 17 2.0 500 2.8 7* 35* 550*
years
31–50 1300 120 15 19 90* 1.4 1.6 17 2.0 500 2.8 7* 35* 550*
years

Note: This table (taken from the DRI reports; see www.nap.edu) presents recommended dietary allowances (RDAs) in bold type and adequate intakes (AIs) in ordinary type followed by an asterisk (*). An RDA is the average daily dietary intake level,
sufficient to meet the nutrient requirements of nearly all (97–98%) healthy individuals in a group. It is calculated from an estimated average requirement (EAR). If sufficient scientific evidence is not available to establish an EAR, and thus calculate an RDA,
an AI is usually developed. For healthy breastfed infants, an AI is the mean intake. The AI for other life stages and gender groups is believed to cover the needs of all healthy individuals in the groups, but the lack of data or uncertainty in the data prevents
one from specifying with confidence the percentage of individuals covered by this intake.
a
As retinol activity equivalents (RAEs). 1 RAE ¼ 1 mg retinol, 12 mg b-carotene, 24 mg a-carotene, or 24 mg b-cryptoxanthin. The RAE for dietary provitamin A carotenoids is twofold greater than retinol equivalent (RE), whereas the RAE for preformed
vitamin A is the same as the RE.
b
As cholecalciferol. 1 mg cholecalciferol ¼ 40 IU vitamin D.
c
Under the assumption of minimal sunlight.
d
As a-tocopherol. a-tocopherol includes RRR-a-tocopherol, the only form of a-tocopherol that occurs naturally in foods, and the 2R-stereoisomeric forms (RRR-, RSR-, RRS-, and RSS-a-tocopherol) that occur in fortified foods and supplements. It
does not include the 2S-stereoisomeric forms (SRR-, SSR-, SRS-, and SSS-a-tocopherol), also found in fortified foods and supplements.
e
As niacin equivalents (NE). 1 mg of niacin ¼ 60 mg of tryptophan; 0–6 months ¼ preformed niacin (not NE).
f
As dietary folate equivalents (DFE). 1 DFE ¼ 1 mg food folate ¼ 0.6 mg of folic acid from fortified food or as a supplement consumed with food ¼ 0.5 mg of a supplement taken on an empty stomach.
g
Although AIs have been set for choline, there are few data to assess whether a dietary supply of choline is needed at all stages of the life cycle, and it may be that the choline requirement can be met by endogenous synthesis at some of these stages.
h
Because 10–30% of older people may malabsorb food-bound B12, it is advisable for those older than 50 years to meet their RDA mainly by consuming foods fortified with B12 or a supplement containing B12.
i
In view of evidence linking folate intake with neural tube defects in the fetus, it is recommended that all women capable of becoming pregnant consume 400 mg from supplements or fortified foods in addition to intake of food folate from a varied diet.
j
It is assumed that women will continue consuming 400 mg from supplements or fortified food until their pregnancy is confirmed and they enter prenatal care, which ordinarily occurs after the end of the periconceptional period – the critical time for
formation of the neural tube.
Source: Reprinted with permission from Dietary reference intakes for calcium, phosphorous, magnesium, vitamin D, and fluoride (1997); Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and
choline (1998); Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids (2000); Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium,
and zinc (2001); Dietary reference intakes for water, potassium, sodium, chloride, and sulfate (2005); and Dietary reference intakes for calcium and vitamin D (2011) by the National Academy of Sciences, courtesy of the National Academies Press,
Washington, DC. These reports may be accessed via www.nap.edu.
Table 3 Dietary reference intakes (DRIs): recommended dietary allowances and adequate intakes, elements (Food and Nutrition Board, Institute of Medicine, National Academies)

Calcium Copper Fluoride Iodine Iron Zinc

Life stage/ (mg Chromium (mg (mg (mg (mg Magnesium Manganese Molybdenum Phosphorus Selenium (mg Potassium Sodium Chloride
group day
1) (mg day
1) day
1) day
1) day
1) day
1) (mg day
1) (mg day
1) (mg day
1) (mg day
1) (mg day
1) day
1) (g day
1) (g day
1) (g day
1)

Infants
0–6 months 200* 0.2* 200* 0.01* 110* 0.27* 30* 0.003* 2* 100* 15* 2* 0.4* 0.12* 0.18*
6–12 260* 5.5* 220* 0.5* 130* 11 75* 0.6* 3* 275* 20* 3 0.7* 0.37* 0.57*
months
Children
1–3 years 700 11* 340 0.7* 90 7 80 1.2* 17 460 20 3 3.0* 1.0* 1.5*
4–8 years 1000 15* 440 1* 90 10 130 1.5* 22 500 30 5 3.8* 1.2* 1.9*
Males
9–13 year 1300 25* 700 2* 120 8 240 1.9* 34 1250 40 8 4.5* 1.5* 2.3*
14–18 1300 35* 890 3* 150 11 410 2.2* 43 1250 55 11 4.7* 1.5* 2.3*
years
19–30 1000 35* 900 4* 150 8 400 2.3* 45 700 55 11 4.7* 1.5* 2.3*
years
31–50 1000 35* 900 4* 150 8 420 2.3* 45 700 55 11 4.7* 1.5* 2.3*
years
51–70 1000 30* 900 4* 150 8 420 2.3* 45 700 55 11 4.7* 1.3* 2.0*
years
>70 years 1200 30* 900 4* 150 8 420 2.3* 45 700 55 11 4.7* 1.2* 1.8*
Females
9–13 years 1300 21* 700 2* 120 8 240 1.6* 34 1250 40 8 4.5* 1.5* 2.3*
14–18 1300 24* 890 3* 150 15 360 1.6* 43 1250 55 9 4.7* 1.5* 2.3*
years
19–30 1000 25* 900 3* 150 18 310 1.8* 45 700 55 8 4.7* 1.5* 2.3*
years
31–50 1000 25* 900 3* 150 18 320 1.8* 45 700 55 8 4.7* 1.5* 2.3*
years
51–70 1200 20* 900 3* 150 8 320 1.8* 45 700 55 8 4.7* 1.3* 2.0*
years
>70 years 1200 20* 900 3* 150 8 320 1.8* 45 700 55 8 4.7* 1.2* 1.8*
Pregnancy
14–18 1300 29* 1000 3* 220 27 400 2.0* 50 1250 60 12 4.7* 1.5* 2.3*
years
19–30 1000 30* 1000 3* 220 27 350 2.0* 50 700 60 11 4.7* 1.5* 2.3*
years
31–50 1000 30* 1000 3* 220 27 360 2.0* 50 700 60 11 4.7* 1.5* 2.3*
years
Lactation
14–18 1300 44* 1300 3* 290 10 360 2.6* 50 1250 70 13 5.1* 1.5* 2.3*
years
19–30 1000 45* 1300 3* 290 9 310 2.6* 50 700 70 12 5.1* 1.5* 2.3*
years
31–50 1000 45* 1300 3* 290 9 320 2.6* 50 700 70 12 5.1* 1.5* 2.3*
years

Note: This table (taken from the DRI reports; see www.nap.edu) presents recommended dietary allowances (RDAs) in bold type and adequate intakes (AIs) in ordinary type followed by an asterisk (*). An RDA is the average daily dietary intake level, sufficient to meet the nutrient
requirements of nearly all (97–98%) healthy individuals in a group. It is calculated from an estimated average requirement (EAR). If sufficient scientific evidence is not available to establish an EAR, and thus calculate an RDA, an AI is usually developed. For healthy breastfed infants, an
AI is the mean intake. The AI for other life stages and gender groups is believed to cover the needs of all healthy individuals in the groups, but the lack of data or uncertainty in the data prevents one from specifying with confidence the percentage of individuals covered by this intake.
Source: Reprinted with permission from Dietary reference intakes for calcium, phosphorous, magnesium, vitamin D, and fluoride (1997); Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline (1998); Dietary
reference intakes for vitamin C, vitamin E, selenium, and carotenoids (2000); and Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc (2001); Dietary reference intakes for water,
potassium, sodium, chloride, and sulfate (2005); and Dietary reference intakes for calcium and vitamin d (2011) by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. These reports may be accessed via www.nap.edu.
Table 4 Dietary reference intakes (DRIs): recommended dietary allowances and adequate intakes, total water and macronutrients (Food and
Nutrition Board, Institute of Medicine, National Academies)

Life stage/ Total watera Carbohydrate Total fiber Fat Linoleic acid a-Linolenic acid Proteinb
group (l day
1) (g day
1) (g day
1) (g day
1) (g day
1) (g day
1) (g day
1)

Infants
0–6 months 0.7* 60* ND 31* 4.4* 0.5* 9.1*
6–12 0.8* 95* ND 30* 4.6* 0.5* 11.0
months
Children
1–3 years 1.3* 130 19* NDc 7* 0.7* 13
4–8 years 1.7* 130 25* ND 10* 0.9* 19
Males
9–13 years 2.4* 130 31* ND 12* 1.2* 34
14–18 years 3.3* 130 38* ND 16* 1.6* 52
19–30 years 3.7* 130 38* ND 17* 1.6* 56
31–50 years 3.7* 130 38* ND 17* 1.6* 56
51–70 years 3.7* 130 30* ND 14* 1.6* 56
>70 years 3.7* 130 30* ND 14* 1.6* 56
Females
9–13 years 2.1* 130 26* ND 10* 1.0* 34
14–18 years 2.3* 130 26* ND 11* 1.1* 46
19–30 years 2.7* 130 25* ND 12* 1.1* 46
31–50 years 2.7* 130 25* ND 12* 1.1* 46
51–70 years 2.7* 130 21* ND 11* 1.1* 46
>70 years 2.7* 130 21* ND 11* 1.1* 46
Pregnancy
14–18 years 3.0* 175 28* ND 13* 1.4* 71
19–30 years 3.0* 175 28* ND 13* 1.4* 71
31–50 years 3.0* 175 28* ND 13* 1.4* 71
Lactation
14–18 years 3.8* 210 29* ND 13* 1.3* 71
19–30 years 3.8* 210 29* ND 13* 1.3* 71
31–50 years 3.8* 210 29* ND 13* 1.3* 71

Note: This table (taken from the DRI reports; see www.nap.edu) presents the recommended dietary allowances (RDAs) in bold type and adequate intakes (AIs) in ordinary type
followed by an asterisk (*). An RDA is the average daily dietary intake level, sufficient to meet the nutrient requirements of nearly all (97–98%) healthy individuals in a group. It is
calculated from an estimated average requirement (EAR). If sufficient scientific evidence is not available to establish an EAR, and thus calculate an RDA, an AI is usually developed. For
healthy breastfed infants, an AI is the mean intake. The AI for other life stages and gender groups is believed to cover the needs of all healthy individuals in the groups, but the lack of
data or uncertainty in the data prevents one from specifying with confidence the percentage of individuals covered by this intake.
a
Total water includes all water contained in food, beverages, and drinking water.
b
Based on g protein per kg of body weight for the reference body weight, for example, for adults 0.8 g kg
1 day
1 body weight for the reference body weight.
c
Not determined.
Source: Reprinted with permission from Dietary Reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (2002/2005) and Dietary
reference intakes for water, potassium, sodium, chloride, and sulfate (2005) by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. The
report may be accessed via www.nap.edu.

Table 5 Dietary reference intakes (DRIs): acceptable macronutrient distribution ranges (Food and Nutrition Board, Institute of Medicine, National
Academies)

Range (percent of energy)

Macronutrient Children, 1–3 years Children, 4–18 years Adults

Fat 30–40 25–35 20–35

n
6 polyunsaturated fatty acidsa (linoleic acid) 5–10 5–10 5–10
n
3 polyunsaturated fatty acidsa (a-linolenic acid) 0.6–1.2 0.6–1.2 0.6–1.2
Carbohydrate 45–65 45–65 45–65
Protein 5–20 10–30 10–35

Macronutrient Recommendation

Dietary cholesterol As low as possible while consuming a nutritionally adequate diet

Trans-fatty acids As low as possible while consuming a nutritionally adequate diet
Saturated fatty acids As low as possible while consuming a nutritionally adequate diet
Added sugarsb Limit to no more than 25% of total energy
a
Approximately 10% of the total can come from longer-chain n
3 or n
6 fatty acids.
b
Not a recommended intake. A daily intake of added sugars that individuals should aim for to achieve a healthful diet was not set.
Source: Reprinted with permission from Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (2002/2005) by the National
Academy of Sciences, courtesy of the National Academies Press, Washington, DC. The report may be accessed via www.nap.edu.

AMDR for an individual can only be calculated once the total
energy expenditure (TEE) is estimated and that it is not based
on experimental data.
The UL is the highest level of daily nutrient intake that is
likely to pose no risk of adverse health effect to almost all
individuals. These are limits set for safety reasons and are not
derived from a continuous distribution like the EAR that per-
mits statistical manipulation of the probabilities of exceeding
the value. UL is determined using a risk assessment model
adapted for nutrients that is set based on expert judgment for
the adverse effect of interest for the nutrient. Table 6 presents
the UL for vitamins, and Table 7 presents the UL for mineral
elements (Figure 2).
The other DRIs describe energy intakes. They include the
resting energy requirement, or REE, and the TEE, an estimate of
the energy expenditure needed to maintain current body weight
and activity levels. There is no RDA for energy since an excess
above one’s energy requirement would result in weight gain. An
individual’s resting energy expenditure only describes needs at
rest. The estimated energy requirement (EER) is the estimated
average dietary intake associated with an individual of a specific
sex, age, height, weight, and activity level. The EER values are
determined from equations that reflect the energy expenditure
of individuals of normal body composition using data from
doubly labeled water experiments. The EER and TEE are the
same under conditions other than growth, pregnancy, or lacta-
tion; the EER prediction equations also account for the deposi-
tion of new tissue during pregnancy or growth and production
of milk in the case of lactation. Many different equations are
used to estimate the TEE or REE, depending on the individual’s
height, weight, age, and current activity level. Some use a mul-
tiplier reflecting physical activity level, ranging from 1 for
sedentary, to 1.48 for moderately active, to higher for the very
active. For hospitalized patients, different multipliers are some-
times used to account for metabolic stresses due to illness. All of
these equations are approximations but useful to obtain a
rough rule of thumb on energy needs.
The DRIs are based on a ‘reference person’ of a given weight
and body composition. Those references are based on typical
characteristics of American and Canadian citizens in these
regards. They present reference values for each nutrient by
age, sex, and physiological condition (pregnancy or lactation)
for healthy people.
Applications
The DRIs are used to assess nutritional adequacy and make
recommendations for dietary planning of individuals and
groups. They are also used as standards for establishing nutri-
ent recommendation for those who are ill. The DRIs for indi-
viduals suffering from disease only change when a specific
nutrient requirement is affected by a disease or condition.
Assessment
The DRIs are used to assess the adequacy of nutrient intakes of
individuals and populations from recalls or records of usual
intake for micronutrients, protein, essential fatty acids, and
carbohydrates. They are also used as a rule of thumb for
Dietary References: US 425
assessing the intakes of individuals, but caution is needed.
Usual intake and not a single day’s intake must be considered.
Moreover, since an individual’s requirement is never known,
only qualitative statements about adequacy are possible. The
EAR is the average requirement for a group, but by definition,
it does not meet the requirements of half of the population. In
contrast, the RDA meets the requirements of virtually all indi-
viduals. Usual intakes at or above the RDA mean a low prob-
ability of inadequacy since the RDA is calculated to meet
97–98% of the needs of the population. Note that intakes
below the RDA may be sufficient for some individuals whose
requirements are low. However, we never know if a given
individual’s requirement is high or low. The likelihood of
nutrient inadequacy increases as usual intakes fall further
and further below the RDA, but intakes below the RDA cannot
be assumed to be inadequate.
Although definitive statements cannot be made, mean intakes
of an individual below the EAR very likely need to be improved
because the probability of inadequacy is by definition at least
50%. An individual’s observed mean intake between the EAR and
the RDA probably needs to be improved, because the probability
of inadequacy is more than 50 but less than 97%. Only if an
individual’s intake has been reported for a large number of days
can one have a considerable degree of confidence that the diet is
inadequate. For nutrients without an EAR or RDA, those who
have intakes above the AI can be considered to have reasonably
good intakes. The AI is an intake at or above which there is low
probability of inadequacy. Therefore, many intakes are already
likely to be in excess of requirements; an intake that is low by the
AI standard may still be perfectly adequate.
The UL is the usual intake above which there is an increased
risk of adverse effects for almost all individuals in the general
population. When assessing nutrient intake, the UL can only
show the likelihood of adverse effects from usual (chronic)
intakes above the UL, not intake on a single day. It is important
to note that with most nutrients, the UL applies to all sources
of dietary intake, but for one nutrient, magnesium, only phar-
macological sources are included.
Dietary assessment of an individual is only one component
of a complete nutritional status evaluation. It should be com-
bined with clinical, biochemical, and anthropometric data to
provide a valid picture of nutritional status, especially in those
who are significantly different than the ‘reference person.’
The goal of assessing nutrient intakes of a population
using the DRI is to determine the prevalence of inadequate
or excessive nutrient intakes within it. There are several
methods available to estimate usual intake distributions
from dietary intake data on two or more days’ worth of
intakes. More information and a link to software are available
through Iowa State University at http://www.cssm.iastate.
edu/software/side/. The statistical techniques are well worked
out, and today, monitoring of dietary adequacy and excess is
done on an ongoing basis in the US National Health and
Nutrition Examination Survey.
Planning
The primary goal of planning an individual’s diet is to devise
an intake pattern that ensures nutrient adequacy while also
minimizing risk of excess and maximizing individual
Table 6 Dietary reference intakes (DRIs): tolerable upper intake levels, vitamins (Food and Nutrition Board, Institute of Medicine, National Academies)

Vitamin Vitamin Vitamin Vitamin Niacin Vitamin Folate Vitamin Pantothenic Choline
Life stage/ A (mg C (mg D (mg E (mg Vitamin K Thiamin Riboflavin (mg B6 (mg (mg B12 acid Biotin (g
group day
1)a day
1) day
1) day
1)b,c (mg day
1) (mg day
1) (mg day
1) day
1)c day
1) day
1)c (mg day
1) (mg day
1) (mg day
1) day
1) Carotenoidsd

Infants
0–6 months 600 NDe 25 ND ND ND ND ND ND ND ND ND ND ND ND
6–12 600 ND 38 ND ND ND ND ND ND ND ND ND ND ND ND
months
Children
1–3 years 600 400 63 200 ND ND ND 10 30 300 ND ND ND 1.0 ND
4–8 years 900 650 75 300 ND ND ND 15 40 400 ND ND ND 1.0 ND
Males
9–13 years 1700 1200 100 600 ND ND ND 20 60 600 ND ND ND 2.0 ND
14–18 2800 1800 100 800 ND ND ND 30 80 800 ND ND ND 3.0 ND
years
19–30 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
31–50 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
51–70 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
>70 years 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
Females
9–13 years 1700 1200 100 600 ND ND ND 20 60 600 ND ND ND 2.0 ND
14–18 2800 1800 100 800 ND ND ND 30 80 800 ND ND ND 3.0 ND
years
19–30 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
31–50 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
51–70 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
>70 years 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
Pregnancy
14–18 2800 1800 100 800 ND ND ND 30 80 800 ND ND ND 3.0 ND
years
19–30 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
31–50 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
Lactation
14–18 2800 1800 100 800 ND ND ND 30 80 800 ND ND ND 3.0 ND
years
19–30 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years
31–50 3000 2000 100 1000 ND ND ND 35 100 1000 ND ND ND 3.5 ND
years

Note: A tolerable upper intake level (UL) is the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. Unless otherwise specified, the UL represents total intake from food, water, and
supplements. Due to a lack of suitable data, ULs could not be established for vitamin K, thiamin, riboflavin, vitamin B12, pantothenic acid, biotin, and carotenoids. In the absence of a UL, extra caution may be warranted in consuming levels above recommended
intakes. Members of the general population should be advised not to routinely exceed the UL. The UL is not meant to apply to individuals who are treated with the nutrient under medical supervision or to individuals with predisposing conditions that modify their
sensitivity to the nutrient.
a
As preformed vitamin A only.
b
As a-tocopherol; applies to any form of supplemental a-tocopherol.
c
The ULs for vitamin E, niacin, and folate apply to synthetic forms obtained from supplements, fortified foods, or a combination of the two.
d
b-Carotene supplements are advised only to serve as a provitamin A source for individuals at risk of vitamin A deficiency.
e
ND ¼ not determinable due to the lack of data of adverse effects in this age group and concern with regard to the lack of ability to handle excess amounts. Source of intake should be from food only to prevent high levels of intake.
Source: Reprinted with permission from Dietary reference intakes for calcium, phosphorous, magnesium, vitamin D, and fluoride (1997); Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline
(1998); Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids (2000); Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc (2001); and
Dietary reference intakes for calcium and vitamin D (2011) by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. These reports may be accessed via www.nap.edu.
Table 7 Dietary reference intakes (DRIs): tolerable upper intake levels, elements (Food and Nutrition Board, Institute of Medicine, National Academies)

Boron Calcium Copper Fluoride Iodine Iron Nickel Selenium Vanadium Zinc Sodium Chloride
Life stage/ (mg (mg Chromium (mg (mg (mg (mg Magnesium Manganese Molybdenum (mg Phosphorus (mg (mg (mg (g (g
group Arsenica day
1) day
1) (mg day
1) day
1) day
1) day
1) day
1) (mg day
1)b (mg day
1) (mg day
1) day
1) (g day
1) day
1) Siliconc day
1)d day
1) day
1) day
1)

Infants
0–6 months NDe ND 1000 ND ND 0.7 ND 40 ND ND ND ND ND 45 ND ND 4 ND ND
6–12 ND ND 1500 ND ND 0.9 ND 40 ND ND ND ND ND 60 ND ND 5 ND ND
months
Children
1–3 years ND 3 2500 ND 1000 1.3 200 40 65 2 300 0.2 3 90 ND ND 7 1.5 2.3
4–8 years ND 6 2500 ND 3000 2.2 300 40 110 3 600 0.3 3 150 ND ND 12 1.9 2.9
Males
9–13 years ND 11 3000 ND 5000 10 600 40 350 6 1100 0.6 4 280 ND ND 23 2.2 3.4
14–18 ND 17 3000 ND 8000 10 900 45 350 9 1700 1.0 4 400 ND ND 34 2.3 3.6
years
19–30 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND 1.8 40 2.3 3.6
years
31–50 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND 1.8 40 2.3 3.6
years
51–70 ND 20 2000 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND 1.8 40 2.3 3.6
years
>70 years ND 20 2000 ND 10 000 10 1100 45 350 11 2000 1.0 3 400 ND 1.8 40 2.3 3.6
Females
9–13 years ND 11 3000 ND 5000 10 600 40 350 6 1100 0.6 4 280 ND ND 23 2.2 3.4
14–18 ND 17 3000 ND 8000 10 900 45 350 9 1700 1.0 4 400 ND ND 34 2.3 3.6
years
19–30 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND 1.8 40 2.3 3.6
years
31–50 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND 1.8 40 2.3 3.6
years
51–70 ND 20 2000 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND 1.8 40 2.3 3.6
years
>70 years ND 20 2000 ND 10 000 10 1100 45 350 11 2000 1.0 3 400 ND 1.8 40 2.3 3.6
Pregnancy
14–18 ND 17 3000 ND 8000 10 900 45 350 9 1700 1.0 3.5 400 ND ND 34 2.3 3.6
years
19–30 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 3.5 400 ND ND 40 2.3 3.6
years
31–50 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 3.5 400 ND ND 40 2.3 3.6
years
Lactation
14–18 ND 17 3000 ND 8000 10 900 45 350 9 1700 1.0 4 400 ND ND 34 2.3 3.6
years
19–30 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND ND 40 2.3 3.6
years
31–50 ND 20 2500 ND 10 000 10 1100 45 350 11 2000 1.0 4 400 ND ND 40 2.3 3.6
years

Note: A tolerable upper intake level (UL) is the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. Unless otherwise specified, the UL represents total intake from food, water, and
supplements. Due to a lack of suitable data, ULs could not be established for vitamin K, thiamin, riboflavin, vitamin B12, pantothenic acid, biotin, and carotenoids. In the absence of a UL, extra caution may be warranted in consuming levels above recommended intakes.
Members of the general population should be advised not to routinely exceed the UL. The UL is not meant to apply to individuals who are treated with the nutrient under medical supervision or to individuals with predisposing conditions that modify their sensitivity to the
nutrient.
a
Although the UL was not determined for arsenic, there is no justification for adding arsenic to food or supplements.
b
The ULs for magnesium represent intake from a pharmacological agent only and do not include intake from food and water.
c
Although silicon has not been shown to cause adverse effects in humans, there is no justification for adding silicon to supplements.
d
Although vanadium in food has not been shown to cause adverse effects in humans, there is no justification for adding vanadium to food and vanadium supplements should be used with caution. The UL is based on adverse effects in laboratory animals and these data could be
used to set a UL for adults but not children and adolescents.
e
ND ¼ not determinable due to the lack of data of adverse effects in this age group and concern with regard to the lack of ability to handle excess amounts. Source of intake should be from food only to prevent high levels of intake.
Source: Reprinted with permission from Dietary reference intakes for calcium, phosphorous, magnesium, vitamin D, and fluoride (1997); Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline (1998); Dietary
reference intakes for vitamin C, vitamin E, selenium, and carotenoids (2000); Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc (2001); Dietary reference intakes for
water, potassium, sodium, chloride, and sulfate (2005); and Dietary reference intakes for calcium and vitamin D (2011) by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. These reports may be accessed via www.nap.edu.
430 Dietary References: US
EAR
1.0
RDA
Risk of inadequacy 0.5
0.0
Observed level of intake
Figure 2 The relationship between different descriptors of dietary reference intakes. Reprinted with permission from Dietary reference intakes: the
essential guide to nutrient requirements, 2006 by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC.
preferences, including meal patterns, culture, religious beliefs,
and taste. Perfectly adequate diets from the nutrient standpoint
that are not eaten are nutritionally useless. When planning for
individuals, the nutrient goal should be at least the RDA or AI
to minimize risk of inadequacies. There is no increased risk of
adverse effects when an individual’s intake is greater than the
RDA as long as it remains below the UL. Similarly, when
planning for populations or particular groups, it is important
to ensure that virtually all individuals exceed the EAR without
exceeding the UL.
Many tools are available for diet planning. The USDA pro-
vides daily food plan guides (http://www.choosemyplate.gov/
myplate/index.aspx) by food groups for those with varied daily
calorie needs. Additionally, the USDA periodically issues the
Thrifty Food Plan (http://www.cnpp.usda.gov/USDAFood-
PlansCostofFood.htm), which is a guide to selecting foods
and menus for individuals using Supplemental Nutrition Assis-
tance Program benefits to maximize the amount and nutri-
tional value of food while receiving benefits.
Challenges, Additional Research, and Future Steps
The development of the DRIs has been important in improving
national assessment and monitoring of dietary intakes and
planning of federal and other programs, but research chal-
lenges remain.
Tolerable Upper Intake Level
The UL is the level of nutrient intake below which adverse
health effects are unlikely. Adverse events have not been col-
lected routinely on a systematic basis in studies of high levels
of nutrient intakes requirements, and therefore, there are
often very little data upon which to base the UL. Moreover,
dose–response studies are rarely done at high levels, and in
some cases, they would be unethical, yet in order to accurately
determine a health risk associated with an intake above the UL,
it is necessary to know the dose–response relationship between
chronic intake of the nutrient and the observed adverse effect
chosen. More data are needed in order to estimate dose–
1.0
Risk of adverse effects
UL
0.5
0.0
response curves for most nutrients. A second problem with
the UL is that the level of severity of the adverse effects upon
which the UL is based varies greatly from one nutrient to
another. For example, exceeding the UL on a chronic basis
for pyridoxine is a major concern since it may produce irre-
versible nerve damage. In contrast, exceeding the UL for cho-
line is likely of lesser concern since the UL is based on a fishy
body odor. A final problem with the UL is its characteristics; it
is based on a toxicological model with many safety factors
that are based on expert judgments but not necessarily on
empirically determined data. The UL is a defined as a sharp
cutoff and is difficult to manipulate from the statistical
standpoint.
Protein Requirement
The current DRI for protein is based on the analysis of nitrogen
balance studies, which have many limitations. Nitrogen bal-
ance may give falsely high estimates because it is easy to over-
estimate nitrogen intake and underestimate excretion due to
incomplete collections and difficult to measure dermal and
miscellaneous nitrogen losses that vary greatly under some
environmental conditions. Protein requirements can also be
determined using other techniques, such as stable isotopes,
carbon balance, and amino acid flux. However, before new
methods are adopted, there is a need to conduct comparative
studies using the proposed techniques to harmonize them with
the nitrogen balance studies of requirements.
One alternative technique that has been proposed to esti-
mate requirements of essential amino acids is the indicator
amino acid oxidation method to determine indispensable
amino acid (IDAA) requirements in humans. It is based on
the idea that when one IDAA is deficient for protein synthe-
sis, then the excess of all other IDAAs will be oxidized,
including the amino acid marked with a stable isotope. Pro-
ponents claim that this method is robust, minimally invasive,
more rapid, and sensitive to changes in amino acid intake
than nitrogen-based studies. Studies using it have identified
amino acid requirements that are much higher than those
based on nitrogen balance.

Better Experimental Data on Subgroups
Many of the DRIs for specific life stage groups have been
extrapolated from adults, due to the lack of data on the specific
nutrient and life stage group. More studies of these groups are
needed in order to set more accurate DRIs.
DRIs for Nonnutrient Bioactives?
Nonnutrient bioactive constituents may have significant health
benefits. They include food components such as fiber, carot-
enoids, glucosinolates, and flavonoids, among others. With
the exception of fiber, DRIs are not established for these
components.
There are many challenges in determining recommendations
for consumption of bioactives including their variable bioavail-
ability and bioactivity, errors in intake measurement, and health
benefits based on surrogate biomarkers of effect rather than
health outcomes. Historically, much of the discussion around
setting recommendations for bioactives was about the paucity
and quality of scientific data available. Phenols, polyphenols,
and flavonoids were excluded from consideration by the DRI
panel at the time the DRIs were last revised because of the lack of
sufficient data. Since then, there has been a substantial increase
in research and available data, although randomized clinical
trials of intakes of these constituents with health outcomes are
still few in number. Some other types of guidance may be more
appropriate than to establish DRI for them.
Chronic Disease Endpoints
More attention needs to be paid to methods for linking nutri-
ent intakes to chronic disease endpoints/outcomes and setting
recommendations based on these.
Updates and Harmonization
It may be time for reevaluation of the DRI development pro-
cess. Perhaps, it should establish criteria for which nutrients are
most in need of revision and then concentrate on revising one
nutrient at a time rather than many nutrients. At present, the
Food and Nutrition Board and Health Canada have received
nominations for many nutrients thought to be in need of
revision, of which four were selected for further consideration
(vitamin E, sodium, magnesium, and omega 3 fatty acids). It
remains to be seen what nutrients will be chosen. Finally, there
is a need to harmonize efforts on setting requirements with
other authoritative bodies in Europe, Australia, New Zealand,
and elsewhere, to avoid needless overduplication of work.
See also: Elderly: Nutrition Requirements; Energy: Intake and Energy
Requirements; Fatty Acids: Determination and Requirements; Infants:
Dietary References: US 431
Nutritional Requirements; Pregnancy: Metabolic Adaptations and
Nutritional Requirements; Protein: Requirements.
Further Reading
Institute of Medicine (2011) Dietary reference intakes for calcium and vitamin D.
Washington, DC: The National Academies Press.
Institute of Medicine (2006) Dietary reference intakes: the essential guide to nutrient
requirements. Washington, DC: The National Academies Press.
Institute of Medicine (2005a) Dietary reference intakes for energy, carbohydrate, fiber,
fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). Washington,
DC: The National Academies Press.
Institute of Medicine (2005b) Dietary reference intakes for water, potassium, sodium,
chloride, and sulfate. Washington, DC: The National Academies Press.
Institute of Medicine (2003a) Dietary reference intakes: applications in dietary planning.
Washington, DC: The National Academies Press.
Institute of Medicine (2003b) Dietary reference intakes: guiding principles for nutrition
labeling and fortification. Washington, DC: The National Academies Press.
Institute of Medicine (2001a) Dietary reference intakes for vitamin A, vitamin K, arsenic,
boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon,
vanadium, and zinc. Washington, DC: The National Academies Press.
Institute of Medicine (2001b) Dietary reference intakes: proposed definition of dietary
fiber. Washington, DC: The National Academies Press.
Institute of Medicine (2000a) Dietary reference intakes for vitamin C, vitamin E,
selenium, and carotenoids. Washington, DC: The National Academies Press.
Institute of Medicine (2000b) Dietary reference intakes: applications in dietary
assessment. Washington, DC: The National Academies Press.
Institute of Medicine (1998a) Dietary reference intakes for thiamin, riboflavin, niacin,
vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington,
DC: The National Academies Press.
Institute of Medicine (1998b) Dietary reference intakes: a risk assessment model for
establishing upper intake levels for nutrients. Washington, DC: The National
Academies Press.
Institute of Medicine (1998c) Dietary reference intakes: proposed definition and plan for
review of dietary antioxidants and related compounds. Washington, DC: The
National Academies Press.
Institute of Medicine (1997) Dietary reference intakes for calcium, phosphorus,
magnesium, vitamin D, and fluoride. Washington, DC: The National Academies
Press.
King JC and Garza C (2007) Harmonization of nutrient intake values. Food and Nutrition
Bulletin 28(S1): S3–S12.
Munro IC (2006) Setting tolerable upper intake levels for nutrients. The Journal of
Nutrition 136: 490S–492S.
Relevant Websites
http://choosemyplate.gov – USDA MyPlate.
http://www.dietitians.ca/Knowledge-Center/Live-Events/Online-Courses/Dietary-
Reference-Intakes.aspx – Dietitians of Canada: Dietary Reference Intakes Online
Tutorials.
http://fnic.nal.usda.gov/dietary-guidance/dietary-reference-intakes – USDA Natural
Agriculture Library: Dietary Reference Intakes.
http://www.iom.edu/About-IOM/Leadership-Staff/Boards/Food-and-Nutrition-Board.
aspx – Institute of Medicine Food and Nutrition Board.
http://ods.od.nih.gov/Health_Information/Dietary_Reference_Intakes.aspx – National
Institutes of Health Office of Dietary Supplements: Dietary Reference Intakes.
 Dietary Surveys: National Food Intake
MC Ocké, CTM van Rossum, and EJ de Boer, National Institute for Public Health and the Environment, Bilthoven, The Netherlands
ã 2016 Elsevier Ltd. All rights reserved.

Introduction long-standing national food intake surveys are probably those in

National food intake surveys are surveys that monitor the food
consumption of groups of individuals that are representative
for a national population or important population groups
within a country. National food intake surveys provide insight
into the consumption of foods, the intake of energy and nutri-
ents, and the exposure to potentially harmful chemical sub-
stances. Regular repetitions of national food intake surveys
show dietary trends in a population.
Apart from national food intake surveys, other sources of
national food data are available in countries. These data from
food balance sheets and household budget and expenditure
surveys are sometimes used as a surrogate for food intake data.
However, each of them provides information about a different
level in the flow of food production to consumption and on
different aggregation levels. Food balance sheets give informa-
tion on the national per capita food supply, and household
budget surveys focus on food acquisition at the level of house-
holds, sometimes supplemented with foods acquired out of
home. The data of food balance sheets and household budget
surveys are thus not directly comparable with data from food
consumption surveys (see Figure 1). Only national food intake
surveys allow to estimate the population distributions of
consumption of foods, intake of energy and nutrients, and
exposure to chemicals and to do this for subgroups within a
population such as by gender and age groups.
Many countries conduct national food intake surveys. For the
European Union, food intake data of 22 member states are
available in the comprehensive food consumption database
of the European Food Safety Authority (EFSA). The number of
countries that carry out food intake surveys on a regular basis
rather than incidentally is however limited. The most well-know
the United States, starting from the National Food Consumption
Survey in 1935 to the ongoing dietary surveys as part of the
National Health and Nutrition Examination Survey (NHANES).
This article highlights major uses of national food intake
data, important aspects about the design and methodology of
food intake surveys and their European harmonization, limita-
tions associated with food intake surveys, and future directions.
Major Uses and Stakeholders of National
Food Intake Data
Data from national food intake surveys together with data on
the composition of foods and information on nutritional sta-
tus are needed to develop and evaluate health, nutrition, and
food policy. Examples are the evaluation of diet from a health
perspective, simulating the impact of dietary policy measures,
describing trends in food and nutrient intakes, and deriving
optimal levels of food fortification. Moreover, the food intake
data are essential to develop food frequency questionnaires,
dietary indexes, and food-based dietary guidelines.
Similarly, data from national food intake surveys in combi-
nation with data on contaminants, additives, or chemical con-
stituents in foods are essential for the assessment of exposure to
hazardous substances, as part of the risk assessment process.
This information is also useful for the evaluation of new food
legislation. Examples are dietary exposure assessment, deriva-
tion of safe maximum fortification levels, communication of
risks associated with the food chain, estimations of the effects of
particular diseases caused by infectious agents that can be trans-
mitted between animals and humans, and identification of core
foods for total diet studies.

Food available but

not acquired

FOOD
FOOD BALANS HOUSEHOLD CONSUMPTION
SHEETS BUDGET SURVEYS SURVEYS
National food Household food
food consumed by
supply acquisition
individuals

Out of home food Foods not

acquisition consumed (by
humans)

Figure 1 Relationships between food supply data, food acquisition data, and food consumption data.

432 Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00237-3


Dietary exposure to food
contaminants, additives
Input for risk
communication
Derive safe maximum
levels for foods
National food
intake data
Develop food frequency
questionnaires
Develop dietary indexes
Describe
diet costs
Figure 2 Examples of uses of food intake surveys.
Additionally, food intake data can be used for the assess-
ment of the environmental impact of diet or to describe dietary
costs (see Figure 2).
Stakeholders of national food intake data are ministries of
health, ministries of agriculture, food industry, dieticians,
patient organizations, branch organizations, journalists, uni-
versities, and national and international health and food safety
organizations like the World Health Organization (WHO),
Food and Agriculture Organization (FAO), and EFSA. Scientists
in public health or nutrition institutes, food safety agencies,
universities, and research departments of the larger food indus-
tries often have an interest to conduct specific statistical
analyses in national food intake databases themselves.
Study Design, Data Collection, and Handling
Two features are critical for the success of national food intake
surveys. The first critical aspect is to obtain a study population
that is representative of the target population. The second
challenge is to collect food intake data that are valid and
precise and contain sufficient detail for their purposes.
Study Population
When planning a national food intake survey, the first step to
be taken is defining the target population. The target popula-
tion is the entire set of individuals to which findings of the
survey are to be extrapolated. Typically, a broad age group
within a national population is defined as the target popula-
tion for a food intake survey. The target population may not be
the same as the study population, that is, the population that
can be defined accurately and reached in a study. In order to
precisely define the study population, it is necessary to agree on
Dietary Surveys: National Food Intake 433
Evaluate nutritional
quality, e.g. % adequate
Describe dietary trends
Simulate impact of food
policies
Derive optimal levels of
food fortification
Derive food-based
dietary guidelines
Describe environmental
impact of diets
exclusion criteria. Institutionalized subjects are often not
included in food intake surveys as their inclusion makes the
sampling procedures and/or the field surveys too complicated.
Also, language barriers are commonly used as exclusion
criteria. In order to obtain a representative study population
for the target population, excluding subjects due to language
barriers or other practical reasons should be kept to a
minimum.
The sampling frame is the list of units from which the
sample is drawn. The availability of sampling frames varies
from country to country. For national food intake surveys,
the most complete lists can be chosen among population
registers, electoral lists, and census lists. Alternative sampling
frames are telephone lists, lists of schools or day care centers,
market research panels, and lists of medical centers or general
practitioners. Great care must be taken when making decisions
on the sampling frame, particularly with regard to bias due to
undercoverage. Overcoverage of the sampling frame normally
can be detected and dealt with during the fieldwork. Specific
subgroups within a population might need a different sam-
pling frame, since electoral list is only suitable for adults and
list of day care centers or schools is especially suitable for
young or older children, respectively.
Sampling from the sampling frame can be done in various
ways. It is considered a good survey practice to use a sampling
design that yields a probability sample. Under such a design,
each sample unit of the sampling frame has a nonzero proba-
bility of being selected. It does not necessarily require that every
person has the same probability of being selected. Stratified
sampling, for example, sampling by subgroups of age and
gender, is often conducted in food intake surveys to ensure
that there are a sufficient number of people in each subgroup
to allow meaningful analysis. For practical reasons, cluster
sampling or multistage sampling can be applied. In cluster
434 Dietary Surveys: National Food Intake

sampling, first, a random sample of naturally occurring units or
groupings within the population is taken (e.g., areas, cities, or
schools). Then, either all members of the chosen clusters or a
random selection from among them is included in the sample.
Multistage sampling is an extension of cluster sampling where a
hierarchy of clusters are chosen going from larger to smaller.
The required size of a food intake survey population
depends on the variability in intake, the statistics of interest
(i.e., mean, percentiles, or variance), and the level of precision
needed. Information on intake variability (from previous stud-
ies) is therefore needed in order to determine the number of
subjects to include in a survey. In the European Food Consump-
tion and Validation (EFCOVAL) project, the sample size per
country was estimated to be, on average, equal to 2000 subjects
for a desired precision of 5% in nutrient and food group intake.
In simple random samples, high percentiles can be assessed
with sufficient accuracy if the minimum sample sizes for the
95th, 97.5th, and 99th percentiles can be estimated to be equal
to 160, 320, and 800, respectively. When results are presented
for different subgroups within a study population, the mini-
mum sample size has to be available for each subgroup.
The expected response rate should be taken into account in
determining the sample size for achieving a certain number of
actual participants. In the case of recruitment in waves through
a year, adjustments in the number of invited participant can be
made according to the actual response rates for specific strata in
previous waves. Moreover, it is important to collect informa-
tion on nonrespondents in order to eventually correct for lack
of representativeness of the study population for the target
population. For active nonparticipants, it is possible to admin-
ister a concise nonrespondent questionnaire (among those
willing); for passive nonparticipant with which no contact
was established, only information from the sampling frame
can be used (see Figure 3).

Sample from sampling

frame, e.g. population
register

Periodic subsample for

recruitment in waves (every
month, season etc,)

Recruitment, e.g. by
invitation letter and brochure

Passive non- Active non-

Participant
participant Participant

First 24-h dietary recall, other

Concise non-response
measurements e.g. general
questionnaire
questionnaire, height/weight

1-6 weeks
in between

Second 24-h dietary recall

Figure 3 Example of the logistic flow of a food intake survey.


Dietary Assessment
Traditionally, four main methods of dietary assessment are
distinguished. These are food records, dietary recalls, food
frequency questionnaires, and dietary history questionnaires.
Food frequency questionnaires capture usual intake, but they
are not quantitatively precise and thus cannot estimate the
percentage of the population with inadequate intakes or too
high intakes. This was clearly demonstrated in a biomarker-
based validation study in the United States, the Observing
Protein and Energy Nutrition (OPEN) Study. The traditional
dietary history also captures usual intake but is extremely
labor-intensive and involves a high subject burden with an
interview of several hours, whereas the modern version of a
dietary history method has similar problems to food frequency
questionnaire with estimating intake quantitatively. Therefore,
food frequency methods and dietary history methods are not
suitable for national food intake surveys.
Well-conducted food records and dietary recalls are better
able to quantify food intake for the specific days they cover.
Since usual intake is of interest for food intake surveys, at least
two repeated nonconsecutive food records or dietary recalls are
required together with statistical modeling of usual intake. With
the statistical modeling, the within-person day-to-day variation
in the intake data can be accounted for. For foods that are
important contributors of nutrients and that are consumed by
less than 50% of the population, additional information on the
frequency of consumption has added value. Examples of these
products are types of dietary supplements and liver.
Whether a food record or dietary recall is the preferred
dietary assessment method for food intake surveys depends
on the survey population. In populations with notably low
response rates, the preferred method is the method with less
respondent burden; this is the dietary recall. The dietary recall
method is also preferred in populations with high illiteracy
levels. In other populations, food records might be preferred.
The food record is a prospective method and therefore does not
have the disadvantage that it relies on memory such as the
retrospective 24 h dietary recall method. However, it is well-
known that people change their diets because of keeping a
record of everything that is consumed. For harmonized pan-
European food intake surveys, EFSA advises to apply dietary
recalls as dietary assessment method, particularly because of
the lower burden to the participants.
In order to obtain intake data that are usable for many
purposes, it is important to collect information on all foods,
beverages, and dietary supplements consumed. The informa-
tion should contain a sufficient level of detail for the different
purposes, for example, information on fat content, sweetening
(sugar and/or artificially sweetened), fortification, preparation
method, conservation method, and package material. Brand
name information can help to ascertain aspects like sweetening
and fortification that are often not known by consumers. It is
also necessary to estimate the consumed amount for each eaten
food. There are different options, which can be used in com-
bination: estimation in household units like glasses or spoons,
estimation in natural units, for example, for eggs or apples,
estimation in commercial units for products like chocolate bars
and sweets, estimation using food models or food photo-
graphs, and estimation in gram or milliliter.
Dietary Surveys: National Food Intake 435
To conduct a high-quality standardized 24 h dietary recall,
it is advised to use a computer-administered software for 24 h
recalls. With the software, the interviewer is guided by auto-
matic response routing to ask tailored questions and prompts
for specific foods. Using multiple steps, the system helps to
ensure completeness and consistency across interviewers.
There are automated links to food lookup tables and data
quality checks. It is of major importance to test any software
before applying it in a survey; preferably, a validation study
should be conducted. Examples of carefully developed and
extensively tested and validated software are the five-step
multiple-pass method used in the NHANES in the United
States and GloboDiet, previously known as EPIC-Soft, used
in food intake surveys of Austria, Belgium, France, Germany,
Malta, the Netherlands, and Switzerland.
Specific subgroups within a population might need a dif-
ferent dietary assessment method. For example, a 24 h dietary
recall is less suitable for young children in case the child is
often not together with the parent or caretaker that is inter-
viewed. Also for older adults with a higher likelihood of
impaired short-term memory, a 24 h dietary recall is less suit-
able. In both age groups, a dietary record or a food diary and
24 h dietary recall could be an option. For less developed
countries, more emphasis is needed for feasible and robust
methods.
Supplemental Information to be Collected from Participants
Depending on the specific aims of a food intake survey, other
information is collected besides the dietary data. All food
intake surveys collect information on background characteris-
tics of the participants, usually including sociodemographic
information and information on dietary practices. This enables
to stratify the results by sociodemographic subgroups and to
judge the representativeness of the study population.
Because of the high prevalence of overweight, additional
information on physical activity and anthropometric informa-
tion like height, weight, and waist circumference is also col-
lected in many food intake surveys. Information on body
weight is also of prime importance if chemical exposure from
the diet is to be compared with health limits. These health limits
are usually expressed per kilogram body weight. Moreover,
some surveys collect information on health and others on
food choice determinants, biological marker for nutritional
status, or aspects important for microbiological risk associated
with food consumption. It is outside the scope of this article to
detail methods for collecting these data.
Administration Protocol
The study design must consider that in order to capture the
interseasonal variability in consumption patterns, subjects
must be uniformly distributed over the four different seasons
and each seasonal sample must represent each survey stratum
(e.g., on age and sex). This can be done by recruitment of
participant in waves throughout the year (see Figure 2). It is
of prime importance to monitor response and the completed
number of participants and to adjust procedures if response or
the completed number of participants falls behind.
436 Dietary Surveys: National Food Intake
The survey calendar must be organized in such a way that
all days of the week are represented proportionally in the food
consumption data. Specific emphasis on an adequate propor-
tion of weekdays and weekend days at population group level
is needed, since this is the main source of variation in intake
over the days. For food intake surveys with nonconsecutive
dietary records or recall, it is advised to have an interval of
1–6 weeks in between both administrations. More than 6
weeks in between, which might be an option theoretically,
might result in losing study participants and is therefore not
advised.
The administration of the interviews is usually done face to
face, although in some surveys, telephone interviews are con-
ducted. Face-to-face interviews can be conducted at a study
center or at the home of the participant. The latter has the
advantage that consumed foods and used household measures
are available to show to the interviewer. Interviewers should
preferably be trained dietitians or nutritionist; although, with
more extensive training on consumed foods and recipes, also
interviewers with other backgrounds are used in practice. During
the survey period, refresher trainings are recommended. More-
over, it is of prime importance to monitor the quality of the
interviewers and provide them feedback to improve their work.
Data Cleaning and Analyses
After data collection, data cleaning is an important phase. It is
recommended to start data cleaning immediately after the first
data are received from the interviewers, rather than waiting
until the survey period is finalized. Data cleaning should
include checking for missing, inconsistent, and extreme values
and considering data-related remarks made by interviewers.
When the food intake data are checked, further handling can
include linkage to nutrient databases for foods and dietary
supplements, databases with concentrations of chemicals in
foods, or other food-related databases. More information on
nutrient databases can be found in this encyclopedia.
Subsequently, statistical analyses can be performed. In these
analyses, survey weights can be used to make the data represen-
tative for the target population. A commonly applied correction
technique for lack of representativeness is weighting adjust-
ment. It assigns an adjustment weight to each survey respon-
dent. Persons with characteristics that are underrepresented get
a weight larger than one, and those in overrepresented groups
get a weight smaller than one. In the computation of survey
results, not just the values of the variables are used, but the
weighted values.
When usual intake is of interest, intake or exposure per
person per day should be calculated and then usual intake
modeling should be applied. In these models, the within-
person variation in intake is removed from the data. Various
tools to model usual intake exist. In the Statistical Program to
Assess Dietary Exposure (SPADE), usual intake can be modeled
for the combination of intake from foods and drinks as well as
dietary supplements measured on two specific days. In the
Monte Carlo Risk Assessment (MCRA) tool, usual exposure to
chemical substances can be modeled, dealing, for example,
with occurrence data below the detection limit and having
the option to check the input data quality.
An important step in data analyses is evaluation of dietary
intake against dietary reference values for nutrients and against
health limits for food chemicals. Procedures for proper evalu-
ation are described elsewhere in this encyclopedia.
Limitations
Two major challenges of food intake surveys are the internal
validity and the external validity.
Internal validity of national food intake surveys is never
perfect, since diet cannot be measured without error; both
random error (including day-to-day variation) and systematic
bias (social desirability) are present in the data. Important
aspects within dietary recalls are the correctness of the reported
foods (both omissions and intrusions occur), the completeness
of food description, and the correctness of portion size estima-
tion. In practice, dietary assessment based on self-reporting
usually leads to underestimation of energy intake. Moreover,
dietary assessment in combination with using food composi-
tion databases has additional limitations for specific compo-
nents in the diet. This is, for example, the case for dietary
exposure assessment of mycotoxins. Since the concentration
of mycotoxins in foods is very variable due to, for instance,
weather and storage conditions, duplicate diet studies or total
diet studies are better equipped to estimate mycotoxin expo-
sure. Another example is estimation of sodium intake. This is
difficult because of the large variation in sodium concentrations
in processed foods and recipes, as well as the difficulty of
estimating discretionary salt intake. Dietary assessment of
sodium (rather than urinary excretion) does however enable
identification of important dietary sources of sodium, which
can inform public health interventions to lower sodium intake.
What is important is whether the dietary assessment
method is suitable for providing useful analytic measurement
for a given purpose and context. For many purposes and in
many contexts, 24 h dietary recall data from national food
intake surveys proved to be useful in helping to address impor-
tant research and policy questions, despite their known errors.
Strategies to optimize the impact of food surveys are to have
more transparency of raw research data, consistent data-staging
techniques, improved data analysis, and reporting indicators
for dietary intake quality such as indicators for underreporting.
External validity is impacted by low response rates that
often occur. In practice, response rates between 26 and 97 are
observed in national food intake surveys, with the majority
between 42% and 71%. Low participation rates and selective
nonparticipation may cause bias to the survey results based on
participants alone. It is known that respondents and non-
respondents might differ in their socioeconomic and demo-
graphic status as well as in their health and lifestyle behaviors.
Therefore, for achieving a representative study population, it is
of utmost importance to use all means to obtain the highest
response rate possible. Strategies to optimize response rates are
choosing data collection methods with a lower burden for the
respondents, providing a suitable incentive, flexibility in
recruiting and participation (times available for the interview,
second call if a no-show, etc.), personal contact in recruitment,
and awareness of the study by media coverage.

Harmonization of National Food Intake Data Across
Countries in the European Union
National food intake surveys in Europe are heterogeneous with
respect to dietary assessment methodology and number of
days for which dietary data are collected. This hampers the
comparison of results across countries, for example, in dietary
exposure assessment using the data of the comprehensive food
consumption database such as conducted by EFSA. Therefore,
EFSA stimulates European Union member states to collect
national food intake data in a harmonized way and prepared
guidance for this in 2009 with an update in 2014. Moreover,
EFSA provides seed money to countries that conduct food
intake surveys in accordance with EFSA guidance. This is called
the EU Menu process. The guidance of EFSA was partly based
on the results of previous European research projects, such as
summarized in the succeeding text.
In the European Food Consumption Survey Method
(EFCOSUM) project, recommendations for reliable and com-
parable pan-European collection of food intake data were for-
mulated. As the most suitable method to collect internationally
comparable data on population means and distributions of
actual intake, the 24 h recall was recommended, to be con-
ducted at least twice. This would also allow for the estimation
of usual intake by a modeling technique that separates intra-
and interindividual intake. GloboDiet, at that time called
EPIC-SOFT, was considered the most suitable software for
a standardized data collection in a pan-European survey. For
a number of micronutrients, that is, vitamin D, folate, sodium,
iron, and iodine, the use of biomarkers was recommended.
Subsequently, the EFCOVAL project further developed and
validated the pan-European food consumption method
recommended by the EFCOSUM project. The validation
study confirmed that the repeated 24 h dietary recall using
GloboDiet in combination with a food propensity question-
naire and modeling of usual intake is a suitable method for a
pan-European food intake survey. This conclusion applied to
healthy adults and possibly to children aged 7 years and older.
As next step, it was recommended to provide and standardize
an implementation plan to facilitate this methodology in
European countries. The plan should account for maintenance
and updates, sampling designs, national surveillance pro-
grams, tailored capacity building and training, and linkage to
food composition and occurrence databases.
The objective of the PILOT-PANEU project was to develop,
test, and evaluate the applicability of tools and procedures for
conducting a pan-European food intake survey including ado-
lescents, adults, and elderly people. The dietary assessment
methodology consisted of two nonconsecutive 24 h recalls per-
formed with GloboDiet methodology. The consortium made a
number of recommendations for the improvement and further
development of the tools and procedures. Assuming that the
recommended modifications are made, the tools and proce-
dures developed and tested were considered suitable for the
EFSA EU Menu process in adolescents, adults, and the elderly.
Similarly, in the ‘Pilot study for the Assessment of Nutrient
intake and food Consumption Among Kids in Europe’
(PANCAKE), the feasibility of tools and procedures for a
pan-European food intake survey among children 0–10 years
was tested. This included two alternative dietary assessment
Dietary Surveys: National Food Intake 437
methods. One method consisted of a 3-day food diary, which
was checked with a parent, and data were entered afterward
using GloboDiet. The alternative method consisted of two
nonconsecutive 1-day food diaries followed by GloboDiet
completion interviews. Both dietary assessment methods with
related tools and administration protocols were evaluated as
feasible. The administration protocol with two 1-day food
diaries with completion interviews was judged to offer more
advantages for the EFSA EU Menu survey in children 0–10
years. It offered more complete description of the consumed
foods, was more suitable to estimate usual intake, and
resembled more the method in adults.
Future Directions
In the future, it is important to improve data collection
methods and to collect food intake data in connection to
other information.
First, improving data collection methods should focus on
methods that have a small participant burden or that are
attractive to participants, so that response rates increase. The
use of modern technologies might help for this purpose,
although for some population groups, it might not be appro-
priate. Additionally, future data collection methods should be
less labor-intensive than current data collection methods, since
the costs of the current methods are high and increase because
of the dynamic and increasingly complex food market. Exam-
ples of promising data collection methods are online 24 h
dietary recalls, devices that automatically take pictures of all
food consumed, and apps for food records using bar codes and
food pictures for portion sizes, or automatic recognition of
photographed consumed food and volumes. These promising
technologies need to be extensively tested in the general pop-
ulation and important subgroups of the population and vali-
dated positively before they can be incorporated in food intake
surveys. At the moment, various interesting developments and
validation studies with modern technologies are ongoing.
Second, it has added value to conduct national food intake
surveys in connection to biomarker and other data collection.
Combining with nutritional status monitoring and collecting
information of biomarker of food intake allow for better inter-
pretation of food intake results and provide insight in the
validity of the results. Combining with data collection on
other lifestyle information has the advantage that risk groups
with multiple unhealthy lifestyles can be identified. Moreover,
in order to get insight in ways to improve dietary intake, it is an
asset to collect food intake data in combination with informa-
tion on determinants of food choice, the food environment,
and economical aspects of diet and health. Such integrated
food intake surveys ask for ingenious study designs so that
the participant burden can be kept acceptable, and not all
information has to be collected from each participant.
Conclusions
Conducting regular national food intake surveys is important
for the development and evaluation of nutritional and food
safety policies. The current best practice in food intake surveys
438 Dietary Surveys: National Food Intake
is to obtain a representative sample from a national population
register. For each participant, at least two nonconsecutive food
records or 24 h dietary recalls combined with a short food
frequency questionnaire need to be collected supplemented
with at least sociodemographic background information. Sta-
tistical modeling should be used to estimate usual intake. For
pan-European harmonization of food intake surveys, dietary
recalls rather than food records are advised. Standardized col-
lection of 24 h dietary recalls with much detail can best be
done using validated software such as GloboDiet.
Overall, study preparation and data collection and data
handling of food intake surveys with much detail in the food
description are labor-intensive and therefore costly. Using the
collected dietary data to their full potential will however lead
to a return on investment. Potential uses are manifold. More-
over, investments can become smaller if the same methods are
used repeatedly and shared with other groups. Output and
lessons can increase if the collected data are easily discovered,
user-friendly to access and understand, and freely available.
The trend toward open data helps here.
This article described that two features are critical for the
success of national food intake surveys. The first is the internal
validity or validity of the collected food intake data. The second
is the external validity or the representativeness of the survey
population for the target population. In the practice of food
intake surveys, we have to deal with suboptimal situation that
diet cannot be measured without error and decreasing
response rates. Future directions should address both these
challenges and time and cost-efficiency.
See also: Dietary Exposure Assessment; Dietary Practices; Dietary
References: US; Food Composition Databases.
Further Reading
Ahuja JK, Moshfegh AJ, Holden JM, and Harris E (2013) USDA food and nutrient
databases provide the infrastructure for food and nutrition research, policy, and
practice. Journal of Nutrition 143(2): 241s–249s.
Andersen LF, Lioret S, Brants H, et al. (2011) Recommendations for a trans-European
dietary assessment method in children between 4 and 14 years. European Journal of
Clinical Nutrition 65(Suppl. 1): S58–S64.
Brussaard JH, Lowik MR, Steingrimsdottir L, et al. (2002) A European food
consumption survey method – conclusions and recommendations. European
Journal of Clinical Nutrition 56(Suppl. 2): S89–S94.
Conway JM, Ingwersen LA, and Moshfegh AJ (2004) Accuracy of dietary recall using
the USDA five-step multiple-pass method in men: an observational validation study.
Journal of the American Dietetic Association 104(4): 595–603.
de Boer EJ, Slimani N, van’t Veer P, et al. (2011) The European Food Consumption
Validation Project: conclusions and recommendations. European Journal of Clinical
Nutrition 65(Suppl. 1): S102–S107.
Dekkers AL, Verkaik-Kloosterman J, van Rossum CT, and Ocke MC (2014) SPADE, a
new statistical program to estimate habitual dietary intake from multiple food
sources and dietary supplements. Journal of Nutrition 144(12): 2083–2091.
Edwards PJ, Roberts I, Clarke MJ, et al. (2009) Methods to increase response to postal
and electronic questionnaires. Cochrane Database of Systematic Reviews (3):
MR000008.
European Food Safety Authority (2014) Guidance on the EU Menu methodology. EFSA
Journal 12(12): 77.
Hebert JR, Hurley TG, Steck SE, et al. (2014) Considering the value of dietary
assessment data in informing nutrition-related health policy. Advances in Nutrition
5(4): 447–455.
Kipnis V, Subar AF, Midthune D, et al. (2003) Structure of dietary measurement error:
results of the OPEN biomarker study. American Journal of Epidemiology 158(1):
14–21 discussion, 22–26.
Kirkpatrick SI, Subar AF, Douglass D, et al. (2014) Performance of the automated self-
administered 24-hour recall relative to a measure of true intakes and to an
interviewer-administered 24-h recall. American Journal of Clinical Nutrition 100(1):
233–240.
Merten C, Ferrari P, Bakker M, et al. (2011) Methodological characteristics of the
national dietary surveys carried out in the European Union as included in the
European Food Safety Authority (EFSA) Comprehensive European Food
Consumption Database. Food Additives and Contaminants. Part A: Chemistry,
Analysis, Control, Exposure & Risk Assessment 28(8): 975–995.
Ocke M, Brants H, Dofkova M, et al. (2014) Feasibility of dietary assessment methods,
other tools and procedures for a pan-European food consumption survey among
infants, toddlers and children. European Journal of Nutrition .
Slimani N, Casagrande C, Nicolas G, et al. (2011) The standardized computerized 24-h
dietary recall method EPIC-Soft adapted for pan-European dietary monitoring.
European Journal of Clinical Nutrition 65(Suppl. 1): S5–S15.
Thompson FE and Subar AF (2013) Dietary assessment methodology. In: Coulston AM,
Boushey CJ, and Ferruzzi MG (eds.) Nutrition in the prevention and treatment of
disease, 3rd ed., pp. 5–46. Amsterdam: Elsevier.
Vandevijvere S, Monteiro C, Krebs-Smith SM, et al. (2013) Monitoring and
benchmarking population diet quality globally: a step-wise approach. Obesity
Reviews 14(Suppl. 1): 135–149.
Relevant Websites
http://www.cdc.gov/nchs/nhanes.htm – Centers for Disease Control and Prevention.
National Health and Nutrition Examination Survey.
http://www.efsa.europa.eu/en/datexfoodcdb/datexeumenu.htm – European Food Safety
Authority. EU Menu.
http://www.rivm.nl/en/Topics/D/Dutch_National_Food_Consumption_Survey –
National Institute for Public Health and the Environment. Dutch National Food
Consumption Survey.
 Drying: Effect on Nutrients, Composition and Health
SV Crowley and JA O’Mahony, University College Cork, Cork, Ireland
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
In their natural state, most foods contain a significant quantity of
water. In foods with a high water activity (aw), spoilage and/or
pathogenic microorganisms can grow; in addition, degradative
physicochemical changes occur more rapidly in high aw foods.
One of the most effective methods of increasing both the shelf
life and safety of foods is through dehydration. Traditional drying
methods, such as sun-drying, are slow and manual in nature, and
can incur losses in yield due to difficult-to-predict factors relating to
the local climate and indigenous wildlife. These traditional
methods of dehydration are still successfully used in developing
countries and also in the production of artisan or minimally pro-
cessed food. However, the modern food supply chain is a global
one, and the increased affluence of consumers has resulted in a
strong demand for the year-round availability of a diverse range of
foods. In industrial countries, this demand has contributed to the
general adoption of mechanized drying technologies (e.g., spray-
and freeze-drying) and a proliferation of food powders.
Food powders are nutrient-dense, low-volume products
that can be easily transported nationally and internationally.
The drying process itself can also be harnessed to encapsulate
nutrients deemed to have specific health benefits, such as
probiotics, fatty acids, and phytochemicals. These encapsu-
lated nutrients can be stored for use as versatile ingredients in
the fortification of a wide range of foods, which could allow
their targeted delivery to a larger proportion of the world
population. In this article, the influence of the drying process
and powder storage on the stability of key nutrients in different
food systems is discussed. Certain dried foods, such as herbs
and spices, are omitted, as the low levels at which they are
consumed were considered to render their nutritional contri-
bution to the average diet negligible. A comprehensive over-
view of the effects of drying on a range of foods and nutrients is
provided, including fruits, fruit juice, vegetables, egg, milk,
wine, algae, infant formulas, enteral formulas, bacteria, and
plant-derived oils. This overview is based on recent research,
which has primarily focused on the effect of drying conditions
(e.g., temperature and flow rate) and postdrying storage con-
ditions (e.g., relative humidity and light exposure) on the
stability of key nutrients. The effect of bulk density on nutrient
dosage in nutritional powders and the role of accelerated sta-
bility tests and fortification overages in formulation design,
two increasingly important topics in modern manufacturing
practices for dried foods, are also highlighted before a general
closing discussion on the link between dried foods and health.
Effect of Drying on Nutrients
Changes in Macronutrients During Drying and Powder Storage
Much of the focus of this article is on the changes that occur in
the micronutrients present in foods during drying and
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00242-7
subsequent storage. Although reactions occurring between
macronutrients, such as those involving proteins and carbohy-
drates during nonenzymatic browning, are of nutritional con-
sequence, individual macronutrients in food are generally not
impacted to a significant degree by the drying process alone. In
this section, the changes that occur in the macronutrient frac-
tion are discussed briefly. The factors influencing the degrada-
tion of macronutrients during the drying of foods are also
summarized in Table 1.
Protein
Protein is naturally present in many common dried products,
including milk and egg powders, and can play a significant role
in the encapsulation of lipids and probiotics. In addition,
advances in processing technology have resulted in an
increased availability of dairy-derived (whey protein and
casein) and plant-derived (soy and rice) protein powders,
bars, and beverages, which are popular product categories
among young adults engaged in athletic activities; on the
other hand, an aging or ‘graying’ global population is likely
to result in increased instances of diseases such as pathological
sarcopenia, which is accompanied by muscle wastage. In both
cases, consumption of adequate quantities of protein is essen-
tial to aid in the process of muscle synthesis. Modification of
the structure of proteins (i.e., denaturation and aggregation)
has been associated with altered biofunctional properties such
as allergenicity and digestibility, but these effects are generally
not attributed to the drying process itself. Additionally, these
structural modifications can occur in predrying processing,
such as the pasteurization and evaporation treatments applied
to some dairy systems. In the presence of carbohydrates, how-
ever, proteins may undergo structural modifications due to the
Maillard reaction, a nonenzymatic browning process. This
aspect is covered in ‘Carbohydrate.’
Carbohydrate
Carbohydrates are important structural components in dried
food matrices. In powders, they are frequently the primary
encapsulating material for other nutrients (e.g., fatty acids
and probiotics). Degradative changes associated with carbohy-
drates of nutritional consequence are mostly limited to the
generation of Maillard reaction products (MRPs) through non-
enzymatic browning reactions between reducing sugars and
amino acids. The generation of MRPs has been linked with
increases in cytotoxicity, mutagenicity, and carcinogenicity;
however, MRPs have also been associated with positive effects
such as increased antioxidant activity. In addition to health
effects, MRPs are associated with color and flavor development
in foods. However, while the sensory attributes that Maillard
browning imbues on certain foods, such as bakery and confec-
tionary products, are often desirable, browning in food pow-
ders is typically considered a defect. Investigations of MRP
generation in infant formula powders have been frequent, as
439
440 Drying: Effect on Nutrients, Composition and Health
Table 1 General properties of the main macronutrients in foods related to the influence of drying on their nutritional quality
Main Drying- Storage-
Macronutrient degradative induced induced
name(s) reaction changes changes
Proteins Denaturation Negligible Negligible
Carbohydrates/ Browning Minor Significant
proteinsa
Lipids Oxidation Significant Significant
a
Nonenzymatic browning requires amino acids in addition to carbohydrates.
b
PUFA, polyunsaturated fatty acid.
they are nutritional products high in both lactose and protein.
MRPs can be generated during spray-drying, with reports
highlighting the impact that spray-drying has had on infant
formulas, resulting in greater loss of lysine than observed
under processes of sterilization; however, it is also worth point-
ing out that higher concentrations of MRPs have also been
measured in sterilized infant formulas compared with spray-
dried formulas. Different drying techniques and feed compo-
sitions can influence the physical properties (e.g., porosity and
morphology) and chemical behavior (e.g., crystallization and
water sorption) of the dried material during storage, which in
turn can influence the generation of MRPs. MRP generation
also occurs in powders during storage. Environmental condi-
tions during the storage of infant formula powders (e.g., rela-
tive humidity, temperature, and light and oxygen exposure)
have a strong impact on the rate of generation of MRPs.
Lipids
Lipids are a primary contributor to the energy density and
texture of foods. Lipid-containing foods are typically emul-
sions, consisting of lipids dispersed in a continuous water
phase (O/W emulsion). Emulsified systems are intrinsically
unstable, and to improve their stability, multiple strategies
are employed, including the reduction in fat globule size
through physical disruption (e.g., indent homogenization
and microfluidization), increasing the viscosity of the contin-
uous phase (e.g., addition of hydrocolloids), and the addition
of amphiphilic molecules that adsorb at the oil–water interface
and reduce interfacial tension (e.g., lecithin). When emulsions
are dried, the continuous phase is removed and a solid carbo-
hydrate/protein matrix forms around the fat. Effective encap-
sulation reduces the levels of free fat at particle surfaces and is
dependent on the properties of the emulsion to be dried, the
nature and level of the encapsulant, and the drying conditions.
Oxidation of specific components in the lipid fraction, namely,
fatty acids and cholesterol, is associated with losses in nutri-
tional quality and is discussed in more detail in ‘Fatty acids’
and ‘Cholesterol,’ respectively.
Some potential
adverse nutritional
Key influencing factors consequences
Predrying thermal processes (e.g., pasteurization and Protein
evaporation) digestibility+
Protein
allergenicity*
Drying technology, drying temperature (spray-drying), feed Cytotoxicity*
composition (type of sugar), storage conditions (relative Mutagenicity*
humidity, temperature, light exposure), crystallization of Carcinogenicity*
sugars Lysine levels in
food+
Emulsion state (e.g., fat globule size distribution, emulsifier Cytotoxicity*
type), encapsulation efficiency, protein/fat ratio, Genotoxicity*
crystallization of sugars PUFAb
biofunctionality+
Changes in Micronutrients During Drying and Powder Storage
Fatty acids
Consumption of foods that are rich in polyunsaturated fatty
acids (PUFAs) has been associated with numerous health ben-
efits. Soybeans are an example of a food that is naturally rich in
PUFAs. Microwave-assisted drying has been reported to result
in less PUFA degradation in soybeans compared to forced-
convection drying, and PUFA degradation was independent
of drying temperature if soybeans were dried to the same
final moisture content. It has been demonstrated that sun-
drying or freeze-drying resulted in higher retention of seaweed
PUFAs compared to oven-drying. Although drying can result in
PUFA degradation, it may be preferable to canning as a method
of preserving seaweed, with one study reporting higher levels
of total PUFAs in five dried seaweeds compared to two canned
varieties.
Infant milk formulas are frequently fortified with PUFAs, as
long-chain PUFAs are abundant in human milk and are
believed to contribute to the development of cognition and
visual perception. Infant formulas contain metals that are
known to be pro-oxidizers, such as iron. Iron-binding proteins,
such as lactoferrin, have been shown to protect against oxida-
tion when added to infant formulas. Methods for the encap-
sulation of PUFA-rich oils (e.g., fish oil) have been developed,
but the protective effect of the encapsulant can diminish dur-
ing processing, and oxidation of PUFAs remains a challenge.
It has been reported that fortification of infant formula with
microencapsulated fish oil resulted in higher peroxide values
after 18 months. Reduced oxidation has been shown in infant
formulas where casein was the dominant protein, compared to
whey protein-dominant formulas, while higher levels of carra-
geenan or lecithin have also been demonstrated to reduce
oxidation in infant formulas. Higher levels of PUFAs, elevated
storage temperatures, and long storage duration generally
result in increased oxidation.
There is ongoing research into the potential for the encap-
sulation of plant-derived oils that are high in PUFAs, such as
walnut and chia oils, using spray-drying, with some recent

research showing that homogenization of a chia oil
emulsion had a greater impact on the effectiveness of sodium
caseinate–lactose encapsulation than the temperature at which
the emulsion was spray-dried. Encapsulation of oils by spray-
drying in the presence of hydroxypropyl methylcellulose, with
or without rosemary oil (antioxidant), improved the oxidative
stability of walnut oil but resulted in greater oxidation in chia
oil; it was suggested that the poor stability of encapsulated chia
oil was linked to chemical damage caused during spray-drying.
Cholesterol
The oxidation of cholesterol with a concomitant increase in the
levels of cholesterol oxidation products (COPs) can occur
during the manufacture of egg powders, which is a concern as
COPs have been linked with disease in humans. In other dried
products, such as spray-dried milk, cream, and infant formula
powders, the levels of COPs are less of a concern. High outlet
air temperatures and the generation of nitrogen oxides in a gas-
fired air heater during spray-drying resulted in increased gen-
eration of COPs. In addition, COPS can form during storage of
egg powders, but this can be controlled by the presence of
antioxidants in the powder, such as ascorbyl palmitate and
tocopherols. Spray-dried egg powders contained more COPs
than freeze-dried varieties, with the generation of COPS being
higher in those powders produced from liquid whole egg than
liquid egg yolk alone.
Phytochemicals
Phytochemicals are plant-derived substances, such as polyphe-
nols and flavonols, which are purported to have various bio-
active properties. Studies have found that the levels of phenolic
compounds in dried kale were highest when freeze-drying was
used compared to air-drying, while blanching before drying led
to reduced levels in kale dried by either method. Recent
research has shown that Irish brown seaweed dried in a hot
air oven at different temperatures (25, 35, or 40  C) increased
the levels of phytochemicals as drying temperature increased
and that increasing temperature of rehydration of dried Irish
brown seaweed in water results in faster rehydration times but
lower levels of phenols.
It has been shown that drying conditions have no impact
on the phytochemical contents of apricots, currants, or prunes,
but do have an effect on grapes, while sun-drying has little
effect on the levels of phenolic compounds in Italian bell
peppers. A comparison of air- and freeze-drying for their ability
to retain phytochemicals in physiologically dropped, un-
matured citrus fruits reported that while air-drying was better
at retaining flavonols, freeze-drying exhibited superior reten-
tion of phenols.
A phenol-rich ingredient, named piraltin, has been manu-
factured by freeze-drying red wine; piraltin contained 70% of
the polyphenols in the wine and was alcohol-free. Phenol
levels in spray-dried bayberry juice, with maltodextrin as
encapsulant, were nearly 100% of those in the original juice;
when stored at 4  C, the levels of polyphenols in the bayberry
juice powder only decreased by <10% at the highest aw studied
(0.44); in addition, air-drying of apple pomace was more
effective than vacuum-drying at preserving the phytochemical
content. The use of maltodextrins with low dextrose equiva-
lents and spray-drying at low inlet temperatures resulted in
Drying: Effect on Nutrients, Composition and Health 441
lower phenol losses during drying of extracts of phenols from
black currant pomace. Encapsulation of a phenol extract
derived from olive pomace by spray-drying at different inlet
temperatures (130 or 160  C), feed flow rates (5 or
10 ml min1), and maltodextrin levels (100 or 500 g l1) has
also been studied, with the highest polyphenol levels at an
inlet temperature of 130  C, a feed flow rate of 10 ml min1,
and a maltodextrin level of 100 g l1; it was also found that the
powder underwent the least degradation of phenols when
stored in dark conditions at low temperatures.
Probiotics
Probiotics are live bacteria or yeasts that are believed to have a
beneficial role in the maintenance of gut health and the pre-
vention of gastrointestinal diseases. In addition, a growing
body of evidence supports a role for probiotics in the promo-
tion of neurological function. Common probiotic strains,
which are frequently added to foods in dried form, include
Bifidobacterium (B.) and Lactobacillus (L.).
Adapting L. paracasei by growing it in a stressful environ-
ment (high heat or salt) resulted in a 16–18-fold increase in
stability when the bacteria were inoculated into milk and
dried. Similar findings have been reported for L. casei Nad
and L. plantarum 8329 spray-dried with skim milk. When L.
salivarius subsp. Salivarius was freeze-dried in distilled water,
only 1% of viable bacteria remained; however, when the same
probiotic was dried in a medium consisting of skim milk,
glucose, and trehalose, >83% of viable bacteria remained;
the survival of the freeze-dried probiotic during storage was
best at the intermediate levels of moisture, which were studied
(2.8–5.6%). Researchers have spray-dried L. paracasei inocu-
lated in a skim milk/yeast medium using an inlet temperature
of 175  C and an outlet temperature of 68  C and reported that
15% of the probiotic was lost during drying; when cheddar
cheese was fortified with the powder, the researchers found
that the probiotic population grew during ripening.
The survival of Saccharomyces boulardii, a probiotic yeast,
was greatest at acid pH and high inlet temperature, deemed
by the authors to be due to optimal protein crust formation
around the yeast in these conditions. Encapsulation of B. ani-
malis subsp. lactis BB-12 in sweet whey resulted in reduced
viability at acid pH and no difference in salt tolerance (bile or
NaCl); when the probiotic was added to a refrigerated dairy
dessert, it showed a consistently higher viability during 6 weeks
of storage. Freeze-drying of B. longum 1941 in different
protein–sugar encapsulants revealed that milk proteins (skim
milk, sodium caseinate, and whey protein concentrate) out-
performed soy proteins and that sugar alcohols (glycerol and
mannitol) outperformed maltodextrins, in terms of postdrying
viability and acid and bile tolerance. Experiments where single
droplets containing L. plantarum A17 in addition to skim milk,
whey protein isolate, lactose, or trehalose were dried revealed
that the dairy ingredients were capable of protecting the bacte-
ria to a greater degree than the sugars during drying, due to the
formation of a crust that limited the thermal load applied to
the encapsulated bacteria.
An interesting approach to the drying of probiotics is their
codrying in the presence of prebiotics. Prebiotics are carbohy-
drates that cannot be digested by humans, but that can be used
as a nutrient source by probiotics, thereby stimulating the
442 Drying: Effect on Nutrients, Composition and Health
growth of the latter. For this reason, they are sometimes col-
lectively referred to as ‘synbiotics.’ Recent studies have shown
that Bifidobacterium species can be effectively encapsulated
using inulin, oligofructose, oligofructose-enriched inulin, or
polydextrose, while Lactobacillus species have been encapsu-
lated using fructooligosaccharide. Other combinatory
approaches at improving the manufacture of probiotics
include the use of spray–freeze-drying, which combines
spray-drying and freeze-drying technologies and has primarily
been used in the production of biopharmaceuticals.
Spray–freeze-drying was successfully used to dry L. casei, with
98% of the probiotic surviving the drying process.
Nucleotides
Nucleotides in the diet are thought to contribute positively to
immune function, gastrointestinal health, and absorption of
other micronutrients. Nutritional sources of nucleotides
mainly include mammalian milks. It has long been known
that human milk has innately high levels of nucleotides, and
this has resulted in efforts to humanize infant formulas
through fortification with nucleotides. Significantly lower
levels of adenosine 50 -monophosphate and cytidine 50 -
monophosphate have been measured in reconstituted skim
milk powder compared to raw milk, indicating that the spray-
drying process may cause degradation of nucleotides. It was
also reported by the same group that pasteurized milk had
lower levels of these nucleotides, and as the skim milk powders
were pasteurized and evaporated before drying, it is difficult to
ascertain the influence of the drying step in isolation; it is also
worth noting that in-container sterilized and powdered milks
were the only samples to contain significantly lower levels of
orotate. A recent study found that pasteurization of human
milk actually increased the levels of free nucleotide monopho-
sphates; on the other hand, high-pressure processing caused a
decrease.
Casein-dominant infant formulas have been shown to have
naturally higher concentrations of nucleotides compared with
whey protein-dominant formulas, whether the latter had been
fortified or not. Additionally, the same author indicated that
minimal degradation of nucleotides occurred in the powders
studied during 1 year of storage. Recently, nucleotides in 11
infant formulas were analyzed and it was found that the major-
ity had levels that were in general agreement with their label
claim; however, one formula was reported to have nucleotide
levels that were 90% less than what the label claims, indicating
major losses during processing. More research is required to
identify the potential role of spray-drying and other processing
steps on the degradation of nucleotides in infant formulas.
Vitamins
Foods such as fruits and vegetables are widely known to be
important natural sources of vitamins, while formulated prod-
ucts such as infant formulas are increasingly fortified with
vitamins. Vitamin D3, which is now routinely added to milk
and infant milk products, was shown to undergo negligible
degradation in the production of spray-dried milk. Vitamin C
was shown to degrade during the air-drying of tomatoes, with
higher drying temperatures resulting in greater losses; immer-
sion in salt or sugar solutions prior to drying was found to
inhibit this degradation. An inverse relationship between
air-drying temperature and vitamin C levels in both tomatoes
and papaya has also been reported.
Decreases in vitamins A, E, and C were measured during
storage of infant formula powders for up to 18 months; the
greatest losses were reported for vitamin C, and levels of all
vitamins decreased more rapidly when powders were stored at
elevated temperatures (40  C vs. 25  C). Tracking the degrada-
tion of vitamins A and E in infant formula powders after the
packaging had been opened revealed that, while the levels of
these vitamins did decrease over 70 d storage, the levels
remained within regulatory limits. Enteral formula powders
underwent reductions in vitamins A, B1, and E during storage
for up to 6 months at 30  C that were sufficient to compromise
the ability of those formulas to satisfy their label claims for
RDAs, particularly in adverse conditions (high aw and lengthy
storage). Efforts to increase the retention of vitamins in pow-
ders by advanced drying techniques (nanospraying or electro-
spraying with encapsulants) have recently been investigated.
Minerals
The greatest potential for loss of minerals in products intended
for drying is when the feed is in its liquid state. Mineral fallout
in neutral-pH liquid products (e.g., cow milk, soy milk, and
infant formula) can be caused by the use of insoluble calcium
salts such as calcium carbonate, phosphate, and citrate, but this
is less of a concern in acid products such as fruit juices. The
stability of insoluble salts in liquids can be improved through
the use of thickeners to increase the viscosity of the solvent or
micronized forms of calcium that sediment less rapidly. Min-
eral loss from food during drying or during postdrying storage
has not been demonstrated to be an issue affecting the nutri-
tional quality of foods.
Other Considerations When Drying Nutritional
Formulations
Influence of Bulk Density on Nutrient Dosage in Nutritional
Powders
Powdered protein supplements are currently very popular
among the physically active. In addition, powders are available
that, once reconstituted, are intended to function as complete
meal replacers, where they are the sole source of sustenance for
the consumer. Critically, these products require the consumer
to add a defined quantity of powder to a defined quantity of
water to create a beverage with defined levels of key nutrients.
In products such as these, infant formula powders being
another example, the manufacturer must ensure that the pow-
der has a consistent bulk density so that the consumer can
scoop the correct quantity of powder into the liquid. In
spray-drying, bulk density is influenced primarily by the vis-
cosity of the feed, degree of aeration of the feed, type of
atomizer, and agglomeration and/or instantization. If pneu-
matic conveying is used to transport powders within a
manufacturing facility, additional care must be taken to pre-
vent agglomerate breakage, which can result in deviation of
bulk density from specified values. The manner in which bulk
density can affect nutrient dosage in these products is illus-
trated in Figure 1.
 Drying: Effect on Nutrients, Composition and Health 443

Multiple factors affect the bulk density of spray-dried powders Scoops are precision engineered for powders with a specific bulk density

-Feed viscosity

- Feed aeration

- Atomizer type

- Agglomeration
Equivalent volume of scooped powder
- Instantisation =

- Pneumatic conveying equivalent mass of nutrients per scoop

if
bulk density is not equivalent

Out-of-spec
bulk density

Figure 1 Influence of bulk density on nutrient dosage in spray-dried nutritional formulations, such as infant formula and meal replacers.

Role of Accelerated Stability Tests and Fortification Overages degradation. Spoiled food is unpalatable and potentially toxic
and generally has poor nutritional value. Dried foods and food
In the development of new nutritional products, the final
powders have shelf lives that far exceed those of undehydrated
formulation and shelf life are determined by the integration
varieties, and it may take many years before any spoilage
of nutrient and physical stability testing data. For extended
eventually occurs. It would be a mistake to simply compare
shelf life powders, the long shelf life necessitates the imple-
the nutritional value of a freshly harvested food with the same
mentation of accelerated stability testing programs that com-
food recently dried, because the relative dynamics of degrada-
plement traditional ambient stability tests. The principal
tion of each over time are also an important factor determining
environmental factors that are modulated during accelerated
their potential health-giving properties.
stability tests are temperature, light exposure/intensity, and
For certain nutrient-dense foods, it may not be possible to
relative humidity. Typical ambient and accelerated stability
produce them in certain regions of the world, or where they
testing storage conditions for powders are 25  C/60% relative
can be produced, the supply may be contaminated or other-
humidity and 40  C/75% relative humidity, respectively. For
wise untrustworthy; in cases such as this, dried foods are a low-
sensitive nutrients (e.g., B vitamins and PUFAs), the formula-
volume product that can be transported from a reliable source
tion overages required are normally determined based on mea-
at low cost and low environmental impact. Some of the
sured losses during processing and stability testing. The
research that has been highlighted in this article has demon-
principal factors affecting the overages required for individual
strated that the nutritional value of foods can indeed depreci-
nutrients vary but are generally determined by inherent stabil-
ate when being transformed into a dried product; however,
ity of the nutrient, regulatory/quality limits, choice of drying
many of these studies have also shown that this effect can be
technology and operating parameters, packaging material,
minimized or even eliminated if the drying process is carefully
transport and storage conditions, and the required shelf life.
controlled.
Some general considerations for nutrient stability testing are
The consumption of bioactive substances (e.g., phytochem-
that ambient and accelerated programs should run in parallel
icals, probiotics, and PUFAs) in sufficient quantities to extract
and stability data should be generated using the actual com-
their purported benefits is difficult to achieve in many regions,
mercial packaging material in all types and sizes that are man-
particularly for the socioeconomically disadvantaged. Encap-
ufactured. These concepts are illustrated in Figure 2.
sulation by drying affords the possibility of a range of
bioactive-fortified foods becoming commercially available.
The availability of such products may alleviate the burden on
Drying and Health: Overview and Future Perspectives individuals to consistently source and acquire the requisite
foods, which may be prohibitively expensive or otherwise
Most foods, in their natural, high-moisture state, are prone to inaccessible. Moreover, it is thought that targeted food fortifi-
rapid deterioration due to microbial spoilage and/or chemical cation strategies may be needed to alleviate region-specific
444 Drying: Effect on Nutrients, Composition and Health
Target = innate + fortification
Fortification = Target – innate
Overage = Target – label claim
Min. regulatory limit
Innate + Fortification
Ingredient
+ Ingredient
A B
Ambient stability tests integrated with
General considerations:
Ambient and accelerated tests should
be run in parallel
Stability data should be generated
using actual commercial packaging
material in all types/sizes to be
manufactured.
Figure 2 Principles of stability testing and fortification overages for sensitive nutrients during formulation of nutritional product.
deficiencies in certain vitamins, for example, vitamin D, the
status of which is thought to be lower in northern latitudes due
to a lack of sunshine; the effective incorporation of such fat-
soluble vitamins into stable food systems is likely to require
effective encapsulation technology.
Dried food is a valuable alternative to fresh food, where the
latter is unsafe or unavailable. The nature of a given food needs
to be considered during the design of appropriate processes for
its dehydration. In general, dried foods should be stored in cool
(<25  C), dark environments and preferably in sealed con-
tainers, to prevent degradation of nutrients. Encapsulation pro-
cesses for the manufacture of different bioactive substances have
been reported as being successful by many researchers. The
challenge now is to incorporate these ingredients into real
food matrices, ensure their stability, and verify that they can
have a positive influence on the health of the modern consumer.
See also: Antioxidants: Role on Health and Prevention; Ascorbic Acid:
Physiology and Health Effects; Ascorbic Acid: Properties,
Determination and Uses; Berries and Related Fruits; Beverage: Health
Effects; Beverage: Patterns of Consumption; Bifidobacteria in Foods:
Health Effects; Bioavailability of Nutrients; Browning: Non-enzymatic
browning; Carbohydrate: Digestion, Absorption and Metabolism; Dairy
Products: Dietary and Medical Importance; Drying: Physical and
Structural Changes; Drying: Principles and Types; Eggs: Composition
and Health Effects; Elderly: Nutrition Requirements; Fatty Acids: Fatty
Acids; Fish Oils: Composition and Health Effects; Food Fortification:
Rationale and Methods; Freeze Drying: Effects on Sensory and
Nutritional Properties; Freeze-drying: The Basic Process; Functional
Foods; Heat Treatment: Principles and Techniques; Infants: Nutritional
Requirements; Lactic Acid Bacteria; Maillard Reaction; Milk Powder;
Milk: Processing of Milk; Milk: Role in the Diet; Oxidation of Food
Components; Papayas; Pasteurization: Effect on Sensory Quality and
Regulatory limits
Max. regulatory limit
Target
Label claim
Overage
Accelerated stability tests
Variables: temperature, light exposure and
intensity, relative humidity
Sensitive nutrients (e.g., B vitamins, PUFAs),
require an overage
Overage is based on losses during
processing and stability testing.
Nutrient Composition; Phenolic Compounds: Occurrence, Classes, and
Analysis; Prebiotics; Preservation of Foods; Probiotics; Protein:
Digestion, Absorption and Metabolism; Protein: Food Sources; Protein
Quality and Amino Acids in Maternal and Child Nutrition and Health;
Protein: Requirements; Solanaceous Fruits Including Tomato,
Eggplant, and Peppers; Sports Nutrition; Storage Stability:
Mechanisms of Degradation; Storage Stability: Shelf Life Testing;
Vitamins: Overview; Whey and Whey Powders: Production and Uses;
Whey and Whey Powders: Protein Concentrates and Fractions.
Further Reading
Demiray E, Tulek Y, and Yilmaz Y (2013) Degradation kinetics of lycopene, b-carotene
and ascorbic acid in tomatoes during hot air drying. LWT – Food Science and
Technology 50: 172–176.
Fang Z and Bhandari B (2011) Effect of spray drying and storage on the stability of
bayberry polyphenols. Food Chemistry 129: 1139–1147.
Foster JA and Neufeld KAM (2013) Gut–brain axis: how the microbiome influences
anxiety and depression. Trends in Neurosciences 36: 305–312.
Frias J, Peñas E, and Vidal-Valverde C (2009) Changes in vitamin content of powder
enteral formulas as a consequence of storage. Food Chemistry 115: 1411–1416.
Fritzen-Freire CB, Pruděncio ES, Amboni RDMC, Pinto SS, Negrão-Murakami AN, and
Murakami FS (2012) Microencapsulation of bifidobacteria by spray drying in the
presence of prebiotics. Food Research International 45: 306–312.
Gharsallaoui A, Roudaut G, Chambin O, Voilley A, and Saurel R (2007) Applications of
spray-drying in microencapsulation of food ingredients: an overview. Food
Research International 40: 1107–1121.
Gil A and Sanchez-Medina F (1982) Effects of thermal industrial processing on acid-
soluble nucleotides of milk. Journal of Dairy Research 49: 295–300.
ICH (2003). ICH harmonised tripartite guideline: stability testing of new drug
substances and products QIA (R2). International conference on harmonisation of
technical requirements for registration of pharmaceuticals for human use (ICH).
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/
guidanceg/ucm073369.pdf.
Indyk H, Littlejohn V, and Woolard DC (1996) Stability of vitamin D3 during spray-
drying of milk. Food Chemistry 57: 283–286.

Korus A (2011) Effect of preliminary processing, method of drying and storage
temperature on the level of antioxidants in kale (Brassica oleracea L. var. acephala)
leaves. LWT – Food Science and Technology 44: 1711–1716.
Manzocco L, Calligaris S, Mastrocola D, Nicoli MC, and Lerici CR (2000) Review of
non-enzymatic browning and antioxidant capacity in processed foods. Trends in
Food Science and Technology 11: 340–346.
Obara A, Obiedziński M, and Kołczak T (2006) The effect of water activity on cholesterol
oxidation in spray- and freeze-dried egg powders. Food Chemistry 95: 173–179.
Drying: Effect on Nutrients, Composition and Health 445
Preidis GA, Hill C, Guerrant RL, Ramakrishna BS, Tannok GW, and Versalovic J (2011)
Probiotics, enteric and diarrheal diseases, and global health. Gastroenterology
40: 8–14.
Vega C and Roos YH (2006) Invited review: spray-dried dairy and dairy-like emulsions
– compositional considerations. Journal of Dairy Science 89: 383–401.
Verma A and Singh SV (2015) Spray drying of fruit and vegetable juices – a review.
Critical Reviews in Food Science and Nutrition 55: 701–719.
 Drying: Physical and Structural Changes
SV Jangam, National University of Singapore, Singapore
AS Mujumdar, McGill University, Ste-Anne-de-Bellevue, QC, Canada
B Adhikari, RMIT University, Melbourne, VIC, Australia
ã 2016 Elsevier Ltd. All rights reserved.
Introduction: Useful Concepts in Food Drying
Food is an important part of our life, and all types of food need
to be preserved one way or another for a number of reasons,
drying being the most important and effective way of preser-
vation. Fresh fruits and vegetables contain high amounts of
water, which results in microbial damage. This reduces the
shelf life of foods substantially, unless it is processed effi-
ciently. A considerable volume of fruit and vegetables is lost
every year because of microbial spoilage. The moisture in a
food product can be reduced to an acceptable level using
several thermal dehydration techniques to minimize the pos-
sibility of microbial damage. In addition to this, the cost for
packaging, transportation, and storage for dried product is
substantially less compared to canned or frozen food products.
Nevertheless, drying is a highly energy-intensive unit operation
because of the high latent heat of evaporation of water
(2260 kJ kg
1 in free state); hence, the cost of reducing mois-
ture to a very low level is expensive. There have been several
developments in recent years in the field of thermal dehydra-
tion to enhance drying rates, reduce energy usage, and mini-
mize environmental impact. However, in the case of food
products, the quality of the dried product is more important,
although other issues such as energy usage and environmental
effects of a selected dryer/drying system cannot be neglected.
The moisture present in solids can be classified as bound or
unbound moisture. The bound moisture exerts vapor pressure
less than that of the pure liquid and the moisture in excess of
bound moisture is the unbound moisture. When wet solids are
subjected to thermal dehydration, two processes occur
simultaneously: removal of surface moisture by evaporation
and transfer of internal moisture to surfaces through diffusion
and its subsequent evaporation. The removal of surface moisture
depends on external conditions such as temperature, relative
humidity of drying air, surface area of the wet material available
for drying, airflow, and pressure. On the other hand, the removal
of internal moisture depends on the physical nature of the solids
present, temperature, and amount of moisture present. This
largely decides physical and structural changes during the drying
of any material. The variation in the airside variables such as
airflow, temperature, and relative humidity becomes less impor-
tant, except when increasing the heat transfer rates to the mate-
rial. In case of food products, drying generally occurs in falling
rate period as most of the moisture present is bound moisture;
hence, physical and structural changes in products are quite
common. This happens as a result of an excessive moisture
gradient from the product interior to the surface, especially dur-
ing the final stages of drying. This causes overdrying and
shrinkage, which, again, result in structural damage.
Drying is a very complex heat- and mass-transferring pro-
cess with several rate processes, including physical and
446 Encyclopedia of Food and Health
chemical transformations. Other factors, which are responsible
for these complexities, are different types of feedstocks,
throughput, quality requirements of a particular product,
and, very importantly, the existence of hundreds of dryer
types. Therefore, selection of dryer or drying system is a crucial
consideration in order to satisfy the demands for a particular
application. Interested readers may refer to either the Handbook
of Industrial Drying or Mujumdar’s Practical Guide to Industrial
Drying for a detailed discussion on classification and selection
of dryers. This article will discuss, in short, some conventional
and advanced drying techniques and their importance with
respect to physical and structural changes in food products.
Importance of Drying in Foods: Benefits and Quality
Importance
Fruits and vegetables – in particular – have many positive
health effects. This food grouping has a high fiber content
and other useful components that help control blood glucose
levels, reduce cholesterol, and probably reduce the risk of
cancers (colon especially). They also contain antioxidants,
vitamins, and minerals, which also have positive health effects.
It is important to preserve all nutritional qualities of such food
products in their dried state as closely as they are in their native
state.
Other important quality attributes are physical and struc-
tural changes, which include shrinkage, puffing, glass transi-
tion, rehydration capacity, density, and mechanical properties.
These properties depend on several factors, such as pre-
treatment process prior to drying, dryer conditions (such as
temperature, pressure, and, to some extent, relative humidity
of drying medium), type of drying medium, type and extent of
heat input, and physical state of the product to be dried. The
selection of drying conditions and dryer type will depend on
the desired and expected properties of the product. The follow-
ing section presents some of the important physical and struc-
tural properties of dried products:
Shrinkage is a change in volume as a result of loss in mois-
ture during drying. Shrinkage is an important consequence of
drying, which determines the rate of drying and quality of the
dried material to great extent. Shrinkage influences product
structure and the rehydration ability of a dried food product.
Shrinkage occurs as a result of capillary collapse due to the
evaporation of water occupying the capillaries. Shrinkage can
be either isotropic or anisotropic. Isotropic shrinkage is
defined as uniform shrinkage of all geometric dimensions of
a material. In anisotropic shrinkage, the extent of shrinkage of
all geometric dimensions of a material is uneven or non-
uniform in nature. As discussed earlier, the outer layer of the
material loses moisture much faster than the interior; hence,
http://dx.doi.org/10.1016/B978-0-12-384947-2.00241-5
 Drying: Physical and Structural Changes 447

the surface layer shrinks. If the surface moisture is removed Table 1 Use of pretreatment on physical properties of dried fruit
very fast, but the diffusivity or the migration of internal mois-
ture to the surface is too slow, then an exterior layer, which is Product Type of pretreatment Findings
essentially impermeable to moisture diffusion, is formed. This Kiwifruit slices of Sucrose syrup Improved texture
phenomenon is known as case hardening. In food drying, case different initial (70% w/w) for 2 h
hardening is quite common and selection of proper drying textures (firm, at 25  C
conditions is very important; however, it may be desirable in medium and soft)
some instances for some products. Apple, banana Sucrose solution Porosity of the
Cracking is another phenomenon that occurs during the (50% w/w) osmotically treated
drying of foods. In this situation, the product surface shrinks followed by air- product decreased
during drying, causing tensile stress at the surface and compres- drying at 70  C as a result of solid
gain; however, it
sive stress inside the material. When the tensile stress exceeds
prevented color
the material strength, cracking occurs. The cracking of material
deterioration;
during drying needs to be avoided as it results in loss of material increases the
and consumer unacceptability. In the case of grains, cracking maximum stress
usually leads to material loss during milling and may also result and maximum
in bacterial attack during storage. The internal microstructure of deformation of the
food products is also very important as it can have a profound dehydrated product
influence on its nutritional value, rheology, and textural attri- Papaya Osmotic dehydration Better maintenance
butes. As the drying can substantially affect the internal struc- followed by hot-air of cell structure of
ture of foods, it is necessary to understand the effect of various (HA) drying of papaya when OD
papaya with a with additives was
parameters on product microstructure. The rehydration ratio
sucrose solution used followed by
is defined and based on the mass of material after drying and
(65 Brix) with HA drying
its corresponding mass after soaking in water. syrup to fruit mass
Drying rates can have dramatic effects on structure of the ratio of 4:1
dried products as drying can cause case hardening. This conse- Salak fruit Thermal pretreatment Increase in the cross-
quently causes the surface to crack, which in turn allows the (blanching) sectional area of
vapor generated within the body to flash out and cause poor cells, which affects
appearance and nonuniform drying. This is undesirable. This the water sorption
phenomenon is product-dependent, and hence, laboratory characteristics
testing is necessary. Discoloration of the product surface can
also occur. Furthermore, gradients in porosity can lead to a
nonhomogeneous product. The use of radiative heat input can characteristics. Table 1 shows the impact that pretreatment has
also result in crust formation if the flux is too high. Drying at on the physical properties of fruit products.
elevated temperatures, if it is elevated enough under the oper- Recently, various approaches have been employed to
ating pressure used, can cause internal boiling of the liquid. enhance the process of osmotic dehydration. The use of micro-
The internal boiling leads to hollow particles in spray-dried wave (MW), pulsed electric fields, ultrasound, and high hydro-
powders. This again enhances the porosity, thereby resulting static pressure has resulted in enhanced osmotic dehydration
in better rehydration ability. The following sections present the rates. It has been shown that the ultrasound pretreatment
effects that operating parameters and dryer types have on the improves the chewability of products such as dried sea cucum-
physical and structural properties of dried foods. ber (low hardness and high springiness) and results in a higher
rehydration ratio as a result of microchannels formed during
ultrasound pretreatment. The use of pulsed vacuum and ultra-
Influence of Various Drying Parameters on Physical sound has also been shown to improve water removal rate,
and Structural Food Modifications favorable glass transition temperature, and color. Some phys-
ical pretreatments include freezing, thawing, bruising, pinning,
Pretreatment drilling holes on surface (to ease moisture diffusion), and
A pretreatment of wet product prior to drying is very important extrusion (to increase the surface area). Each one of these
for foods. This is used to achieve physical or chemical changes techniques has been used selectively to either enhance drying
in order to enhance the drying rates and product quality. rates or improve final product quality.
Blanching and osmotic treatment are used for several food
products, especially for fruits and vegetables. During osmotic
Temperature and Humidity
dehydration, a product to be dried is treated with an osmotic
solution; water from the food material migrates outward and is As explained earlier, the external drying conditions determine
replaced by the solute from the osmotic solution. The presence removal of surface moisture; however, the diffusion of internal
of this solute also serves as a preservative. Although osmotic moisture determines the drying rate when internal moisture
dehydration is mainly useful to improve product quality, needs to be removed. It was also highlighted that the drying of
including color, microstructure, and enhanced drying rate, most food products shows a falling rate period; hence, the
however, it may sometimes have adverse effects on physical increase in temperature can elevate internal moisture diffusion.
448 Drying: Physical and Structural Changes

However, the use of very high temperatures may harm the Pressure
quality of a dried product. Several studies have been carried
The pressure used during drying can have considerable influence
out in an attempt to understand the effect of temperature and
on the physical structure of the dried material. A dryer can be
initial moisture on the physical properties of material. In gen-
operated either at atmospheric, at subatmospheric, or with vari-
eral, low-temperature drying results in lower shrinkage and
able pressure cycles. Most commonly used dryers operate at
higher rehydration compared with drying at a higher temper-
atmospheric pressure. However, if there is a restriction on the
ature, as it results in crispy crust formation. Some studies also
drying temperature used, because of heat sensitivity of a particular
observed increases in porosity with increase in temperature,
product, then drying needs to be carried out at subatmospheric
which resulted in better preservation of the structure at inter-
pressures. The moisture can be removed at lower temperatures
mediate temperatures. This is due to the fact that both the
when vacuum is used, although operating a dryer at sub-
longer drying time at lower temperatures and the use of very
atmospheric pressure increases processing cost considerably. Dry-
high temperatures can result in development of wrinkled struc-
ing under vacuum can open up voids within a material with a
ture. Figure 1 shows scanning electron microscopy (SEM)
nonrigid structure. This increases the porosity and enhances rehy-
photographs of carrot dried using hot-air drying; higher tem-
dration ability. This has been observed by a number of researchers
perature affects the pore diameter. In the case of some prod-
using a variety of drying methods, such as freeze-drying (FD),
ucts, such as gelatin films, crystallinity and glass transition
MW vacuum drying, and superheated steam drying. Some
temperature are important. The lower drying temperature can
researchers have used variable pressures to achieve porous struc-
result in higher crystallinity and higher glass transition temper-
tures, such as successive pressure drop (déshydratation par
ature of the dried product, which is important for better water
détentes successives, DDS) and controlled instantaneous pressure
absorption and lower levels of hysteresis.
drop (détente instantanée contrôlée, DIC). All these ideas and
The relative humidity of a drying medium also has an effect
related findings will be discussed in more detail in the section on
on physical properties of the dried material. In addition to
advanced drying techniques in this article.
temperature, the humidity also decides the rate of drying. If
the drying medium is highly saturated with moisture, then the
drying rates are slower; hence, either temperature rise or reduc-
Drying Medium
tion of relative humidity is necessary. Besides this, relative
humidity can also affect product structural properties if the The choice of drying medium for food products is very impor-
glass transition temperature is affected by higher relative tant as it can have considerable effects on product quality. The
humidity, especially products containing sugars. most commonly used drying medium is air as it is the least

(a) (b)

(c)

Figure 1 SEM photographs showing cross section of carrot cube undergoing hot-air drying (a) fresh sample; (b) hot-air drying at 60  C; (c) hot-air
drying at 80  C.
 Drying: Physical and Structural Changes 449

expensive to use. However, the use of air can damage product and related applications are discussed in the following
quality in terms of browning (as a result of unwanted enzy- sections.
matic or nonenzymatic reactions when in the presence of
oxygen), shrinkage, porosity, and texture. Modified atmo-
spheric drying can be carried out by replacing air with an Effect of Drying Methods
inert medium such as carbon dioxide, nitrogen, or steam.
This is very useful for many products, especially food products. It is extremely important to select an appropriate dryer or
The use of superheated steam was proposed in 1990s and has drying system for each food product as most are highly heat-
been developed over the years as an important drying tech- sensitive. An incorrect choice of drying system and/or drying
nique for foods. There are several published reports on use of conditions adversely affects the physical and nutritional prop-
inert atmosphere during drying, although the system needs to erties of the dried food. This section discusses conventional
be a closed cycle in order to minimize the volume of the drying and recently developed drying techniques, as well as their
medium used, thereby reducing processing cost. An extensive effects on the physical structure of foods.
review on drying of foods using heat pump dryers has been
published, which addresses the use of inert atmosphere for
drying. This article elaborates on the improvement of quality Conventional Drying Techniques
attributes of dried products, such as reduction in browning,
There are several techniques traditionally used for food drying,
increased porosity, and production of less firm products com-
such as open sun drying, solar cabinet drying, and hot-air
pared with products obtained using heat pump drying (HPD)
drying; however, these techniques result in unacceptable phys-
employing air. The improved structure of the products dried
ical and nutritional properties of foods. The products from
using an inert atmosphere also resulted in faster rehydration. It
these dryers can be used to partly satisfy domestic demands,
is certainly useful to use an inert atmosphere for foods, but one
although one cannot sustain the ever-growing international
needs to consider several aspects such as added cost, improve-
markets. Table 2 lists several conventional dryers used for
ment in quality, and possible returns.
food drying. This list is based on type of feedstock used,
although the dryers can be classified based on several other
parameters.
Mode of Heat Input For particulate materials, a variant of fluid bed is the most
popular choice because of the requirement of very high heat
The classification of dryers based on the mode of heat input is
and mass transfer rates as a result of vigorous mixing between
most common. Dryers are classified as direct and indirect.
particles and drying medium. A major limitation of fluidized
Direct dryers, also known as convective dryers, are by far the
bed dryers can be particle size and the feedstock particle size
most commonly used in industry, although efficiencies are
distribution. In the case of some food products, attrition results
low. In direct dryers, the drying medium contacts the material
in unacceptable size distribution. In fluidized beds, the non-
to be dried directly and supplies the heat required for drying by
uniform distribution of drying gas also results in nonuniform
convection. Drying gas temperatures may range from 40  C to
quality of drying products in terms of shrinkage, moisture
very high temperatures, the temperature selected being depen-
content, and rehydration ratio. Traditionally, tray drying is
dent upon the food material to be dried. As described earlier,
popular, but throughput and energy efficiency are major issues.
the selection of dryer temperature is very important in order to
Drying time can be very long, in the case of tray dryers, and
minimize structural food damage. As described previously in
hence, it can have adverse effects on product quality, especially
this article, the drying of foods falls in the falling rate period.
with respect to physical structure. Though continuous tray
Hence, one can use intermittent heat supply at the end of the
dryers were also used, these dryer types are not popular
drying when the moisture transport from inside to the surface
anymore.
is very slow.
Spray drying is one of the techniques used extensively in
On the other hand, indirect dryers involve the supply of
food-related industries for liquid feedstocks. As heat contact
heat to the wet solids without direct contact with the heat
time is very short and the rate of evaporation is high, spray
transfer medium. The heat is transferred from the heat transfer
drying produces a high-quality product, although cost may
medium to the wet solid by conduction, and the use of vacuum
also be high due to energy consumption. The liquid droplets
or gentle gas flow is necessary in order to remove the evapo-
rated moisture. Heat transfer surfaces may range in tempera-
ture from 40  C (as in FD) to very high temperatures; the Table 2 Classification of conventional dryers based on the type
of feedstock employed
selection of which is dependent upon the specific application.
The application of indirect dryers in foods generally uses vac- Type of feedstock Dryer types
uum as most of the foods processed are heat-sensitive.
Heat can also be supplied by radiation (using electric or Particulates Tray, fluidized bed dryers, belt conveyor, vibrated
natural gas-fired radiators) or volumetrically by placing the wet bed, rotary, screw conveyor, vacuum, freeze
solid in dielectric fields in microwave or radio-frequency (RF) Sheets, extruded Impingement, infrared, microwave, conduction
ranges. These options, especially MW dryers, are worth consid- material
Liquids Spray, variant of fluidized bed dryer with a bed of
ering as devices to speed up food drying in the tail end of the
inert particles, drum
falling rate period in order to reduce changes to physical struc-
Paste Paddle, fluidized bed, drum, rotary
ture as a result of prolonged drying times. Some of these dryers
450 Drying: Physical and Structural Changes
containing solids are dried in seconds as a result of the highly
efficient heat and mass transfers. The final product from a spray
dryer is in the form of powder. However, granules or agglom-
erates are conveniently produced by combining a fluidized bed
dryer or by recycling the fine particulates. There are several
factors that decide the physical form of the spray-dried prod-
uct, mainly atomization, chamber design, the type of contact
between drying gas and droplets, and drying air conditions.
SEM images of dried product are generally used to examine
particle morphology. The use of very high temperatures can
result in crust formation. There are several reports on SEM
analysis of spray-dried food products, such as skimmed milk,
fruit juices, and other extracts showing the effect of operating
parameters, atomization, and chamber design on morphology
of dried powder. Figure 2 shows SEM images of spray-dried
powder of fermented mixed juice of carrot and watermelon at
different operating conditions.
Spray drying is also commonly used in the food industry for
microencapsulation. Microencapsulation is defined as a pro-
cess by which one material, or a mixture of materials, is coated
or entrapped within another material or system. The coated
material is called the active or core material, and the coating
material is called the shell, wall material, carrier, or encapsu-
late. It is necessary to obtain a rigid structure to protect the
active ingredients trapped within the carrier. The selection of
drying conditions and the type of carrier and its composition
are extremely important in order to achieve efficient microen-
capsulated material.
Other techniques for drying of liquid materials are drum
drying and drying on a bed of inert particles, which has grown
in popularity in recent times. For sheets and extruded food
products, mainly belt conveyors or batch tray dryers are used
in combination with infrared (IR), microwave, or conductive
heating. In the case of pasty materials, paddle dryers or fluid-
ized bed of inert particles are more commonly used.
Recently Developed Drying Techniques
Each traditional drying technology described earlier can have
numerous variants, which possess some unique characteristics.
It is highly unusual to have a variant that only presents advan-
tages without limitations. So, final selection is always a com-
promise based on the product specification. Selection of dryers
is a very complex process. There are several reasons why few
truly innovative technologies find commercial adoption, the
most important being quality in terms of physical structure of
the dried material. The following are some of the more recently
developed drying techniques and their influence on the struc-
tural properties of the final dried products.
Heat pump drying
Although HPD is not a very new technology, there has been
continuous improvement in the design and application of
HPD technologies for food drying. HPD consists of a refriger-
ation cycle to recover heat from dryer exhaust air and reheat it
to the required temperature before it enters back into the dryer.
Detailed discussion on HPD and its operation can be found
elsewhere. The HPD system was traditionally used for the
recovery of heat; however, HPD is used in food drying to
improve product quality by utilizing some of the other
advantages it offers. The primary advantages of employing
the HPD system include providing a wide range of operating
conditions (humidity and temperature), possibility of using
inert gas as drying medium, and combining with other heat
modes. For heat-sensitive food products with long drying
times, HPD is very useful as drying can be carried out using
relatively lower temperatures in combination with de-
humidified air. As presented in several published studies, the
use of inert gas has resulted in better physical properties of the
dried material. A closed-loop HPD system can substantially
reduce the operating cost, as the same inert gas can be recycled,
with some top-up as necessary. Some of the applications of
inert gas food drying include drying of apple, ginger, guava,
papaya, lecithin, etc.
The most common HDP system used is mechanical com-
pression with low coefficient of performance. Some research
has focused on improving process efficiency, including multi-
stage heat pumps, chemical heat pumps, or use of heated pipes.
The chemical heat pump has good commercial potential but
needs thorough research. Another way of improving the perfor-
mance of heat pump systems is using external heating modes
such as microwave, RF, IR, or solar energies. This is useful
during the final stages of drying in order to minimize possible
damage to the physical structure of the foods. Solar-assisted
HPD is extremely useful for agricultural products. Another
approach of reducing product damage, as well as reducing the
overall cost of drying, is by varying the operating cycle of HPD,
such as through intermittent or time-dependent approaches.
For more detailed discussion of recent technological advances
and possible R&D opportunities in HPD, readers may refer to
related book chapter and review articles listed in reference.
Refractance window drying
The refractance window drying is a relatively new method used
to convert pulverized fruits and vegetables, pastes, and also
thin slice of fresh fruits. In this technology, the IR energy
coming from hot water (90–97  C) is used to dry the thermally
sensitive food materials. An IR-transparent plastic film is essen-
tially allowed to float on top of thin layer of heated water body.
The IR energy refracted through the plastic film due to the
presence of ‘window’ of moisture in the product across the
film is responsible for drying. The conduction of heat through
the thin plastic film also assists with drying. This is a relatively
energy-efficient system as the IR only gets refracted when the
wet product is present across the film. The refractance gradually
declines as the product dries. The loss of heat in this system (in
the absence of product) is due to conduction. It is reported that
this drying method preserves the color, texture, and antioxi-
dant contents compared with other drying methods of similar
heat intensity. This method is attracting increased research
attention.
Filtermat spray drying
This is a relatively newly developed drying method (GEA Tech-
nologies, Denmark) in which spray drying is combined with
Filtermat drying. This technology is especially designed to dry
difficult-to-dry sugar-rich products. The Filtermat component
involves a porous conveyor with controlled linear speed. The
drying section is usually divided into different sections. The
partially dried particles are deposited on this Filtermat, and
 5 kV ×1, 500 10 µm 10 30 SE I 5 kV ×1, 500 10 µm 10 30 SE I

(a) (b)

Drying: Physical and Structural Changes

5 kV ×1, 500 10 µm 10 30 SE I 5 kV ×1, 500 10 µm 10 30 SE I

(c) (d)

Figure 2 Scanning electron micrographs of spray-dried powder illustrating the variation in particle size and surface roughness when produced at different operating conditions (T, temperature; F, flow rate;
P, pressure): (a) T ¼ 140  C, F ¼ 3.5 ml min
1, P ¼ 2 bar, maltodextrin ¼ 17.5%; (b) T ¼ 160  C, F ¼ 5 ml min
1, P ¼ 3 bar, maltodextrin ¼ 10%; (c) T ¼ 140  C, F ¼ 3.5 ml min
1, P ¼ 2 bar,
maltodextrin ¼ 12.5%; and (d) T ¼ 120  C, F ¼ 5 ml min
1, P ¼ 1 bar, maltodextrin ¼ 15%. Reproduced from Mestry, A. P., Mujumdar, A. S. and Thorat, B. N. (2011). Optimization of spray drying of an
innovative functional food: fermented mixed juice of carrot and watermelon. Drying Technology 29(10), 1121–1131.

451
452 Drying: Physical and Structural Changes
the hot air is allowed to pass through the pores of the conveyor
and through the interstices of the deposited mass of particles. In
the second stage, dehumidified cold air is used to further dry
and cool the product. The final product requires certain degree
of grinding. The difference in this method over fluidized bed
drying is that the particles are not fluidized. The deposited
agglomerates are allowed to dry as thin layer of porous cake.
This type of dryer can be suitable for producing baby formula
powders.
Intermittent drying
Intermittent drying is an attractive concept due to its potential
to enhance energy efficiency and achieve better product quality
as a result of uniform drying. In intermittent drying, drying
airflow, temperature, or humidity is varied, or other alternative
parameters such as system pressure and type of external heat
source (microwave, IR, or RF) are varied with time in order to
suit the requirements. Sometimes, cycles of different heating
modes can be used. The main objective of intermittent drying
is to allow internal moisture to migrate to the surface of the
material during nonactive period – known as the tempering
period. During the active period, the heat is supplied, while
during tempering period, the supply of drying medium is
completely stopped. As the moisture content at the surface is
increased during tempering period, the rate of drying increases
substantially during the following active period. However, as
the drying rate is very small during the tempering period, the
overall drying time may increase slightly, although this is com-
pensated by the overall energy consumption.
Another intermittent drying approach is through the
application of a stepwise change in operating conditions.
This is especially important in the case of food products, as
drying rates are very slow during final processing stages. This
is due to the fact that drying is diffusion-controlled in the
final stages; hence, the effect of external conditions is negli-
gible. The use of this stepwise strategy of changes in the
operating conditions is very useful as a lot of energy can be
saved. At the same time, the product being dried is exposed to
severe drying temperatures for a shorter time, and this results
in less product shrinkage and less physical product damage.
The concept of intermittent drying can be applied to various
food products, using different drying methods, including the
use of a tray dryer, conveyor dryer, vibrating bed dryer, and
fluidized bed dryer.
Variable pressure methods
Drying can be carried out using either a controlled instanta-
neous pressure drop method or a cyclic pressure drop method.
In a standard controlled instantaneous pressure drop (DIC)
treatment, the product is subjected to high pressure, generally
using gas or steam, while temperature is maintained by one of
the various means available. This is followed by an instanta-
neous drop in pressure, usually assisted by vacuum technolo-
gies, which leads to autovaporization of water and sometimes
volatiles. This process can be easily controlled and results in
very good product quality, especially with respect to product
expansion ratio and porosity and also in the enhancement of
product texture. Swell drying is a combination of hot-air drying
and the DIC texturizing process. The reduction in drying time,
using swell drying, results in the significant enhancement of
product quality. In a dehydration process, followed by suc-
cessive decompression (DDS), the thermosensitive product
undergoes a series of cycles, during which it is placed at a
particular pressure for a defined time, followed by an instan-
taneous drop in processing pressure. The product is main-
tained at lower pressures for some time, before the
following cycle begins, and each decompression step results
in partial removal of water by autovaporization. The
amount of water removed depends on various factors, such
as operating conditions and the type of water present in the
material dried.
The major advantage of these techniques is the considerable
reduction in drying time that results compared to normal
vacuum drying or hot-air drying techniques used for food
products. The reduction in drying time always helps control
the physical properties of dried products. The techniques of
instantaneous and cyclic pressure drop have been successfully
applied to various food products, including grains, fruits,
vegetables, and fish. The DIC method is specifically used to
tackle the problem of product shrinkage.
Superheated steam drying
Superheated steam is an attractive drying medium for some
food products since the net energy consumption can be min-
imized if the exhaust (also superheated steam) can be utilized
elsewhere in the plant. The quality of superheated steam-dried
products tends to be better than that from conventional hot-air
dryers. Superheated steam also allows pasteurization,
sterilization, and deodorization of food products. This is par-
ticularly important for food and pharmaceutical products that
require a high standard of hygienic processing. In addition,
drying rates are higher in the case of superheated steam drying,
in both constant and falling rate periods and under certain
conditions, which are important to control in order to create
the structural changes required in foods. Mujumdar had dis-
cussed the principles, advantages, and limitations as well as
diverse applications of superheated steam drying technologies
in a number of publications, including the Handbook of Indus-
trial Drying.
Some products are not stable at 100  C, then one option is
to lower the operating pressure. The application of low-
pressure superheated steam drying is used for products, such
as potato chips, carrot, and longan. SEM images shows consid-
erable improvement in structure (porosity), less shrinkage, and
high rehydration ratio compared to hot-air drying. As an exam-
ple, SEM images of fresh and dried carrot using hot air and the
application of low-pressure superheated steam drying (LPSSD)
are shown in Figure 3. Although one can obtain good dried
product quality at low pressures, the process is still not popular
due to the requirement for large equipment owing to low
drying rates, but perhaps it may be necessary to use supple-
mentary heat sources in order to improve the process. Much
research and development is needed in this area in order to
progress the drying technologies discussed.
Advances in FD
FD is undoubtedly the most acceptable drying technique for
foods because it probably generates the best product quality, as
 Drying: Physical and Structural Changes 453

(a) (b)

(c)

Figure 3 Microstructure of (a) fresh carrot, (b) carrot dried until reaching equilibrium moisture content using hot-air drying, (c) carrot dried using
low-pressure superheated steam drying. Reproduced from Kerdpiboon, S. and Devahastin, S. (2007). Fractal characterization of some physical
properties of a food product under various drying conditions. Drying Technology 25(1), 135–146.

the frozen product is not allowed to melt during drying. It
prevents undesirable shrinkage and produces materials with
high porosity, ultimately resulting in better product rehydra-
tion properties. There are several reports in the literature show-
ing SEM images of freeze-dried products, clearly indicating that
FD enhances powder microstructure compared with that pro-
duced using conventional hot-air drying. However, the indus-
trial application of FD for food drying is limited because of the
high associated processing cost. There has been significant
effort to develop new techniques in order to achieve similar
product quality obtained from FD, although it is difficult to
achieve some characteristics, which only FD can achieve such
as rehydration, color, and texture. The cost of FD can be
reduced by improving the drying rates using external heating,
but without allowing the product to melt. This can be achieved
by either using magnetic and electric fields, using atmospheric
FD, or using microwave in combination with FD (MFD).
Application of ultrasound during the freezing stage also greatly
improves the drying rate and product quality of freeze-dried
products. A review on the use of electric and magnetic fields
during freezing and their application to FD suggests better
control on the ice crystals and, ultimately, on product quality.
The use of microwave FD for products such as fruit, vegetable,
and some marine-derived products has resulted in better dry-
ing rates and product quality, particularly in relation to prod-
uct microstructure, shrinkage, and rehydration ratio. Figure 4
shows comparison of SEM images of banana chips dried using
FD and microwave FD. A detailed discussion on FD and
technological advances in the area can be found elsewhere in
this book.
Hybrid drying
In the case of some food products, many conventional drying
techniques may not provide the expected final moisture con-
tents at sufficiently acceptable drying rates. In such cases, a
454 Drying: Physical and Structural Changes
HV Mag WD Spot Det 400.0µm
5.0 kV 200x 13.1 mm 4.0 ETD
(a)
Figure 4 Scanning electron micrographs of (a) FD banana chips and (b) MFD banana chips. Reproduced from Jiang, H., Zhang, M., Mujumdar, A. S.
and Lim, R.-X. (2013). Analysis of temperature distribution and SEM images of microwave freeze drying banana chips. Food Bioprocess
Technology 6, 1144–1152.
combination of dryers or drying technologies can be used. In
recent years, hybrid drying is popular as it can reduce drying
time substantially and provide more flexibility. Hybrid drying
systems may adopt more than one drying technique or more
than one heating mode in the same dryer. Microwave drying
offers advantages in enhancing drying kinetics, precise control,
fast start-up and shutdown times, quality of dried product,
smaller footprint of equipment, etc. IR drying is also useful in
removing final traces of product moisture at a faster rate. These
techniques are typically combined with other drying methods
to overcome the limitations of uneven heating, resulting in
unacceptable physical structure, for example, overdrying at
the corners and edges of products.
Spray FD is another hybrid drying technique used to pre-
pare highly porous particles. This technique involves the spray-
ing of a liquid feed in a cryogenic atmosphere, followed by FD
of these frozen droplets. The technique of spray FD has been
used for selected food application; however, necessary research
and development work is needed to minimize associated costs.
Concluding Remarks
Drying of foods is an extremely important commercial and
preservation process, and the approach used to dry foods will
impact differently upon the physical and structural properties
of the products in question. Drying rate and operating condi-
tions can have considerable effect on structure of the dried
product. To a certain extent, the structure can be controlled
by selection of an appropriate drying technique and
HV Mag WD Spot Det 400.0µm
5.0 kV 200x 13.1 mm 4.0 ETD
(b)
manipulation of operating conditions, although it is necessary
to take into consideration cost-effectiveness and suitability of
each method employed. Appropriate changes in existing dry-
ing systems and the use of some innovative dryers can give
improved quality of dried foods such as porosity, rehydration
ratio, texture, and microstructure.
New drying technologies such as microwave or RF-assisted
drying, pulse combustion drying, intermittent batch drying,
and superheated steam drying at subatmospheric pressure
may be applied in specific cases to obtain desired product
characteristics under optimal drying conditions. Experimental
validation at laboratory or pilot scale is necessary as it is not
possible to predict the effect of drying conditions on product
characteristics with high reliability.
See also: Drying: Principles and Types; Freeze Drying: Effects on
Sensory and Nutritional Properties.
Further Reading
Fernandes FAN and Rodrigues S (2008) Application of ultrasound and ultrasound-
assisted osmotic dehydration in drying of fruits. Drying Technology 26(12):
1509–1516.
Gharsallaoui A, Roudaut G, Chambin O, Voilley A, and Saurel R (2007) Applications of
spray-drying in microencapsulation of food ingredients: an overview. Food
Research International 40(9): 1107–1121.
Islam MR, Ho JC, and Mujumdar AS (2003) Convective drying with time-varying heat
input: simulation results. Drying Technology 21(7): 1333–1356.
Jangam SV (2011) An overview of recent developments and some R&D challenges
related to drying of foods. Drying Technology 29(12): 1343–1357.

Jangam SV and Mujumdar AS (2011) Heat pump assisted drying technology

overview with focus on energy, environment and product quality. In: Tsotsas E and
Mujumdar AS (eds.) Modern drying technologyvol. 4: pp. 121–162. UK: Wiley
Interscience.
Jiang H, Zhang M, Mujumdar AS, and Lim R-X (2013) Analysis of temperature
distribution and SEM images of microwave freeze drying banana chips. Food and
Bioprocess Technology 6: 1144–1152.
Kerdpiboon S and Devahastin S (2007) Fractal characterization of some physical
properties of a food product under various drying conditions. Drying Technology
25(1): 135–146.
Kudra T and Mujumdar AS (2009) Advanced drying technologies, 2nd ed. Boca Raton,
FL: CRC Press.
Kumar P and Mujumdar AS (1990) Superheated steam drying – a state of the art survey.
In: Mujumdar AS (ed.) Drying of solids. India: SaritaPrakashan.
Maache-Rezzoug Z, Rezzoug SA, and Allaf K (2002) Development of a new drying
process – dehydration by cyclical pressure drops (D.D.S.): application to the
collagen gel. Drying Technology 20(1): 109–129.
Mestry AP, Mujumdar AS, and Thorat BN (2011) Optimization of spray drying of an
innovative functional food: fermented mixed juice of carrot and watermelon. Drying
Technology 29(10): 1121–1131.
Minea V (2013) Heat-pump-assisted drying: recent technological advances and R&D
needs. Drying Technology 31(10): 1177–1189.
Mounir S, Allaf T, Mujumdar AS, and Allaf K (2012) Swell drying: coupling instant
controlled pressure drop DIC to standard convection drying processes to intensify
transfer phenomena and improve quality – an overview. Drying Technology 30(14):
1508–1531.
Mujumdar AS (2006) Some recent developments in drying technologies appropriate for
post-harvest processing. International Journal of Postharvest Technology and
Innovation 1: 76–92.
Mujumdar AS (2007) Handbook of industrial drying, 3rd ed. Boca Raton, FL: CRC
Press.
Drying: Physical and Structural Changes 455
Mujumdar AS (2014) Handbook of industrial drying, 4th ed. Boca Raton, FL: Taylor and
Francis.
Mujumdar AS and Law CL (2010) Drying technology: trends and applications in
postharvest processing. Food and Bioprocess Technology 3(6): 843–852.
Nido CI and Tang J (2007) Refractance window dehydration technology: a novel contact
drying method. Drying Technology 25(1): 37–48.
Nindo CI, Sun T, Wang SW, Tang J, and Powers JR (2003) Evaluation of drying
technologies for retention of physical quality and antioxidants in asparagus
(Asparagus officinalis, L.). Lebensmittel-Wissenschaft & Technologie 36: 507–516.
Rahman MS and Perera CO (1999) Drying and food preservation. In: Rahman MS (ed.)
Handbook of food preservation, pp. 173–216. New York: Marcel Dekker.
Woo MW and Mujumdar AS (2010) Effects of electric and magnetic field on freezing and
possible relevance in freeze drying. Drying Technology 28(4): 433–443.
Zhang M, Jiang H, and Lim RX (2010) Recent developments in microwave-assisted
drying of vegetables, fruits, and aquatic products
drying kinetics and quality
considerations. Drying Technology 28(11): 1307–1316.
Relevant Websites
http://www.abcar-dic.com/index.php?id_site¼2&id_page¼12 – Swell drying and
applications.
http://www.aces.uiuc.edu/vista/html_pubs/DRYING/dryfood.html – General article on
drying of foods.
http://www.analytica-world.com/en/whitepapers/126349/microwave-freeze-drying-of-
fruits-vegetables.html – Microwave freeze drying and its applications.
http://www.arunmujumdar.com/e-books.htm – Prof. Arun S. Mujumdar’s Research
Groupebooks.
http://www.foodtech-portal.eu/index.php?title¼Superheated_steam_drying –
Superheated steam drying.
 Drying: Principles and Types
JM Barat and R Grau, Universitat Politècnica de València, Valencia, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Drying is one of the oldest methods used for food preservation.
In fact, it is quite evident that initially, drying occurred by
chance and under natural conditions. Most of the raw material
used as food comes from biologically active sources. Conse-
quently, very few foodstuffs are inorganic in nature, such as
table salt. Since water is needed for life, it implies that most raw
materials destined for food consumption have a high water
content and, ultimately, high water activity (aw) values. Water
activity is a thermodynamic term that is related with the degree
of freedom of water.
Although the first objective for using drying was food pres-
ervation, nowadays, drying is also used to reduce food volume
and/or weight in order to optimize its storage and transporta-
tion (e.g., milk powder) and to avoid the need for refrigeration
or freezing conditions for preservation (e.g., dry-cured ham)
and is used for food product development (e.g., snacks).
Food drying occurs when the water activity of the air sur-
rounding food is lower than its aw. One characteristic of the
drying process is that the liquid water of food is transferred to
the gaseous state in drying air at temperatures below the boil-
ing point through an evaporation process.
The water activity of the air surrounding food is equal to its
relative humidity (RH) divided by 100 and is usually lower
than the original aw value of the animals and plants used as
food sources. Thus, when an animal or plant dies, a natural
drying process occurs, during which the water activity value of
food naturally decreases, thereby making it more stable.
Some characteristic water activity values of raw and dried
food products are presented in Table 1.
Sometimes, drying is used in combination with other pre-
serving techniques. The most common technique is addition of
solutes, which contribute to further aw reduction due to their
interaction with the water present in food. The most widely
used solutes are NaCl and sucrose. NaCl is normally used with
meat and fish products (e.g., dry-cured ham and dry-salted
cod), while sucrose is habitually employed with processed
fruit products, like jams and marmalades.
Water Activity and Stability
The water activity of food is related with its stability. The original
water activity of plants and animals is high enough to allow
reactions that permit life (such as solute movement and bio-
chemical reactions). This fact implies that when an animal or
plant dies, the spoilage process begins quickly since high aw
values would promote microbial growth and allow chemical
and biochemical reactions to proceed because of solute move-
ment and the activity of natural reagents and enzymes.
Reduced aw of food implies lower kinetics of spoilage reac-
tions, and a time comes when the spoilage reaction stops
456 Encyclopedia of Food and Health
altogether when a low enough aw point has been reached
within the food.
The only exception to this behavior is during fat oxidation.
The kinetics of fat oxidation is initially diminished with a
reduction in aw of food, until a value (usually in the 0.2–0.3
range) is reached when oxidation kinetics begins to increase
due to the direct exposure of fats to oxygen as a consequence of
the removal of most of the water molecules that create a
protective layer on the fat surface.
Thus, as a general pattern, the preservation behavior of food
increases with the reduction of water activity, up to 0.2–0.3.
Water Activity and Equilibrium
As mentioned in ‘Introduction,’ water activity is a thermody-
namic concept. This means that, in the absence of other forces,
movement of water will occur from points with a higher aw to
points with a lower aw. It is important to bear this in mind,
since a very common mistake is to consider that food systems
remain at equilibrium when the moisture at all points is the
same, instead of the true condition, which is when the aw of all
its points is the same.
So, food also tends to its internal equilibrium and with the
water activity of its surrounding air, which is the basis of the
drying processes.
At equilibrium, the water activity at each point within the
food is the same and equals that of the surrounding air. Within
the range of high water activity values, predictive equations can
be used to estimate the water activity of foods depending on
their composition: water and solute concentrations. Within
this range, food behaves similarly to the liquid phase con-
tained within its structure, and models developed for nonionic
or ionic solutes can be used, for example, Norrish for nonionic
solutes, Pitzer and Bromley for ionic solutes, or Ross for a
combination of solutes. If water activity values are low, predic-
tive equations for solutions are not useful because the interac-
tion of water with nonsoluble food compounds plays a major
role. This is why sorption isotherms must be used, which
represent the water activity of food versus the water content
of food on a dry basis. The main disadvantage of using sorp-
tion isotherms is that they must be experimentally determined
for each food type, owing to the variance in food composition
and structure. Figure 1 shows a typical sorption isotherm,
which relates the moisture of a certain food with a particular
water activity.
The sorption isotherm can also be used to determine the
equilibrium moisture of a certain food with the air used for
drying, which is achieved at the end of a certain drying process
if the air RH remains constant.
In commercial situations, the drying process usually fin-
ishes far from equilibrium conditions with the drying air
because the drying rate is very low close to equilibrium.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00240-3
 Drying: Principles and Types 457

Table 1 Characteristic water activity values of raw and dried food pressure of air and its temperature (eqn [1]), and its value
products increases when temperature rises, so the water activity of air
reduces:
aw raw food aw dried food
pws ¼ eð77:345þ0:0057 T7235=T Þ =T 8:2 [1]
Raw fruit and vegetables Dried fruits and vegetables
Apples: 0.99 aw within the 0.6–0.7 range where pws ¼ water vapor saturation pressure (Pa), e ¼ the con-
Bananas: 0.98 stant 2 718, and T ¼ dry bulb temperature of moist air (K).
Figs: 0.97 Nonetheless, heating air contributes not only to reduce its
Grapes: 0.98 water activity but also, consequently, to increase the gradient
Oranges: 0.99
between food and air. Air heating implies an increase in the kg
Carrots: 0.99
Tomatoes: 099
of water that each kg of dry air can take from food. Finally, an
Fresh meat: 0.99 Dry-cured hama: 0.87–0.92 increase in transport coefficients occurs because they are
Salamia: 0.82 temperature-dependent, usually following the Arrhenius equa-
Beef jerkya: 0.80 tion pattern.
Fresh fish: 0.99 Dry-salted coda: 0.65–0.75 Apart from using air heating to reduce water activity, the
Milk: 0.99 Milk powder: 0.20 direct removal of water from air can be used if food products
Aged cheesea: 0.87–0.89 are sensitive to high temperatures. In this case, water removal
Coffee: 0.99 Instant coffee: 0.20 can be done by passing air through hygroscopic media or by
a
The final aw of these products is due not only to drying but also to the combined effect
water condensation at temperatures below the dew point of air,
with salt. followed by heating.
Understanding air thermodynamics is the most important
in drying processes.

Water Transport Through Drying Processes

Moisture
g w/g d.m. As previously highlighted, drying occurs if the water activity of
food is higher than that of air and, more specifically, if the
water activity on the food surface is higher than the surround-
ing air. Thus, a typical drying process implies transporting the
water inside food towards its surface, evaporating air on the
0 aw 1
food surface, and transporting water vapor from the food
Figure 1 Typical sorption isotherm (w, water; d.m., dry matter). surface to the bulk of the drying air:

• Transporting water inside food: transport of water inside

When drying moist products begins, the decreased water
activity in food is due mainly to the concentration of the solutes
present in the food liquid phase. A time arrives when the liquid
phase disappears and the interaction of water molecules with
the solid compounds (insoluble or precipitated) of food occurs,
and this plays a key role in the water activity of food.
Water Activity of Air
The water activity of air is equal to its RH divided by 100. On
most occasions, the water activity of air is lower than the water
activity of raw material used as food, which means that a
natural drying process occurs, usually when food is stored
without protection. Nevertheless, the reduction of air RH is
usually desirable to increase the difference of aw of food and air
to enhance the drying process.
A number of actions can be performed to modify relative air
humidity, of which the most common is to increase air tem-
perature. This is why most drying techniques imply heating air
by different means. Air RH is the water vapor pressure of air at a
certain pressure divided by the saturated vapor pressure of air
at the same pressure, at the system’s temperature multiplied by
100. There is a direct relationship between saturated vapor
food can occur in the liquid state (by diffusion, capillary
forces, etc.) or as vapor.
– The movement of water in the liquid state usually occurs
in products with a free liquid phase. This is a fast process
that implies food shrinkage equal to the volume of water
lost from the food product.
– The movement of water in the vapor state during drying
usually occurs when food shrinkage is lower than the
volume of water lost during drying. This implies that
voids with a gas phase inside food appear. Thus, water
needs to be in the gas state to leave food. A consequence of
this type of movement is that the drying rate is very low.
• Transporting water inside drying air: transporting water
inside air usually occurs by convection, since drying air is
normally at a rate in the dryer that ensures convection. On
rare occasions, water vapor moves by diffusion and is usu-
ally associated with storage conditions more than drying
conditions.
In a real process, the transported water inside food is equal to
the transported water inside drying air. In most drying pro-
cesses, the limiting factor defining the drying rate is the trans-
port inside the food itself, because the food structure is more
limiting to water transport than air. This implies that the drying
rate decreases with time, since reduced moisture inside food
458 Drying: Principles and Types
Food moisture
Drying rate /
temperature
IP CRP
Figure 2 Typical drying curve (IP, initial period; CRP, constant rate period; FRP, falling rate period).
implies a lower transport coefficient (higher viscosity, higher
shrinkage, smaller quantity of liquid phase, lower water activ-
ity, etc.). This period is called the falling rate period (FRP). The
typical shape of this period is shown in Figure 2.
In products with a large amount of water, which can be
easily evaporated, a constant rate period (CRP) can be
observed at the beginning of drying. This is typical in the
drying processes of liquid food, such as coffee or milk.
Finally, a very short drying period can be observed at the
very beginning of the drying process, with an increasing drying
rate (initial period) due to both the initial increase in temper-
ature on the food surface and the mobilization of the water in
the food product.
Figure 2 shows what can be considered a whole drying rate
curve with the three typical periods.
Product and Process Variables Affecting Drying
In accordance with previous explanations, the mass transfer rate
in drying processes is dependent on water activity gradients,
which is the driving force of the process, and the mass transfer
coefficients, which are dependent on the properties of the air or
product involved in the drying process (eqn [2]):
Vi ¼ Ki Aðaw1  aw2 Þ=Dx
where Vi ¼ mass transfer rate in the i phase (food, interphase,
or air), Ki ¼ mass transfer coefficient in the i phase, A ¼ mass
transfer area, aw1 ¼ water activity of point 1, aw2 ¼ water activity
of point 2, and Dx ¼ distance.
The mass transfer rate equation can be applied to the trans-
port of the water inside food (Vf), in the interphase of food
with air (Vint), and in bulk air (Va).
In a drying process, it can be considered that the following
equality Vf ¼ Vint ¼ Va is fulfilled. The only exception to be
considered is when case hardening occurs on the food surface
because transporting the water in the food interior can be faster
than on the food surface, with consequent moisture accumu-
lation below the crust.
Thus, product and process variables are those that influence
the value of the mass transfer coefficients (K), mass transfer
area (A), distance (Dx), and water activity gradients (aw1aw2):
Food
temperature
FRP
food moisture
• Product variables:
– Type of surface: Most food that is dried is non-
homogeneous (exceptions occur in slurries, liquid, or
minced food). It is usual to find a surface that is resistant
to water transfer, such as skin in tomatoes and grapes or
skin and fat layers with hams. Sometimes, surface treat-
ments are performed on food to enhance water transfer
through the food surface, for example, treatment with
acids, skin and fat perforation, or skin removal.
– Internal food structure: The internal structure of foods has a
direct influence on the transport coefficient of the water
inside food. For instance, the presence of an intact cellular
structure or internal fat layers represents barriers to water
transport. Sometimes, treatment of food is possible to
reduce the presence of internal barriers, such as freezing
and thawing of cellular structures or milling, thereby lead-
ing to a product with a higher mass transfer coefficient.
– Food shape and size: The mass transfer process inside
food is limited by the maximum distance that water must
cross to leave food (Dxmax) – the radius in a sphere or
cylinder and half-thickness in a layer dried through both
sides. If possible, the shape and size of food should be
modified to reduce the aforementioned maximum dis-
tance. The variable-specific surface (surface/volume) is
related with the total time needed to dry a product
[2]
since the water contained in a certain volume leaves
through the product surface; thus, the drying process is
faster when the specific surface is higher. The higher the A
value for the same sample volume, the higher the V
value.
– Temperature: Usually, the transfer coefficient of a prod-
uct is exponentially temperature-dependent. This means
that an increase in the temperature of the food product
results in an increased mass transfer coefficient. There are
some exceptions, mainly due to discontinuities in prod-
uct structure as a result of temperature. This happens in
meat products containing fat pieces. If temperature rises,
fat can melt and an additional barrier inside food
appears, which results in a slower drying process.
• Air variables:
– Temperature: As mentioned earlier, the mass transfer
coefficient (K) is dependent on temperature, which is

higher for rising temperatures. Thus, an increase in air
temperature implies a higher mass transfer rate in air.
– Air velocity: The mass transfer coefficient depends on air
velocity since a rise in this value implies increased turbu-
lence. Nevertheless, a time arrives when the water trans-
fer rate does not increase at faster air velocities, because
the limiting transfer rate of the process becomes the
water transfer through the food interior.
– RH: As previously explained, the water activity of air
equals its RH divided by 100. For the same aw of food,
lowered RH implies that the aw gradient increases, with
the consequent rise in the water transfer rate. Neverthe-
less, case hardening can occur at a low RH in air due to
excess food surface drying, which can slow the drying
process.
• Type of drying: Apart from the product and air variables,
there are other processing conditions that strongly influ-
ence the result of the drying process, such as type of contact
of air and food, heating procedures, pressure, and
movement. These processing conditions are closely related
with drier design and are also related with the physico-
chemical properties, size, and shape of the food and/or
with other food characteristics. This point will be explained
in more detail in section ‘Drying Equipment and
Techniques (Types of Drying).’
Temperature and Drying
In most drying processes, air is heated by solar energy or by any
other source of energy, such as electric resistance, or mainly
through the combustion of gas, oil, or food industry by-
products. As mentioned earlier, the rise in temperature results
in lower RH of air (reduced aw), an increase in the maximum
capacity of air to take water vapor per kg of dry air, and higher
mass transfer coefficients.
Nevertheless, temperature has a direct influence on the qual-
ity of dried food and is, therefore, a very important variable.
– Food temperature during drying: When food comes into
contact with hot air, there is a tendency to equalize its
temperature. Nevertheless, if food is very moist and the
water inside food is transferred very quickly, food remains
in the CRP, and the food temperature tends to equal the
adiabatic saturation temperature, which, usually, is signifi-
cantly lower than air temperature. This is because the energy
transported from air to the food surface, due to temperature
gradients, is used completely for changing its state from
liquid to gas, and it returns to air as latent heat in water
vapor.
When the food surface becomes dry and food is unable to
transport as much water as drying air demands, it is known
as the FRP, and the temperature on the food surface begins to
rise to air temperature.
Temperature affects food properties in some ways:
– Vitamins and other nutritious compounds can be affected by
high temperatures.
– Food can be cooked at a high temperature (this can happen
with fruit and vegetables and meat and fish products).
Drying: Principles and Types 459
– Microorganisms can grow if food is maintained at high aw
values for long periods of time at temperatures within the
3–40  C range (e.g., in dry-cured ham production).
– Temperature can affect the Maillard reaction, sugar carameli-
zation, etc.
These are some examples of changes that can occur in food
because of air temperature, and a detailed analysis of the effect
of drying temperature on the type of food and the processing
conditions utilized is recommended.
Drying Equipment and Techniques (Types of Drying)
There are many types of driers, which are usually optimized for
each product type. The following is a list of the characteristics
of dryers to be considered for design purposes:
– Air contact with food: Air can flow in parallel or perpendic-
ular to food through the bed of the product to be dried.
– Stirring food: Food can be static or stirred throughout dry-
ing. Stirring the product implies a faster, more homoge-
neous drying process. Stirring the product can be done
mechanically or by means of the drying air.
– Air rate: The air rate is an influencing parameter in the drying
process. Nevertheless, using a very high air rate can lead to
stirring of products (fluid bed dryers) or even to product
transport while drying (pneumatic drying).
– Heating system: The most common procedure is heating air
before it enters the drying chamber by means of a heat
exchanger. There are other alternatives, such as intermittent
heating inside the drying chamber by means of a heat
exchanger, radiation heat, and use of microwaves to heat
the food product.
– Use of solar energy to heat air in drying processes: solar
dryers can range from very simple (solar drying through
direct exposure to the sun) to very sophisticated systems,
where the only difference with other dryers is that solar
energy is used, which can accumulate in thermal fluids and
be used in a heat exchanger with or without being combined
with other sources of energy.
– Pressure: Although the drying processes imply working at
atmospheric pressure, the use of a vacuum pressure to
improve the drying kinetics, by reducing heat damage, is
an alternative in dryer designs.
– Air recirculation: Partial air recirculation is a usual practice
to increase the dryer’s energy yield due to the increased
amount of water removed per kg of dry recirculated air.
– RH control: In most dryers, no control of RH exists. Never-
theless, controlling RH is an option, which is used mainly
when drying at low temperatures.
There are many possible combinations of all the afore-
mentioned parameters, and consequently, many different
types of dryers exist. Nevertheless, the list in the succeeding
text describes the most widely used dryers in the food industry:
– Spray dyer: It is a dryer for liquids. Liquid is sprayed at the
top of a cylinder, and droplets are dried on their way to the
bottom of the dryer, which is conical in shape.
– Drum dryer: It consists of one or two cylinders that are
heated in their interior. The food to be dried (mainly viscous
460 Drying: Principles and Types
products, such as potato puree or hydrolyzed baby food) is
dosed onto the drum surface and dried by contact with the
hot drum surface. The sheets of drum-dried product are
removed from the cylinder surface and milled.
– Drying chambers: It is for small production batches or very
long drying processes. Discontinuous drying is possible
using drying chambers. Food products can be arranged on
trays (e.g., apricot halves) or individually hung (e.g., dry-
cured ham).
– Belt dryer: The products to be dried are continuously trans-
ported inside the drying chamber placed on a conveying
belt. This can be used with many types of products, such as
breakfast cereals covered later with honey and washed fruits
and vegetables.
– Rotary drier: Food comes into contact with heated air on its
way through a cylinder that rotates to facilitate the move-
ment of food and to facilitate it coming into contact with the
air circulating inside the cylinder. It can be used with prod-
ucts that are hard enough to resist impacts inside the cylin-
der or when there is no need to maintain the food shape
and/or structure, such as seeds before solvent oil extraction.
– Fluid bed dryer: Food floats in the air that circulates upward.
When food is dry enough, due to its lowered density, it is
transported by air to the collecting unit.
– Pneumatic dryer: Food is transported and dried simulta-
neously by a fast air current. It can be employed for foods
such as sugar and flour.
– Column dryer: These are vertical dryers used with free-
flowing products, such as cereals. The products to be dried
are placed into the column at the bottom and descend by
gravity, while they are well mixed with hot air by various
procedures, such as perforated cylinders.
– Superheated steam dryers: These dryers use superheated
steam instead of air. This offers the potential for heat
recovery and also enables operations with alternative heat
sources while providing enhanced safety and improved envi-
ronmental benefits.
– Paddle dryer: It is a dryer where heat is transferred by contact
with heated paddles that are also used to stir food. It can
be used with powdered, granulated, or pasty products
(e.g., milk powder).
– Other types of drying: flash drying, freeze drying, and vac-
uum drying.
When talking about drying food, we commonly refer to the
operation that implies loss of water due to gradients of aw. This
implies water passing from liquid to vapor by evaporation at
temperatures below the boiling point. Nevertheless, some dry-
ing processes are based on the change of state of water due to
boiling. The most well-known drying techniques based on this
are the following:
– Flash drying: Food is heated at temperatures above the boil-
ing point at atmospheric pressure. Water is maintained in
the liquid state because high pressure is used. When the
system is decompressed, water from food immediately evap-
orates, which leads to a porous, dry product, such as inflated
rice for breakfast cereals.
– Vacuum drying: This process is based on lowering the water
boiling temperature through use of low pressure. This results
in the temperature of food being lowered in the process,
thereby resulting in less thermal damage. One example is
dried sausages. In this process, air scarcely moves due to its
removal as a vacuum is used.
– Freeze drying: This is a particular vacuum drying situation
based on water sublimation from frozen food by applying
vacuum conditions. The drying temperature is very low, and
some original food properties are well maintained, such as
freeze-dried coffee extracts.
Changes in Product Properties Due to Drying
Drying implies not only reducing the aw of food but also
altering the characteristics of food products. As regards texture,
hardness usually increases because the dry matter concentra-
tion increases. If moisture is low enough, the product can pass
from a glassy to a vitreous state at room temperature, which
implies a dramatic change in textural properties. If very fast
drying is employed, case hardening can occur due to the very
strong dehydration of the food surface, which leads to a hard
and very dry surface and is an important barrier for water
transport.
Drying also affects food structure due to shrinkage, as well
as other properties such as color and flavor. Sometimes, these
changes are positive, but may be negative in others, which
depends on the product and the objective of the drying
process.
See also: Drying: Effect on Nutrients, Composition and Health; Drying:
Physical and Structural Changes; Freeze Drying: Effects on Sensory
and Nutritional Properties; Freeze-drying: The Basic Process;
Preservation of Foods.
Further Reading
Baker CGJ (1997) Industrial drying of foods. London: Blackie Academic & Professional.
Brennand CP (1994) Home drying of food. Logan, UT: Utah State University
Cooperative Extension. http://extension.usu.edu/files/publications/publication/FN-
330.pdf.
Cazier JB and Gekas V (2001) Water activity and its prediction: a review. International
Journal of Food Properties 4(1): 35–43.
Chen XD and Mujumdar AS (2008) Drying technologies in food processing. West
Sussex, UK: Wiley-Blackwell, 352 pp.
Crank J (1975) The mathematics of diffusion. Ely House, London: Oxford University
Press.
Doe P, Sikorski Z, Haard N, Olley J, and Pan BS (1998) Basic principles. In: Doe PE
(ed.) Fish drying and smoking. Production and quality, pp. 13–45. Lancaster, PA:
Technomic Publishing Co., Inc.
Gou P, Comaposada J, Arnau J, and Pakowski Z (2005) On-line determination of water
activity at the lean surface of meat products during drying and its relationship with
the crusting development. Drying Technology 23(8): 1641–1652.
Heldman DR and Hartel RW (1997) Principles of food processing. New York: Chapman
and Hall.
IFT/FDA (2003) Factors that influence microbial growth. Comprehensive Reviews in
Food Science and Food Safety 2: 21–32.
Leistner L (1995) Principles and applications of hurdle technology. In: Gould GW (ed.)
New methods of food preservation, pp. 1–21. London: Blackie Academic &
Professional.
Merts, I., Lovatt, S. J. and Lawson, C.R. (1998). Diffusivity of moisture in whole muscle
meat measured by a drying curve method. In: IIR Proceedings Series Refrigeration
Science and Technology, Sofia, Bulgaria, pp. 473–479.
Mujumdar AS (2014) Handbook of industrial drying, 4th ed. Boca Raton, FL: CRC
Press, 1348 pp.
 Drying: Principles and Types 461

Rahman MS (1999) Handbook of food preservation. Boca Raton, FL: CRC Press,
809 pp.
Toldrá F (2002) Dry-cured meat products. Trumbull, CT: Food & Nutrition Press,
pp. 27–62.
Tsotsas E and Mujumdar AS (2014) Modern drying technology, 5 volume set.
Chichester: Wiley-Blackwell, 1984 pp.
Relevant Websites
http://www.fao.org/waicent/faoinfo/food-safety-quality/cd_hygiene/cnt/cnt_en/sec_3/
docs_3.2/Design%20mech%20dryers.pdf (29/08/2014) – FAO.
http://www.klmtechgroup.com/PDF/ess/
PROJECT_STANDARDS_AND_SPECIFICATIONS_dryers_systems_Rev01.pdf –
KLM Technology group.
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E
Eating Disorders
CC Schreyer, Johns Hopkins School of Medicine, Baltimore, MD, USA
S Makhzoumi, University of Maryland, Baltimore, MD, USA
JW Coughlin and AS Guarda, Johns Hopkins School of Medicine, Baltimore, MD, USA
ã 2016 Elsevier Ltd. All rights reserved.
Eating Disorders Are Motivated Behavioral Disorders
Eating disorders are motivated behavioral disorders and are
characterized by (i) increasingly driven and ritualized behav-
iors that include dieting, purging, excessive exercise, and
intermittent binge eating, (ii) narrowing of the behavioral
repertoire with increased time spent on disordered eating
behaviors, (iii) excessive preoccupation with food, (iv) pro-
gressive functional impairment, and (v) ambivalence toward,
or frank avoidance of, treatment. Individuals diagnosed with
eating disorders typically exhibit body dissatisfaction and a
tendency to base self-evaluation on weight and shape.
As behaviors such as dieting or binge eating are repeated,
learning and the conditioning of environmental cues and feeling
states associated with disordered eating result in increasingly
automatic and inflexible behavioral response patterns. Over
time, repetition of these abnormal behavioral patterns deregulates
homeostatic hunger and satiety signaling between the gut and
the brain altering levels of neuropeptides and hormones
such as cholecystokinin, pancreatic polypeptide, leptin, ghrelin,
glucagon-like polypeptide-1, and opioid peptides. Some of these
secondary changes are increasingly believed to further contribute
to the maintenance and driven quality of disordered eating. In
Anorexia Nervosa (AN), for example, starvation results in both
physiological and psychological consequences that perpetuate the
anorectic state. The landmark 1940s Ancel Keys’ Minnesota Star-
vation Experiment focussed on the effects of starvation in 36 male
conscientious objectors. These men developed many of the symp-
toms and behaviors typical of AN including depressed mood,
apathy, preoccupation with food, eating rituals, increased obses-
sionality, and an overvalued concern with ascetic goals. Some
exhibited increased drive to exercise and many developed binge
eating behavior during the refeeding phase of the experiment.
Although unable to mimic all aspects of eating disorders,
experimental animal models including the activity-based
model of AN and rodent models of Bulimia Nervosa (BN)
that alternate intermittent food restriction with access to palat-
able food have confirmed that physiological consequences of
sustained or intermittent food deprivation can alter hunger and
satiety signaling, influencing food reward and eating behavior.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00243-9
Diagnostic Criteria of Eating Disorders
The Diagnostic and Statistical Manual of Mental Disorders, 5th
Edition (DSM-V), provides the specific criteria to identify
symptoms and diagnose individuals with an eating disorder.
Anorexia Nervosa
The primary features of AN include a significantly low body
weight driven by caloric restriction and/or purging behaviors
coupled with an intense fear of fatness or weight gain and the
presence of body image disturbance such that the individual
either places undue emphasis on the importance of their
weight or shape or minimizes that his or her weight is danger-
ously low. A ‘significantly low body weight’ is defined as less
than what would be expected given the individual’s age, sex,
health status, and developmental level. Both the World Health
Organization (WHO) and the Center for Disease Control
(CDC) define the lower level of ‘normal’ weight in adults as a
body mass index (BMI) of 18.5 kg m 2, and suggested
BMI ranges for assigning a level of severity to AN in the
DSM-V are as follows: mild (BMI > 17 kg m 2), moderate
(16–16.99 kg m 2), severe (15–15.99 kg m 2), and extreme
(BMI < 15 kg m 2). Fear of fatness need not be verbally
acknowledged if the patient is of significantly low weight and
consistently engages in behavior that prevents weight gain.
Patients may recognize that they are too thin but remain overly
concerned with the size of specific body parts, such as the
stomach, hips, or thighs. Frequent or obsessional body check-
ing and weighing are often used to evaluate weight and shape.
Individuals with AN are divided into two subtypes based on
an assessment of disordered eating behaviors in the past 3
months. Patients who engage solely in restricting behaviors
and excessive exercise, without regular (weekly) binge eating
or purging behaviors, are diagnosed with AN, restricting
subtype, whereas those whose restricting behaviors are accom-
panied by at least weekly binge eating and/or purging behav-
iors are diagnosed with the binge eating/purging subtype of
AN. The 12-month prevalence rate among females is low
(0.4%) and approximately 10% of cases are male. Of note,
463
464 Eating Disorders
AN has the highest standardized mortality rate of any psychi-
atric condition with fivefold risk of any cause of death and 18-
fold risk of death by suicide compared to an age-matched
female population.
Bulimia Nervosa
BN is characterized by recurrent episodes of binge eating and
inappropriate compensatory behaviors. A binge is defined as
eating a larger amount of food than typically normal in a
discrete period of time, associated with a sense of loss of
control over eating. Lack of control is defined as the inability
to refrain from eating or to stop eating once an individual has
begun. Compensatory behaviors are employed to prevent
weight gain and include self-induced vomiting; misuse of lax-
atives, diet pills, or diuretics, restricting, and excessive exercise.
Both the binge eating episodes and compensatory behaviors
must occur on average at least once a week for 3 months. As in
AN, excessive emphasis on body shape and weight as a mea-
sure of self-evaluation and self-esteem is a core criterion of BN.
Individuals with BN are typically of normal weight or over-
weight; if significantly underweight, the diagnosis of AN-purging
subtype trumps the diagnosis of BN. Within BN, the level of
severity depends on the frequency with which individuals
engage in compensatory behaviors and ranges between mild
(1–3 episodes per week), moderate (4–7 episodes per week),
severe (8–13 episodes per week), and extreme (14 or more
episodes per week). BN is slightly more common than AN,
with 12-month prevalence rates of 1–5% in females. Similar to
AN, male patients represent approximately 10% of cases.
Binge Eating Disorder
Binge Eating Disorder (BED), defined as recurrent consumption
of large amounts of food in a fairly short period of time accom-
panied by feelings of loss of control (without the compensatory
behaviors seen in BN), has recently been changed from a provi-
sional diagnosis in need of further research in the Diagnostic and
Statistical Manual of Mental Disorders, 4th Edition (DSM-IV), to a
unique psychiatric condition in the DSM-V. Binge episodes are
associated with eating until uncomfortably full, eating rapidly,
eating alone, eating in the absence of hunger, and/or feeling
disgusted with oneself after binge eating. The DSM-V requires
that at least three of these five features accompany binge epi-
sodes on average once a week or more for 3 months to make a
diagnosis. Binge episodes differ from overeating in that they are
often secretive, associated with shame, and occur in the context
of negative affect and cognitions.
BED is associated with decreased quality of life and
increased lifetime co-occurrence of medical (e.g., irritable
bowel syndrome, fibromyalgia, type 2 diabetes, and obesity)
and psychiatric disorders, including major depressive disorder,
bipolar disorder, anxiety disorders, substance use disorders,
and body dysmorphic disorder. Although not all individuals
with BED are overweight or obese, approximately 30% of those
seeking weight loss treatment meet criteria for BED in compar-
ison to 2–5% prevalence rates in community samples. BED is
particularly prevalent among those pursuing bariatric surgery,
ranging from 10% to 50%, and preoperative BED may
indirectly predict postsurgical weight loss mediated by postop-
erative eating behavior.
Other Eating Disorders
Avoidant/Restrictive Food Intake Disorder (ARFID) is an eating
or feeding disturbance in which individuals avoid or restrict
their oral intake of food in the absence of an underlying
medical condition. This disturbance results in one or more of
the following: significant weight loss, or in children, a failure to
meet expected weight gain, nutritional deficiency, dependence
on oral nutritional supplements or enteral feeding, or a signif-
icant impairment in psychosocial functioning. The avoidance
or restriction of food in ARFID is not in response to scarcity of
food or cultural and developmental norms such as picky eating
in preschool-aged children. Typically, the avoidance/restric-
tion results from a lack of interest in eating or food, or an
avoidance of eating due to sensory characteristics of food,
such as texture or smell. Unlike other eating disorders, individ-
uals with ARFID do not experience a disturbance in the per-
ception of body shape or weight.
In addition to these disorders, DSM-V distinguishes a fifth
broadly defined category of Other Specified Feeding or Eating
Disorders. The clinical presentations of these syndromes may
be behaviorally similar to AN, BN, or BED and cause signifi-
cant impairment and distress in the individual’s life. However,
the symptoms do not meet full diagnostic criteria for one of the
earlier mentioned conditions, or there is insufficient informa-
tion to make a diagnosis. Examples of these disorders include
atypical AN, subclinical BN or BED, purging disorder, and
night eating syndrome. In atypical AN, all the criteria for AN
are met; however, despite significant weight loss, the
individual’s weight is within or above the normal range. Sub-
clinical BN or BED may be diagnosed when the individual
engages in binge eating or inappropriate compensatory behav-
iors less than once a week and/or for less than 3 months.
Purging disorder is characterized by recurrent purging behav-
iors with the goal of preventing weight gain in the absence of
binge eating. Night eating syndrome is defined as recurrent
episodes of nocturnal eating in which an individual consumes
an excessive amount of food after an evening meal or will
eat after awakening from sleep. These episodes of eating
are not in response to external influences or related to a med-
ical or psychiatric disorder. Finally, unspecified Feeding or
Eating Disorder may be diagnosed if a patient presents with
symptoms of disordered eating that cause significant distress or
impairment in functioning and is most commonly used when
there is insufficient information to determine a diagnosis.
Disordered Eating Behaviors
Certain disordered eating behaviors are observed cross diag-
nostically. Further, these behaviors may occur in nonclinical
samples and potentially signify the need for early intervention.
Though restricting behaviors are thought to be primarily asso-
ciated with AN, they are seen in other eating disorders as well,
especially BN. Individuals who engage in restricting behaviors
attempt to limit their dietary intake with the goal of weight loss
or to prevent weight gain. Common restricting behaviors

include reducing the amount and frequency of meals and food
portions, avoiding calorie-dense foods, and narrowing one’s
food repertoire such that consumption is limited to specific
low-calorie ‘safe’ food items. In addition, individuals will often
engage in prolonged periods of fasting. For example, after a
binge episode, individuals may forgo food for an extended
period of time to prevent weight gain.
Binge eating and purging behaviors also occur trans-diag-
nostically. Binge eating episodes are defined as consuming an
unusually large amount of food (about two meals worth) in a
discrete period of time coupled with a sense of loss of control
over eating. When a sense of loss of control is associated with
eating small amounts of food, this is termed a subjective binge
episode. Subjective binge episodes are more frequently
reported by patients with binge eating/purging type of AN
than by those with BN. Common purging behaviors include
self-induced vomiting and laxative, enema, or diuretic abuse.
Uncommon but important additional purging behaviors
employed in the service of weight loss by individuals with
eating disorders can include insulin omission in type I dia-
betics and wasting expressed breast milk in postpartum
mothers. The frequency of purging behavior use varies, with
some individuals purging exclusively after binge eating epi-
sodes, whereas others may purge after consuming small
amounts of food or after every meal.
Additional behaviors are observed across all eating disorder
diagnoses. These include chewing and spitting and grazing.
Chewing and spitting out food before swallowing is common,
often associated with a sense of lack of control over eating, and
can involve large amounts of food and feelings of shame/guilt
afterward. This behavior may be utilized as a way to taste
‘forbidden foods’ without ingesting calories. In other cases,
chewing and spitting is best conceptualized as an alternative
to bingeing and purging and is perceived as less medically
risky, with lower likelihood of electrolyte abnormalities or
other medical complications than vomiting. Grazing is the
unplanned, repetitive ingestion of small amounts of food
that may occur in a compulsive manner, such as repeatedly
eating from a container of food when walking past it. Although
grazing is frequently observed in nonclinical and eating disor-
der samples, when accompanied by a sense of loss of control
over eating, it is associated with increased psychopathology.
Grazing behavior has received significant attention in the bar-
iatric surgery literature, particularly since grazing postsurgically
is associated with suboptimal weight loss and/or weight regain,
and efforts are currently under way to determine the best way
to define grazing both clinically and for research purposes.
Excessive exercise is a frequently observed behavior employed
in the service of weight loss among individuals diagnosed with
AN or BN and is commonly defined by dysfunction in the quality
and quantity of exercise. Unlike normal exercise, excessive exer-
cise manifests as a strict and compulsive adherence to exercise
regimes, preoccupation with exercise, feelings of guilt if exercise is
not completed, and exercising despite illness, injury, or physical
complications. These behaviors are utilized to prevent weight
gain, influence one’s shape and/or weight, or reduce feelings of
distress related to food consumption. In addition, individuals
may engage in increased frequency, duration, and intensity of
exercise. Excessive exercise is associated with increased psycho-
logical distress, psychopathology, and risk of relapse.
Eating Disorders 465
Laboratory Studies of Food Consumption/
Macronutrient Selection
Laboratory studies of eating behavior have consistently shown
that individuals with eating disorders will engage in disordered
eating under controlled laboratory conditions. Compared to
healthy controls, patients with AN select fewer food items, eat
fewer calorie-dense foods, and have a lower total caloric intake
in laboratory test meals in which they are presented with a
large variety of foods of different calorie densities. Macronutri-
ent selection primarily consists of carbohydrates ( 55%) and
protein ( 30%), with limited caloric intake from fat (<20%).
Following treatment, weight-restored patients with AN show
increased caloric intake in test meals but continue to eat fewer
calorie-dense foods and have a more limited repertoire of
food items compared to healthy controls. Further, studies
of implicit and explicit liking (pleasure associated with food)
and wanting (desire for food) show that current AN and
recently weight-restored patients with AN report decreased
explicit and implicit wanting of higher-calorie food items com-
pared to healthy controls. Of concern, avoidance of calorie-
dense foods and limited food choices are associated with
increased risk of relapse in this population.
Laboratory studies of binge eating typically offer partici-
pants either single-item or multi-item food arrays and instruct
participants to ‘eat a normal meal’ (meal paradigm) or ‘eat as
much as you would like’ (binge paradigm). Individuals with
BN and obese individuals with BED consume significantly
more calories and significantly more calories from fat than
healthy controls using normal meal and binge eating test par-
adigms. Patients with BN also eat more rapidly than controls
during laboratory binge eating episodes and report higher
caloric intake for binge eating episodes than individuals with
BED. Binge size is positively correlated with BMI in patients
with BED, but this has not been observed in BN samples.
Nutritional and Medical Complications of Eating
Disorders
Complications of eating disorders are best thought of as conse-
quences of starvation, purging behaviors, or both. Those indi-
viduals most at risk are therefore those with the binge/purge
subtype of AN, who are both underweight and engage in purg-
ing behaviors. Medical complications can affect all body sys-
tems and several are severe and potentially life-threatening.
Cardiovascular complications of starvation include severe
bradycardia and orthostatic hypotension, which can result
from fluid restriction, vagal instability, or decreased venous
return attributable to atrophy of peripheral muscles. A frequent
cause of orthostatic hypotension in individuals who purge is
dehydration resulting from vomiting or laxative or diuretic
abuse. In very low weight patients, starvation-induced atrophic
cardiomyopathy can lead to mitral valve prolapse as the mitral
valve becomes too big for the heart. Cardiac arrhythmias are
often the result of electrolyte imbalances but can also be due to
abuse of caffeine or ephedra in the service of weight loss.
Of all metabolic complications, the most common and the
ones of highest concern are hypokalemia, because of its
466 Eating Disorders
association with lethal heart rhythms, and severe hypoglycemia.
Acid–base imbalances include metabolic alkalosis in individuals
who vomit and metabolic acidosis in laxative abusers. Less fre-
quently, starved patients may develop hypomagnesemia, hypo-
calcaemia, or hyponatremia. The latter can be due to sodium
depletion in starvation, diuretic abuse, or water intoxication.
Excessive water ingestion is motivated by a desire to increase
satiety, make weight, or ‘eliminate toxins.’ Severe hyponatremia
or hypoglycemia can result in seizures as the presenting symp-
tom. Oral repletion of electrolytes is the safest initial route to
stabilize patients, although adjunctive judicious intravenous
repletion may be indicated in severe electrolyte deficiency.
Gastrointestinal complications are very common in eating
disorders. One study found that 98% of eating disorder inpa-
tients met criteria for a coexistent functional gastrointestinal
disorder (FGID), one-half of whom reported three or more
FGIDs. The most commonly reported of these were irritable
bowel syndrome, functional abdominal bloating, functional
constipation, functional heart burn, functional dysphagia, and
functional anorectal pain. Even in a nonclinical college student
population, severity of dieting has been associated with
increased abdominal symptoms.
State-dependent consequences of starvation, including
delayed gastric emptying and delayed gastrointestinal transit
times, are associated with early satiety and abdominal
discomfort, which impede patients’ efforts to increase their
food intake. Gastrointestinal symptoms in AN dramatically
improve with refeeding alone. Delayed gastric emptying is
also well documented in BN, and a recent study found that
gastric emptying normalized faster in restricting subtype of AN
than in those individuals with AN who also engaged in binge/
purge behaviors. The use of proton pump inhibitors or meto-
clopramide may be of some help in select cases; however,
improvement is limited without concomitant increase in nutri-
tional intake and weight gain. Gastrointestinal complications
arising from laxative abuse can include colonic dysmotility,
development of hemorrhoids, and rectal prolapse.
In individuals who purge by vomiting, enamel erosion of
the lingual surface of the teeth, parotid gland enlargement, and
reflux esophagitis are common, and there is risk for a Mallory–
Weiss tear presenting with hematemesis. Acute gastric dilata-
tion is a potentially life-threatening complication of refeeding
in which marked dilation of the stomach can result in infarc-
tion by compression of the mesenteric artery or rupture of the
stomach. It is best treated conservatively with nasogastric
decompression. Both pancreatitis and transaminitis can be
seen as consequences of starvation and both may transiently
worsen with refeeding before improving. High cholesterol is
common in AN and has been attributed to depressed
cholesterol-binding proteins or to low triiodothyronine levels
that inhibit cholesterol breakdown.
Starvation is associated with multiple reversible endocrine
abnormalities. These include hypercortisolemia, growth hor-
mone resistance, low insulin-like growth factor 1 (IGF-1), hypo-
thalamic amenorrhea, and sick euthyroid syndrome. All are
reversible with refeeding and hormone replacement therapy is
not indicated. As the typical age of onset of AN is peripubertal,
there are developmentally relevant medical complications
besides amenorrhea. Both stature and breast development can
be stunted, and catch-up growth is limited following recovery
from prolonged adolescent AN. This information may be help-
ful in motivating a reluctant teen to gain weight.
Osteopenia and osteoporosis occur rapidly in AN with up
to 50% of patients demonstrating abnormal DEXA scans
within 6 months of diagnosis. Osteoporosis is one of the
most common, serious, and irreversible complications of AN
and is thought to result from the combination of low estrogen,
high cortisol, and low IGF-1. Unlike in menopause, estrogen is
not protective of bone loss in AN, whereas refeeding clearly
improves bone density in adolescent AN and prevents contin-
ued bone loss. Most experts agree that hormone replacement
should not be routinely prescribed in AN as it results in
monthly withdrawal bleeding, which exacerbates any underly-
ing nutritional anemia and can contribute to the mistaken
belief by patients that their reproductive function is normal.
Prescription of calcium and vitamin D is recommended as well
as weight-bearing exercise following weight restoration. Data
on bisphosphonates are still lacking, and caution is warranted
in using these compounds in women of reproductive age since
they leach from bone during pregnancy and there is concern
about their teratogenic potential.
Besides nutritional anemia, at low weights, hematologic
complications of severe AN include hypocellular bone marrow
on biopsy and pancytopenia. Leukopenia in severely cachectic
patients is associated with immunocompromised status and
increased risk of opportunistic infections, and thrombocytope-
nia can cause easy bruising or bleeding.
Renal complications are not common, but can include
impaired creatinine clearance in AN binge/purge subtype rela-
tive to AN restricting type. This usually reverses with refeeding
although occasional cases with renal failure attributed to laxa-
tive abuse are well described. Renal failure is presumed to be
the result of chronic hypokalemia and chronic intravascular
volume depletion.
Brain changes in AN are most notable for the development
of atrophy, visible on MRI, which reverses at least partially with
refeeding. Patients with AN have also been found to have state-
dependent neuropsychological deficits in a number of
domains including set shifting, delayed discounting, gambling,
and attention.
In general, information on medical complications may
serve as a therapeutic tool to increase patient engagement in
treatment. Reviewing lab results and providing patients copies
of any abnormal tests or scans while supportively but clearly
stating that these are medical consequences of their behavior
may help increase motivation to change and engage patients in
behavioral treatment. Somatically preoccupied patients may be
in denial about their eating disorder and be overly concerned
about functional gastrointestinal symptoms. These patients
often avoid eating disorders specialty treatment, presenting
instead to medical providers with somatic complaints that are
consequences of their eating behavior. In such cases, the clini-
cian can maintain an agnostic stance about diagnosis while
formulating behavioral treatment as physiological retraining
aimed at reinstating normal homeostatic mechanisms of hun-
ger and satiety. This explanation may be more acceptable to
these patients than an eating disorder diagnosis. Educating
family members on how they can work with health-care pro-
viders to help contain the patient’s behavior and how this will
ultimately return control to the patient is also helpful.

Treatment of Eating Disorders
Levels of Care
Treatment options for patients diagnosed with eating disorders
include outpatient individual, family, or group psychotherapy,
partial hospitalization, and residential or inpatient treatment.
Inpatient hospitalization and partial hospitalization are
reserved for the most severe cases, and a high proportion of
these patients have very low BMI and are diagnosed with AN.
These patients may require hospitalization for medical stabili-
zation, refeeding, and monitoring cardiac irregularities and
dehydration or for co-occurring psychiatric conditions such
as depression with suicidal ideation. Patients with purging
behaviors may be admitted due to electrolyte imbalances asso-
ciated with frequent vomiting or laxative abuse especially
hypokalemia.
Generally, the first step for patients diagnosed with an
eating disorder is outpatient evaluation and treatment. While
a wide variety of therapies have been used with this popula-
tion, the most efficacious outpatient therapy modalities
include family-based therapy (FBT) for adolescent patients
with AN and cognitive behavioral therapy (CBT) for patients
diagnosed with BN and BED. When indicated, admission
should ideally be to a behavioral specialty program for the
treatment of eating disorders as these programs are capable of
helping patients interrupt unhealthy behavioral patterns
and normalize eating with the help of an expert multidisciplin-
ary team using a structured behavioral protocol and group
therapy approaches. Inpatient treatment goals include medical
stabilization, normalization of eating behaviors, weight resto-
ration in low weight patients, and relapse prevention. Length
of stay varies by patient and is influenced by a number of
factors including co-morbidity, BMI at admission, insurance
coverage, patient dropout, and rate of weight gain. For under-
weight patients with AN, a goal weight is determined by the
treatment team (generally a BMI of 20 or above for adults).
Most expert programs are able to achieve rates of weight gain of
1–2 kg per week.
Pressure from insurance companies to decrease inpatient
hospital-based costs has resulted in expansion of residential
and partial or day hospitalization programs. Partial hospital
programs may be stand-alone treatment centers or hospital-
based step-down programs integrated with an inpatient unit.
Patients in partial hospital programs typically engage in treat-
ment activities such as supervised meals and individual and
group psychotherapy and may also participate in meal-based
occupational therapy sessions such as restaurant outings, meal
preparations, and grocery shopping in order to practice newly
acquired social eating skills in real-world settings.
Nutritional Rehabilitation
Nutritional rehabilitation includes weight restoration as well
as normalization of eating behaviors. Clinically, this requires
patients to consume appropriate quantities of foods of a
variety of calorie densities without engaging in binge eating
or compensatory behaviors, such as purging or excessive exer-
cise. Though not the sole focus of treatment, weight restora-
tion is the primary goal of treatment for AN as it is associated
Eating Disorders 467
with superior outcomes including long-term weight mainte-
nance, correction of medical complications, and improve-
ment in both eating disorder psychopathology and
comorbid psychiatric symptomatology. Weight restoration is
also associated with decreased frequency of disordered eating
behaviors, reduced likelihood of readmission, and increased
rate of recovery at both short and long-term follow-up and is
often used as a marker of readiness to progress to a lower level
of treatment.
Refeeding Syndrome and Medical Monitoring
Refeeding syndrome comprises a constellation of potentially
life-threatening metabolic and clinical changes that result from
rapid oral, enteral, or parenteral feeding. These are most likely
to occur with parenteral refeeding and least likely with oral
nutritional rehabilitation. In the absence of access to an expert
behavioral program capable of restoring weight in patients
through oral refeeding alone, the use of enteral feeds may be
indicated to initiate weight restoration. Enteral feeding should
not be the initial choice however as it does not address the fear
of consuming calorie-dense foods central to the phenomenol-
ogy of AN. Furthermore, although some specialty programs
advocate use of supplementary nasogastric night feeds, the
outcomes of these programs are not superior to those of expert
behavioral programs that rely on oral nutrition alone. Finally,
parenteral feeding should be resorted to rarely if at all given the
high risk of line sepsis and refeeding complications especially
in those with AN who are severely malnourished and
immunocompromised.
The hallmark of refeeding syndrome is hypophosphatemia,
which occurs within 1–3 days of an increase in food intake.
Hypophosphatemia results from the intracellular movement of
phosphate for the formation of ATP and other anabolic
demands and places patients at risk for potentially lethal car-
diac arrhythmias. Postprandial hypoglycemia is also common
during refeeding as is the development of peripheral edema,
which can lead in the extreme to congestive heart failure, ileus,
or anasarca.
Edema is most common in individuals who abuse laxatives
or diuretics and who self-induce vomiting once they stop purg-
ing. Most edema can be managed conservatively; however,
chronic volume depletion from severe purging can result in
pseudo-Bartter’s syndrome marked by secondary hyperaldos-
teronism, salt retention, hypokalemia, and marked edema.
Judicious use of spironolactone can alleviate severe edema. In
low weight patients who do not purge, dependent edema
during refeeding can result from low albumin, fragile capil-
laries, and third spacing of fluid into the interstitial compart-
ment. Care should be taken not to rapidly replete intravascular
fluid as this can worsen edema in these cases. For patients who
abuse laxatives, these can be stopped abruptly and need not
be tapered, although compliance with laxative discontinuation
is difficult outside a structured inpatient setting. When
compliant, however, most patients do well with a bowel regi-
men of a stool softener and bulk laxative, with the possible
addition of an osmotic laxative agent as well as education and
reassurance that their bowels will normalize over a period of a
few weeks.
468 Eating Disorders
Other potential risks of refeeding include two very serious
neurological complications associated with severe AN,
Wernicke’s encephalopathy due to thiamine deficiency and
central pontine myelinolysis, which most commonly results
from overly aggressive correction of hyponatremia. Wernicke’s
encephalopathy is especially a risk in patients with comorbid
alcohol abuse. Thiamine should be repleted IV or IM in
patients who are severely malnourished (BMIs of <14) before
initiating refeeding or administering IV dextrose.
Since relief from medical symptoms is a common goal for
both patient and provider, formulating behavior change as
the path to symptom relief can make weight gain and treat-
ment more acceptable to patients and help engage them in
therapy. It is important to appreciate that medical complica-
tions can play a sustaining role in the maintenance of eating
disorders.
Psychological and Behavioral Treatment
Family-based therapy
FBT based on the ‘Maudsley model’ is generally considered to
be the most efficacious treatment for AN in adolescent patients
with less than 3 years of illness. The Maudsley model of family
therapy consists of training parents to take control of their sick
child’s eating schedule. Utilizing an agnostic stance toward the
origin of AN, parents are informed of the grave nature of their
child’s illness and are encouraged to exclusively focus on refeed-
ing their child, usually via the provision of high-calorie meals
and supplements, meal and bathroom supervision, and the
development of a system of rewards and consequences to
increase compliance with eating. Regular family therapy ses-
sions are used to support parental refeeding efforts and improve
communication skills among family members. As symptoms
begin to remit, patients gradually regain privileges based on
their ability to make appropriate food choices and maintain
their goal weight. When compared to ego-oriented individual
therapy (focussed on building autonomy and improving self-
mastery), FBT has been associated with better rates of weight
gain and an increased likelihood of resuming menstrual func-
tioning, which may lower the risk of osteoporosis. Following
achievement of recovery, FBT has low relapse rates of 10%
compared to 25–30% often reported following inpatient hos-
pitalization, though the latter group may include more severe
cases. Attrition is also much lower with only 10–15% of
patients dropping out of treatment compared to drop-out
rates of 50% or more from other treatment modalities.
Exposure and response prevention
A potentially promising yet understudied intervention in AN is
exposure with response prevention (EX/RP). EX/RP in AN
targets eating related anxiety by exposing patients to in vivo
eating challenges while preventing them from engaging in
ritualized compulsive behavior. Patients are encouraged to
practice these skills between sessions and monitor their anxiety
and compulsive rituals, including excessive exercise. Weight-
restored inpatients with AN who received 12 sessions or EX/RP
demonstrated increased caloric intake in a laboratory test meal
compared with inpatients who received a cognitive-based
intervention following weight restoration. In particular, EX/
RP may be beneficial in relapse prevention as it focuses on
achieving normative eating practices in real-world settings.
Psychological treatment for BN and BED
Fortunately for patients diagnosed with BN, outpatient treat-
ment recommendations are quite clear: CBT for BN, a form of
psychotherapy, was given the top grade from the National Insti-
tute for Health and Clinical Excellence in 2004. The goal of CBT
for BN (CBT-BN) is to disrupt the diet–binge eating cycle by
regulating the eating pattern, introducing avoided foods into the
diet, and eliminating restrictive behaviors. It further addresses
cognitive aspects of BN, including excessive concern about
weight and shape, perfectionism, rigid thinking, and low self-
esteem. It can be delivered in a variety of formats, including
individual and group therapy, as well as in a guided self-help
version. A recent treatment review reported that one-third to
one-half of all patients treated with CBT-BN endorsed abstinence
of behavioral symptoms (e.g., binge eating and purging behav-
iors) at the end of treatment and that these gains were generally
maintained 1 year post treatment. CBT-BN was shown to be
more effective than fluoxetine alone, and patients reported
increased satisfaction with CBT-BN compared to medication.
In a meta-analysis of treatment for BN, CBT-BN demonstrated
superiority to nutritional counseling, supportive therapy, and
interpersonal psychotherapy (IPT), a form of treatment that
helps individuals resolve interpersonal problems and overcome
their eating disorder indirectly by better managing negative feel-
ings associated with interpersonal conflict. Although research on
an eating disorder-specific model of IPT is in a much earlier stage
than research on CBT, IPT has yielded comparable recovery rates
to CBT in the long term for BN.
Although many individuals with BED are overweight or
obese and seek weight loss treatment, CBT for BED (CBT-BED)
is the most studied and established treatment for BED. Like CBT-
BN, CBT-BED focuses on disrupting the binge eating cycle by
promoting normative and structured eating patterns, developing
skills for managing urges/triggers to binge, and encouraging the
use of cognitive strategies to tackle problematic ways of thinking
that maintain binge eating. A meta-analysis examining psycho-
logical and pharmacological treatments for BED found that
psychotherapy and structured self-help based on CBT were supe-
rior to pharmacotherapy and weight loss treatment. Pharmaco-
therapy and weight loss treatment produced medium and
moderate effects on binge eating, respectively. Individual versus
group formats of CBT-BED do not significantly differ when
examining their effect on reductions in binge eating frequency
and recovery and relapse at follow-up. When examining pre-
dictors of outcome in CBT-BED, rapid response during early
treatment (the first four therapy sessions) is predictive of more
favorable long-term treatment outcomes. Furthermore, those
who cease binge eating during treatment tend to lose the most
weight in the long but not in the short term; therefore, it is
generally agreed that treating binge eating should be the first
phase of treatment in BED before treating weight.
Conclusion
Eating disorders are motivated behavioral disorders marked by
serious morbidity and mortality. A range of restricting, binge

eating, and purging behaviors occur across diagnoses and are
accompanied by body image disturbance in AN and BN and
often in BED, although body image disturbance is notably
absent in ARFID. Medical and nutritional complications may
be severe and life-threatening and include cardiac arrhythmias,
electrolyte imbalances, pancreatitis, transaminitis, anemia, and
osteoporosis. FGIDs are also common in this population, with
the majority of patients presenting with one or more FGID.
Discussion of medical complications with patients may serve
to increase motivation to engage in treatment. Behaviorally
oriented treatments that emphasize weight restoration or
maintenance, as well as normalization of eating behaviors,
are associated with the best outcomes. In particular, FBT for
adolescents with AN and CBT for individuals with BN and BED
are the best evidenced current treatments. Weight restoration
in AN and the resumption of normative eating practices across
diagnoses are associated with decreased rates of relapse.
See also: Adolescent Nutrition; Anemia: Causes and Prevalence;
Appetite Control in Humans: A Psychobiological Approach; Enteral
Feeding; Glucose: Metabolism and Regulation; Malnutrition:
Prevention and Management; Obesity: Causes and Prevalence; Obesity
Management; Osteoporosis; Parenteral Nutrition; Satiety.
Further Reading
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predictors of functional gastrointestinal disorders in patients with eating disorders.
Scandinavian Journal of Gastroenterology 40: 929–935.
Crow SJ, Peterson CB, Swanson SA, Raymond NC, Specker S, Eckert ED, and
Mitchell JE (2009) Increased mortality in bulimia nervosa and other eating
disorders. American Journal of Psychiatry 166: 1342–1346.
Eating Disorders 469
Eddy KT, Dorer DJ, Franko DL, Tahilani K, Thompson-Brenner, and Herzog DB (2008)
Diagnostic crossover in anorexia nervosa and bulimia nervosa: implications for
DSM-5. American Journal of Psychiatry 165: 245–250.
Eddy KT, Swanson SA, Crosby RD, Franko DL, Engel S, and Herzog DB (2010) How
should DSM-V classify eating disorder not otherwise specified (EDNOS)
presentations in women with lifetime anorexia or bulimia nervosa? Psychological
Medicine 40: 1735–1744.
Fairburn C (2013) Overcoming binge eating, 2nd ed. New York: Guilford Press.
Grilo C and Mitchell J (eds.) (2010) The treatment of eating disorders: a clinical
handbook. New York: Guildford Press.
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Lawson EA and Klibanski A (2008) Endocrine abnormalities in anorexia nervosa. Nature
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Vocks S, Tuschen-Caffier B, Pietrowsky R, Rustenbach SJ, Kersting A, and Herpetz S
(2010) Meta-analysis of the effectiveness of psychological and pharmacological
treatments for binge eating disorder. International Journal of Eating Disorders
43: 205–217.
Relevant Websites
http://www.nationaleatingdisorders.org/ – NEDA.
http://www.nimh.nih.gov/health/publications/eating-disorders-new-trifold/index.
shtml – NIMH.
 Eggs: Composition and Health Effects
ML Fernandez, University of Connecticut, Storrs, CT, USA
CJ Andersen, Fairfield University, Fairfield, CT, USA
ã 2016 Elsevier Ltd. All rights reserved.
Egg Composition
The basic components of eggs are the white and the yolk,
which differ greatly in their ratio of fluid, physical
characteristics, and nutrient content. A current existing mis-
conception is that the egg white is more healthful than the
yolk due to its high protein content. However, the egg yolk is a
much more important source of nutrients, some of which exert
protective effects that go beyond nutrition. While eggs from a
range of different birds are produced for human consumption,
this article will present data relevant to chicken eggs – the most
common and significant type of egg consumed in the human
diet. The egg white is composed of 88% water, 11% protein,
0.2% fat, and 0.8% ash, while the egg yolk is made of 48%
water, 17.5% protein, 32.5% lipids, and 2% ash. Regarding
micronutrient composition, one large egg contains 186 mg
cholesterol, 126 mg choline, 0.2 mg riboflavin, 0.5 mg
vitamin B12, 24 mg folate, 0.1 mg vitamin B6, 41 IU vitamin
D, 270 IU vitamin A, 0.5 mg vitamin E, 99 mg phosphorus,
and 0.9 mg iron. These nutrients are distributed between the
egg white and the yolk. The percent distribution of the different
nutrients is presented in Table 1.
Eggs have been reported to be a nutrient-dense food with
high-quality protein, which is present in both the egg white
and the yolk. Kwashiorkor and other types of malnutrition
have been correlated with lower egg consumption in underde-
veloped countries. Eggs can also be part of weight loss diets due
to their effect in suppressing appetite and decreasing caloric
intake during the next 24 h.
In addition to its cholesterol content, a highly controversial
topic regarding heart disease risk, the egg yolk also contains the
carotenoids lutein and zeaxanthin, which are key antioxidants
in protecting against oxidative damage, low-density lipopro-
tein (LDL) oxidation, and age-related macular degeneration
(AMD). Further, the egg yolk has other micronutrients absent
from the egg white including iron, vitamin E, and vitamin A.
Egg yolk is also a good source of choline, a nutrient that plays a
major role in normal fetal development and cognitive function
and may possibly protect against Alzheimer’s disease. There-
fore, eggs contain major nutrients that are important through-
out the life cycle, in addition to other components that have
been shown to protect against chronic diseases such as AMD,
sarcopenia, and Alzheimer’s disease. However, the perceived
concern of eggs being a source of dietary cholesterol is highly
associated with decreased consumption and dietary recom-
mendations in the United States limit and target eggs as a
source of dietary cholesterol.
Egg Sources and Production
Eggs are produced in many countries throughout the world. As
of 2006, China was the largest producer of eggs, followed by
470 Encyclopedia of Food and Health
the United States, India, and Japan. The number of cases of eggs
produced in United States in 2012 was 223.7, 58% of which
were allocated to retail, 31.9% for food preparation, 9% for
institutions, and 3.8% for export. There are a number of factors
that determine egg production, including time of the year, color
of the eggshells, and health-related concerns due to eggs’ cho-
lesterol content. However, health concerns are decreasing as the
public becomes more aware of the healthful benefits of eggs and
the lack of correlation between egg intake and heart disease. A
closely controlled breeding program results in maximum egg
production. Other factors that need to be considered are (1)
laying more eggs early although in the long run, this can result
in smaller eggs; (2) resistance to disease, and (3) favorable
genetic factors. Today, the Single White Leghorn hen dominates
the egg industry although in certain regions such as New
England, consumers prefer the brown-shelled eggs; thus, the
Rhode Island Red, New Hampshire, and Plymouth Rock hen
breeds predominate in this region.
The production of eggs can be classified into four main
categories: (1) conventional cage eggs, (2) cage-free eggs, (3)
free-range eggs, and (4) organic eggs. In the first category, hens
live in communal cage systems, which vary depending on the
size of the bird and the facility. These eggs are normally collected
by an automatic collection system. For the second category, hens
live on indoor floor operations and are housed in a barn or
poultry house, and they may be allowed to roam outdoors.
Depending on the farmer, egg collection may or may not be
automated. In the third category of free-range hens, animals are
raised outdoors or have access to the outdoors; shelter is pro-
vided during inclement weather. All of these housing systems
participate in handling and care practices. Organic eggs are
produced according to specific standards that exist in many
countries. For example, in the United States, the United States
Department of Agriculture (USDA) dictates the organic stan-
dards in relationship to methods, practices, and substances
used in the production of crops. In addition, antibiotics and
growth hormones are prohibited. All organic systems are cage-
free. It is important to note that the nutrient content of eggs is
not affected by the way the hens are raised. Other important
facts regarding production are that only 5% of eggs come from
cage-free systems and they are generally more expensive. In
addition, eggs from conventional systems have been found to
have lower bacterial levels than that those from cage-free or free-
range hens. Eggs need to be produced under the best sanitary
conditions since the moment an egg is laid; physical and chem-
ical changes begin to occur that can reduce freshness.
Patterns of Consumption
Eggs are consumed throughout the world in various forms.
Item penetration for eggs in the United States is 93%, which
http://dx.doi.org/10.1016/B978-0-12-384947-2.00246-4
 Eggs: Composition and Health Effects 471

Table 1 Percent distribution of the various components of eggs accumulation of cholesterol in vital organs including the
between the white and yolka liver, adipose tissues, and heart arteries leading to fatty liver,
inflammation, and atherosclerosis. However, it is important to
Component (% of total) White Yolk
make a clear distinction between dietary cholesterol and choles-
Weight 66 34 terol in our bodies. Since our body produces about 78% of the
Volume 40 60 cholesterol handled on a daily basis, dietary cholesterol only
Calories 15 52 comprises 22% of the total. Therefore, it is important to make a
Water 64.7 35.3 clear distinction between dietary cholesterol and circulating cho-
Protein 57 43 lesterol or cholesterol accumulated in diverse organs.
Fat 0.6 99.4 A large egg contains between 186 and 230 mg of cholesterol,
Cholesterol 0 100 which has resulted in the perception that eggs, being a high
Vitamin A 0 100
source of this dietary component, may lead to heart disease
Vitamin E 0 100
risk and other problems. In the next paragraphs, some strong
Vitamin D 0 100
Riboflavin 66.6 33.3 evidence that the cholesterol in eggs should not be a concern for
Folate 4 96 healthy populations is presented.
Choline 0.4 99.6
Selenium 40 60 Health effects
Iron 0 100 Epidemiological studies
Lutein 0 100 Epidemiological studies do not support an association
Zeaxanthin 0 100 between egg consumption and coronary heart disease (CHD)
a risk. This could partly be explained by the presence of other
These percentages do not take into account the egg shell.
Source: http://www.eggnutritioncenter.org/.
nutrients in eggs that are known to be protective against oxi-
dative stress and dyslipidemias. In addition, the response to
dietary cholesterol is highly variable. Some individuals
means that eggs can be found in almost every household. respond by having no changes in plasma cholesterol following
According to a consumer research conducted by Miller-Zell, a dietary challenge as high as three eggs per day for 12 weeks,
85% of consumers view eggs as very or somewhat healthy. This while others could have a higher response than 0.05 mmol l 1
research also concluded that individuals are more likely to for each additional 100 mg of dietary cholesterol, which is
consume eggs if they are told how to prepare nutritious dishes considered the average response. However, all those individ-
with eggs rather than providing information on the nutritional uals who are responders to a dietary cholesterol challenge have
benefits of eggs. increases in both LDL cholesterol (LDL-C) and high-density
According to the USDA’s Economic Research Service 2000 lipoprotein cholesterol (HDL-C), resulting in a maintenance of
data, it was estimated that more than 70 billion eggs were eaten the LDL-C/HDL-C ratio, a central marker of CHD risk.
in the United States, corresponding to an annual 250 eggs per The main epidemiological studies addressing the effects of
person. These data include not only eggs that were eaten indi- specific foods and nutrients on the development of chronic
vidually as main dishes but also eggs that were incorporated disease – including Framingham Heart Study, the Nurses’
into other foods as ingredients or beverages. According to Health Study, National Health and Nutrition Examination
USDA Continuing Survey of Food Intakes by Individuals Survey (NHANES), and the Lipid Research Clinics Prevalence
data, eggs are used in more than 900 different foods in the Follow-up Study – do not support a relationship between
United States. These data are supported by the fact that the dietary cholesterol/egg intake and increased risk for CHD.
primary dietary sources of eggs are processed foods or mixed Further studies conducted in Japan in which more than 9000
dishes that use eggs or egg components as ingredients. This is individuals were evaluated reported an inverse association
true regardless of whether individuals are eating at home or between egg consumption and heart disease risk. There are,
away. Findings from USDA surveys also revealed that per capita however, some epidemiological data reporting that egg intake
consumption of eggs increases from low- (<185% of poverty can adversely affect heart disease risk, but these are mostly in
line) to high-income ( 300% of poverty line) groups and that people with documented heart disease or diabetes. In sum-
men consume more eggs than women on average. Further, per mary, the preponderance of the epidemiological evidence
capita egg consumption increases with overweight status and from the past 14 years does not support a relationship between
obese weight status in adults. egg intake and risk for CHD.

Nutrients in Eggs
Cholesterol
Cholesterol, a sterol and a lipid-soluble compound, has essen-
tial functions in the body, among which are to provide perme-
ability and fluidity to cell membranes and to be the precursor
of bile acids, steroid hormones, and vitamin D. Despite these
key functions, an excess of cholesterol in the body leads to a
number of metabolic dysregulations that can result in an
Clinical studies
A number of clinical studies conducted in recent years have
demonstrated that eating two or three eggs per day for 4–12
weeks does not increase the risk for heart disease. Individuals
from these studies did not have changes in their LDL/HDL
ratio or even had improvements in this ratio; this is possibly
due to the consistent finding that egg intake increases HDL.
Further, studies have been conducted with individuals having
metabolic syndrome, a population with an increased risk for
both diabetes and heart disease. These individuals were
472 Eggs: Composition and Health Effects
prescribed a carbohydrate-restricted diet (25% of total energy)
and were randomly allocated to consume either three eggs per
day for 12 weeks or the equivalent amount of egg substitute.
After the intervention, individuals consuming the eggs did not
have increases in LDL, but they presented a consistent increase
in HDL; therefore, there was an improvement in the LDL/HDL
ratio. Another finding in all these clinical interventions is that
egg intake favors the formation of the large LDL, which is less
atherogenic, and the large HDL involved in reverse cholesterol
transport.
Cholesterol and dietary recommendations
A very interesting observation is that a number of countries
including the European Union, Japan, Korea, India, Canada,
and New Zealand, among others, do not have an upper limit
for dietary cholesterol in their guidelines; thus, their dietary
guidelines do not restrict egg consumption. In the United
States, both the USDA and the American Heart Association
consider that consuming one egg per day is appropriate for
healthy populations, which still results in a cautionary and
confusing message for most individuals. It is very clear that
most of the recently published data support the lack of effect of
whole egg consumption on heart disease. Dietary recommen-
dations should not target the elimination of a food that not
only has a substantial amount of nutrients but also provides
additional health benefits. In fact, a recent report on the man-
agement to reduce cardiovascular disease states that “There is
insufficient evidence to determine whether lowering dietary
cholesterol reduces LDL-C,” a statement that clearly supports
both recent epidemiological data and clinical studies.
Protein
Eggs are known to be one of the best sources of high-quality
protein. The 2010 USDA National Nutrient Database for Stan-
dard Reference reported that one large egg contains 6.3 g of
protein on average. Egg protein is distributed between the egg
yolk and egg white portions, with egg white containing more
protein than egg yolk. On average, a large egg containing 6.3 g
of protein in total will provide 3.6 g of protein in the egg white,
in addition to 2.7 g of protein in the egg yolk. In total, one egg
provides approximately 13% of one’s recommended daily
intake for protein.
Egg protein composition
Eggs are a rich source of essential amino acids and are partic-
ularly abundant in glutamine, leucine, and asparagine/aspartic
acid. The majority of egg protein can be found in the white
(57% of total protein) rather than in the egg yolk (43% of total
protein).
Egg whites contain a number of different proteins that exert
a range of biological and antimicrobial activities. The predom-
inant protein in egg whites is ovalbumin, comprising approx-
imately 54% of total egg white protein. Conalbumin, the
second most predominant egg white protein, binds metal
ions, whereas flavoprotein and avidin are known to bind ribo-
flavin and biotin, respectively. A number of egg white proteins
have proteinase inhibitor activity, including ovomucoid,
ovomacroglobulin, and ovoinhibitor. Ovomucin possesses
antiviral properties, whereas lysozyme exerts antimicrobial
N-acetylmuramidase enzyme activity to disrupt Gram-positive
bacteria cell walls. Together, these proteins protect eggs against
microbial contamination and spoilage.
Egg yolk additionally contains a variety of proteins that
have biological functions. The predominant proteins in egg
yolk fractions include lipovitellins, livetins, and phosvitin.
Lipovitellins represent HDL in egg yolk, whereas livetins cor-
respond to proteins found circulating in chicken blood, such as
serum albumin, a2-glycoprotein, and g-globulin. Phosvitin
contains a relatively high amount of phosphorous and strongly
binds multivalent cations such as iron and calcium. The ability
of phosvitin to bind heavy metal ions supports antioxidant
activity and protects against lipid oxidation. Egg yolks addi-
tionally contain antibodies referred to as immunoglobulin Y,
which have promising therapeutic potential in human and
veterinary medicine.
Health effects
Eggs confer a number of health effects due to their protein
content. High-quality protein from eggs supports skeletal mus-
cle synthesis and maintenance, which is particularly important
in athletes and aging populations to protect against sarcopenia.
Individuals who consume eggs for breakfast have also been
shown to have increased satiety and consume fewer calories
throughout the day. Compared to consumption of a bagel
breakfast, men who consumed eggs had lower plasma levels
of glucose, insulin, and ghrelin – an appetite-regulating hor-
mone. An additional study found that whole egg intake during
carbohydrate restriction improves insulin resistance as deter-
mined by the homeostasis model assessment of insulin resis-
tance (HOMA-IR) equation. Therefore, eggs represent a
nutrient-dense addition to low-glycemic and weight loss diets.
The antimicrobial activity of egg white proteins may also
confer anti-inflammatory properties to humans. For example,
egg white-derived hen egg lysozyme has been shown to reduce
intestinal inflammation in an experimental model of colitis.
Egg intake has also been shown to reduce proinflammatory
markers in the plasma of overweight and metabolic syndrome
subjects, including C-reactive protein, monocyte chemoattrac-
tant protein-1, and tumor necrosis factor-a. Conversely, indi-
viduals with an egg allergy display hypersensitivity and
immune reactivity to egg-derived proteins, thus initiating a
proinflammatory immune response.
Lutein and Zeaxanthin
Egg yolks are an important source of two highly bioavailable
antioxidant carotenoids, lutein and zeaxanthin. Since lutein
and zeaxanthin are embedded in the lipid matrix, these carot-
enoids have been shown to be more bioavailable in eggs when
compared to vegetable sources, despite a concentration of
200–300 mg per egg versus concentrations up to 1500 mg
per serving of spinach, broccoli, or kale. Table 2 shows the
content of lutein and zeaxanthin in different food sources
including eggs.
Biological roles of carotenoids
Egg yolks are an important source of highly bioavailable lutein
and zeaxanthin, two carotenoids that are selectively removed
from circulation by the eyes and protect against AMD and

Table 2 Lutein concentration in selected foods
Food
Kale (raw)
Kale (cooked)
Spinach (raw)
Spinach (cooked)
Collards (cooked)
Turnip greens (cooked)
Green peas (cooked)
Corn (cooked)
Broccoli (cooked)
Broccoli (raw)
Romaine lettuce (raw)
Green beans (cooked)
Papaya (raw)
Egg (cooked)
Orange (raw)
Data from U.S. Department of Agriculture, Agricultural Research Service, USDA Nutrient
Data Laboratory. http://www.ars.usda.gov/nutrientdata.
cataract formation. This selection is indicative of an important
photoprotective role. These carotenoids exert their action by
absorbing blue light and reducing its ability to reach the retina
by an estimated 40–90%, thereby reducing the generation of
reactive oxygen species, which damage photoreceptors and
retinal pigment epithelium. The concentration of these carot-
enoids in the retina can be measured by quantifying macular
pigment density in the eye.
Health effects
Epidemiological studies also indicate an inverse relationship
between intake of lutein and zeaxanthin and both cataract and
AMD. A major clinical trial, Age-Related Eye Disease Study 2
demonstrated that lutein and zeaxanthin are more protective
against AMD than other supplements including b-carotene. In
addition, studies conducted in animal models have shown potent
antioxidant properties of lutein as documented by its effects in
decreasing the susceptibility of LDL to oxidation, reducing liver
injury, protecting against early atherosclerosis development, and
reducing oxidative stress in the liver and eyes. Cell studies have
also shown that lutein or zeaxanthin supplementation protect eye
lens protein, lipid, and DNA from oxidative damage.
Studies evaluating the effects of egg intake on plasma carot-
enoids have demonstrated a substantial increase of plasma lutein
and zeaxanthin following egg consumption. Lutein and zeaxan-
thin circulate in plasma transported by LDL and HDL. Egg intake
has been demonstrated to result in the formation of larger LDL
and HDL particles. These larger particles facilitate the circulation
of lutein and zeaxanthin in plasma and support the findings of
increased plasma concentrations of these carotenoids. More
importantly, studies conducted in both hypercholesterolemic
and healthy individuals have demonstrated that macular pig-
ment density is significantly increased following egg consump-
tion indicating that eggs could be used to protect against AMD.
Choline
Choline is a water-soluble organic compound and natural
amine that has a wide variety of biological functions. The
Eggs: Composition and Health Effects 473
Food and Nutrition Board of the Institute of Medicine first
established Dietary Reference Intakes for choline in 1998
mg/serving after recognizing its role as an essential nutrient. The body is
26.5/1 cup capable of de novo choline synthesis via hepatic catabolism of
23.7/1 cup phosphatidylcholine; however, this process fails to adequately
3.7/1 cup meet daily nutrient needs.
20.4/1 cup
14.6/1 cup Choline in the diet
12.2/1 cup
Despite choline being essential in our diets, many individuals
4.1/1 cup
fail to consume adequate amounts.
1.5/1 cup
0.8/1/2 cup According to population-based data from NHANES,
1.3/1 cup 2003–04, it has been estimated that only one in ten Americans
1.1/1 cup (adults and older children) is meeting the adequate intake
0.9/1 cup guidelines for daily choline intake.
0.3/1 large Eggs are known to be one of the best sources of dietary
0.2/1 large choline, in addition to beef and chicken liver, beef steak, milk,
0.2/1 large cod, and peanut butter. According to the 2010 USDA National
Nutrient Database for Standard Reference, one large, raw, fresh
egg contains approximately 126 mg of choline. The majority of
choline is located in the yolk, which on average is estimated to
contain 116 mg of choline, with approximately 0.4 mg in the
egg white.
Biological roles of choline
Choline provides structural integrity to cell membranes, in
addition to playing roles in cell signaling, muscle functioning,
neurotransmitter synthesis, and hepatic transport of lipids to
the circulation via lipoproteins. In addition, choline is essential
for proper fetal development, particularly in regard to brain
and spinal cord development. This is attributed to the role of
choline in folate metabolism and its effects on regulating neu-
ral stem cell proliferation and apoptosis. However, this biolog-
ical role of choline has also led to its association with an
increased risk of developing certain types of cancer.
Deficiency in choline can result in fatty liver and muscle
damage, whereas choline deficiency during pregnancy
increases risk of fetal neural tube defects. Reduced intake of
choline has also been associated with increased plasma homo-
cysteine due to a weakened capacity to handle methionine,
resulting in an increased risk of developing cardiovascular
disease. Given its significant neurological roles, choline is
also thought to protect against the development of Alzheimer’s
disease and dementia while promoting integration of learning
and memory.
Health effects
A number of health effects have been attributed to diets rich in
choline, with eggs often serving as one of the primary dietary
sources of choline across the world. Diets higher in choline
have been shown to be protective against neural tube defects
and cognitive impairments. Choline ingestion from egg intake
has also been shown to undergo metabolism to trimethyla-
mine N-oxide by gut microflora, which has been associated
with an increased risk of major adverse cardiovascular events.
However, choline-rich diets are also associated with lower
levels of homocysteine, suggesting a protective effect against
cardiovascular disease. Diets high in choline, in addition to egg
intake, have also been associated with increased risk of devel-
oping lethal prostate cancer. Conversely, dietary choline and
474 Eggs: Composition and Health Effects

Not associated
• Protects against with increased
Cholesterol
malnutrition in risk for heart
children and disease
sarcopenia in Protein
adults
• Promotes
satiety

• Cognitive function
• Fetal development
• Lipid metabolism Choline Lutein/Zeaxanthin
and transport
• Muscle function
Protect against
oxidative stress and
Vitamin A age-related macular
Vitamin E Other Nutrients degeneration
Vitamin D
Iron
Selenium
Riboflavin

Nutrients in Egg Yolk ( ) and Egg White ( )

Figure 1 The nutrient content of egg yolk (cholesterol, protein, lutein/zeaxanthin, choline, vitamin A, vitamin E, vitamin D, selenium, riboflavin, and iron (*))
and egg white (protein, selenium, riboflavin, and iron ({)). The specific function of each nutrient as related to health is described.

egg intake has been shown to be protective against the devel-
opment of breast cancer.
Other Nutrients in Eggs
Eggs are also a good source of fat-soluble vitamins including
vitamin A, vitamin E, and vitamin D in addition to iron, all of
which are located in the yolk. Since they are part of the lipid
matrix of eggs, they can be more easily incorporated into
micelles and be absorbed into the intestine where they are
incorporated into chylomicrons and distributed throughout
the body. Eggs are also good sources of riboflavin and sele-
nium, which are distributed between the egg white and the
yolk. One large egg provides 5% of the daily requirements of
vitamin A and 5% of the daily requirements of iron. It is also a
good source of vitamin E. In a study conducted in children,
where they were allocated to consume either two whole eggs
per day for 4 weeks or an equivalent amount of egg whites,
dietary records indicated that the amount of vitamin E they
consumed was almost three times more during the whole egg
period.
The nutrients present in eggs and their contribution to
nutrition and health are depicted in Figure 1.
Conclusions
Eggs are a rich source of key nutrients and should therefore be
considered part of a healthy diet. Not only eggs provide a well-
known source of high-quality protein, but also they contain
dietary constituents that promote health across the life
spectrum, protecting individuals against chronic disease. Evi-
dence derived from epidemiological data and clinical studies
support the notion that there is no association between egg
consumption and heart disease risk. The yolk is a good source
of choline that could be beneficial for pregnant women, for
fetal and infant development, and for protection against
Alzheimer’s disease in the elderly.
All populations of children, young adults, and the elderly
should benefit from the excellent protein quality of eggs that
supports optimal growth in children and adolescents, while
additionally protecting the elderly against sarcopenia. In addi-
tion, the carotenoids lutein and zeaxanthin protect against
AMD and oxidative stress. Overall eggs should be part of a
healthful diet.
See also: Adolescent Nutrition; Antioxidants: Characterization and
Analysis; Cholesterol: Properties, Processing Effects, and
Determination; Choline: Properties and Determination; Functional
Foods; Mediterranean Diet.
Further Reading
Andersen CJ and Fernandez ML (2013) Dietary approaches to improving
atheroprotective HDL functions. Food and Function 14: 241–254.
Ballesteros MN, Cabrera RM, Saucedo MS, and Fernandez ML (2004) Dietary
cholesterol does not increase biomarkers for chronic disease in a pediatric
population at risk from Northern Mexico. American Journal of Clinical Nutrition
80: 855–861.
Blesso CN, Andersen CJ, Barona J, Volk B, Volek JS, and Fernandez ML (2013a) Effects
of carbohydrate restriction and dietary cholesterol provided by eggs on clinical risk
factors of metabolic syndrome. Journal of Clinical Lipidology 7: 463–471.

Blesso CN, Andersen CJ, Barona J, Volek JS, and Fernandez ML (2013b) Whole egg
consumption improves lipoprotein profiles and insulin sensitivity to a greater extent
than yolk-free egg substitute in individuals with metabolic syndrome. Metabolism
62: 400–410.
Drewnowski A (2010) The Nutrient Rich Foods Index helps to identify healthy,
affordable foods. American Journal of Clinical Nutrition 91: 1095S–1101S.
Eckel RH, Jakicic JM, Ard JD, et al. (2014) 2013 AHA/ACC guideline on lifestyle
management to reduce cardiovascular risk. Journal of the American College of
Cardiology 63: 2960–2984.
Fernandez ML (2010) Effects of eggs on plasma lipoproteins in healthy populations.
Food and Function 1: 156–160.
Fernandez ML (2012) Rethinking dietary cholesterol. Current Opinions in Medicine and
Nutrition Metabolic Care 15: 17–21.
Fernandez ML and Calle MC (2010) Revisiting dietary cholesterol recommendations:
does the evidence support a 300 mg/d limit? Current Atherosclerosis Reports
12: 377–383.
Howell WH, McNamara DJ, Tosca MA, Smith BT, and Gaines JA (1997) Plasma lipid
and lipoprotein responses to dietary fat and cholesterol. American Journal of
Clinical Nutrition 65: 1747–1764.
Hu FB, Stampfer MJ, Rimm EB, et al. (1999) A prospective study of egg consumption
and risk of cardiovascular disease in men and women. Journal of the American
Medical Association 281: 1387–1394.
Eggs: Composition and Health Effects 475
Jensen HH, Batres-Marquez SP, Carriquiry A, and Schalinske KL (2007) Choline in the
diets of the US population: NHANES, 2003–2004. FASEB Journal 21: lb219.
Krinsky NI, Landrum JT, and Bone RA (2003) Biologic mechanisms of the protective
role of lutein and zeaxanthin in the eye. Annual Reviews in Nutrition 23: 171–201.
Ratliff JC, Leite JO, DeOgburn R, Puglisi M, VanHeest J, and Fernandez ML (2010)
Consuming eggs for breakfast influences plasma glucose and ghrelin, while
reducing caloric intake during the next 24 hours in adult men. Nutrition Research
30: 96–103.
U.S. Department of Agriculture, Agricultural Research Service (2010). USDA National
Nutrient Database for Standard Reference, Release 23, Nutrient Data Laboratory
Home Page: http://www.ars.usda.gov/nutrientdata.
Zeisel SH (2006) Choline: critical role during fetal development and dietary
requirements in adults. Annual Reviews in Nutrition 26: 229–250.
Relevant Websites
http://www.eggnutritioncenter.org/ – Egg Nutrition Center.
http://www.ers.usda.gov/ – United States Department of Agriculture, Economic
Research Service.
 Eggs: Use in the Food Industry
CG Belyavin, Chris Belyavin (Technical) Ltd, Newport, UK
ã 2016 Elsevier Ltd. All rights reserved.
This article is reproduced from the Encyclopedia of Food Sciences and Nutrition, volume 4, pp. 2000–2004, ã 2003, Elsevier Science Ltd., with an
updated Bibliography section supplied by the Editor.
Introduction
The majority of eggs produced in the world are sold in-shell for
consumers to utilize in the home in traditional ways or for baking.
In the United Kingdom, only 17% of eggs produced go into egg
products, and in the United States, the figure is nearer 30%.
Traditionally, surplus eggs not required for the shell egg
market (normally the smaller sizes) and downgraded eggs
were turned into egg products. Today, farmers have specific
contracts to produce eggs for further processing, and any egg
that is damaged cannot be used for this purpose. There are
essentially three types of egg products, liquid, dried, and frozen,
with small markets for peeled, hard-boiled, and hard-boiled
pickled eggs. The main products are used widely in the bakery
and confectionery trades along with other industries. Figure 1
illustrates the many uses to which processed whole eggs can be
put and also shows the way in which whole eggs can be used to
produce value-added products for the food industry without
going through the process of pasteurization with or without
subsequent drying. Value-added products in the United States
are often made with pasteurized eggs.
The world trade in liquid eggs is in excess of 110 000 tonnes
traded annually, some 80% of which are traded between EU
member countries. Outside of this area, the only significant
major market is Japan, which purchases around 15 000 tonnes
a year. The Netherlands is the number one exporter closely
followed by Belgium, both of them accounting for about
45% of the total world trade.
Shell eggs
Value-added products
Scrambled egg
Egg macmuffins
Boiled eggs Dried
Pickled eggs
Sandwich fillings
Whole egg
Egg roll (long egg)
Albumen
Omelette mix
Yolk
Egg and apple drink
Burger egg/fried egg
Figure 1 Eggs in the food industry.
476 Encyclopedia of Food and Health
Some 28 000 tonnes of dried eggs is marketed internation-
ally. Japan and European countries are the major buyers, with
the United States being the major seller.
When setting up an egg processing plant, it should ideally be
located close to its supply of eggs. A constant supply of eggs is
important in the efficient operation of the plant. The correct
location enables the eggs to arrive at the plant in good condi-
tion, thus ensuring that the resulting product is of good quality.
Liquid Egg Products
The production of liquid egg products takes place in specialist,
large plants specifically designed for the purpose. Such a plant
could process in excess of 36 million eggs a year. Mechanical
equipment is used to break the eggs automatically and, if neces-
sary, separate the egg yolk from the albumen. Such systems can
handle up to 108 000 eggs an hour with electronic detectors to
make sure that no yolk ends up in the white at the time of
separation. A temperature of 10  C is optimum for breaking and
separating.
Further processing of eggs into liquid product is not a
means of disposing of poor-quality eggs. In fact, the increasing
concern about food safety and quality has meant that only
surplus first-quality eggs and uncracked seconds are used in
breaking plants.
At the time of breaking, each egg will be visually inspected,
either automatically or by an operator, to ensure that no
Pasteurized
Liquid
Whole egg
Fortified
Albumen
Yolk plain
blends
Bakeries
Restaurants
Cake mixes/meringues
Sweets
Salad dressings/mayonnaise
Icecream
Noodles
Bady food
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 Eggs: Use in the Food Industry 477

undesirable material is passed into the further processing line.

Any eggs seen to be unsuitable will be rejected from the system
at this stage and disposed of. The contents from the broken
eggs are then passed through a sieve to remove any remnants of
shell, the chalazae, which are the strands of protein that hold
the egg yolk in place, and any other undesirable material.
About 50% of total liquid egg production in the United
States is in the form of whole egg (yolk and albumen), and
some may be fortified by the addition of extra yolk to increase
the ratio of yolk to albumen over and above that found in a
normal egg. The balance is divided between production of
albumen and that of yolk. Liquid yolks may be sold as plain
yolk or special blends such as sugared yolk or salted yolk. Not
only such products meet the needs of specific customers but
also the addition of these agents to yolk helps to maintain the
consistency of the yolk, as without these natural additives, it
tends to separate into a thin liquid surrounding a congealed
core if it is frozen. The whole egg tends to be standardized to a
solids content of 24.7%. The separate egg white should not
contain more than 0.03% yolk on a liquid basis, and yolk
tends to be standardized to a solids content of 43%.
In the past, all these products would have been frozen
Figure 2 A liquid egg pasteurization plant. Courtesy of Oasters–Fridays
rapidly in a blast air freezer and held in this form until used.
(Cranbrook) Ltd.
However, there has been a tendency in large-scale plants to
chill the products and handle them as a fresh food, transport-
ing them in refrigerated vehicles to their point of use. In this
case, whole eggs and yolk liquids are chilled to 4.4  C and egg
albumen is chilled to < 7  C. In this form, the shelf life of the
product is only about 4 days, but the costs of canning and
freezing are avoided. Freezing is still adopted for small-scale
use of liquid eggs.
Bakeries as a group are the largest users of whole eggs and
separate albumen. Mayonnaise manufacturers and salad dress-
ing makers use large quantities of salted yolks, and ice cream
makers use sugared yolks. Noodle makers and processors of
baby foods use plain yolks.
Liquid eggs or egg products are pasteurized (Figure 2) dur-
ing their manufacture to prevent organisms such as salmonella
from becoming a major health hazard during their subsequent
use and particularly after the eggs are removed from storage.
Legislation to enforce this was introduced in the United
Kingdom in 1964 and in the United States in 1966. The pas- Figure 3 Liquid egg being packed into small cartons for retail sale.
teurization process in the United Kingdom involves subjecting Courtesy of Oasters–Fridays (Cranbrook) Ltd.
the product to a temperature of 64.4  C (148  F) for 2.5 min
for whole eggs. Comparable figures for the United States are expensive way of producing liquid eggs because of the cost of
60  C (140  F) for 3.5 min. Albumen is more susceptible to the packaging. In these cases, liquid whole eggs would be used
heat, and so, a slightly lower temperature is used, whereas for making scrambled eggs and omelets, for example.
the reverse is the case for yolk, where any salmonella is more Over the last 10 years, the production of egg products has
heat-resistant. The alpha-amylase test is routinely used to test been seen as the way in which any decline in egg consumption
for adequate pasteurization. can be reversed. Consequently, a more diverse range of prod-
Traditionally, liquid egg products were packed in metal ucts are now available, including burger eggs, fried eggs, and
cans or transported by large tankers. Today, modern packaging meringue mix.
machinery enables the product to be put in large plastic bags
contained in rigid outers, and each bag can contain 500 kg of
liquid egg, which is drained from the container by means of a Freezing of Liquid Egg
tap inserted at the bottom. The bag is then disposed of and the
outer is returned to the manufacturer to be refilled. The prod- If the liquid egg is going to be frozen, to maintain a quality
uct can be put into smaller cartons (Figure 3) that can be product, it is important that the freezing process should be
supplied to retail outlets for direct sale to consumers or larger rapid and that the frozen product should be stored at a low
cartons for supplying to hospitals and institutions. This is an temperature. Cans of chilled eggs should be subjected to an air
478 Eggs: Use in the Food Industry
blast at 30 to 40  C to ensure total freezing within 24 h,
producing a uniform product without coagulation of the yolk.
Subsequent storage should be at 10 to 20  C.
Production of Egg Powder
The production of dried egg products is a further step beyond
pasteurization. Spray drying is now the most common method
for producing egg powder. However, some egg white is pan-
dried and some whole egg is freeze-dried. Dried egg products
have a number of advantages, which are listed in the succeed-
ing text.
(1) They can be handled and stored with ease and at low cost.
(2) They are ready to use immediately with no thawing.
(3) They are easy to handle in a hygienic way.
(4) They are easy to remove from the container without
scraping.
(5) No bacterial growth can occur in powder at room
temperature, provided it is kept dry.
(6) There is good uniformity.
(7) There is precise control over the amount of water used in
formulation.
(8) There is no loss when used because the dried egg is
usually added directly to the batch.
(9) No special transfer or storage equipment is needed.
(10) Because the moisture content is reduced from around
74% to 2–4% by weight, there is a reduction in weight
and volume and a concentration of food value.
A possible disadvantage of powder compared with liquid egg is
a loss of ‘fresh flavor’ and a loss of certain functional properties
such as aerating power, unless treated with nonreducing sugars
prior to heating. This, however, limits its use because of the
sweetened nature of the powder.
There are now several different types of dried egg products
available that can be summarized under four major headings:
dried egg white, dried plain whole egg and yolk, dried blends
of whole egg and yolk with carbohydrates, and special types of
dried egg products.
The growth in egg drying is well illustrated by reference to
the United States. Prior to 1941, <1% of the eggs produced
went to drying plants. Government purchases stimulated dry-
ing operations to such an extent that 20 400 tonnes were
produced in 1941, 107 000 tonnes in 1942, 118 000 tonnes
in 1943, and 146 000 tonnes in 1944. The current annual
volume is over 180 000 tonnes. A 30 dozen case of shell eggs
will yield about 18 kg of liquid whole egg or 4.5 kg of dried
product containing 2.5% moisture.
The handling of shell eggs prior to drying is no different
from the handling of eggs that are to be kept liquid. The liquid
material is put through a clarifier to remove any bits of shell
and is then screened to remove the chalazae and vitelline
membranes. Pasteurization is important, not only to control
salmonella infection but also to preheat the liquid so as to
ensure a low-moisture powder that will not show any scorch-
ing. A typical egg-drying plant is shown in Figure 2.
The liquid egg is then pumped under a pressure of
17 250–34 500 kPa (2500–5000 lbf in 2) to nozzles through
which it is released into a large chamber where it immediately
comes into contact with a stream of air that has been heated to
temperatures of 120–175  C. The enormous surface area cre-
ated by atomization causes instantaneous evaporation of most
of the moisture from the egg material and the formation of
powder that falls to the floor as a fine powder, while the moist
air passes on out of the drying chamber at a temperature of
65–71  C. It is important that the spray dryer is designed so
that the product stays at relatively low temperatures through-
out the drying system and that the dried product is removed
from the hot air stream as rapidly as possible.
Modern dryers produce a powder in which the moisture
content does not exceed 2%. The temperature of the powder as
it leaves the dryer may be 65  C or higher, and this must be
reduced quickly to <29  C if the product is to have good
keeping qualities. Since the powder is extremely hygroscopic,
the temperature must be reduced by contact cooling, rather
than by exposure to cold air. Two different powder-cooling
methods are commonly used: first, pneumatic conveying
with cooling air and, second, mixing the powder with carbon
dioxide in the form of dry ice. After cooling, the powdered eggs
are then packed immediately in sealed containers. Many of
them are packed in carbon dioxide to remove the oxygen and
lower the pH value in order to improve the keeping quality of
the product.
Egg white can be dried in pans, rather than through a spray
technique. The pan-dried egg white can be in the form of flake
or granular albumen or can be milled to a powder form. This
form of egg albumen tends to have good whipping properties
once reconstituted.
The chief users of dried egg products are cake mix manu-
facturers, sweet makers, and manufacturers of meringue pow-
ders and, to a lesser extent, mayonnaise makers. Most dried egg
products made for commercial use are packed in polythene-
lined boxes or drums. A common pack size would contain
25 kg of product.
Quality of Dried Egg Products
The quality of dried egg or dried egg products can be affected
by a number of factors. Important are the quality of the eggs
broken out to make the original product, handling methods,
sanitation practices, conditions during processing, pasteuriza-
tion procedures, drying, and conditions under which the prod-
ucts are held in storage. Glucose (0.3–0.5%) is removed from
the liquid egg white prior to drying by fermentation using a
yeast or bacterial culture or by oxidation to gluconic acid using
a glucose oxidase–catalase enzyme system. With glucose
removed, dried egg whites are completely stable. If glucose is
not removed, the product would be unstable because reducing
groups from the glucose would combine with amino acids in
the protein, leading to a condensation reaction that would be
followed by browning and the development of insoluble pro-
teins. There could also be the development of off-odors and the
loss of some functional properties during storage. Plain whole
egg and yolk can be pasteurized and dried without removal
of the glucose, and consequently, these are the least stable of
the egg products.
The physical properties important in relation to dried egg
products are bulk density, dispersibility, solubility, and recon-
stituted viscosity.

Storage of Dried Egg Products
One of the real advantages of dried egg products is their ease of
storage. Most are relatively stable when stored at room temper-
ature. Dried egg whites can be held under almost any storage
condition for an indefinite period of time. Dried products
containing whole egg and yolk should be under refrigeration
if held for long periods of time. Some of these are relatively
stable at room temperature, particularly those where the natu-
ral glucose has been removed prior to drying.
Other Uses of Eggs in the Food Industry
Although liquid eggs and dried eggs still account for the major-
ity of eggs used in the food industry, there are other products,
which have evolved during the last 10–15 years, that are com-
mercially available.
The mass production of hard-boiled eggs has developed in
recent years. Normally, white eggs are used in the process,
rather than brown, because the shells can be more easily
removed from white eggs than from brown, resulting in less
damage to the finished product. In countries where brown egg
production predominates, such as the United Kingdom, this
means that white eggs may have to be imported for the process
from countries such as Denmark. However, in recent years,
flocks of white egg-laying hens have been established in the
United Kingdom specifically to supply the processing industry.
Again, only good-quality eggs are used in the process so
that the final product is also of good quality. Normally, in
European Union countries, small eggs would be used (under
53 g) for boiling as there is little demand for these sizes of eggs
in the shell egg market.
The eggs are gradually boiled as they move around a trough
arrangement containing high-temperature water, with the
objective of them being hard-boiled by the time they have
completed a single circuit of the trough. The eggs are then
removed, deshelled, and cooled. Once hard-boiled, the eggs
can be used for a number of products. They can be pickled in
jars in plain vinegar or with spices. They can be used in pies or
salads and a major use in this form is in airline meals. Eggs
damaged during the boiling or shelling processes would not
necessarily be discarded. They can be diverted to the manufac-
ture of sandwich fillings, such as egg mayonnaise, which can
be packed into 5 kg tubes for distribution to sandwich manu-
facturers and the catering trade.
Problems associated with large-scale boiling of eggs, apart
from that of deshelling, include optimizing the temperature for
both the boiling and cooling processes, effluent discharges,
and the prevention of black rings occurring at the junction
between the yolk and the albumen. This is associated with
the breakdown of sulfur-containing amino acids in the albu-
men producing hydrogen sulfide, which reacts with iron
released from the yolk to form iron sulfide.
Hygiene Measures
In all egg processing plants producing egg products for human
consumption, a strict code of practice relating to hygiene is
Eggs: Use in the Food Industry 479
essential. Conventional egg products must now meet strict
chemical, physical, and functional specifications, which
include moisture, fat, protein, ash, glucose, reconstituted vis-
cosity, whipping ability, and functional performance in the
foods in which they are used. Also included are microbiolog-
ical standards for total plate count, coliform, yeast and mold,
Escherichia coli, salmonella, coagulase-positive staphylococcus,
and Clostridium perfringens.
Many plants have a resident food technologist responsible
for the implementation of a quality control program using an
in-house quality control and microbiology laboratory. Every
batch of product produced would be tested for contamination,
and production plants would be swabbed twice a week to
test for the presence of undesirable organisms within such a
program.
See also: Clostridium: Food Poisoning by Clostridium perfringens;
Clostridium: Occurrence and Detection of Clostridium perfringens;
Drying: Principles and Types; Eggs: Composition and Health Effects;
Freezing Theory.
Further Reading
Abdou AM, Kim M, and Sato K (2013) Functional proteins and peptides of hen’s egg
origin. In: Hernandez-Ledesma B and Hsieh CC (eds.) Bioactive food peptides in
health and disease. Rijeka, Croatia: Intech.
Anonymous (1998) Watt poultry statistical yearbook. Mount Morris, IL: Poultry
International Watt.
Austic RE and Nesheim MC (1990) Poultry production. Philadelphia, PA: Lea &
Febiger, pp. 280–284.
Bergquist DH (1981) New development in drying egg products. In: Beuving G,
Scheele CW, and Simons PCM (eds.) Quality of eggs, pp. 7–14. Beekbergen, The
Netherlands: Spelderholt Institute for Poultry Research.
Capetillo GO (1981) Egg de-hydration plant. In: Beuving G, Scheele CW, and
Simons PCM (eds.) Quality of eggs, pp. 15–23. Beekbergen, The Netherlands:
Spelderholt Institute for Poultry Research.
Deeming DC and Fergusson MWJ (2009) Egg incubation. Cambridge, UK: Cambridge
University Press.
Hauber ME (2014) The book of eggs. Chicago, IL: The University of Chicago Press.
McNamara DJ (2012) Impact of egg consumption in development or prevention of heart
disease. In: Moghadasian MH and Eskin NAM (eds.) Functional foods and
cardiovascular disease. Boca Raton, FL: CRC Press.
Miranda JM, Anton X, Redondo-Valbuena C, et al. (2015) Egg and egg-derived foods:
effects on human health and use as functional foods. Nutrients 7: 706–729.
Perić L, Rodić V, and Milošević N (2011) Production of poultry meat and eggs as
functional food – challenges and opportunities. Biotechnology in Animal Husbandry
27: 511–520.
Roux M (2006) Eggs. Canada: John Wiley & Sons.
Sahin N, Akdemir F, Orhan C, Kucuk O, Hayirli A, and Sahin K (2008) Lycopene-
enriched quail egg as functional food for humans. Food Research International
41: 295–300.
Sim JS and Sunwoo UH (2006) The amazing egg. Nature’s perfect functional food for
health promotion. Edmonton, Canada: University of Alberta Hospitals.
Relevant Websites
http://animalscience.ucdavis.edu/Avian/sim.pdf – Eggs nutritional significance.
http://www.foodtimeline.org/foodeggs.html – Food timelines on eggs.
 Elderly: Nutrition Requirements
R Chernoff, University of Arkansas for Medical Sciences, Little Rock, AR, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
It is well known that the population of elderly people is expand-
ing rapidly; it has been estimated that by 2030, 70 million
people will be age 60 years or older, and, by 2050, the popula-
tion older than age 65 is projected to be 88.5 million. The 85 þ
population is anticipated to grow from 4.2 to 6.6 million in
2020, then to 8.9 million by 2030, to 19 million by 2050. An
increase in the prevalence of disabilities and diseases is one of
the natural consequences that accompany advanced aging.
Functional impairment in older adults is associated with
poor nutritional status or malnutrition, which is frequently
also associated with the perception of quality of life, particu-
larly among women. Disability is associated with frailty and
frailty is linked to loss of muscle strength and weight. Frail
individuals are not always cachectic but may be overweight or
obese. Data from the Health and Retirement Study, a prospec-
tive study conducted by the University of Michigan, demon-
strate that excess body weight is associated with less Activities
of Daily Living (ADL) and Instrumental Activities of Daily
Living (IADL) impairment. The challenge of determining
nutritional requirements in elderly individuals is associated
with an increasingly diverse impact of physiological aging
and a lack of adequate and appropriate standards against
which nutritional status can be measured.
Energy Requirements
As humans age, there are changes in body composition; lean
body mass, total body water, and bone density decrease over
time. Fat stores may increase or remain the same, but fat com-
partments shift and tend to become denser around major organs.
These body composition changes contribute to a decrease in the
energy required to maintain homeostasis including body weight
and function. To avoid excess weight gain, caloric intake must be
reduced. The Baltimore Longitudinal Study of Aging demon-
strated a decrease in energy intake from 2700 kcal at age 30 to
2100 kcal by age 80 in male subjects. This observation is impor-
tant because as basal energy needs decrease and activity becomes
more sedentary, less energy substrate is required to maintain lean
body mass and support energy expenditure. However, reducing
total caloric intake can increase the individual’s at risk for a
deficient intake of other essential nutrients.
Decreased energy expenditure is associated with chronic dis-
ease and increasingly sedentary behavior. There is a decrease in
consumption of calories with age as well as a decrease in energy
expenditure. However, if older adults do not reduce their caloric
intake as they age and become more sedentary, weight gain
results. Weight gain may serve to further exacerbate disabling
conditions; however, heavier patients may have better survival
and recovery from atherosclerotic heart disease, stroke, and other
potentially life-threatening conditions.
480 Encyclopedia of Food and Health
Requirements for energy to maintain weight are often linked
to energy consumption, but research indicates that this might
not be reasonable in estimating energy needs for elderly indi-
viduals. Prediction equations for energy requirements do not
give reliable estimates of energy requirements in elderly people,
and indirect calorimetry should be employed when actual
energy needs must be defined rather than grossly estimated.
The clinical challenge is to continue to meet all nutrient require-
ments in a smaller caloric base of food consumed regardless
of weight.
Data indicate that the relationship between obesity and mor-
tality becomes stronger with age. Perhaps, more pervasive is the
role that obesity has in functional consequences; older adults
who are physically limited because of obesity (body mass index
of 30) tend to be more sedentary. Sedentariness contributes to
weight gain, more severe obesity, and possibly mortality.
Undernutrition
Undernutrition, a deficit of essential nutrients, including energy
substrate and protein, is a common finding in older adults. In
elderly individuals, measuring nutritional status is somewhat
challenging because there are no population reference standards
against which to compare. Alternate approaches to measure
undernutrition or malnutrition abound, but it has been well
documented that undernutrition exists and is a therapeutic chal-
lenge in older adults regardless of their health status. In elderly
people, loss of reserve capacity is associated with undernutrition.
Reserve capacity is the ability of an organism to respond to
trauma, acute illness, or other physiological insults; for patients
who lose this ability, an insult may lead to a rapid decline.
Weight loss has been associated with adverse outcomes.
Calorie Restrictions
Evidence that caloric restriction increases life span in research
models ranging from single-cell organisms to nonhuman pri-
mates has had positive results. Evaluating the animal calorie
restriction models in humans is challenging due to the poorly
defined mechanisms of calorie restriction and longevity and
the potentially difficult ethical issues in human research. Skep-
ticism about the positive effects of calorie restriction in
humans is related to the real-life, nonlaboratory, controlled
environment situation under which people live.
Protein Requirements
Protein intake tends to be highest in young adults and progres-
sively declines with advancing age. The decrease in protein
intake is likely to be the result of reduced energy intake and
not a change in the proportion of energy consumed as protein.
Data indicate that although the acceptable macronutrient
http://dx.doi.org/10.1016/B978-0-12-384947-2.00247-6

distribution range for protein is 10–35% of energy, very few
older adults usually consume more than 20% of their energy
intake as protein. In normal aging, changes in body composi-
tion occur that result in a shift toward decreased muscle mass
and increased fat mass.
The loss of fat-free mass with advancing age theoretically
results in a relatively lower protein requirement (per kilogram
of body weight) compared to a younger adult. However, at
present, there is no consensus regarding the protein needs of
elderly persons, although there is some research suggesting that
the Recommended Dietary Allowances (RDAs) should be higher
than the current level. Short-term nitrogen balance studies are
inconclusive, whereas the limited longer-term nitrogen balance
studies suggest that the RDA is not sufficient to completely meet
the metabolic and physiological needs of elderly persons.
Fat Requirements
A minimum of 10% of total energy intake should be derived
from fat to ensure an adequate dietary intake of fat-soluble
vitamins and essential fatty acids. Recommendations for dietary
fat intake are the same for older adults as for younger adults: 30%
or less of total diet with saturated fats providing from 8% to 10%,
polyunsaturated fats being  10%, and monounsaturated fats
making up the difference. The recommendation for dietary cho-
lesterol intake is 300 mg day1 or less. Total cholesterol is gen-
erally considered an important risk factor for coronary heart
disease; however, the impact of cholesterol profiles in elderly
people is unknown. In a 5-year prospective study of 4066 men
and women older than age 71 years, elevated total cholesterol
levels were associated with a similar pattern of death from coro-
nary heart disease as seen in younger adults. The subject group
with the lowest total cholesterol levels (<160 mg dl1) had the
highest incidence of preexisting cardiovascular disease, the high-
est risk factors for cardiovascular disease, the highest indexes of
poor health, and the highest crude coronary heart disease mor-
tality. Currently, it appears that elevated total cholesterol levels
remain a risk factor for death from coronary artery disease in
elderly persons. High-density lipoprotein cholesterol fraction
values <35 mg dl1 predict coronary heart disease mortality
and occurrence of new events in individuals older than 70
years. Specific recommendations for dietary fat modifications
must be made on an individual basis based on a complete profile
of cardiovascular and stroke risk factors.
Carbohydrate Requirements
Carbohydrate is the third major macronutrient; it includes the
simple sugars and complex carbohydrates. Requirements for
dietary carbohydrate generally approach 55–60% of total energy
intake. The complexity of dietary carbohydrate can be as impor-
tant as the percentage of calories contributed by carbohydrate-
rich food. Consumption of food with the indigestible dietary
fiber removed can contribute to gastrointestinal disorders com-
monly encountered in older adults, including constipation,
diverticular disease, diabetes, and hyperlipidemia.
Dietary fiber, mainly composed of plant polysaccharides
and lignin, is resistant to human digestive enzymes. One
Elderly: Nutrition Requirements 481
example of such fiber, wheat bran, increases fecal bulk and
decreases gut transit time and intraluminal pressure within the
colon. These effects help to reduce the incidence of constipa-
tion and the formation of colonic diverticula. However, solu-
ble fibers such as gums and pectins increase the viscosity of
intestinal contents, increase gut transit time, and decrease the
rate of small intestinal absorption. Consumption of fibers such
as guar, pectin, and tragacanth leads to reduced insulin secre-
tion in normal subjects, increased carbohydrate tolerance in
diabetics, improved lipid profiles, and reduced glycosylated
hemoglobin (HbA1c). The mechanism of the positive effect
seems to be related to delayed gastric emptying and reduced
rate of absorption of carbohydrate in the small intestine. Puri-
fied fibers such as guar, pectin, oat bran, and high-fiber foods,
including cereals, starchy vegetables, and beans, have been
reported to lower serum lipids. Decreases in total cholesterol,
LDL cholesterol, and triglycerides without changes in HDL
cholesterol have been reported. At present, it seems prudent
to have a daily dietary fiber intake of 25–35 g day1 and to
include a variety of fibers from fresh fruits, vegetables, legumes,
and whole-grain products. Every effort should be made to
encourage older adults to have a varied dietary intake of
carbohydrate-containing foods daily. This strategy will increase
the likelihood of adequate consumption of micronutrients
as well.
Fluid Needs in Older Adults
In older adults, fluid intake often does not receive the attention
it deserves. Inadequate fluid consumption can lead to rapid
dehydration, which has its own potentially risky consequences
including hypotension, constipation, nausea, vomiting, muco-
sal dryness, decreased urinary output, elevated body tempera-
ture, and mental confusion. Total body water decreases with
age, shrinking from 75% body weight in infants to 55% body
weight in elderly adults associated with the reduction in total
lean body mass.
Older adults are at risk of dehydration, particularly when
there are climate challenges (extreme heat or cold). Water acts
as a thermal buffer to protect the individual from hypo- or
hyperthermia. Elderly people have decreased sweating and
changes in other thermoregulatory responses, making them
more vulnerable to periods of environmental shifts in tempera-
ture. Insensible losses through skin can range from 0.3 l h1
when sedentary to 2.0 l h1 with exercise; this may lead to a
loss as high as 6 l day1 in extreme heat. Decreased plasma
volume and osmolality can result in death during periods of
extreme environmental heat. Adequate hydration in older adults
affects their ability to be physically active; cognition, alertness,
and short-term memory are affected by mild dehydration. The
challenge comes from the fact that thirst mechanisms and sensi-
tivity are compromised in elderly people. The decrease in fluid
intake is the result of alterations in mechanisms that control
thirst sensitivity, particularly a decrease in osmoreceptors and
baroreceptors and secretion of vasopressin.
Environmental factors, hormonal changes, climate
(temperature and humidity), availability of water and food,
medical problems, diuretic abuse, polypharmacy, and avolun-
tary decrease of fluid intake may all affect fluid balance. Along
482 Elderly: Nutrition Requirements
with disordered thirst control mechanisms and inadequate
intake of fluid for maintenance, older adults who have fever,
infection, immobility, dementia, coma, fluid loss due to hemor-
rhage, diarrhea, vomiting, incontinence, or diabetes insipidus or
have acute illness can become chronically dehydrated. The
kidneys’ ability to concentrate urine or efficiently conserve
water also can affect fluid balance.
Many older adults, particularly those who are
institutionalized, are not consuming adequate amounts of
fluid. Although the usual stated requirement for fluid intake
is eight 8 ounce glasses of fluid per day, data to support this
recommendation are lacking. Actual need is probably in the
range of six 8 ounce glasses of fluid per day.
Methods commonly used to determine fluid needs in indi-
viduals include 30 ml kg1 body weight with a minimum of
1500 ml day1, 1 ml kcal1 consumed, or 100 ml kg1 for the
first 10 kg of actual weight, 50 ml kg1 of the next 10 kg actual
body weight, and 15 ml kg1 of remaining weight. The first
method (30 ml kg1 body weight) approximates fluid needs
most accurately in institutionalized elderly persons; additional
research needs to be conducted to better estimate actual needs
of older adults. Strong and chronic encouragement for oral
fluid intake might be necessary to maintain hydration status.
Vitamin Requirements
Water-Soluble Vitamins
Vitamin B1 (thiamine)
Some studies suggest that older adults might have a higher
requirement for vitamin B1, but lower energy requirements
for older adults may contribute to an increase in requirement.
Based on what is presently known, the recommended amount
appears to be adequate for all adults independent of age.
Depending on income, race, and socioeconomic status, up to
70% of elderly adults have low levels of vitamin B1 intake.
Vitamin B2 (riboflavin)
Vitamin B2 acts with its coenzymes flavin mononucleotide and
flavin adenine dinucleotide in many electron transfer reactions.
Because of the many different roles of the flavoenzymes in
intermediary metabolism, the important role of riboflavin
(such as in antioxidant defense or in the modulation of homo-
cysteine metabolism) is often forgotten. Dairy products and
cereal products are, for older adults, the most important sources
of riboflavin. In various populations around the world, the high
prevalence of riboflavin deficiency is mainly caused by an inad-
equate intake of dairy products.
There might be a role for riboflavin in the pathogenesis of
age-related cataracts because this vitamin might be important
as a photosensitizing agent. Despite the potential involvement
in some disease states, there is no evidence that there is a higher
need for vitamin B2 in older adults.
Niacin
Data about niacin status in elderly adults are very limited.
Niacin intake in elderly adults varies widely and is associated
with the variables of socioeconomic background, race, age,
health status, and institutionalization. Nutritional intake of
niacin is related to the mortality of elderly people; however,
this relation does not necessarily imply causality. The nutrient
density of niacin was adequate in nursing home diets so that
low intakes become the major determinant of recommenda-
tions for this vitamin. For niacin, low dietary intake is key to a
niacin-insufficient diet. Encouragement of higher energy and
protein consumption is the best strategy to increase niacin
status because this approach improves niacin and protein
intake.
Vitamin B6
The intake of vitamin B6 varies widely among elderly persons as a
function of age, socioeconomic level, and state of health. Studies
report intakes below the RDA in up to 50% or even 90% of
elderly populations. Even in healthy, free-living, elderly adults,
61% of women consumed <1 mg of vitamin B6 per day, and
54% of elderly men consumed < 1.1 mg (corresponding to 55%
of the 1989 RDA). Between 10% and 50% of the US population
older than 51 years consumed less than the Estimated Average
Requirement (EAR) of vitamin B6, <1.4 mg day1 for men and
<1.3 mg day1 for women. (The EAR corresponds to the daily
nutrient intake that is estimated to meet the requirement of half
the healthy population of the corresponding age group.)
It has been suggested that an age-related increase in activity of
the alkaline phosphatase, considered to be of major importance
in the degradation of pyridoxal-5-phosphate (PLP), and low
intakes of vitamin B6 are the major causes of age-related decline
in plasma PLP levels. Abnormalities of vitamin B6 can be
corrected by the ingestion of vitamin B6 supplements in most
elderly persons. A few studies report the inability of some older
adults (up to 20%) to correct their biochemical vitamin B6 status
on supplementation. These observations are suggestive of age-
related changes in vitamin B6 requirements of older adults. The
effects of diseases and comorbidities seem to be the likely causes
of unresponsiveness to supplements. The bioavailability of vita-
min B6 from food is low; the RDA, based on the intake vitamin
B6 from food sources, might be even higher than assumed. The
present recommendations do account for these alterations of
the metabolism with aging.
Both retrospective and prospective studies have described a
relationship between the risk of coronary artery disease,
myocardial infarction, and the intake of vitamin B6. This rela-
tionship is, however, strongest for folic acid.
Folate
Since the 1989 RDA was published, the recommended intakes
of folate have been increased for older women and men. Folate
intakes in older adults vary considerably as a result of diet, but
most elderly people ingest amounts close to the RDA. Before
fortification of cereal grains with folate (i.e., before 1 January
1998), the prevalence of low folate intakes varied between 0%
and 50%. The median intake in the population at large before
mandatory cereal folate fortification was 250 mg day1, which
is well below the present recommendation. The average folate
intake has increased by  80–100 mg day1 due to the fortifi-
cation of cereals.
A high intake of alcohol alone or in combination with an
inadequate diet is one of the most important factors contribut-
ing to clinical folate deficiency. In elderly subjects, the presence
of both low folate intake and elevated plasma homocysteine
level represents a rather prevalent clinical situation. Elevated

levels of plasma homocysteine are mainly attributable to an
impairment of folate, vitamin B6, and/or vitamin B12 status. In
most people, folate seems to be the major determinant of
plasma homocysteine levels. Perhaps, homocysteine is only a
surrogate factor and its relationship to etiologies of chronic
disease is more complicated. Presently, the best strategy to man-
age progression of chronic disease risk is the consumption of a
well-balanced diet.
As a result of the US government-mandated folic acid forti-
fication of cereal grain products, a shift in the distribution
of folate intakes and consequently the biochemical status of
folate occurred. It has been reported that a higher supplemen-
tal folate intake (>400 mg day1) was associated with an
increased cognitive decline in elderly subjects. This effect was
noticeable in older people who also had a vitamin B12 defi-
ciency or low vitamin B12 plasma concentration as reported in
other studies. Adequate intake (AI), whenever possible, from
food, based on present guidelines should be more strongly
encouraged, but it should be remembered that larger amounts
of folate from fortified foods and/or supplements are not good
for everyone.
Vitamin B12
Vitamin B12 requirements for older persons (i.e., > 51 years)
can be obtained through consumption of vitamin B12-fortified
foods or vitamin B12-containing supplements. Fortified foods
and supplements contain crystalline vitamin B12 and absorp-
tion is not affected by aging. Intake of vitamin B12 in free-living
elderly people varies, and 0–50% of older adults have been
reported to have (dietary nonsupplemental) intakes below the
present recommendation. Dietary intake varies in older adults
as a function of food choices. Nearly 70% of vitamin B12 intake
in elderly subjects is from meat, eggs, and fish.
It has been shown that atrophic gastritis, common among
older adults, decreases the bioavailability of dietary vitamin
B12. In food, vitamin B12 is bound to a protein carrier and
must be released before it can be absorbed. Several mechanisms
can lead to the malabsorption of protein-bound vitamin B12 in
atrophic gastritis. Because of decreased or lack of gastric acid
production, protein digestion is impaired, and vitamin B12
cannot be released from its protein carrier. Another potential
factor in atrophic gastritis is that bacterial overgrowth in the
upper gastrointestinal tract can bind vitamin B12 and make the
vitamin unavailable. However, it has been shown that malab-
sorption of the protein-bound vitamin B12 in subjects with
atrophic gastritis can be reversed by treatment with tetracycline;
the absorption of crystalline vitamin B12 is not impaired in
atrophic gastritis. Most elderly people can absorb vitamin B12
from fortified food. Vitamin B12 deficiency could be a major
undetected problem in apparently healthy elderly people since
the symptoms of deficiency are vague. There is a controversy
over the lower limit of normal plasma levels of vitamin B12, so
elderly subjects with low plasma levels of vitamin B12 should be
monitored more closely.
‘Pernicious dementia’ and other changes in cognitive func-
tion caused by a deficiency of vitamin B12 have been described
in the literature. Patients with different forms of cognitive
impairment may have impaired vitamin B12 status; however,
this is mainly the result of low intakes. It has been reported that
anemia in old age is more likely related to folate deficiency
Elderly: Nutrition Requirements 483
than to vitamin B12 deficiency. The new Dietary Reference
Intakes (DRIs) for vitamin B12 have been increased compared
to earlier recommendations because a large fraction of elderly
people malabsorb this vitamin from food sources (i.e., protein-
bound vitamin B12); it is recommended that individuals older
than 50 years consume foods fortified with B12 or take a B12
supplement.
Biotin
Biotin deficiency occurs rarely; the recommendation for the AI
for biotin has been set at 30 mg day1 for all adults indepen-
dent of age, but there are hardly any data about elderly adults.
Since aging and excessive alcohol consumption can be linked
to an alteration in biotin metabolism, it is likely that elderly
individuals may be biotin-insufficient. Present evidence,
although minimal, suggests that biotin status is not a major
concern for older adults.
Vitamin C (ascorbic acid)
Vitamin C deficiency disease, scurvy, is rare but as new meta-
bolic properties of vitamin C are described, an AI of vitamin
C-rich foods remains a key for health maintenance, especially
in elderly adults. There are no age-related changes in absorption
and metabolism at median level intakes; it can be assumed that
any deficiency is related to poor intake. Dietary vitamin C
intakes vary mainly as a function of the presence of illness
associated with overall poor nutrient intake, institutionaliza-
tion, and age.
The intake of different dietary supplements, especially
including vitamin C, has increased and contributes consider-
ably to the nutrient intake of large segments of the elderly
population. Institutionalization, hospitalization, and illness
lead to a decrease of vitamin C intakes contributing to low
plasma levels.
It has been reported that lower ascorbic acid levels are seen
more frequently in elderly men than in elderly women; sex
differences in plasma ascorbic acid concentration might be
caused by a lower tubular reabsorption of vitamin C in elderly
men. Healthy centenarians had significantly lower vitamin C
plasma concentrations because of lower intakes.
Low levels of vitamin C in healthy elderly people can be
corrected easily by the administration of oral ascorbic acid sup-
plements, but when stopped, there may be a rapid decrease in
the blood levels. Elderly people with low vitamin C levels do not
necessarily show clinical symptoms, so it has been concluded
that there is no real need to raise the ascorbic acid levels in the
blood through supplementation. Because of the functions of
vitamin C, this conclusion might be incorrect. In view of what
is known or not yet known about the significance of low plasma
levels, a continuous, adequate daily intake should be ensured.
Ascorbic acid can be important in the pathogenesis of
chronic diseases of aging. Ascorbic acid has been found to be
potentially important in the prevention of atherosclerosis,
cancer, senile cataract, lung diseases, cognitive function, and
various degenerative diseases. Vitamin C supplements are pro-
moted to decrease lipid levels, thereby reducing the risk of
atherosclerosis. However, a 5-year vitamin C supplementation
trial had no markedly favorable effects on the serum lipid and
lipoprotein profile in an adult population.
484 Elderly: Nutrition Requirements
Vitamin C supplements do not prevent any of the chronic
diseases of aging, but neither do pharmacological doses of
vitamin C have a protective effect. The current recommendation
for ascorbic acid seems to be adequate for older adults,
although some individuals might find it difficult to achieve
this intake level from diet alone. Nevertheless, intake should
be strongly encouraged in elderly individuals who are institu-
tionalized, ill, or of lower socioeconomic status. An increased
consumption of fresh fruits and vegetables should be encour-
aged in elderly people.
Fat-Soluble Vitamins
Vitamin A
Meeting vitamin A needs by an increased ingestion of
carotenoids might actually be a safe strategy for older adults.
The vitamin A intake of many elderly people is below the
present recommendations, but their vitamin A plasma levels
remain well within normal limits. Vitamin A intake varies
widely, and up to 70% of elderly people, depending on age,
income, sex, race, geographic location, and socioeconomic
status, have vitamin intakes below two-thirds of the 1989 RDA.
There is evidence that vitamin A status is generally not
impaired in elderly people. In the Women’s Health Study, vita-
min A status was associated with an increased risk of hip fractures
only in postmenopausal women with vitamin D deficiency. This
is a well-known phenomenon and bone symptoms are a known
feature of vitamin A toxicity. Nevertheless, it is probably wise not
to encourage high vitamin A intakes in older adults, especially in
the presence vitamin D deficiency. A more reasonable strategy
for older adults is to get vitamin A from a higher intake of
carotenoid-containing foods. An increased intake of carotenoids
from fruits and vegetables was found to be associated with a
decreased cardiovascular mortality in elderly patients.
High vitamin A supplement use by older adults might
contribute to toxicity in elderly individuals. Acute hypervita-
minosis A is rare, but chronic toxicity can be seen in this age
group. Increased consumption of carotenoids from food and
vegetables to improve vitamin A status should be encouraged
in older adults.
Vitamin D
For men and women older than 70 years, the AI is 15 mg day1
(600 IU). Skin synthesis is important compared to intake from
food sources. Cutaneous synthesis of vitamin D ranges
between 80% and 100% of the total vitamin D content of the
body. It has been suggested that, with adequate sun exposure,
dietary sources of the vitamin become unnecessary. Fortified
foods, such as milk, contain considerable amounts of vitamin
D, which would cover the needs of older adults. Some of these
products are, however, not regularly consumed by elderly peo-
ple or are consumed only in small quantities. Even a marine-
based diet (e.g., salmon, sardines, and mackerel) seems to
be insufficient for the maintenance of an adequate vitamin D
status.
Data reported from the NHANES III survey indicate that
older adults (71 years and older) report low intakes of vitamin
D from food. Food fortification can lead to an improved
vitamin D nutriture in elderly people.
Dietary vitamin D intake is usually not able to prevent
vitamin D insufficiency especially during the winter months
when people have low sun exposure. Plasma and serum levels
of 25-hydroxyvitamin D3(25[OH]D3) decline with age in all
populations. Along with low intake, decreased synthesis in the
skin, decreased 1-a-hydroxylation in the kidney, decreased
sunlight exposure, decreased plasma-binding capacity for vita-
min D and vitamin D metabolites, and drug/nutrient interac-
tions all contribute to inadequate vitamin D nutriture. Dietary
fortification, enrichment of food, and ingestion of vitamin D
supplements have a high potential for improving vitamin D
status; the consequences are prevention of bone loss and
decreased fracture risk.
Vitamin E
Age-related changes resulting from oxidative reactions are found
in most organ systems; vitamin E and other antioxidants would
seem to have a role to play in aging and the aging process.
However, the consequences of a deficiency do not necessarily
imply that pharmacological doses of antioxidant vitamins will
have a protective effect on aging and related diseases. Neverthe-
less, all large supplementation trials have led to disappointing
results regarding the role of vitamin E in the modulation of age-
related diseases. Current evidence suggests that there is no
impairment of vitamin E absorption with aging.
Because of the antioxidative properties of vitamin E, the
effects of the vitamin in the prevention of atherosclerosis, cancer,
cataracts, central nervous system disorders such as Alzheimer’s
and Parkinson’s disease, immune function, impaired glucose
tolerance, and many more conditions have been proposed.
Despite all these potential relationships, the intake of high
doses of supplements cannot be recommended.
Vitamin K
Data about vitamin K status in older adults are scarce. Poor
vitamin K status has been associated with different chronic
diseases involving calcification processes (e.g., osteoporosis
and atherosclerosis). It is assumed that a dietary intake of
1 mg kg1 of body weight per day should be sufficient to main-
tain normal blood clotting in adults. Recommendations are
based on the maintenance and function of normal clotting
mechanisms. In elderly people who have atrophic gastritis
with a bacterial overgrowth, vitamin K status does not appear
to be affected when assessed by measuring the resistance to
warfarin therapy. Intake of vitamin K varies over a wide range,
primarily due to diet composition. Studies indicate that up to
one-third of elderly adults do not consume the recommended
amount of phylloquinone.
Although the function of vitamin K is usually associated
with blood coagulation, research conducted during the past
few years identified vitamin K as an important factor in bone
metabolism and thus in the development of osteoporosis.
Combined treatment of vitamin K2 and bisphosphonates in
postmenopausal women with osteoporosis demonstrated that
the combination treatment is more effective in the prevention
of new vertebral fractures than a single treatment of bispho-
sphonate alone. A high dose of vitamin K might have positive
effects on fracture risk. More studies are needed to clarify the

effect of vitamin K on bone health and at this time do not point
to any specific recommendation regarding an ideal intake of
vitamin K for bone-related issues.
Minerals
Sodium, potassium, calcium, and magnesium are considered
essential minerals for humans. Older people may have chal-
lenges in maintaining levels of these minerals within normal
limits because of decreases in renal function, by other renal
mechanisms designed to conserve or excrete excessive amounts
of these minerals, and certain medications or disease states. The
DRIs and the Dietary Guidelines for Americans serve as guides
for the intakes of these nutrients from foods and supplements
(e.g., for calcium). It has been suggested that the dietary intakes
of sodium are too high and the intakes of potassium, calcium,
and magnesium are too low among older adults.
Sodium
Dietary sodium is available through many sources, particularly
processed, preserved, and canned foods. It is actually a greater
challenge to avoid dietary sodium for individuals on sodium
restrictions.
Potassium
Age alone does not appear to affect the ability to maintain the
concentration gradient of potassium. Intracellular potassium
stores can be depleted in a variety of clinical conditions
commonly seen in elderly patients (metabolic and respiratory
acidosis, congestive heart failure, cirrhosis, and uremia) with
serum potassium concentrations remaining within normal
limits. Dietary potassium is found in a wide variety of foods,
and there are rarely disturbances of potassium status due to
dietary factors.
Calcium
Both hypocalcemia and hypercalcemia occur with more fre-
quency in older adults because specific disease conditions that
cause challenges to calcium balance are more common in
elderly people. More importantly, the age-related loss of bone
calcium occurs in both sexes, but more commonly in women,
it is related to a variety of variables such as diet, hormone
levels, bone density, and other factors. Nevertheless, this loss
leads to the development of osteoporosis and an increased risk
of fractures. Dietary factors play an important role in the age-
related loss of bone calcium. In older individuals, calcium
intake is generally low and is associated with reduced absorp-
tion of calcium. Lack of vitamin D (hypovitaminosis D) is
often associated with increased age. Because vitamin D is the
major regulator of intestinal calcium absorption, the cumula-
tive effect of a calcium intake deficit and an inadequate intake
and generation of active vitamin D is a negative calcium bal-
ance. This stimulates parathyroid hormone (PTH) release,
increasing bone calcium resorption and risk for osteoporotic
fractures, a major cause of morbidity and mortality in the
elderly population.
Elderly: Nutrition Requirements 485
Magnesium
Poor magnesium status has been associated with numerous
age-related conditions. Gastrointestinal disorders and alcohol
consumption are the most commonly encountered problems
in older adults. Hypomagnesemia produces neuromuscular
and psychiatric symptoms, including neuromuscular hyperir-
ritability, tetany, seizures, muscle weakness, vertigo, gross
tremors, and mental changes (e.g., irritability and aggressive-
ness). Linking these symptoms with hypomagnesemia may
be elusive because serum levels will generally remain within
normal limits.
Barriers to Maintaining Adequate Nutrition
Ingesting an adequate diet may be affected by a variety of other
secondary factors associated with advancing age. Oral health
status; swallowing difficulties; changes in vision, smell, and
taste; socioeconomic factors; polypharmacy; drug/nutrient
interactions; cognitive status (delirium, depression, and
dementia); and functional status all constitute potential bar-
riers to maintaining adequate nutritional status and the
consumption of a nutrient-dense, nutritionally adequate diet.
For elderly adults who are living on fixed income such as
social security or other retirement incomes, food can be a
significant part of their living expenses. Living conditions,
cooking facilities, transportation, and seasonal variations in
the price of specific foods, for example, fresh fruits and vege-
tables, can have an impact on nutritional status.
Polypharmacy is a problem among many elders who are
taking multiple medications. There are often drug/drug, drug/
nutrient, and drug/herbal product interactions that may affect
appetite, taste, the efficacy of medication treatments and may
be the etiology of gastrointestinal disturbances. A review of the
prescription, over-the-counter, and herbal remedies is war-
ranted on an annual basis to minimize the effect that drugs
may have on the nutritional status of an elderly person.
Cognition may have a profound impact on dietary intake;
dementia, delirium, and depression will affect appetite, the
perception of food, activity level, and the ability to feed oneself.
Activity level, and need for energy calories, may be quite
impacted by dementia, with some individuals becoming hyper-
active and others becoming very sedentary.
Depression may have a longer and more profound effect on
nutritional status than dementia. Depression has an impact on
appetite, weight loss, and poor oral intake. It may be associated
with a broad range of problems associated with chronic dis-
ease, medications, grief, loneliness, and others.
Conclusion
Encouraging older adults to eat more complex carbohydrates
rather than simple carbohydrates can contribute to adequate
dietary fiber intake. Reduction of saturated fat, with an
increased emphasis on consumption of poly- and monounsat-
urated fats, and decreased overall fat intake compensate for
lower basal energy requirements and a decreased activity level
often encountered in older adults. Providing adequate protein
486 Elderly: Nutrition Requirements
of high biological value is essential in older adults as is meeting
fluid needs. Attention to overall fluid intake in healthy free-
living, chronically ill, functionally dependent, or acutely ill
elderly people is key to maintaining optimal health in any
person, but particularly important for this age group. Needs
for these macronutrients (carbohydrate, fat, protein, and fluid)
can be met with awareness of individual requirements,
imagination, and attention to daily intake.
Not all micronutrients are affected by aging although
chronic disease may be a factor in the adequacy of intake,
digestion, absorption, or metabolism of various vitamins and
minerals. The water-soluble vitamins (B complex and vitamin
C) require attention because they cannot be stored and are
involved in many metabolic pathways that affect overall health
and nutritional status. Among the fat-soluble vitamins, vita-
mins A and D require some attention. Vitamin D has been an
issue in recent years due to indications that many adults have
low levels of dietary vitamin D, particularly older adults.
Barriers to adequate consumption of a nutritious diet
including, but not limited to, cognitive status, oral health,
deficits in sensory perception and functional status, socioeco-
nomic factors, swallowing disorders, polypharmacy, and other
changes that occur with age must all be assessed. Screening
older adults for potential risks for nutrition problems is a
highly recommended, cost-effective, and preventive strategy.
The specific recommendations concerning nutrient intakes
and food-based dietary guidelines for older adults – with par-
ticular emphasis on guidelines for promoting physical activity
among older and prevention of chronic noncommunicable
diseases – can contribute to healthy aging.
See also: Antibiotics and Drugs: Drug–Nutrient Interactions;
Beverage: Health Effects; Bioavailability of Nutrients; Dietary
References: US; Energy: Intake and Energy Requirements; Malnutrition:
Concept, Classification and Magnitude; Malnutrition: Prevention and
Management; Osteoporosis; Protein: Requirements; Single Cell
Proteins; Trace Minerals and Trace Elements.
Further Reading
Bollwein J, Volkert D, Diekmann R, et al. (2013) Nutritional status according to the mini
nutritional assessment (MNA) and frailty in community dwelling older persons: a
close relationship. The Journal of Nutrition, Health & Aging 17(4): 351–356.
Chernoff R (ed.) (2014a) Geriatric nutrition: the health professional’s handbook, 4th ed.
Sudbury, MA: Jones & Bartlett.
Chernoff R (2014b) Nutritional screen monitoring tools. In: Berdanier C, Dwyer J, and
Heber D (eds.) Handbook of nutrition and food, 3rd ed. Boca Raton, FL: CRC Press.
Doets EL, Cavalaars AEJM, Dhonukshe-Ruthen RAM, Veer PV, and de Groot LCPGM
(2011) Explaining the variability in recommended intakes of folate, vitamin B12, iron
and zinc for adults and elderly people. Public Health Nutrition 15: 906–915.
EURONUT-SENECA-Investigators (1991) Nutritional status: blood vitamins A, E, B6,
B12, folic acid, and carotene. European Journal of Clinical Nutrition 45(Suppl. 3):
63–82.
Ford ES and Dietz WH (2013) Trends in energy intake among adults in the United
States: findings from NHANES. American Journal of Clinical Nutrition 97: 848–853.
Frankenfield D, Roth-Yousey L, and Compher C (2005) Comparison of predictive
equations for resting metabolic rate in healthy, non-obese individuals: a systematic
review. Journal of the American Dietetic Association 105(5): 775–789.
Institute of Medicine (1997) Dietary references intakes for calcium, phosphorus,
magnesium, vitamin D, and fluoride. Washington, DC: National Academy Press,
pp. 1–432.
Institute of Medicine (2000) Dietary reference intakes for vitamin C, vitamin E, selenium
and carotenoids. Washington, DC: National Academy Press.
Institute of Medicine (2001a) Dietary reference intakes for vitamin A, vitamin K, arsenic,
boron, chromium, copper, iodine, manganese, molybdenum, nickel, silicon,
vanadium, and zinc. Washington, DC: National Academy Press.
Institute of Medicine (2001b) Dietary reference intakes for thiamin, riboflavin, niacin,
vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington,
DC: National Academy Press.
Institute of Medicine (2004) Dietary references intakes for water, potassium, sodium,
chloride, and sulfate. Washington, DC: National Academy Press pp. 1–617.
Institute of Medicine Panel on Macronutrients and Standing Committee on the Scientific
Evaluation of Dietary Reference Intakes (2005) Dietary reference intakes for energy,
carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids.
Washington, DC: National Academies Press.
Jensen GL and Hsaio PY (2010) Obesity in older adults: relationship to functional
limitation. Current Opinion in Clinical Nutrition and Metabolic Care 13: 46–51.
Masters RK, Powers DA, and Link BG (2013) Obesity and US mortality risk over the
adult life course. American Journal of Epidemiology 177(5): 431–442.
Maxmen A (2012) Calorie restriction falters in the long run. Nature 488.
Nutrition and the Ageing Population, A Review of Current Policy and Science, Food and
Health innovation, 2012. http://www.foodhealthinnovation.com/media/5810/
nutrition_and_the_ageing_population.pdf.
Popkin BM, D’Anci KE, and Rosenberg IH (2010) Water, hydration and health. Nutrition
Reviews 68(8): 439–458.
Ross CA, Caballero B, Cousins RJ, Tucker KL, and Ziegler TR (eds.) (2012) Modern
nutrition in health and disease, 11th ed. Philadelphia, PA: Williams & Wilkins.
Vincent GK and Velkoff VA (2010) The next four decades: the older population in the
United States: 2010 to 2050. Washington, DC: US Dept of Commerce, Economics
and Statistics Administration, US Census Bureau, Current Population Reports,
pp. 25–1138.
WHO (2002). Keep fit for life, Meeting the nutritional needs of older persons. http://
www.who.int/nutrition/publications/olderpersons_nutritionalneeds/en/.
WHO (2012). Strategy and action plan for healthy ageing in Europe, 2012–2020, WHO
Regional Committee for Europe.
World Health Assembly resolution (2012). WHA65.3 on strengthening
noncommunicable disease policies to promote active ageing. Geneva: World Health
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 Emerging Foodborne Enteric Bacterial Pathogens
SJ Forsythe, Nottingham Trent University, Nottingham, UK
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Compared to clinical infectious diseases, the number of cur-
rently recognized foodborne pathogens seems small. In part,
this is due to general procedures of food production, for
example, cooking, acidification, and dehydration, which over
the centuries and in different cultures have been refined and
become a self-proved effective means of food preparation.
However, with changes in the sourcing of food ingredients,
the need for large-scale food preparation practices and longer
storage periods along with changes in eating habits has ensured
a continuation of food-related illnesses. Such infections are an
important cause of morbidity and mortality worldwide and
have a major impact on public health. The true worldwide
burden of diseases caused by foodborne pathogens is largely
unknown. At best, we have data indicating trends in infections
for a few pathogens in a few number of developed countries.
The European Food Safety Authority has defined ‘emerging
risk to human, animal and/or plant health’ as a risk resulting
from a newly identified hazard to which a significant exposure
may occur or from an unexpected new or increased significant
exposure and/or susceptibility to a known hazard. Hence, in
order to detect such risks, adequate surveillance and monitor-
ing programs need to be in place. There are many food safety
monitoring systems at national and international levels. These
are summarized in Table 1. Examples are the ‘Rapid Alert
System for Food and Feed’ (RASFF) in Europe and WHO–
INFOSAN. In general, these systems identify problems such
as the increased reported occurrence of a foodborne hazard
retrospectively. Consequently, any intervention is initially reac-
tive instead of predictive.
In order to determine the global health burden of foodborne
illness, the WHO set up the Foodborne Disease Burden Epide-
miology Reference Group. This is composed of internationally
renowned experts in a broad range of disciplines relevant to
global foodborne disease epidemiology; http://www.who.int/
foodsafety/foodborne_disease/ferg/en/index3.html. They have
estimated that in 2005, 1.8 million people died from diarrheal
disease, and these were mainly attributable to contaminated
food and water sources. In the United States, foodborne illnesses
affect up to 80 million people and cost an estimated US$5
billion on an annual basis. Additionally, our understanding of
such infections is changing with the recognition of previously
unrecognized and previously well-recognized pathogens, which
have increased in their prevalence or become associated with
new food vehicles.
The major organisms associated with foodborne disease are
bacteria due to their ease of isolation and identification com-
pared to viruses and fungi. However, with improved global
surveillance systems, as well as improved isolation and identifi-
cation methods, previously unrecognized foodborne infectious
organisms are being recognized. With a global economy and the
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00248-8
international transportation of food, the need to attribute
human infections to food vehicles is warranted for their control.
The study of emerging foodborne enteric pathogens is both
exciting and daunting at the same time. The opportunity to
recognize and define an emergent pathogen leading to its con-
trol and reduced risk is an admirable goal, yet to publicize the
emergence of an enteric pathogen based on limited informa-
tion and initiate action could be regarded as scaremongering
and alarmist. Given the limited number of organisms, which
are routinely monitored in food, it may take considerable time
before there is sufficient body of evidence of its significant
presence such that it might be argued that by the time the link
between the clinical presentations and food as a vector has been
established, the organism will no longer be ‘emerging’ and
therefore outside the scope of the study. The word ‘emerging’
implies the emergence de novo of the pathogen, yet for some, the
term ‘recently recognized’ is more appropriate as the organism
had been previously described, but the link with foodborne
disease may be new. Some organisms in this article are ‘emer-
gent’ or ‘recently recognized’ pathogens for which food has
been established as a vector, but some will not be so strongly
associated but could be foodborne. It may be argued that the
latter should not be included – however, this begs the question
of how will we ever know unless we look. Our powers of
microbial epidemiology are still mainly limited to bacteria as
we are still largely culture-dependent. In time, next-generation
sequencing (NGS) will enable real-time genomic analysis of
infections, but for now, our future predictions remain misty
and unclear.
Illness caused by foodborne pathogens represents an
important economic and public health burden worldwide.
Based on a yearly case estimate and recent US census data, an
estimated 25% of the US population is affected by foodborne
illness each year. Viral foodborne pathogens are thought to
contribute to 50% of all foodborne illness cases in the United
States. Rates of foodborne illness in many developed countries
are similar. While exact numbers are difficult to estimate, it is
thought that the burden of foodborne illness in developing
countries is even higher.
Since 1996 in the United States, FoodNet has been an active
surveillance program for laboratory-confirmed infections due
to seven commonly reported bacterial foodborne pathogens:
Salmonella, shiga toxin-producing Escherichia coli O157, Shigella
spp., Vibrio spp., Yersinia spp., Campylobacter spp., and Listeria
monocytogenes. This surveillance covers 15% of the US popula-
tion. Over the period 1996–2005, there had been 121 536 cases
including 552 deaths, for which 215 and 168 were due to were
among persons infected with Salmonella and Listeria, respec-
tively. Useful as this surveillance is, it does not cover the myriad
of foodborne pathogens and toxigenic agents.
In the United States, there are an estimated 9.4 million cases
of foodborne illness, which can be attributed to 31 known
487
488 Emerging Foodborne Enteric Bacterial Pathogens
Table 1 Early identification systems for emerging foodborne hazards
Country Organization Activity
The United FSA Food Standards Agency
Kingdom (FSA)
The ISIS Day-to-day changes in
Netherlands frequency of all
communicable diseases
European European Centre for Provides a structured and
Disease Prevention systematic approach to
and Control (ECDC) the control of
communicable diseases
and other serious health
threats in partnership
with national health
protection agencies.
Issues warnings to EU
member states through
the early warning and
response system (EWRS)
Rapid alert system on Centralized reporting of
food and feed food hazards (physical,
(RASFF) chemical, biological)
EU: FVO Food and Veterinary Office
(FVO) promotes effective
control systems in food
safety and checks on
compliance
The United CDC: FoodNet Determines the exact
States burden of foodborne
diseases, monitors
foodborne disease
trends in the United
States, and relates
foodborne disease to
specific foods
CDC: eFORS Electronic Foodborne
Outbreak Reporting
System
CDC: PulseNet National network of public
health laboratories that
undertake pulsed-field gel
electrophoresis analysis
of bacteria that may be
foodborne
International WHO: GPHIN Global Public Health
Intelligence Network
WHO: INFOSAN International Network of
Food Safety Authorities
Network disseminates
urgent information
OIE Office International des
Epizooties (OIE) sets
standards for sanitary
practices in the diagnosis
of diseases in animals
and animal products. This
includes the World
Animal Health
Information System
(WAHIS)
After Marvin, H. J. P., Kleter, G. A., Prandini, A., Dekkers, S., and Bolton, D. J. (2009)
Early identification systems for emerging foodborne hazards. Food and Chemical
Toxicology 47, 915–926.
pathogens (2011 data). Of these, 14 pathogens account for
95% of the illnesses and hospitalizations and 98% of deaths.
The associated costs are estimated as $14.0 billion (ranging
from $4.4 to $33.0 billion) of which 90% are attributable to
five pathogens: nontyphoidal Salmonella enteric, Campylobacter
spp., L. monocytogenes, Toxoplasma gondii, and norovirus.
However, there were an additional estimated 38.4 million
cases (80%), which were due to ‘unspecified agents, including
known agents with insufficient data to estimate agent-specific
illness, known agents not yet recognized as causing foodborne
illness, substances known to be in food but of unproven path-
ogenicity, and unknown agents.’ These unattributed cases
resulted in  71 878 hospitalizations and 1686 deaths.
Although this author is not claiming that all these cases are
due to emerging foodborne pathogens, the article illustrates
that there is still scope for previously unrecognized pathogens
to be discovered. Toxin-induced gastroenteritis due to Bacillus
cereus is a well-recognized foodborne pathogen, yet the lack
diagnostic tools means its incidence is highly likely to be
underestimated and not an emergent pathogen as such.
The microbiological safety of food is dependent upon mul-
tiple variable ‘from farm to fork’ along the food chain. The
combined advances in the Internet and culture-independent
(i.e., DNA sequencing) methodologies are enabling improve-
ments in monitoring and surveillance, though the scenario is
constantly changing with increased global transportation of
foods and food ingredients, aging populations, and changes
in eating habits. Against this background, this article reviews
the current situation regarding emergent enteric foodborne
pathogens. In particular, reference will be made to previously
unrecognized vehicles, for example, fresh produce, and serious
issues to public health following the acquisition of antimicro-
bial resistance. Additionally, previously unknown foodborne
pathogens, many of which are zoonotic, are being recognized
through improved methods of surveillance and identification.
This article will not consider well-recognized foodborne
pathogens: Campylobacter jejuni, Salmonella enterica serovars,
E. coli O157 and O104, Vibrio cholerae, V. parahaemolyticus,
Yersinia enterocolitica, Listeria monocytogenes, Clostridium perfrin-
gens, Staphylococcus aureus, and Bacillus cereus. For information
on these organisms, please consult the specific chapters as
listed at the end under ‘See also.’ Such organisms are often
formally controlled via microbiological specifications whether
in international policies or producer–distributor agreements.
Essentially, improvements in the microbiological safety of
foods have been largely driven by public demand in response
to disease outbreaks, and consequently, less-recognized path-
ogens are overlooked. Focussing on a particular potential
source of infection will invariably lead to discoveries, as evi-
denced by the emphasis of bats as vehicles of bacterial disease,
which has opened a whole field of previously unrecognized
bacterial and virus route of transmissions. Awareness and sur-
veillance of viral foodborne pathogens are generally poor and
primarily limited to norovirus, hepatitis A, and rotaviruses.
Although many foodborne parasites are recognized, only Asca-
ris, Cryptosporidia, and Trichinella are effectively monitored in
foods. Their epidemiology of such parasites through the food
chain is poorly understood. The issues of viral and parasitic
foodborne pathogens are not considered in this article due to
the lack of information.

Bacterial Enteric Pathogens
Aeromonas Species
Description: Aeromonas genus, composed of 17 hybridization
groups and 14 phenospecies
Phenotypic differentiated groups: A. hydrophila, A. caviae, and A.
sobria
Clinical presentations: Traveler’s diarrhea
Sources: Water, fish, shellfish, meats, dairy products, and fresh
vegetables
Detection methods: Various culture media, for example, starch
ampicillin agar (SAA) and BIBG
The genus Aeromonas was first proposed in 1936 for rod-
shaped Enterobacteriaceae, which possessed a polar flagellum.
Since then, there have been many taxonomic revisions, and
currently, it forms the family Aeromonadaceae. They are Gram-
negative, rod-shaped, facultative anaerobic, nonspore-forming
bacteria, which are widely distributed in the soil and aquatic
environments. Members of the genus are divided into three
phenotypically differentiated groups: Aeromonas hydrophila,
Aeromonas caviae, and Aeromonas sobria. Currently, the genus
is composed of 17 DNA hybridization groups or genomospe-
cies and 14 phenospecies.
The Aeromonas genus includes psychrophiles and
mesophiles and is associated with various diseases of warm-
and cold-blooded animals. They have been isolated from fish,
shellfish, meats, dairy products, and fresh vegetables. However,
only few foodborne outbreaks have been documented. Some
studies have shown that some motile Aeromonas species are
becoming food- and waterborne pathogens of increasing
importance. They have been associated with several foodborne
outbreaks and are progressively being isolated from patients
with traveler’s diarrhea.
A. hydrophilia is present in all freshwater environments and
in brackish water. It has frequently been found in fish and
shellfish and in market samples of red meats (beef, pork, and
lamb) and poultry. The organism is able to grow slowly at 0  C.
It is presumed that given the ubiquity of the organisms, not all
strains are pathogenic. Some strains of A. hydrophila are capable
of causing illness in fish and amphibians as well as in humans
who may acquire infections through open wounds or by inges-
tion of a sufficient number of the organisms in food or water.
A. hydrophila may cause gastroenteritis in healthy individuals or
septicemia in individuals with impaired immune systems or
various malignancies. A. caviae and A. sobria also may cause
enteritis in anyone or septicemia in immunocompromised
persons or those with malignancies.
There is controversy as to whether A. hydrophila is a cause of
human gastroenteritis. The uncertainty occurs because volun-
teer human feeding studies (1011 cell dose) have failed to
demonstrate any associated human illness. However, its pres-
ence in the stools of individuals with diarrhea, in the absence of
other known enteric pathogens, suggests that it has some role in
disease and has been included in this survey of foodborne
pathogens. Similarly, A. caviae and A. sobria are putative patho-
gens associated with diarrheal disease, but as of yet, they are
unproved causative agents. General symptoms of A. hydrophilia
gastroenteritis are diarrhea, abdominal pain, nausea, chills
and headache, dysentery-like illness, and colitis. Additional
Emerging Foodborne Enteric Bacterial Pathogens 489
symptoms may include septicemia, meningitis, endocarditis,
and corneal ulcers.
Two types of gastroenteritis have been associated with A.
hydrophila: a cholera-like illness with severe diarrhea and a
dysenteric illness characterized by loose stools containing
blood and mucus. The infectious dose of this organism is
unknown. A. hydrophila may spread throughout the body in
the bloodstream and cause a general infection in persons with
impaired immune systems. Those at risk are individuals suffer-
ing from leukemia, carcinoma, and cirrhosis and those treated
with immunosuppressive drugs or who are undergoing cancer
chemotherapy.
A. hydrophila produces a cytotonic enterotoxin, hemolysin,
acetyl transferase, and phospholipase. Together, these viru-
lence factors may account for the organism’s pathogenicity.
The detection of genes responsible for Aeromonas virulence is
a vital tool in establishing the potential pathogenicity of the
bacteria, as these virulence genes may act alone or in synergy in
the establishment of infections.
A number of selective and differential isolation media have
been developed for the recovery of Aeromonas species from the
environment, foods, and clinical specimens. Since comparative
studies demonstrate that no single medium results in the opti-
mal recovery of aeromonads, combinations of different isolation
media and methods are used including direct plating, membrane
filtration, and multiple tube tests for determining most probable
numbers. SAA and bile salts–inositol–brilliant green (BIBG)
agar with initial enrichment in alkaline peptone water (APW)
or tryptose broth containing ampicillin (TSB-30, ampicillin
30 mg l 1) are recommended simultaneously with commer-
cially available media such as Aeromonas medium (Ryan’s
medium). Starch glutamate ampicillin penicillin (SGAP-10)
medium has been used to detect aeromonads from foods. The
isolation of aeromonads from contaminated samples such as
feces requires the use of selective and differential media such as
MacConkey agar, cefsulodin–irgasan–novobiocin (CIN) agar,
and blood agar with ampicillin (10 mg l 1 ampicillin). To facil-
itate recovery of aeromonads from highly contaminated samples
such as feces, enrichment broths such as APW are incubated
overnight and subcultured onto CIN agar and blood agar with
ampicillin. Culture plates are then incubated aerobically at 35  C
for 24–48 h. Aeromonas species produce distinctive colonies,
with or without hemolysis on blood agar. Colonies are screened
by carrying out oxidase test and identified using biochemical
methods or commercially available bacterial identification kits.
Arcobacter Species
Description: Arcobacter butzleri, A. cryaerophilus, and A. skirrowii
are the primary species of interest.
Clinical presentations: Persistent and watery diarrhea.
Sources: Contaminated drinking water and food.
Detection methods: mCCDA and CAT.
Campylobacter jejuni and C. coli are well recognized as the major
causes of human gastroenteritis. Meanwhile, a number of other
Campylobacter species and related organisms have also been
recognized as causing animal if not human cases of gastroen-
teritis. These include Campylobacter concisus and Arcobacter spe-
cies. While there are a relatively few reported cases of infection
490 Emerging Foodborne Enteric Bacterial Pathogens
due to Arcobacter, this is probably due in part to the lack of
routine use of appropriate isolation media.
A number of reviews on Arcobacter spp. have demonstrated
that the organism is a cause of human pathogen and is
associated with food products. One of the first significant
studies of Arcobacter started in 1995 in Belgium where the
WHO Centre for Campylobacter specifically looked for non-
jejuni/Campylobacter coli-like organisms (CLOs) in fecal sam-
ples. It is plausible that A. butzleri and A. cryaerophilus, which
were first isolated from aborted bovine fetuses and later from
porcine fetuses, will in the future be more fully recognized as of
significant human importance. Currently, there is increasing
awareness of their role as veterinary pathogens, but there are
relatively few human cases.
The Arcobacter genus was formerly known as aerotolerant
campylobacters and CLOs. But improved methods of taxo-
nomic evaluation have led to the recognition of the genus
Arcobacter. There are currently 17 recognized Arcobacter species,
and these have been isolated from various sources. Of particu-
lar interest are A. butzleri, A. cryaerophilus, and A. skirrowii, as
these have been associated with human cases of diarrhea, the
probable transmission routes being through the ingestion of
contaminated drinking water and food. These three species are
also veterinary pathogens causing porcine abortions. A. butzleri
serotypes 1 and 5 are regarded as the primary human
pathogens; however, no epidemiological studies have yet
shown the transmission of the organism through the food
chain to humans. The situation is, however, reminiscent of C.
jejuni and L. monocytogenes. These organisms were recognized
as veterinary pathogens many years before the medical micro-
biologists used suitable isolation media to samples from
patients suffering from gastroenteritis. Arcobacter species have
been isolated from a range of food sources (Table 2) using a
range of methods, some of which may favor certain species
more than others. Water may also be a vector of Arcobacter
transmission to animals and humans.
In general, Arcobacter spp. are more aerotolerant and have
lower growth temperature limit than Campylobacter spp. On
blood-based agars, Arcobacter spp. produce round, off-white or
grayish colonies, 2–4 mm in diameter after 3 days of incuba-
tion. The colonies are generally small, nonpigmented, and
Table 2 Incidence of Arcobacter in animals and animal products
Source % n Site
Pigs 0–89.9 299a Ground pork
7 100 Pork
Cows 1.5 68 Minced beef
2.2 90 Beef
Poultry 96.8 125 Chicken carcasses
24.1 220 Turkey meat
Ducks 50 10 Carcasses
52 170 Carcasses
Water 100 10(4)b
a Description: Campylobacter concisus, a fastidious Campylobacter
Only tested for A. butzleri.
b
All samples were positive using FISH, whereas Arcobacter was only isolated from four
samples.
Adapted from Forsythe, S. J. (2006). Arcobacter. In: Motarjemi, Y. and Adams, M.
(eds.), Emerging foodborne pathogens. Cambridge: Woodhead Publishing, references
therein.
convex with entire edges. Swarming has been reported on
fresh agar. Brain Heart Infusion Agar supplemented with
0.6% (w/v) yeast extract and 10% (w/v) blood agar has been
used recently for routine culturing.
Arcobacter isolates can be presumptively identified by their
shape (small comma-shaped or spiral rods) and motility (dart-
ing or corkscrew motility). They can be easily distinguished
from Campylobacter and related genera by their ability to grow
in air at 25  C. The main phenotypic traits used for Arcobacter
species differentiation are catalase activity, nitrate reduction,
cadmium chloride susceptibility, microaerophilic growth at
20  C, and growth on MacConkey agar and in the presence of
3.5% NaCl and 1% glycine. Organic acids and amino acids are
used as carbon sources. Hydrogen is not required for growth.
All Arcobacter isolates hydrolyze indoxyl acetate. A simple diag-
nostic characteristic useful for the presumptive identification of
Campylobacter jejuni, C. coli, and Arcobacter spp. is the cadmium
chloride test.
Arcobacters appear resistant to antimicrobial agents typi-
cally used in the treatment of diarrheal illness caused by Cam-
pylobacter spp., for example, erythromycin, other macrolide
antibiotics, tetracycline, and chloramphenicol. Isolation of
arcobacters requires selective media such as mCCDA and CAT.
Identification can subsequently be achieved using 16S rRNA
probes and genotyped.
The occurrence of arcobacter-related diseases may be under-
estimated due to the lack of surveillance and optimized detec-
tion procedures. Examination of human and veterinary clinical
specimens for the presence of Arcobacter species is rarely per-
formed, and in most cases, suboptimal procedures are used. In
addition, little is known about the risk factors for human
infection. The most extensive study to date on this matter was
undertaken on a total of 67 599 stool samples over an 8-year
period. A. butzleri was the fourth most common CLO isolated.
It was more frequently associated with symptoms of a
persistent and watery diarrhea than C. jejuni.
The isolation of A. butzleri was associated with cases of
persistent and watery diarrhea and less associated with bloody
diarrhea compared to C. jejuni. The pathogenicity and viru-
lence mechanisms of Arcobacter spp. are still poorly understood
and have only been studied in A. butzleri, A. cryaerophilus, A.
skirrowii, and A. cibarius. Analysis of the A. butzleri strain RM
4018 genome shows the presence of several putative virulence
genes in the organism, such as ciaB, cj1349, and cadF. These are
homologous to genes associated with pathogenicity in other
closely related organisms. The ciaB gene encodes an invasion
protein injected directly into the cytoplasm of the host cells
through a secretion system. The cj1349 gene encodes for pro-
teins that enable adhesion to host cells by binding specifically
to fibronectin, and the CadF protein also induces the internal-
ization of bacterial cells by the activation of GTPases.
Campylobacter concisus
species
Clinical presentations: Isolated from healthy and diarrheal
individuals
Sources: Contaminated food and water
Detection method: No specific method available to date
 Emerging Foodborne Enteric Bacterial Pathogens 491

Despite considerable efforts in surveillance and improvements

2002 Outbreak at the University of Tennessee investigated
in isolation methods, only about half of the reported intestinal
by CDC and FDA. Fatal case linked to the use of
infectious cases are attributable to a named organism. While a
powdered formula
large number of cases are related to C. jejuni and C. coli
FDA Cronobacter (and then E. sakazakii) detection
infections, there is evidence that other Campylobacter species
method; Bacteriological Analytical Manual
may also be causative agents. Epidemiological evidence is
2004 1st FAO–WHO risk assessment meeting on
poor, but it is reasonable to assume that more fastidious
microbiological safety of PIF
Campylobacter species, such as C. concisus, will not be recovered
Development of first chromogenic differential agar
as efficiently as the better understood C. jejuni. In common with
2006 2nd FAO–WHO risk assessment meeting
other campylobacters, C. concisus is a Gram-negative, curved,
ISO/TS 22964 method released
microaerophilic bacterium. It can be isolated from the human
2008 3rd FAO–WHO risk assessment meeting
oral cavity and fecal samples from healthy and diarrheic
Revised Codex Alimentarius Commission guidelines
patients. As a consequence, its primary pathogenic potential is
for microbiological specifications for PIF released, to
uncertain. In many diarrheal cases, C. concisus is isolated as the
include Cronobacter as a named pathogen
only potential intestinal pathogen, and hence, the need to
Cronobacter genus defined, composed of 5 species
determine its role in human enteric disease is evident.
2009 Open-access seven-loci multilocus sequence typing
Studies to date suggest C. concisus is composed of various
(MLST) scheme established to replace ambiguous 16S
genotypes, which may vary in their pathogenic potential.
rDNA analysis; www.pubMLST.org/cronobacter
However, considerable further work is needed to determine
2010 First whole sequence for C. sakazakii published; isolate
the risk of C. concisus to human health and vehicles of infec-
from the University of Tennessee outbreak
tion. Given the habitat of the organism as being in the
2011 C. sakazakii sequence type 4 (clonal lineage)
intestinal tract of healthy individuals, it is plausible that
association with neonatal meningitis determined
contaminated food and water could be the sources of infec-
C. condimenti and C. universalis recognized
tion. The organism has been included here to raise awareness
2012 First pan-genome analysis of Cronobacter genus based
of this emerging pathogen and its possible contribution to
on 14 genomes
foodborne infections.
Revised FDA method of detection; Bacteriological
Analytical Manual
2013 www.pubMLST.org/cronobacter expanded to include
the following:
Cronobacter spp., Formerly Enterobacter sakazakii
(a) Tax-MLST established, extension of MLST to
Description: Cronobacter genus. Seven recognized species include genotyping of ompA and rpoB
proposed: C. condimenti, C. dublinensis, C. malonaticus, C. (b) Cronobacter BIGSdb searchable repository for all
muytjensii, C. sakazakii, C. turicensis and C. universalis. Gram- published Cronobacter sp. genomes established
negative, frequently motile, facultative aerobic rods; mem-
bers of the Enterobacteriaceae.
Before 1980, yellow-pigmented Enterobacter cloacae-like
Clinical presentations: Infections in all age groups, particularly
strains were called Enterobacter sakazakii. Despite earlier spo-
neonates, infants <6 months of age, and the elderly. Life-
radic cases of neonatal and infant infections, the organism
threatening symptoms in neonates and infants <6 months
was highlighted following an outbreak in a university
of age include meningitis, necrotizing enterocolitis, and
hospital in Tennessee (the United States). One neonate died
respiratory infections.
from meningitis, two died from suspected infections (tracheal
Sources: Infections in neonates associated with contaminated
aspirate-positive), and seven were colonized (six fecal-positive
reconstituted powdered infant formula (PIF). Ubiquitous
and one urine-positive) with the same strain according to
in nature, particularly plant material. Human carriage.
pulsed-field gel electrophoresis (PFCE) profiling. The source
Detection methods: Cultivation on chromogenic agar, followed
was attributed to powdered formula. Since such neonates
by sequencing of fusA or rpoB gene for phylogenetic
only have a limited number of exposure routes, the
analysis.
FAO–WHO instigated three risk assessments that have
Cronobacter is possibly the archetypal recently recognized bac- directly contributed to the 2008 Codex Alimentarius Com-
terial pathogen, which has been associated with foodborne mission revisions in the microbiological specifications of PIF.
infection, albeit via one particular food source: PIF. The con- Prior to this, the number of Enterobacteriaceae (except for
siderable progress in better understanding and control of this Salmonella) permitted in PIF was <100 cfu g 1. These specifi-
organism is undoubtedly due to policy issues. Using seven-loci cations had not been revised since 1979. However, following
multilocus sequence analysis, the close relatedness of the the concern of the serious implications following Cronobacter
Cronobacter species is shown in Figure 1. According to phylo- infections of neonates, the microbiological criteria changed
genetic analysis, these major divisions formed about 41 mil- such that Cronobacter should not be detectable in 10 g test
lion years ago, and further, host adaptation has occurred as will volume quantities of PIF.
be considered later with the specific case of C. sakazakii In order to implement the specifications, improved under-
sequence type 4. standing of the organism and development of detection
Short timeline: Pre-2002, various outbreaks in neonatal methods are needed. The focussed attention showed Enterobac-
intensive care units and sporadic cases reported. ter sakazakii should be regarded as a separate genus and was
492 Emerging Foodborne Enteric Bacterial Pathogens

100 C_sakazakii
99 C_sakazakii-ST4
100 C_malonaticus
C_universalis
100 84 C_turicensis

77 C_muytjensii
C_dublinensis
97
C_condimenti

100 F. pulveris
F. helveticus
S. turicensis

0.01
Figure 1 Maximum likelihood tree of the seven-loci multilocus sequence typing (3036 base pair concatenated length) for the members of
Cronobacter, Franconibacter, and Siccibacter genus, showing the sequence type for each species type strain and the neonatal meningitis-associated
sequence type 4. The tree was drawn using MEGA5.2 (http://www.megasoftware.net) with 1000 bootstrap replicates. Adapted from Holy, O. and
Forsythe, S. J. (2014). Cronobacter species as emerging causes of healthcare-associated infection. Journal of Hospital Infections 86,169–177.

Table 3 Isolation of Cronobacter spp. from throat swabs of outpatients according to age groups

Patient age (years)

Year <1 1–4 5–9 10–14 15–44 45–64 65–74 >75 Total

2005 1 5 1 1(1) 2 0 2 0 12(1)

2006 (1)a 1(2) 0 5 2 0 (3) (2) 8(8)
2007 2 9 0 2 2(1) (1) (1) (1) 15(4)
2008 0 1(1) 2 2(1) 0 0 (2) (1) 5(5)
2009 (2) 4 1 2 1 (2) 1 (1) 9(5)
2010 0 5 2(1) 0 (2) 1(1) (2) 2 10(6)
2011 (1) 0 2 0 0 0 0 0 2(1)
Total number of Cronobacter isolates 3(4) 25(3) 8(1) 12(2) 7(3) 1(4) 3(8) 2(5) 61(30)
% of isolates/age group 4.9 41 13.1 19.7 11.5 1.6 4.9 3.3 100.0
Number of patients sampledb 808 3404 2651 1867 18 223 10 744 3888 3538 45 123
Incidence of Cronobacter isolates/1000 patients sampled 8.7 8.2 3.4 7.5 0.5 0.5 2.8 2.0 2.0
a
Numbers in parentheses indicate additional isolates from normally sterile sites, sampled according to the clinical presentation of the patient.
b
Total number during the period 2005–2011.
Reproduced from Holy and Forsythe (2014).

named Cronobacter; in 2013, there were seven recognized spe-
cies. Only four of which are associated with human infections:
C. sakazakii, C. malonaticus, C. turicensis, and C. universalis. It is
highly probable that the number of infections has probably
been unreported due to misidentification. The species can be
grouped, with the mostly clinically relevant being group 1
(C. sakazakii and C. malonaticus, which form the majority of
clinical isolates in all age groups) and group 2 (C. turicensis and
C. universalis, which have been less frequently reported). The
other species are primarily environmental commensals and are
probably of little clinical significance. Table 3 shows an age
profile of Cronobacter isolated using throat swabs from over
45 000 outpatients during the period 2005–2011. The organ-
ism was isolated from every age group, with a higher frequency
from children < 14 years of age.
In 2011, Joseph and Forsythe announced the strong associ-
ation between neonatal meningitis cases and the clonal lineage
of C. sakazakii ST4. This was established using retrospective
study of 41 clinical strains from 1953 to 2008, collected from
seven countries. This association was confirmed later in the
year by the analysis of a number of highly publicized cases in
the United States. The reason for the association of one
sequence type out of >200 STs in the genus is unclear as no
particular virulence traits have been determined in C. sakazakii
ST4. However, it is known that this sequence type is frequently
(24% of strains) isolated from the infant formula and milk
powder manufacturing plants in Australia, Germany, Switzer-
land, and Ireland and therefore may represent a particularly
persistent clonal variant, resulting in increased neonatal
exposure.
Cronobacter can invade human intestinal cells, replicate in
macrophages, and invade the blood–brain barrier. The route of
infection is probably through attachment and invasion of the
intestinal cells. Whole genome sequencing has revealed a large
 Emerging Foodborne Enteric Bacterial Pathogens 493

number of plausible virulence factors, though many require 2013, they were reclassified as Cronobacter and therefore
further laboratory studies for confirmation. These are should have been positively detected, however in 2014 there
summarized in the succeeding text, and further details can be were removed from the genus and formed the new genera
obtained from the original publications. Due to the increasing Franconibacter and Siccibacter (Figure 1).
number of Cronobacter genomes being sequenced, for up-to- It should be noted that the emphasis of controlling Crono-
date information, the reader should consult the Cronobacter bacter spp. in PIF has given the false impression that this is the
BIGSdb, which is a searchable (BLAST) repository of all pub- only route of infection. In fact, the C. malonaticus type strain
lished Cronobacter genomes (see ‘Relevant Websites’). was isolated from breast abscess, there is human carriage and
A number of fimbria clusters have been identified in the the organism is recovered from a wide range of foods (Table 4),
genomes of Cronobacter species. Many fimbria clusters are and the organism has been isolated from the nasogastric feed-
common to all species, though there are two interesting excep- ing tubes of neonates not receiving reconstituted infant
tions. C. sakazakii is the only Cronobacter species encoding for formula.
b-fimbriae, whereas the genomes of the other species encode
for curli fimbriae. This may reflect evolution to the host eco-
system. A number of iron assimilation mechanisms have been
Enteroaggregative Escherichia coli
found in Cronobacter species, which might enable the organism
to utilize iron from breast milk and infant formula. Five puta- Description: The enteroaggregative Escherichia coli (EAEC) are a
tive type VI secretion system clusters have been identified pathotype within the E. coli species. Gram-negative, facul-
Cronobacter sp. genomes. These may be involved in adherence, tative rods. The genus is a member of the Enterobacteria-
cytotoxicity, host-cell invasion, growth inside macrophages, ceae family.
and survival within the host. It has been proposed that the Source: Contaminated food.
outer membrane proteins ompA and ompX have roles in Cro- Clinical presentations: Watery diarrhea with or without blood
nobacter penetrating the blood–brain barrier. The mechanism and mucus, abdominal pain, nausea, vomiting, and low-
(s) leading to the destruction of the brain cells is unknown and grade fever can be persistent.
could in part be a host response. The organism also encodes for Detection methods: No specific isolation procedures are
a number of hemolysins. available.
C. sakazakii is unique in the Cronobacter genus in its utiliza-
tion of exogenous sialic acid, and this may have clinical signif-
Table 4 Survey of dry food products and food ingredients from
icance. The ability to utilize sialic acid could be a major
which Cronobacter spp. have been isolated
evolutionary host adaptation since the compound is found in
breast milk, mucin, and gangliosides. Sialic acid is also an Food product or Number of Total number of
ingredient in PIF due to its association with brain develop- ingredient positive samples samples %
ment. C. sakazakii is also able to grow on the ganglioside
GM1 as a sole carbon source. Levels of endotoxin due to the Powdered infant 2 82 2
presence of heat-stable lipopolysaccharide (LPS) have been formula
Follow-up formula 3 91 3
measured in infant formula. In rat pups, LPS enhances the
Dry infant food 24 199 12
translocation of Cronobacter across both the intestines and the Milk powder 3 72 4
blood–brain barrier and therefore indicates that its presence in Milk powder and 1 55 2
infant formula could increase the risk of bacteremia in neo- derived products
nates. It has been speculated that frequent LPS contamination Starches 40 1389 3
of PIF (known to disrupt tight junctions) might contribute to Corn, soy, wheat, and 14 78 18
the invasiveness of Cronobacter across the blood–brain barrier. rice
The organism also has a number of heavy metal resistance Rice flour 6 16 38
factors and biofilm formation, which might enable it to resist Saengsik 41 86 48
disinfectants in food production environments. Dry food ingredients 15 66 23
Herbs and spices 40 122 33
Due to the high profile and changes in microbiological
Spices 14 71 20
specifications for PIF for target age <6 months, a number of Nuts 2 2 100
isolation and identification methods were developed Instant soups 2 13 15
post-2002. Tea 3 5 60
Isolation of the organism from PIF largely resembles that Confectionary 3 42 7
for Salmonella, with the use of stages for preenrichment, enrich- Chocolate products 11 37 30
ment, presumptive isolation, and confirmatory tests. It should Seeds 14 34 41
be noted that the FDA and ISO methods for the detection of Dried fish (inc. 13 50 26
Cronobacter are not up to date with respect to the taxonomy of shrimp)
the genus and not all species will necessarily be recovered by Sunsik 17 36 47
Tofu 4 11 36
these techniques. In fact, a well-known phenotyping kit man-
Desiccated foods 18 115 16
ufacturer still retains the name Enterobacter sakazakii instead of
Cronobacter spp. in its online database. In addition, E. helveti- Adapted from Forsythe, S. J., Dickins, B., and Jolley, K. A. (2014). Cronobacter, the
cus, E. pulveris, and E. turicensis have been used as negative emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole
control cultures as they were regarded as close relatives. In genome sequence analysis. BMC Genomics 15, 1121 and references therein.
494 Emerging Foodborne Enteric Bacterial Pathogens
The well-known gastrointestinal bacterium Escherichia coli
includes a number of pathotypes defined according to their
clinical presentation or adherence pattern on Hep-2 cell line.
EAEC self-aggregate and form a biofilm on intestinal mucosa
cells (Hep-2 cell line) with a ‘stacked-brick’ adherence pheno-
type. The first EAEC infection case was described in 1987 in a
child with acute diarrhea in Lima, Peru. These days, EAEC is
estimated to be the second most common cause of traveler’s
diarrhea. Unconfirmed reports have linked EAEC with the
development of irritable bowel syndrome. EAEC is a heteroge-
neous cluster of E. coli strains, which is an important cause
worldwide of acute or persistent diarrhea in children and
adults. The reason for its inclusion in this article is that since
the late 1990s, EAEC has received increasing attention as a
cause of acute, often persistent, and watery diarrhea and
malnutrition in children and HIV-infected persons living in
developed countries.
EAEC outbreaks have been described in children and adults
in the United Kingdom and France. The carriage of the organ-
ism by children < 5 years of age was higher in those with a
history of diarrhea. Similarly in Brazil, EAEC was found to be
more frequently associated with diarrhea in children < 2 years
of age. There has been an increasing rise in the proportion of
childhood diarrheal cases in which EAEC are implicated, and
this indicates that EAEC are important emerging agents of
pediatric diarrhea. It is believed that EAEC not only may be
causing acute and persistent diarrheal disease but also may
be persisting in the human intestine subclinically, inducing
chronic inflammation in the absence of dysentery. This persis-
tent infection could result in chronic disruption of gut function
resulting in malnutrition and restricted childhood
development.
EAEC route of transmission can be through contaminated
food. In Italy, two consecutive EAEC outbreaks (affecting 24
individuals) were linked to contaminated unpasteurized
cheese. A study of Mexican tabletop sauces identified 44% of
sauces from Guadalajara (Mexico) contained viable EAEC cells,
compared to 0% of sauces in restaurants in Houston (the
United States). In the largest reported outbreak so far, 2697
(40.6%) Japanese children who consumed infected school
lunches had severe diarrhea, and EAEC was found in 10% of
cases. Serotyping of EAEC is not applicable in the diagnosis of
diarrheagenic E. coli infections. In the United Kingdom, 97
EAEC strains were serotyped to 40 different O types, and in
another study, 93 out of 143 EAEC strains could be serotyped
to 47 different serotypes. Finally, many of the EAEC strains
autoagglutinate and so are described as nontypable or O
rough.
EAEC pathogenesis involves three stages: (1) adherence to
the intestinal mucosa by aggregative adherence fimbriae and
adherence factors, (2) increased production of mucus that
enhances EAEC adherence to the surface of enterocytes, and
(3) the release of toxins and elicitation of an inflammatory
response, mucosal toxicity, and intestinal secretion. The best-
studied virulence factor is AggR. This is a transcription activator
carried on pAA, a large 60 MDa plasmid. AggR controls the
expression of fimbriae (adherence factors), a dispersin protein,
and at least two pathogenicity islands on the EAEC chromo-
some. Dispersin is an antigenic antiaggregative protein, which
is encoded by the aap gene in the pAA plasmid. It is regulated
by aggR, modulates fimbrial adhesion, and facilitates penetra-
tion of the microorganism through intestinal mucus by bind-
ing to LPS and altering the electrostatic properties of the EAEC
outer membrane surface. Although several other virulence
factors are detectable using PCR probes, none exhibit 100%
specificity to EAEC strains. Other putative virulence factors
include a yersiniabactin system, a complex carbohydrate-
specific lectin, enterotoxins, and cytotoxins. The large E. coli
O104 outbreak in 2011 was due to a hybrid EAEC.
Escherichia albertii
Description: Escherichia albertii, species closely related to E. coli.
Gram-negative, facultative nonmotile rods. The genus is a
member of the Enterobacteriaceae family.
Sources: Human feces, birds, animals, and environment.
Clinical presentation: Diarrhea.
Detection methods: No specific isolation procedures are avail-
able. May originally be identified by routine diagnostic
protocols as enteropathogenic E. coli (EPEC) or enteroh-
emorrhagic E. coli (EHEC).
Little is known about the host or geographic distribution of
E. albertii; however, it is carried by 1% of wild birds and is
associated with mass mortality events among birds in the
Northern Hemisphere and the cause of mortality in captive
birds and poultry. The author does not know of any confirmed
cases of foodborne infections due to this organism or even
isolation from food. Nevertheless, the organism has been
tentatively included in this article as an emerging enteric food-
borne pathogen based on its association with poultry and
misidentification with EPEC and EHEC in order to raise
awareness.
The recognition of Escherichia albertii as an emerging
human enteric pathogen typifies the value in reevaluating pre-
vious identification schemes and anomalies. The organism has
previously been misidentified as eae-positive Hafnia alvei, as
well as strains of eae-positive E. coli, E. coli serotype O86, EPEC,
and EHEC.
In 1991, clinical isolates of H. alvei were found to differ
from representative H. alvei strains by phenotypic and geno-
typic assays. This variation included the carriage of an intimin
gene homologous to the eae gene encoding for the attaching-
effacing phenotype of EPEC. Following the use of 16S rDNA
sequence analysis and DNA–DNA hybridization, the new
Escherichia species, Escherichia albertii, was proposed for the
previously eae-positive H. alvei strains. In addition to the pos-
session of intimin, E. albertii also produces a cytolethal dis-
tending toxin. Using MLST and virulence gene sequences (eae
and cdt), it is proposed that the E. albertii lineage includes
Shigella boydii (serotype 13) and other previously nontypable
clinical isolates. This lineage shared a common ancestor with
E. coli and Shigella pathogenic groups 28 million years ago
(mya), and also the E. albertii lineage was established before
the radiation of pathogenic E. coli and Shigella. In essence, E.
albertii is not a newly emerged pathogenic variant of E. coli–
Shigella, but a previously unrecognized pathogen, which was
confused with other more familiar, closely related organisms.
E. albertii strains are nonmotile, fermented D-glucose (with
gas), D-mannitol, and D-mannose, but does not ferment lactose

or other sugars. All strains are positive for the eaeA gene, but the
presence of other virulence-related genes is variable, namely,
cdtB, phoE, ehxA, and stx2f. Therefore, there is variability within
this lineage, which may cause confusion and hinder accurately
identifying E. albertii from other members of the Escherichia
genus. Although the number of accurately recorded outbreaks
due to E. albertii is uncertain, it is plausible that some outbreaks
have been misattributed to E. coli as shown in the reassessment
of a food poisoning outbreak of gastroenteritis.
Plesiomonas shigelloides
Description: Plesiomonas shigelloides is the only recognized spe-
cies in the genus Plesiomonas. Gram-negative, facultative
aerobic rods; oxidase-positive. The genus is a member of
the Enterobacteriaceae family.
Clinical presentations: Three forms of gastroenteritis, (i) a secre-
tory, watery type; (ii) an invasive, dysentery-like type; and
(iii) a subacute or chronic form, lasting between 2 weeks
and 3 months.
Sources: Freshwater and estuarine water. Wide range of hosts:
amphibians, birds, fish, and animals.
Detection methods: No specific isolation procedures are
available.
P. shigelloides is the only species in the genus Plesiomonas and is
the only oxidase-positive member of the Enterobacteriaceae
family. It is recognized as an emerging water- and foodborne
enteric pathogen and a major cause of traveler’s diarrhea in
China and Japan. The organism can cause three types of gas-
troenteritis: (i) a secretory, watery type; (ii) an invasive,
dysentery-like type; and (iii) a subacute or chronic form lasting
between 2 weeks and 3 months. There are additional reports of
extraintestinal infections: meningitis in neonates, bacteremia,
sepsis, and septic shock with high fatality rates. The natural
environment for the organism is freshwater and estuarine
water. It is also found in amphibians, birds, fish, and animals.
Outbreaks are generally related to the consumption of contam-
inated seafood (crabs, oysters, and fish) or untreated water.
The pathogenicity of P. shigelloides is poorly understood,
although there are reports of secreted toxins in vitro: cholera-
like toxin, thermostable and thermolabile toxins, b-hemolysins,
and cytotoxin complex. The somatic antigen of P. shigelloides
may also play a role in pathogenicity, since the gene encoding
the most common type, O17, shares almost complete identity
with the smooth antigen gene of Shigella sonnei. An acute food-
borne outbreak in Cameroon was postulated to be due to a
preformed toxin in the food, due to the short incubation period.
There is no specific medium for the direct isolation of
P. shigelloides. In general Enterobacteriaceae media, the organism
produces nonlactose-, nonsucrose-fermenting colonies. The
organism does not grow on thiosulfate–citrate–bile salts–
sucrose medium, but does grow on CIN medium (normally
used to isolate Yersinia and Aeromonas species), producing opa-
que colonies without a pink center indicating that mannitol is
not fermented. It has a minimum growth temperature of 8  C.
Providencia alcalifaciens
Description: Gram-negative rods. The genus is a member of the
Enterobacteriaceae family.
Emerging Foodborne Enteric Bacterial Pathogens 495
Clinical presentation: Traveler’s diarrhea.
Sources: Contaminated food and water.
Detection method: A selective medium has been formulated but
possibly not evaluated.
Providencia alcalifaciens is primarily recognized as possible
cause of traveler’s diarrhea in developing countries. But in
1996, there was a large outbreak of foodborne infection caus-
ing acute gastroenteritis, involving 290 patients, in Fukui,
Japan. No previously recognized enteropathogens were
detected in the fecal samples of the patients. However, PFGE-
indistinguishable P. alcalifaciens strains were recovered from 7
of 18 samples. Additionally, a specific antibody against the
isolated strain was found to be elevated in the patients’ sera.
In vitro virulence studies of the strains using the Caco-2 cell line
showed strains could invade human intestinal cells and fluid
accumulation in rabbit ileal loop experiments. Together, these
results indicate that P. alcalifaciens was probably the causative
agent of the outbreak.
A selective medium for the recovery of the organism from
feces has been published. But to the author’s knowledge, no
large-scale trials of method evaluations have been undertaken.
Shigella sonnei
Description: Gram-negative facultative aerobic rods. The
Shigella genus is a member of the Enterobacteriaceae family.
Clinical presentations: Dysentery, fever, and stomach cramps.
Sources: Water and food contaminated with feces and flies.
Detection methods: No specific isolation procedures are available.
Shigella spp. can be regarded as pathotypes of Escherichia coli
with the ability to invade the human gut mucosa and cause
dysentery. Infection is due to the ingestion of the organism via
fecally contaminated food or water and can be due to poor
personal hygiene by food handlers. S. sonnei is well known as a
primary cause of dysentery in developed countries and is now
emerging as a problem in the developing world. It is replacing
the more diverse Shigella species S. flexneri in areas that are
undergoing economic development and improvements in
water quality.
Whole genome sequence analysis shows that the current S.
sonnei population descends from the most recent common
ancestor <500 years ago. Evidence to date indicates the species
primarily diversified in Europe into four distinct lineages with
unique characteristics. This was followed by the spread and
establishment of a single, rapidly evolving, multidrug-resistant
lineage into Asia, Africa, and America, termed ‘pandemic line-
age III.’ This lineage is now a dominant cause of dysentery in
many endemic areas of the world.
The emergence of S. sonnei has been attributed to the acqui-
sition of virulence plasmid pINV B, encoding the Plesiomonas
shigelloides-related O antigen. This has been difficult to confirm
since the plasmid is easily lost during laboratory subculturing
prior to analysis. Similarly, it has been proposed that previous
exposure to P. shigelloides (via contaminated water) may offer
some protection from S. sonnei infection since the O antigens
are indistinguishable and cross-react. This could account for
the increased incidence in S. sonnei infections following
improvements in water quality due to the reduced passive
cross protection by exposure to P. shigelloides.
496 Emerging Foodborne Enteric Bacterial Pathogens
Ones to Watch
Antibiotic Resistance of Recognized Foodborne Bacterial
Pathogens
A detailed consideration of antibiotic resistance and their wide-
spread use in human and veterinary medicine, agriculture, and
aquaculture animal husbandry and veterinary usage are out-
side the scope of this article. Nevertheless, antibiotic-resistant
enteric foodborne pathogens are an emerging public risk. Anti-
microbial resistance has been described in well-known patho-
gens, some of which are foodborne: Salmonella, Campylobacter,
Shigella, and Vibrio spp., methicillin-resistant Staphylococcus
aureus, E. coli, and enterococci. The resistance of animal- and
food-associated bacteria to antimicrobial agents has been
repeatedly reported. Bacterial resistance inevitably develops to
each new class of antibiotics. A variety of different antimicro-
bial resistance phenotypes result from the acquisition of exter-
nal genes that can provide resistance to an entire class of
antimicrobials. These genes are frequently associated with
large transferable extrachromosomal DNA elements, plasmids,
on which there may be other mobile DNA elements such as
transposons and integrons.
Plasmids in the incompatibility group IncA/C are of con-
siderable interest with respect to multiple antibiotic resistance
in enteric pathogens of humans and animals. Although these
large, low-copy number plasmids have been known for over 40
years, they appear to be of increasing relevance in the spread of
antibiotic resistance. The plasmids have at least three hotspots
for the integration of mobile genetic elements. They were first
identified from multidrug-resistant Aeromonas hydrophila and
Vibrio spp. in cultured fish. This coincided with the use of
antibiotics for cultured fish. A survey of clinical isolates of
Salmonella newport from 1940 to 2000 revealed that IncA/C
plasmids and their multiresistance phenotype emerged after
1980, the reservoir being environmental bacteria, followed by
the acquisition of antibiotic gene modules according to anti-
biotic exposure. IncA/C plasmids which have acquired the
blaNDM-1 gene encoding the New Dehli metallo-b-lactamase
have been disseminated in clinical E. coli and K. pneumoniae.
Another issue with increasing antibiotic resistance is the
coresistance to microbiocides, such as triclosan and quaternary
compounds. It has been reported that exposure of Salmonella
enterica and Escherichia coli O157 to sublethal concentrations
of antibacterial agents contributed to their development of
adaptive resistance to both biocides and antibiotics. Benzalk-
onium chloride-resistant Salmonella enterica serovar Virchow
showed elevated resistance to chlorhexidine. E. coli O157
Table 5 Foodborne outbreaks investigated using whole genome sequencing, genomic epidemiology
Organism Source Size of outbreak
Salmonella Seven egg farms 193 cases
Typhimurium
Salmonella Spiced salami 300 people in 44 US states
Montevideo
E. coli O104 Bean sprouts >3600 infections and >40 deaths in Germany and >100 in
other countries
L. monocytogenes Ready-to-eat meat 22 deaths and at least 57 illnesses
1/2a products
acquired high levels of resistance to triclosan after only two
sublethal exposures and, when adapted, repeatedly demon-
strated decreased susceptibilities to various antimicrobial
agents, including chloramphenicol, erythromycin, imipenem,
tetracycline, and trimethoprim. However, the specific mecha-
nisms of such coresistance remain poorly understood.
Revelations from Whole Genome Sequencing
Clinical and food microbiology is moving from being culture-
dependent to culture-independent. This gives the promise of
earlier recognition of attributable organisms. In order to
achieve this, there needs to be (a) accessibility to centralized
information and (b) consistent interpretation of the DNA
sequence. From mid-2013, the FDA has started to sequence
100 000 foodborne pathogens, with 30 completed in Decem-
ber 2013. The genome sequence data will have open access and
are being deposited in the National Center for Biotechnology
Information and hence shared with other countries for inter-
national surveillance.
The major advantages of culture-independent testing are
that it is usually faster than culture-based methods and can
simultaneously provide subtyping information as required for
epidemiological studies and accessible for further analysis.
MLST can be predicted from the genome sequence and is not
limited to the conventional seven loci. BIGSdb has been imple-
mented for a number of bacterial pathogens such as Campylo-
bacter jejuni and Cronobacter spp. A number of retrospective
foodborne outbreaks have been studied using NGS although
as yet few in real time (Table 5).
Conclusions and Future Studies
Compared to clinical infectious diseases, the number of cur-
rently recognized foodborne pathogens seems small. In part,
this is due to general procedures of food production, which
over the centuries have been refined and self-proved effective
means of food preparation, for example, cooking, acidification,
and dehydration. However, with changes in sourcing of food
ingredients, large-scale food preparation practices, longer
storage, and changes in eating habits have ensured a continued
of food-related illnesses. The major organisms associated with
foodborne disease are bacteria due to the ease of isolation and
identification compared to viruses and fungi. However, with
improved global surveillance systems, as well as improved iso-
lation and identification methods, previously unrecognized
Comment References
Retrospective Hawkey et al.
Retrospective Allard et al. and
Lienau et al.
Real time Mellmann et al.
Retrospective Gilmour et al.

foodborne infectious organisms are being recognized. With a
global economy and transportation of food, the need to con-
tinue to attribute human infections to food vehicles is war-
ranted for their control. This article considers a range of viral
and bacterial enteric pathogens, which may be foodborne.
Some like Cronobacter are currently associated with one food
product, reconstituted infant formula, and was previously
unrecognized as a bacterial genus, whereas others such as Shi-
gella are far better understood. Some recently recognized enteric
pathogens are due to increased awareness (such as Cronobacter);
others are adaptations of existing pathogens. Selective pressures
due to the use of antibiotics will drive further changes in the
microbiota, which may have severe consequences yet in the
future. Being able to predict the future emergence of foodborne
enteric pathogens is not possible at this time because of their
diversity in virulence and pathogenicity properties, their ability
to adapt, the diversity of foods, and the variation in suscepti-
bility to infection by the human population. The best available
approach to recognizing the emergence of a foodborne enteric
pathogen emergence is through improved surveillance schemes
leading to prompt, informed, and appropriate level of control.
In part, this will be met by the expansion of genomic epidemi-
ology whereby cases and outbreaks are investigated using whole
genome sequencing.
The recognition, control, and monitoring of potential
enteric pathogens require a reliable and robust detection sys-
tem. It is probable that as newly described enteric pathogen is
studied, then its description and taxonomy are likely to change
as hitherto, it was poorly described and any strains in culture
collections could be the more easily recovered variants and
only a subpopulation of the taxonomic unit.
It is clear that the major challenge ahead is to establish
effective communication between public health, veterinary,
and food safety experts, which will bring together multidisci-
plinary skills and multipathogen expertise. Such collaboration
is essential to monitor changing trends in foodborne infec-
tions, in order to detect emerging pathogens and to predict
their risk to human health such that appropriate control mea-
sures can be implemented.
Emerging Foodborne Enteric Bacterial Pathogens 497
See also: Clostridium: Food Poisoning by Clostridium perfringens;
Clostridium: Occurrence and Detection of Clostridium botulinum and
Botulinum Neurotoxin; Clostridium: Occurrence and Detection of
Clostridium perfringens; Escherichia coli and Other Enterobacteriaceae:
Occurrence and Detection; Food Poisoning: Classification; Food
Poisoning: Epidemiology; Food Poisoning: Tracing Origins and
Testing; Foodborne Pathogens; Salmonella: Detection; Salmonella:
Properties and Occurrence; Salmonella: Salmonellosis.
Further Reading
Allard MW, Luo Y, Strain E, et al. (2012) High resolution clustering of Salmonella
enterica serovar Montevideo strains using a next-generation sequencing approach.
BMC Genomics 13: 32.
Forsythe SJ (2008) Other Gram-negative bacterial pathogens. In: Blackburn C and
McClure P (eds.) Foodborne pathogens: Hazards, risk analysis and control.
Cambridge: Woodhead Publishing.
Forsythe SJ (2006) Arcobacter. In: Motarjemi Y and Adams M (eds.) Emerging
foodborne pathogens. Cambridge: Woodhead Publishing.
Forsythe SJ (2010) The microbiology of safe food, 2nd ed. Chichester: Wiley-Blackwell.
Gilmour MW, Graham M, Van Domselaar G, et al. (2010) High-throughput genome
sequencing of two Listeria monocytogenes clinical isolates during a large foodborne
outbreak. BMC Genomics 11: 120.
Hawkey J, Edwards DJ, Dimovski K, Hiley L, Billman-Jacobe H, Hogg G, and Holt KE
(2013) Evidence of microevolution of Salmonella Typhimurium during a series
of egg-associated outbreaks linked to a single chicken farm. BMC Genomics
14: 800.
Lieneau EK, Strains E, Wang C, et al. (2011) Identification of a salmonellosis outbreak
by means of molecular sequencing. The New England Journal of Medicine
364: 981–982.
Mellmann A, Harmsen D, Cummings CA, et al. (2011) Prospective genomic
characterization of the German enterohemorrhagic Escherichia coli O104:H4
outbreak by rapid next generation sequencing technology. PLoS One 6: e22751.
Relevant Websites
www.pubMLST.org/cronobacter – Searchable (BLAST) repository of all published
Cronobacter genomes.
 Emulsifiers: Types and Uses
R Miller, Kansas State University, Manhattan, KS, USA
ã 2016 Elsevier Ltd. All rights reserved.

Introduction (3) hydrophilic/lipophilic balance (HLB), and (4) functional

groups that compose them.
Surfactants, also called surface-active agents, are molecules that
migrate to the interface between two phases (solid, liquid, or
gas). The three most common two-phase systems in food prod- Charge in Aqueous Systems
ucts are emulsions, foams, and dispersions (Table 1). Emulsions
There are four classifications of emulsifiers based on the charge
are composed of two immiscible liquids. One liquid is dispersed
of the hydrophilic group when it solubilizes in water. They
within the other as individual droplets and is called the discon-
may be anionic, cationic, nonionic, or amphoteric. Anionic
tinuous phase, while the other liquid surrounds the droplets and
emulsifiers form negatively charged ions. They are effective in
is called the continuous phase. The emulsion behaves like the
the neutral to alkaline pH range and are also sensitive to ionic
continuous phase. The typical food emulsions are oil-in-water
strength and so do not function well in high salt-containing
(o/w), which consists of discontinuous droplets of oil dispersed
systems. The salt shields the negative charges so the anionic
in a continuous water matrix, and water-in-oil (w/o), which is
emulsifiers become ineffective and the emulsion breaks down
composed of discontinuous droplets of water dispersed in a
rapidly. Sodium stearoyl-2-lactylate (SSL) is an example of
continuous oil matrix. When describing emulsions, the ‘oil’
an anionic emulsifier. Cationic emulsifiers form positively
component may be actual oil or some other ingredient that is
charged ions in water and are functional in the acidic pH
not soluble in water. Examples of o/w emulsions are homoge-
range. They are somewhat toxic and so are not used as food
nized milk and ice cream, while common w/o emulsions include
additives. Nonionic emulsifiers have no net charge and form
margarine, salad dressing, and mayonnaise. Emulsions can be
no ions. They are highly effective and are compatible with all
formed mechanically by shaking or stirring; however, they are
other types of emulsifiers. They are relatively insensitive to pH
not stable for long periods of time and will separate into the two
and salt concentrations. Mono- and diglycerides (MDGs) are
separate components relatively quickly. Emulsifiers are a type of
nonionic. Amphoteric emulsifiers form both positively and
surfactant that is used to stabilize emulsions.
negatively charged ions when dissolved in water. They are not
affected by pH values except some are not effective near the
isoelectric point (pH at which protein is uncharged). Lecithin
Types of Emulsifiers is an amphoteric emulsifier.

There are many types of emulsifiers. They are classified

according to (1) charge in aqueous systems, (2) solubility,
Solubility
The solubility of an emulsifier governs the type of emulsion
Table 1 Interfaces in food systems that will be formed. The phase in which the emulsifier is least
soluble becomes the continuous phase. A key attribute of
Food system Continuous phase Discontinuous phase emulsifiers is that they are amphiphilic. This means one end
of the molecule is lipophilic and the other end is hydrophilic
Emulsion Liquid Liquid
(Figure 1(a)). The lipophilic end, referred to as the tail, is
Foam Liquid Gas
Dispersion Liquid Solid
usually a long-chain fatty acid taken from a food grade fat or
oil. It is not soluble in water but is soluble in oil and is called

oil water
“tail”
lipophilic
nonpolar
oil soluble
water oil

“head”
hydrophilic
polar group
water soluble
(a) (b) (c)

Figure 1 Emulsifier molecules contain a lipophilic ‘tail’ and a hydrophilic ‘head’ (a). Orientation of the emulsifier molecules in water-in-oil (b) and
oil-in-water (c) emulsions.

498 Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00249-X

 Emulsifiers: Types and Uses 499

oil-loving. The hydrophilic end is often referred to as the head serves as a useful guide for classifying and selecting emulsifier
and is polar. Thus, it easily dissolves in water and is termed systems. Selecting the ideal emulsifier or combination of emul-
water-loving. Emulsifiers that are easily soluble in water (more sifiers often requires experimental work.
hydrophilic) produce o/w emulsions. The emulsifier interacts
strongly with the water, thus reducing the surface tension of
the water so that it approaches zero. The water no longer forms Functional Groups
droplets and becomes the outer (continuous) phase of the emul-
Food emulsifiers are created by alcoholysis or direct esterifica-
sion, leaving the oil dispersed as discrete droplets (inner or
tion of edible fatty acids taken from animal or vegetable sources
discontinuous phase) to form an o/w emulsion (Figure 1(b)).
with polyols (i.e., glycerol, propylene glycol, and sorbitol).
Lipophilic emulsifiers are not water-soluble and interact with the
Further processing by reaction with ethylene oxide or esterifi-
oil phase. As a result, the surface tension of the water is not
cation with organic acids produces a wide range of emulsifiers
significantly reduced so it remains as discrete droplets in the
with differing properties. The most commonly used food emul-
oil, producing a w/o emulsion (Figure 1(c)).
sifiers include MDGs, stearoyl lactylates, sorbitan esters, poly-
glycerol esters, sucrose esters, and lecithin. They find use in a
wide array of food products (Table 3).
Hydrophilic/Lipophilic Balance MDGs are the most commonly used food emulsifiers,
Several methods of classifying emulsifiers are available. The composing about 75% of total emulsifier production. The
most commonly used is HLB. HLB is an index of the solubiliz- largest use is in yeast-raised baked goods to increase shelf
ing properties of emulsifiers and indicates the type of emulsion life, followed by cake and cake icings. Another major use is
(o/w or w/o) that the emulsifier is best suited for (Table 2). in margarine manufacture. MDGs are produced by heating
HLB values can be calculated based on the molecular structure triglycerides and glycerol with an alkaline catalyst. The
of the emulsifier or determined empirically. The values range
from 0 to 20, but some emulsifiers have been shown experi-
Table 3 Common food emulsifiers and uses
mentally to have higher values. Emulsifiers with low HLB
values are more oil-soluble (lipophilic), while those with Emulsifier Food products
higher values are more water-soluble (hydrophilic). In general,
emulsifiers with HLB values of 3–6 are lipophilic and best Mono- and diglycerides Bread, cake, pasta, frozen dessert, icing,
suited for w/o emulsions. Emulsifiers in this range include topping, peanut butter, margarine,
dehydrated potatoes, shortening, coffee
MDGs and propylene glycol monostearate (PGMS). Hydro-
whitener, and pasta
philic emulsifiers with HLB values of 10–18 are best suited
Glycerol monolaurate Bread, whipped topping, frosting, glaze,
for o/w emulsions. Examples include diacetyl tartaric acid and cheese products
esters of monoglyceride (DATEMs) and lecithin. The effective- Ethoxylated Bread, whipped topping, icing, frozen
ness of emulsifiers is affected by processing conditions and monoglyceride dessert, and coffee whitener
ingredients such as sugar, salt, and protein. It has been reported Diacetyl tartaric acid Bread, extruded products, icing,
that the strength of the interaction is more important than the esters of monoglyceride margarine, and salad dressing
number of reactive groups in determining the hydrophilic Succinylated Bread
properties of emulsifiers. However, HLB can provide an esti- monoglyceride
mate of the hydrophilic nature of various emulsifiers and Calcium stearoyl-2- Bread, egg whites, and dehydrated
lactylate potatoes
Sodium stearoyl-2- Bread, pasta, dehydrated potatoes, and
Table 2 Hydrophilic/lipophilic balance (HLB) values of common food lactylate coffee whitener
emulsifiers Propylene glycol esters Cake, whipped topping, dehydrated
potatoes, and shortening
HLB Sorbitan esters Whipped topping, cake, cake mix, cocoa,
Emulsifier Abbreviation value icing, filling, and coffee whitener
Polysorbate 60 Whipped topping, cake, cake mix, cocoa,
Mono- and diglycerides MDGs 4 icing, filling, coffee whitener,
Glycerol monolaurate 7 shortening, salad dressing, and
Ethoxylated monoglyceride EMG 13 edible oil
Diacetyl tartaric acid esters of DATEMs 9 Polysorbate 65 Ice cream, frozen custard, ice milk,
monoglyceride sherbet, frozen dessert, icing, cake, cake
Succinylated monoglyceride SMG 5 mix, whipped topping, filling, and coffee
Lactylated monoglyceride LMG 3 whitener
Sodium stearoyl-2-lactylate SSL 22 Polysorbate 80 Ice cream, frozen custard, ice milk,
Propylene glycol monostearate PGMS 3 sherbet, frozen dessert, gelatin mix,
Sorbitan monostearate 6 shortening, baked goods, bakery mixes,
Sodium stearoyl-2-lactylate SSL 15 filling, icing, topping, and frying oil
Polysorbate 65 11 Sucrose esters Bread, bakery mixes, frozen dessert,
Polysorbate 80 15 whipped milk products, and ice cream
Sucrose monostearate 5 Lecithin Baked goods, chocolate, cooking spray,
Calcium stearoyl-2-lactylate CSL 8 instant foods, and margarine
500 Emulsifiers: Types and Uses
resulting product is a mixture of about 45% monoglycerides,
45% diglycerides, and 10% triglycerides. The monoglyceride
concentration can be increased to about 90% using molecular
distillation to produce distilled monoglycerides. MDGs can
also be processed to produce emulsifiers with various func-
tionalities. Ethoxylated monoglycerides (EMGs) are formed
by treating monoglycerides with ethylene oxide gas. DATEMs
are produced from the reaction of monoglyceride with diacetyl
tartaric acid anhydride. Succinylated monoglycerides (SMGs)
result from the reaction of distilled monoglycerides with suc-
cinic anhydride. Stearoyl lactylates are the reaction products
of stearic acid and lactic acid, which are further converted
into the calcium or sodium salts and become calcium
stearoyl-2-lactylate and SSL. SSL is one of the most common
anionic emulsifiers used in the food industry. Sorbitan mono-
stearate, a sorbitan ester, is produced by reacting sorbitol and
stearic acid. It can then be reacted with ethylene oxide to
produce polyoxyethylene sorbitan monostearate (polysorbate
60), polyoxyethylene sorbitan tristearate (polysorbate 65),
and polyoxyethylene sorbitan monooleate (polysorbate 80).
Polysorbate 60 is the most widely used in the group. Polygly-
cerol esters result from reacting fatty acids with polymerized
glycerol of three to ten molecules. Sucrose esters are derived
from sucrose and edible tallow and consist of the mono-,
di-, and triesters of sucrose with fatty acids. Emulsifiers
that solidify into the stable alpha crystalline form are called
alpha-tending and include acetylated monoglycerides (AMGs),
lactylated monoglycerides (LMGs), and propylene glycerol
monoesters (PGMEs). Lecithin is a mixture of phosphatides
including phosphatidylcholine, phosphatidylethanolamine,
and inositol phosphatide. It is a natural component found in
animal and vegetable products. Egg yolk, which contains about
20% phospholipid, is well known for its emulsifying ability. It is
too expensive to isolate lecithin from egg yolk; therefore, most
commercial lecithin is derived from soybeans, which contain
about 2% lecithin. Many other emulsifiers and emulsifier blends
are available for specific applications.
Mechanism of Action in Food Products
An o/w emulsion will be used as an example of how an
emulsion is formed. When water and oil are mixed vigorously
(i.e., whipped, stirred, and shaken), the oil will form into
individual droplets dispersed within the water. The oil droplets
are the discontinuous phase because they are separated by the
water. The water (also referred to as the aqueous phase) is the
continuous phase where each water molecule is touching
another water molecule. Although an emulsion was formed,
it is not stable. Within a short period of time, the oil droplets
will bump into each other, and the two droplets will merge
into a single, larger droplet. This is called coalescence. These
larger oil droplets have less surface area and higher surface
tension. Ultimately, all of the oil droplets will coalesce and
rise out of the water to form into a separate layer floating on
top of the water. The rate at which the oil droplets rise out of
the emulsion is inversely proportional to their size; thus, larger
droplets rise faster than smaller droplets. In the case of a w/o
emulsion, the water is present as individual droplets
(discontinuous phase) within the continuous oil phase. Emul-
sifiers are added to promote emulsion formation and
stabilization.
Emulsifiers do not create emulsions. An emulsion is typi-
cally created using mechanical means such as mixing or stirring.
However, one of the roles of emulsifiers is promoting the
formation of smaller oil droplets during emulsion creation,
which slows the rate of coalescence. When the appropriate
emulsifier is added into the oil and water mixture, the mole-
cules orient at the interface between the water and the oil
droplets. The hydrophilic heads are attracted to the water,
whereas the lipophilic tails are repelled by the water and
attracted to the oil. As a result, the lipophilic tail will react
with the oil droplet, while the hydrophilic head will react
with the water at the surface of the oil droplet; thus, it reacts at
the interface between the two immiscible phases. When this
happens, the surface tension of the oil droplet decreases
considerably, which decreases the surface tension and allows
the droplets to be subdivided into smaller droplets during mix-
ing. The effectiveness of lowering surface tension varies
between different emulsifiers.
The second role of emulsifiers is stabilizing the emulsion
once it has formed. This is the main reason that emulsifiers are
used in food systems. Even though smaller droplets are more
stable, emulsion stabilization by emulsifiers is not directly the
result of their ability to lower interfacial tension and allow for
the creation of smaller droplets. Rather, emulsifiers stabilize
emulsions by accumulating at the interface of the two phases
where they form a barrier that prevents the droplets of the
discontinuous phase from coalescing. The mechanism by
which the barrier forms is determined by the properties of
the emulsifier. One mechanism of stabilization is due to like-
charged particles repelling each other. Anionic emulsifiers
carry negative charges that repel each other to keep the droplets
apart. SSL, SMG, and DATEM are anionic emulsifiers. They are
not highly effective in systems with high salt levels because the
salt shields the charge, which suppresses the repulsive forces
and leads to rapid coalescence and breakdown of the emul-
sion. Although not used in food systems, cationic emulsifiers
carry positive charges that also repel each other to stabilize the
emulsion. In nonfood systems, anionic and cationic emulsi-
fiers should not be used in combination as the opposite
charges would interact, thus rendering the emulsifiers inactive.
Another form of stabilization is employed by emulsifiers that
contain a large hydrophilic portion such as EMG. The large
hydrophilic head holds a large amount of water and creates a
layer of ‘bound’ water around the droplet, which prevents
coalescence. These emulsifiers are not sensitive to salt. Simple
steric hindrance is a third type of stabilization. The emulsifier
forms a solid layer at the interface, which physically prevents
coalescence even if the droplets touch each other. This occurs
in alpha-tending emulsifiers such as PGME.
Food Uses of Emulsifiers
In the food industry, the term ‘emulsifier’ is used generically and
includes surfactants that are not technically classified as emulsi-
fiers. Additives categorized as emulsifiers in the food industry
provide a broad range of functions in a wide range of food

products including emulsification, stabilization, antistaling,
crumb softening, foaming, fat crystal modification, mouthfeel,
agglomeration, defoaming, wetting, solubilizing, and stickiness
control. Many food products contain emulsifiers that perform
many different functions (Table 3). A few are discussed here.
Dairy Products
Emulsifiers are added to ice cream to shorten the freezing time,
improve whipping, and produce a dry, stiff texture that melts
slowly. The ice cream mix is an o/w emulsion. Emulsifiers stabi-
lize the emulsion and prevent the fat globules from agglomerat-
ing in the mix. When the mix is churned into ice cream, the
emulsifiers function to destabilize the natural milk protein film
that surrounds the milk fat globules so they can surround the air
bubbles and stabilize into a frozen foam. MDGs, sucrose esters,
and polysorbates are commonly used in ice cream.
Nondairy whipped toppings are whipped cream replacers
made using vegetable fats. They may be frozen or in pressur-
ized cans. In these products, emulsifiers are added as destabi-
lizers to promote aeration. In this role, the emulsifier causes
the fat globules to agglomerate during whipping. This is done
to create a fat network in the continuous aqueous phase, which
stabilizes the air bubbles and imparts stiffness to the foam.
Polysorbate 60 and alpha-tending emulsifiers such as LMG and
propylene glycol monoesters are commonly used in the pro-
duction of commercial nondairy whipped toppings.
Coffee whiteners are liquid or powdered cream replacers.
Emulsifiers are added to improve their dispersion in hot coffee.
Typical emulsifiers used include distilled monoglycerides,
DATEM, polysorbate 60, and sorbitan monostearate.
Salad Dressings
Mayonnaise is an o/w emulsion that contains at least 65%
vegetable oil. The emulsion is stabilized by the high level of
lecithin in the egg yolk. FDA standards of identity do not allow
the addition of other emulsifiers.
Pourable salad dressings are w/o emulsions that contain at
least 30% vegetable oil. Typically, consumers shake the bottle to
form the emulsion before pouring the dressing over their salad.
Polysorbates and DATEMs are commonly used to enhance emul-
sification when the bottle is shaken.
Margarine
Margarine is a w/o emulsion containing about 80% fat. It is
usually made with MDGs of long-chain fatty acids and lecithin.
The MDGs promote the formation of the emulsion, while the
lecithin is added to increase the solubility of the MDGs in the
oil blend. Distilled monoglycerides with a high concentration
of unsaturated fatty acids and lecithin are used in low-calorie
spreads, which are also w/o emulsions that contain at least
50% water and 40% fat.
Shortening
Shortening consists of a fat blend that contains emulsifiers.
All-purpose shortenings contain about 1% distilled monoglyc-
erides. High-ratio or emulsified shortenings, for use in cakes
Emulsifiers: Types and Uses 501
where the levels of sugar and liquid are present at higher levels
than the flour, contain several emulsifiers including distilled
monoglycerides, LMG, and PGME. These shortenings allow
better air incorporation during mixing and bind more sugar
and water.
Pasta
SSL and distilled monoglycerides are added into pasta products
to aid in extrusion. They are also used in pasta for soups or
canned products to reduce stickiness and improve the texture
of the cooked product.
Baked Products
In the baking industry, the term ‘emulsifier’ is used to describe
a broader group of surfactants, which includes those that are
active in foams (air-in-water) and dispersions (liquid-in-solid)
and in emulsions (liquid-in-liquid). Thus, the term emulsifiers
will be used in this section to encompass the common surfac-
tants used in baked products.
Doughs and batters are complex systems that contain mul-
tiple interfacial interactions that occur simultaneously, some of
which change forms as the products are heated. Emulsifiers
play many different roles in these products. Most batters and
doughs contain a polar, continuous phase that consists of an
aqueous (water) solution of salts, sugars, water-soluble pro-
teins, and other solubilized formula ingredients, which can be
a component of an emulsion, a foam, and a dispersion simul-
taneously in a single batter or dough. For example, the contin-
uous aqueous phase may form an emulsion in which a lipid in
some form (oil or solid fat) constitutes the nonpolar, discon-
tinuous phase. It may be the continuous phase in an emulsion
with a nonpolar, discontinuous polar discontinuous phase of
lipid in some form (oil or solid fat). Almost all batters and
doughs contain a foam of the continuous aqueous phase with
a discontinuous phase of air bubbles, which are incorporated
during mixing. Furthermore, the continuous aqueous phase
makes a dispersion with the insoluble formula ingredients,
which include the starch and gluten in the flour.
In cake batters, distilled monoglycerides and alpha-tending
emulsifiers such as AMG and PGMS function as aerating agents
to speed up the whipping rate and facilitate the incorporation
of many small air cells. This leads to a more stable emulsion
and results in a cake with a larger volume and fine, uniform
crumb grain. Alpha-tending emulsifiers are critical in cakes
made using a one-stage mixing procedure or prepared from
boxed cake mixes where liquid shortening or oil is used
because they form a solid film at the oil–water interface,
which stabilizes the emulsion and also keeps the liquid oil
from breaking the emulsion or foam that is prepared during
mixing.
Emulsifiers used in yeast-leavened bread products are also
active in the solid–liquid interface (dispersion) where they
function as crumb softeners and dough strengtheners. As
crumb softeners, they interact with starch granules and gluten
protein polymers to impart a softening effect on the crumb
grain and extend the shelf life of the product. Monoglycerides
are the primary crumb softener used in bread. Other common
crumb softeners include DATEM, sucrose esters, and PGMS.
502 Emulsifiers: Types and Uses
Dough strengtheners interact with the gluten protein and alter
the rheology of the dough to produce a stronger gluten struc-
ture. Anionic emulsifiers such as DATEM, SSL, SMG, EMG, and
polysorbates are good dough strengtheners. Some of the emul-
sifiers used in baking have only one or the other of the
functionalities, while others have both. For example, SSL func-
tions mainly as a dough strengthener, but it also plays a minor
role promoting the emulsification, incorporation, and subdi-
vision of air cells during mixing and crumb softening.
See also: Aeromonas; Bread: Chemistry of Baking; Food Additives:
Classification, Uses and Regulation; Rheological Properties of Food
Materials; Stabilizers: Types and Function; Triacylglycerols: Structures
and Properties.
Further Reading
Norm V (2015) Emulsifiers in food technology, 2nd ed. Oxford: Blackwell.
Pomeranz Y (1991) Additives. In: Pomeranz Y (ed.) Functional properties of food
components, 2nd ed., pp. 339–367. San Diego, CA: Academic Press.
Schuster G and Adams WF (1984) Emulsifiers as additives in bread and fine baked
products. In: Pomeranz Y (ed.) Advances in cereal science and technology, vol. VI,
pp. 139–287. St. Paul, MN: AACC.
Stauffer CE (1990) Emulsifiers and dough strengtheners. In: Stauffer CE (ed.)
Functional additives for bakery foods, pp. 69–124. New York: AVI.
Stauffer CE (1999) Emulsifiers. St. Paul, MN: AACC International, Inc.
Relevant Websites
www.emulsifiers.org – European Food Emulsifiers Manufacturers Association (EFEMA).
www.faia.org.uk – Food Additives and Ingredients Association (FAIA).
 Energy Metabolism
JA Coss-Bu, Baylor College of Medicine, Houston, TX, USA
NM Mehta, Harvard Medical School, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Regulation of Energy Metabolism
Energy metabolism represents the most important body func-
tion and has an effect on energy expenditure. It is paramount to
basal metabolism, growth, and physical activity in humans.
Energy derived from nutrients drives the critical cellular func-
tions in humans and is essential for survival. Using complex
biochemical pathways, chemical energy stored in the macronu-
trients (carbohydrate, fat, and protein) is transformed into
other forms of energy such as heat and adenosine triphosphate.
The regulation of metabolism at the cellular level relies on a
complex neurohormonal system, which responds to a variety to
stimuli and controls the utilization of substrate in individual
cells. The overall aim of this regulatory process is to maintain
energy balance, and it is important to understand these path-
ways that convert food into usable fuel.
The importance of the central nervous system (CNS) in the
regulation of body weight has been known for the past 100
years. Several factors have been identified, including mechani-
cal, hormonal, neural, and metabolic, that act as peripheral
signals in sending information to the CNS to regulate substrate
metabolism. The CNS processes these signals and integrates a
response to modify the control mechanisms that maintain
energy balance. It has been proposed that energy balance is
maintained by the regulation of body fat through a feedback
mechanism with specific processes mediated. In these pro-
cesses, the hormones leptin and insulin send signals to the
CNS about the body fat stores and participate in body weight
control. Both leptin and insulin cross the blood–brain barrier
and interact with receptors at the hypothalamus; two types of
neurons in this area have been described to have opposite
effects (anorexigenic vs. anabolic) to the action of leptin and
insulin concentrations, and it is by this mediating effect that
energy balance is achieved.
Other factors have been described in the CNS regulation of
energy balance, including signals from the gastrointestinal tract
and from the nutrients (i.e. glucose, fatty acids, and amino
acids). Among the satiety hormones secreted by the gut in
response to presence of substrate are cholecystokinin, amylin,
peptide YY, apolipoprotein A, glucagon-like peptide-1, and
ghrelin. The concentrations of the different substrates send
feedback to the CNS about the energy status of the body. The
observation that lower or higher blood glucose levels induce
changes in feeding behavior led to the well-known fact that the
CNS uses several sugar molecules as signals to regulate food
intake. Although neurons get most of their energy supply from
the oxidation of glucose rather than fat, studies in animals have
demonstrated that administration of fatty acid synthase inhib-
itors produces anorexia and weight loss. Recently, studies have
shown the effect of intake of different amounts of amino acids
in the central regulation of satiety, with diets high on protein
having an effect of decreasing food intake. On the other hand,
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00254-3
administration of several amino acids (i.e., leucine and valine)
has been shown to have opposite effects on food intake.
In summary, the CNS is an important and vital component
of the regulation of energy metabolism. Hormonal secretion
and feedback regulation by the CNS, gastrointestinal factors,
and substrate intake and metabolism are important in achiev-
ing energy homeostasis.
Components of Energy Expenditure
In order to understand energy balance, it is important to review
the total energy expenditure (TEE) during health and illness. In
healthy individuals, TEE can be partitioned into three main
components: basal metabolic rate (BMR), thermogenesis, and
physical work. BMR represents 60–70% of TEE, while thermo-
genesis represents 10%, and physical work accounts for
20–30% of TEE. During conditions of stress, the proportions
of these components vary depending on the severity of the
insult, amount of substrate intake, and physical activity. Diet-
induced thermogenesis (DIT) is the amount of energy required
to absorb, process, and store nutrients and represents an
increase in energy expenditure with respect to postabsorptive
state (Figure 1). BMR is difficult to measure. However, indirect
calorimetry (IC) allows a measurement of a close approxima-
tion of this value and has been described in more detail later in
this article. Resting energy expenditure (REE) is the energy
expenditure measure by IC in true resting state. Energy supply
greater than two to three times the REE gives rise to a DIT that
increases from 10% up to 20% after the first week of illness with
resultant increases in energy expenditure and higher ventilatory
and cardiocirculatory demands on the patient; therefore, it is
important not to supply excessive calories in relation to mea-
sured energy expenditure or BMR during the acute phase of the
injury or surgical stress. With age, energy expenditure is reduced
and this in addition to decreased exercise activity explains the
increase in weight gain commonly seen during adulthood. On
average, the rate of basal metabolism per body weight is
reduced from infancy to adulthood by 1.5–2 times (Figure 2).
For many years, the concept of the increased energy expen-
diture seen in infants has been attributed to the energy cost of
growth. In early infancy, the growth velocity is maximal at
approximately 6 months of age; this process slows down con-
siderable by the age of 1 year. It has been described in infants
less than 6 months that approximately 7–8% on the energy
used by the body is utilized for growth purposes; this amount
decreases to less than 2% of the energy expenditure by the time
the child becomes older than 1 year of age when the BMR
reaches about 55 kcal kg
1 day
1. There is a reduction in the
rate of basal metabolism that starts after the age of 3 years. This
change is explained by changes in body composition and an
uneven growth of organs, for example, the metabolic rate of
503
504 Energy Metabolism

100% changes in body composition and an uneven growth of organs,

Adaptive energy 10%
for example, the metabolic rate of the brain represents 12% of
the total in the newborn, while in adults, it is only 2% of their
80% Total energy expenditure 30% body mass. The last peak of BMR occurs at the age of 14 years,
and after this period, the BMR is stabilized at the level of
60% Diet induced thermogenesis 10% an adult.
An important component of energy expenditure is the
energy dissipated by physical activity; this expenditure in addi-
40%
tion to the BMR and the thermal effect of food represents the
TEE. The explanation for the decline in TEE with age is not well
Basal metabolic rate 60%
20% understood. With aging, there is an increase in body fat mass,
making body composition changes an important reason for
0%
this change. Another important factor in creating energy imbal-
ances and eventually weight gain is the ability to oxidize fat,
Figure 1 Components of energy expenditure. with an increase in body fat stores if oxidation of fat is
decreased and vice versa.
For many years, the concept of the increased energy expen-
55 diture seen in infants has been attributed to the energy cost of
growth. Growth velocity (as measured by grams/day or grams/
BMR in kcal / sq. m / h

50 kg/day) is highest in neonates and early infancy and starts to

Boys
45
40 Girls
Men
35
Women
30
5 10 15 20 30 40 50 60 70 80 90
Age in years
Figure 2 Change in basal metabolic rate with age. Reproduced with
permission from Mitchell, H. H. (1962). Comparative nutrition of man and
domestic animals, vol. 1, pp. 3–90. New York: Academic Press.
the brain represents 12% of the total in the newborn, while in
adults, it is only 2% of their body mass. The last peak of BMR
occurs at the age of 14 years, and after this period, the BMR is
stabilized at the level of an adult.
BMR defined as the energy expenditure after a 12 h fasting
period represents 50–70% of TEE, and it does decrease over
time by approximately 1–2% per decade. Many studies have
reported that the BMR is lower after adjustment for fat-free
mass in older individuals. The two changes that could explain
a lower metabolic rate during aging is the relative increase of fat
mass in relation to the decrease of fat-free mass. Multiple
studies have indicated that BMR is lower in the elderly when
compared to rates in younger individuals even after taking into
consideration changes in body composition associated with
age. BMR defined as the energy expenditure after a 12 h fasting
period represents 50–70% of TEE, and it does decrease over
time by approximately 1–2% per decade. Many studies have
reported that the BMR is lower after adjustment for fat-free
mass in older individuals. The two changes that could explain
a lower metabolic rate during aging are the relative increase of
fat mass in relation to the decrease of fat-free mass. Multiple
studies have indicated that BMR is lower in the elderly when
compared to rates in younger individuals even after taking into
consideration changes in body composition associated with
age. There is a reduction in the rate of basal metabolism that
starts after the age of 3 years. This change is explained by
slow down in the second half of infancy. In infants less than 6
months, approximately 7–8% on the TEE is allocated for
growth purposes; this amount decreases to less than 2% of the
energy expenditure by the time the child becomes older than 1
year of age when the BMR reaches about 55 kcal kg
1 day
1.
In summary, multiple studies have shown that dysregula-
tion of energy intake occurs in healthy older individuals
increasing the risk of energy imbalance; in addition, studies
have indicated a dysregulation of energy expenditure and sub-
strate oxidation associated with aging that results in changes in
body weight. In periods of sickness, when energy expenditure
varies and the individual is dependent on artificial nutrition, it
is important to be aware of the balance between energy intake
and requirement.
Metabolic Response to Stress
The profound metabolic response to critical illness, including
injury, surgical stress, or inflammation, is not always predict-
able and varies in intensity and duration between individuals.
The energy cost imposed by this metabolic response may be
proportional to the severity and duration of the stress but
cannot always be accurately estimated. Importantly, nutri-
tional support itself cannot alter the metabolic stress response.
Failure to recognize existing nutritional deficiencies and pro-
vide adequate nutrition support during the acute phase of the
illness may exacerbate preexisting malnutrition or result in new
nutritional deficiencies. Both overfeeding and underfeeding
should be avoided in order to decrease metabolic imbalances
and development of malnutrition in critically ill patients. The
nutritional plan should be individualized and customized to
each patient during their hospital admission. A complete
understanding of the metabolic events involved in the meta-
bolic stress is essential for planning appropriate nutritional
support plans in critically ill patients.
The hormonal and cytokine profile of the stress response to
critical illness is characterized by increased serum levels of
insulin, glucagon, cortisol, catecholamines, and proinflamma-
tory cytokines. Elevated serum counterregulatory hormone

Ketones
Fuel for brain
Lipolysis
≠ Fatty acids
Trauma
Sepsis
Critical Muscle
illness breakdown
Burn
Surgery
Glycolysis
Ø utilization
Hyperglycemia
Figure 3 Basic pathways of the metabolic stress response to injury. Reproduced with permission from Mehta, N. and Jaksic, T. (2008). The critically ill
child. In: Duggan, C., Watkins, J. B. and Walker, W. A. (eds.) Nutrition in pediatrics (4th ed.), pp. 663–673. Hamilton, ON: BC Decker.
concentrations induce insulin and growth hormone (GH)
resistance, resulting in the catabolism of endogenous stores
of protein, carbohydrate, and fat. This autocannibalism
ensures a steady supply of essential substrate intermediates
and energy necessary to support maintenance energy and
micronutrient needs in addition to driving the ongoing meta-
bolic stress response. The sine qua non of this acute metabolic
response is a hypermetabolic, hypercatabolic state resulting in
the loss of endogenous muscle stores with increased glucose
and free fatty acid oxidation, elevated energy expenditure, and
increased protein breakdown (Figure 3).
Among the changes at the CNS seen during the acute met-
abolic response are alterations in the hypothalamic–anterior
pituitary axes, resulting in increased cortisol secretion, elevated
concentrations of GH, increased reverse T3 secretion, increased
prolactin, and decreased testosterone. The degree of alterations
in the levels of these hormones is proportional to the severity
of the insult and produces significant changes in body homeo-
stasis. Cytokines are endogenous proteins that act as mediators
of local and systemic injury or inflammation. They are synthe-
sized by several tissues and white blood cells in response to
stimulation by endotoxin and viral particles. These mediators
are critical in modulating a number of responses by promoting
tissue repair, prioritization of substrate for protein synthesis,
and anti-inflammatory actions. This response when aug-
mented secondary to an overwhelming insult or inadequate
response by the host immune response can lead to an
increased risk of injury-induced morbidity and mortality. Tis-
sue injury elicits the release of proinflammatory cytokines
(TNF-a, interleukin (IL)-1, IL-6, and IL-8). Once these peptides
are released by activated macrophages and endothelial cells,
signals are enacted to release anti-inflammatory cytokines,
mainly IL-10. Many clinical studies have reported the use of
drugs aimed to attenuate the proinflammatory response or
Energy Metabolism 505
Tissue repair
Wound healing
Loss of lean Acute inflammatory
Proteins
body mass
Protein synthesis
Amino
acids
Gluconeogenesis
Urea
≠≠ Glucose
Fuel for
brain,
RBC, and
kidneys
medications to augment the anti-inflammatory reaction with
mixed results, highlighting the inherent difficulties in the con-
trol of the inflammatory response.
During the acute metabolic response, there is a significant
increase in the concentrations of the counterregulatory
hormones (oppose the anabolic effects of insulin) including
catecholamines, glucagon, and cortisol. The actions of catechol-
amines include an elevation of the metabolic rate, hyperglyce-
mia induced by hepatic glycogenolysis, and suppression of
pancreatic secretion of insulin. The known actions of glucagon
are glycolysis and gluconeogenesis with a resultant elevation in
the serum concentrations of lactate and alanine; this results in
the endogenous regeneration of glucose. The effects of cortisol
are at the muscle level with significant proteolysis and elevation
of metabolic rate in conjunction with the action of catechol-
amines. The other important effect of cortisol is the increase in
gluconeogenesis by the incorporation of alanine and glutamine
from muscle proteolysis and gut sources, respectively. Also, cor-
tisol has been shown to increase insulin resistance and oppose
the anabolic effects of insulin.
It has been postulated that the attenuation of the effects of
anabolic hormones during the acute metabolic response rep-
resents a compensatory mechanism. Insulin is a potent ana-
bolic hormone with multiple actions including glycogenesis,
lipogenesis, and protein synthesis. When a state of increased
insulin resistance is present, glucose production, lipolysis, and
proteolysis are increased, conditions usually seen in critically
ill adults and children. Recent evidence suggests an anti-
inflammatory effect of insulin administration; this finding
has been reported in animal and clinical studies. GH, also
known as somatotropin or somatropin, is a peptide hormone
that stimulates growth, cell reproduction, and regeneration in
humans and other animals. GH is a stress hormone that raises
the concentration of glucose and free fatty acids and stimulates
506 Energy Metabolism
production of insulin-like growth factor 1 (IGF-1). The actions
of GH are aimed toward anabolic conditions, and its synthesis
and secretion are stimulated by IGF-1. During stress
conditions, the secretory pattern of GH and IGF-1 is sup-
pressed by a cytokine-mediated mechanism. Clinical studies
in children with burns have reported that the exogenous
administration of recombinant GH has increased protein syn-
thesis and nitrogen balance and improved clinical outcomes.
Substrate Adaptations During the Acute Metabolic
Response
The changes in substrate homeostasis seen after the acute met-
abolic response are aimed to preserve life, despite the fact that
in many instances, this response is maladaptive. During
periods of starvation not associated with stress, endogenous
mobilization of adipose stores is the main source of energy for
metabolic purposes. This mobilization of body fat stores is
preceded by the use and eventual depletion of glycogen stores;
the time it takes for glycogen reserves to be depleted is usually
few hours for infants and children and up to 24 h in adults. It is
during this initial period of lack of substrate intake that the
energy needs are supplied mainly with glycogen and protein.
After a period longer than 48–72 h, an increase in fat store
mobilization is ensued with subsequent utilization of fat as the
sole source of energy, with the exception of tissues that are
dependent mainly on glucose for metabolic fuel. Also, a
decrease in energy expenditure is seen aimed to preserve lean
body mass. Unfortunately, patients with limited or smaller
protein stores (i.e., children and malnourished adults) will be
metabolically compromised in less time.
The acute metabolic response seen with injury, trauma, and
sepsis results in important alterations in substrate metabolism.
In addition to the use of glycogen stores, gluconeogenesis is
increased and provides the bulk of carbohydrates for the
glucose-dependent tissues; protein breakdown provides substrate
for the synthesis of acute phase reactants and gluconeogenesis.
The hyperglycemic response seen is secondary to the uncon-
trolled need to synthesize new glucose and to the increased
insulin resistance at the peripheral tissues associated to the dys-
function of the intracellular glucose transport protein 4 (GLUT4).
The acute metabolic response in the critically ill subject is
hypercatabolic resulting in proteolysis, increased protein turn-
over, and decreased protein synthesis. These changes have been
shown by the use of stable isotopes in studies that included
patients with sepsis and extensive burns. The response of the
acute ill patient in terms of protein catabolism is different from
the subject under starvation conditions in that the former has
negative protein balance secondary to an exaggerated proteol-
ysis as opposed to decreased synthesis in the starved subject.
This catabolic response is not attenuated by the provision of
adequate nutrition support and persists until the acute process
is resolved. Much of this loss of lean body mass as it relates to
commonly measured outcomes, including length of intensive
care unit (ICU) stay and length of hospitalization, has been
debated by the experts. Even though the exaggerated catabolic
response is not reversed by the provision of substrate in the
form of glucose, protein, and fat, adequate supplementation of
protein is recommended to increase protein synthesis and
attenuate the net negative protein balance.
Glucose is the primary fuel for the CNS and minimal
amounts are required to maintain function. Provision of
glucose in excess of the capacity of oxidation, usually
8–10 mg kg
1 min
1 in neonates and 4–6 mg kg
1 min
1 in
adults, has been associated with lipogenesis, hepatic steatosis,
and hyperglycemia. It has been reported that up to 150% of
the glucose oxidized under stress conditions is provided by
gluconeogenesis, and this response is not attenuated by the
exogenous administration of glucose. Exogenous administra-
tion of insulin does not increase the rate of glucose oxidation
in critically ill patients because the rate of maximal glucose
oxidation has been reached in addition to increased endoge-
nous levels of insulin. The resultant hyperglycemia seen in
these patients is a result of a combination of factors including
increased glucose oxidation and hepatic glucose production
and decreased peripheral uptake of glucose.
Assessment of Energy Requirement for the
Hospitalized Patient
Methods to Estimate Energy Needs
Adequate nutrition support is important giving the high prev-
alence of malnutrition in the hospitalized patient. Several
studies have reported malnutrition rates ranging from 30% to
50% upon admission to the hospital, with more than 50% of
these already malnourished patients having a worsening nutri-
tional status if it is left untreated. It has been reported that up
to one-third of hospitalized adults patients will develop mal-
nutrition after admission to the hospital. Malnutrition has
been reported to worsen outcomes, including immune sup-
pression, infection, longer hospital stay, increased morbidity,
and decreased survival. Critically ill children differ in their
energy needs from healthy children in terms of underlying
metabolism and growth, comorbidities, and preexisting energy
reserve, and therefore, it is difficult to estimate energy needs in
this population. In addition, frequent monitoring of energy
expenditure is necessary to accommodate any variations in
energy expenditure throughout the course of illness. In the
absence of metabolic equipment to measure energy needs in
the ICU, caution should be used when reference values are
used to estimate energy needs in this population.
Reference values
The BMR reference values include the following:
1. The Harris–Benedict equation, (Harris–Benedict 1919) one
of the most widely used to estimate BMR. This equation is
widely used in adults and has never been validated for
children.
2. The Food and Agriculture Organization, the World Health
Organization, and the United Nations University (FAO/
WHO/UNU). These equations were based on data derived
from 6100 individuals under a variety of conditions and
represent BMR.
3. Talbot published guidelines in 1938 for the estimation of
BMR based on measurements made in children. These
values were based on studies performed by the author.
 Energy Metabolism 507

Table 1 Prediction equations

Equation Gender Age Basal metabolic rate estimate

Harris–Benedict Children [22.1 þ (31.05  Wt) þ (11.6  Ht)]

Female [665.0955 þ (9.5634  Wt) þ (1.8496  Ht)
(4.6756  age)]
Male [66.473 þ (13.7516  Wt) þ (5.0033  Ht)
(6.755  age)]
FAO/WHO/UNU Female 0–3 years [(61  Wt)
51]
3–10 years [(22.5  Wt) þ 499]
10–18 years [(12.2  Wt) þ 746]
Male 0–3 years [(60.9  Wt)
54]
3–10 years [(22.7  Wt) þ 495]
10–18 years [(17.5  Wt) þ 651]
Schofield Female 0–3 years [(16.252  Wt) þ (10.232  Ht)
413.5]
3–10 years [(16.969  Wt) þ (1.618  Ht) þ 371.2]
10–18 years [(8.365  Wt) þ (4.65  Ht) þ 200.0]
18–30 years [(13.623  Wt) þ (2.83  Ht) þ 98.2]
Male 0–3 years [(0.167  Wt) þ (15.174  Ht)
617.6]
3–10 years [(19.59  Wt) þ (1.303  Ht) þ 414.9]
10–18 years [(16.25  Wt) þ (1.372  Ht) þ 515.5]
18–30 years [(15.057  Wt)
(0.1  Ht) þ 705.8]
Estimated energy requirements Female and male 0–3 months [89  Wt
100] þ 175
4–6 months [89  Wt
100] þ 56
7–12 months [89  Wt
100] þ 20
Female 3–8 years 135.3
[(PA  10  Wt) þ (30.8  Age) þ (934  Ht)] þ 20
9–18 years 135.3
[(PA  10  Wt) þ (30.8  Age) þ (934  Ht)] þ 25
Male 3–8 years 88.5
[(PA  26.7  Wt) þ (61.9  Age) þ (903  Ht)] þ 20
9–18 years 88.5
[(PA  26.7  Wt) þ (61.9  Age) þ (903  Ht)] þ 25

FAO, Food and Agriculture Organization; WHO, World Health Organization; UNU, United Nations University. Basal metabolic rate estimate in kcal per day; weight (Wt) in kg; height (Ht)
in cm; physical activity (PA).

4. Schofield equations. These equations were based on data
from the FAO/WHO/UNU report with some additional
data (Table 1). It is important to mention that the use of
these reference values in critically ill patients could lead to
underfeeding or overfeeding because of the variability of
the metabolic state of patients during their stay in the ICU.
Correction factors
The use of stress and activity factors in addition to the BMR
reference values has been reported to overestimate and under-
estimate energy needs in critically ill patients, reinforcing the
concept that IC should be used as a strategy to measure energy
needs in this population of patients. Several studies conducted
in critically ill patients on mechanical ventilation that used
different BMR equations (i.e., Harris–Benedict, Schofield, and
Talbot) and correction factors of 1.3 and 1.5 to estimate energy
needs were found to have significant differences when com-
pared to measured energy expenditure by IC, emphasizing the
concept that IC is the only method to accurately measure
energy needs in critically ill patients.
Predictive equations
The use of regression equations based in multiple variables
(sex, weight, height, body temperature, heart rate, inotrope
dose, sepsis, days of admission to the ICU, minute ventilation,
etc.) has been reported in both adults and children admitted to
the ICU and has shown to be more accurate than reference
values when compared to measured energy expenditure by IC.
Caution should be given when using predictive equations
because of the dynamic nature of the metabolic condition of
critically ill patients.
Methods to Measure Energy Needs
The measurement of energy expenditure in humans has been
attempted in the last century by physiologists. These methods
include direct calorimetry, IC, and the isotope dilution tech-
nique. The use of direct calorimetry requires the measurement
of heat produced by the body while the subject is placed in an
insulated and tight chamber. The principles used are similar to
those in a bomb calorimeter (Figure 4). IC is the method by
which metabolic rate and substrate utilization are estimated
from respiratory gas exchange measurements and urinary
nitrogen excretion. It has been regarded as the gold standard
for accurate measurement of REE. IC measures whole body
oxygen consumption (VO2) and carbon dioxide production
(VCO2). Then, the VO2 and VCO2 values are converted to a
caloric equivalent based on equations developed by Weir:
REE ¼ [VO2 (3.941) þ VCO2 (1.11)]  1440. The use of IC
and urinary nitrogen allows the calculation of ‘net’ oxidation
rates for carbohydrates, protein, and fat by the use of the
Conzolazio formulas. The VCO2/VO2 ratio is known as respi-
ratory quotient (RQ), and it is well known that the conversion
of glucose to fat elevates the RQ and reflects the proportion of
substrate utilization in the body. The npRQ represents the ratio
of glucose and fat utilization by excluding the participation of
protein and varies in value from 0.70 to 1.0, with values >1.0
indicating net fat biosynthesis from glucose (lipogenesis).
508 Energy Metabolism
Figure 4 The bomb calorimeter. This diagram shows the bomb
calorimeter designed by the French chemist Pierre Eugene Marcellin
Berthelot (1827–1907). It is used to measure the heat of chemical
reactions. Artwork from the tenth volume (second period of 1892) of the
French popular science weekly ’La Science Illustree.’ From the Science
Photo Library.
The correct interpretation of IC results implies an under-
standing of the assumptions and technical considerations of
this methodology. There are several sources of error and many
technical difficulties in applying this methodology in the ICU
including (1) model of calculation and assumptions, (2) calo-
rimetric factors used, (3) leak around endotracheal tube, (4)
inspired oxygen concentration above 0.60, (5) use of high
levels of positive end expiratory pressure, (6) unstable gas
analyzers, (7) inability to reach steady state, and (8) human
factors. Studies in mechanically ventilated adults and children
have concluded that the use of an abbreviated IC protocol of
3–5 min duration is enough to achieve steady state and obtain
reasonable accuracy, while other authors have concluded that
energy expenditure results of a 30 min IC test are comparable
to 24 h test results.
The potential useful clinical applications of IC in criti-
cally ill patients can be summarized as follows: (a) assess-
ment of energy expenditure in patients who fail to
adequately respond to estimated nutritional needs, (b)
assessment of energy expenditure in patients with single or
multiple organ dysfunction who need prolonged ICU care
and artificial nutritional support, (c) assessment of the
effects induced by artificial nutrition on the cardio-
circulatory and respiratory systems in mechanically venti-
lated patients with acute and chronic respiratory failure,
and (d) monitoring of VO2 while weaning from mechanical
ventilation. Targeted measurements of REE are currently
recommended and are included in the American Society
for Parenteral and Enteral Nutrition (ASPEN) Clinical
Guidelines: Nutrition Support of the Critically Ill Child. In
summary, IC has brought understanding of how energy is
utilized during critical illness; this has yet to be translated
into improving patient outcomes. Studies examining the role
of simplified IC technique, its role in optimizing nutrient
intake, its ability to prevent overfeeding or underfeeding in
selected subjects, and the cost–benefit analyses of its appli-
cation in the ICU are needed.
TEE that takes into account physical activity can be mea-
sured using doubly labeled water technique (DLW). The
isotope dilution technique uses stable isotopes (2H2O,
H218O, and NaH13CO3) to measure energy expenditure. The
DLW method is based on the differences in turnover rates of
2
H2O and H218O in body water. The DLW technique has been
validated against IC and is now considered to be a gold stan-
dard for measurements of TEE under free-living conditions.
Sources of measurement error include analytic inaccuracies in
the mass spectrometric determination of isotopic enrichment,
biological variations in the isotope enrichment, isotopic frac-
tionation during formation of carbon dioxide and during
vaporization of water, the calculation of total body water,
and the assumption or calculation of the 24 h RQ. The use of
the DLW method is not possible in the critically ill child
because of the fluid shifts and imbalances.
The carbon dioxide production (VCO2) during respiration
has long been used as an index of substrate oxidation and
energy expenditure. The isotopic dilution technique allows
the 13C from infused labeled bicarbonate (NaH13CO3) tracer
to be diluted by metabolically produced carbon dioxide. By
measuring the degree of isotopic dilution in expired air or
blood, VCO2 rates can be calculated. The assessment of energy
expenditure must involve knowledge of the amount of
energy released per liter of carbon dioxide produced, or the
energy equivalents of CO2, which constitutes the food
quotient, which serves as a surrogate for RQ under conditions
of nutrient balance. Two important concerns in relying on this
technique relate to (1) errors in quantifying the tracer dose of
labeled bicarbonate and (2) the possibility that the labeled
bicarbonate does not adequately trace total CO2 formed in
the mitochondria. Another limitation is the need to know
accurately the labeled bicarbonate correction factor(s) required
for the physiological condition investigated (recovery factor).
In summary, isotopic tracer techniques and IC should be con-
sidered as complementary techniques, in particular since the
tracer techniques require the measurement of carbon dioxide
production obtained by IC. However, it should be kept in
mind that the assessment of substrate oxidation by IC may
involve large errors in particular over a short period of time.
By using IC, energy expenditure is calculated with less error
than substrate oxidation rates.
In summary, energy needs must be carefully evaluated dur-
ing hospitalization particularly during the course of critical
illness by measuring the energy expenditure. If IC is available
and the patient meets the conditions for a measurement test,
IC provides an accurate way to measure REE. IC could be used
in specific patient populations (targeted IC; Table 2) to prevent
unintended underfeeding and overfeeding. In the absence of
measured REE, equations to estimate energy needs may be
used, but caution should be exercised in order to avoid incor-
rect estimation.

Table 2 Criteria for high risk for metabolic alterations and candidates
for targeted indirect calorimetry per American Society for Parenteral and
Enteral Nutrition (ASPEN)
1. Underweight (BMI <5th percentile for age) or at risk for overweight
(BMI >85th percentile for age) or overweight (BMI >95th
percentile)
2. Children with >10% weight gain or loss during ICU stay
3. Failure to consistently meet prescribed caloric goals (for >5 days)
4. Failure to wean or need to escalate respiratory support
5. Need for muscle relaxants for >7 days
6. Neurological trauma (traumatic, hypoxic, and/or ischemic) with
evidence of dysautonomia
7. Oncological diagnoses (including children with stem cell or bone
marrow transplant)
8. Children with thermal injury
9. Children requiring mechanical ventilatory support for >7 days
10. Children suspected to be severely hypermetabolic (status
epilepticus, hyperthermia, systemic inflammatory response
syndrome, dysautonomic storms, etc.) or hypometabolic
(hypothermia, hypothyroidism, pentobarbital, midazolam
coma, etc.)
11. Any patient with ICU length of stay >4 weeks may benefit from
indirect calorimetry to assess adequacy of nutrient intake
BMI, body mass index; ICU, intensive care unit.
Source: Mehta, N. M. and Compher, C. (2009). JPEN Journal of Parenteral and Enteral
Nutrition (33(3)), pp. 260–276, copyright @ 2009 by Elsevier. Reprinted by Permission
of SAGE Publications.
Energy Provision for the Hospitalized Patient
In the critically ill patient, the goal of nutrition support is to
provide adequate needs and prevent underfeeding and over-
feeding. Multiple complications do occur attempting to convert
a catabolic condition in an anabolic state including increased
carbon dioxide production, hyperglycemia, hypertriglyceride-
mia, and fatty liver. Recent studies in critically ill adults indicate
worse outcomes occur with low energy intake in patients with
either low or very high body mass index. The inability to deliver
adequate energy intake, resulting in negative protein–energy
balance, has been associated with increased morbidity and
mortality rates in adult ICU patients. Cumulative energy deficit
also is associated with an increasing number of complications
including increased length of ICU and hospital stay.
The magnitude of the stress response and the presence of
malnutrition amplify the complications associated with under-
nutrition in the management of the critically ill patient admit-
ted to the ICU. Nitrogen deficits accrue over short periods of
time; therefore, it is important to remember this situation
when malnourished patients in the ICU develop an acute
metabolic stress response (i.e., sepsis and injury), because the
level of nitrogen deficit to be tolerated is less compared to
patients with the same level of stress and normal nutritional
status; thus, delaying nutrition support in the malnourished
patient beyond 5–7 days can potentially increase morbidity
and mortality in this population. Another important aspect
in the provision of nutrition support to patients admitted to
the ICU is the prescription of overnutrition. This excessive
nutrition comes as carbohydrates and protein. Hyperglycemia
secondary to increased insulin resistance in addition to over-
supply of dextrose can impose additional respiratory work with
Energy Metabolism 509
eventual development of fatty liver. Also, excessive amounts of
protein in patients with underlying renal disease increase the
risk for uremia-associated complications.
Enteral nutrition should be the feeding method of choice in
critically ill patients because it replicates the normal pattern of
nutrient consumption and hormonal homeostasis, maintains
and improves gastrointestinal integrity, and reduces the inci-
dence of multiorgan failure. Early enteral nutrition appears to
be well tolerated in the general ICU population and is associ-
ated with early attainment of nutritional goals. The impact of
early enteral nutrition and optimal energy balance might be
most relevant in patients with preexisting malnutrition, who
cannot afford added nutritional worsening during the course
of the acute illness. Enteral nutrition is preferable to parenteral
nutrition, but if enteral nutrition route is contraindicated or
not tolerated, parenteral nutrition should be started after
patient condition has stabilized. If enteral nutrition is partially
tolerated or the rate of advancement is slow and does not cover
energy and protein needs, then supplemental parenteral nutri-
tion should be considered.
Recent studies in critically ill patients have found an asso-
ciation between the use of parenteral nutrition and increased
morbidity and mortality. Since critically ill patients are highly
dependent on substrate availability, have lower reserves,
and are at risk of malnutrition, parenteral nutrition should be
considered. Current recommendations agree on initiating
parenteral nutrition if enteral nutrition has failed or it is con-
traindicated. If low-volume enteral nutrition is started and is
inadequate to cover nutritional needs, supplemental parenteral
nutrition should continue and be gradually decreased as
enteral nutrition is advanced until full volume of enteral
feeds covering nutritional requirements is achieved.
The use of clinical practice guidelines developed by multi-
disciplinary expert consensus and based on clinical evidence
can help to improve nutrition support practices in the ICU.
Most recent clinical guidelines for the critically ill patient
by the American Society for Parenteral and Enteral Nutrition
recommend to provide energy intake equivalent to BMR; this
is based on extensive studies that reported measured energy
expenditure in the patient admitted to the ICU. Regarding the
provision of protein supplementation during periods of stress,
current recommendations are to supplement 15–20% of the
total nutrient intake to be provided as protein, with
protein intakes of 1.5–2.0 g kg
1 day
1 for adult patients and
1.5–3 g kg
1 day
1 for infants and children.
The nutritional assessment of critically ill patient upon
admission to the ICU should aim to identify those with
existing malnutrition, and a tailored and individualized nutri-
tional plan should be implemented in order to prevent
underfeeding and overfeeding in these patients. Serial assess-
ment of energy expenditure, by using IC, will guide energy
intake and prevent cumulative imbalances. The most impor-
tant aspect of the provision of nutrition support during
periods of stress is the preservation of lean body mass; the
amount of lean body mass has been shown to be associated
with mortality. There are multiple barriers to the implemen-
tation of adequate nutrition with many of these barriers being
preventable. Implementation and utilization of clinical nutri-
tion guidelines may help achieve nutrition goals in the hos-
pitalized patient.
510 Energy Metabolism
See also: Amino Acids: Metabolism; Carbohydrate: Digestion,
Absorption and Metabolism; Cholesterol: Absorption, Function and
Metabolism; Fatty Acids: Metabolism; Fructose: Sources, Metabolism,
and Health; Glucose: Metabolism and Regulation; Protein: Digestion,
Absorption and Metabolism.
Further Reading
Institute of Medicine (2005) Dietary reference intakes for energy, carbohydrate, fiber, fat,
fatty acids, cholesterol, protein, and amino acids. Washington, DC: National
Academies Press.
Kyle UG and Coss-Bu JA (2012) Nutrition support in critically ill children: underdelivery
of energy and protein compared with current recommendations. Journal of the
Academy of Nutrition and Dietetics 112: 1987–1992.
McClave SA and Martindale RG (2009) Guidelines for the provision and assessment of
nutrition support therapy in the adult critically ill patient: Society of Critical Care
Medicine (SCCM) and American Society for Parenteral and Enteral Nutrition (A.S.P.
E.N.). JPEN Journal of Parenteral and Enteral Nutrition 33: 277–316.
Mehta NM (2011) Nutrient metabolism and nutrition therapy during critical illness.
In: Furhman BP and Zimmerman JJ (eds.) Pediatric critical care, 4th ed.,
pp. 1073–1088. Philadelphia, PA: Elsevier Saunders.
Mehta NM and Compher C (2009) A.S.P.E.N. clinical guidelines: nutrition support of
the critically ill child. JPEN Journal of Parenteral and Enteral Nutrition 33: 260–276.
Mehta NM and Duggan CP (2009) Nutritional deficiencies during critical illness.
Pediatric Clinics of North America 56: 1143–1160.
Mongardon N and Singer M (2010) The evolutionary role of nutrition and metabolic
support in critical illness. Critical Care Clinics 26: 443–450.
Orellana RA and Coss-Bu JA (2015a) Energy and macronutrient requirements in the
critically ill child. In: Goday PS and Mehta NM (eds.) Pediatric critical care nutrition,
1st ed., pp. 33–58. New York: The McGraw-Hill Companies.
Orellana RA and Coss-Bu JA (2015b) Impact of infection-nutrient interactions in infants,
children and adolescents. In: Pammi M, Vallejo JG, and Abrams SA (eds.)
Nutrition–infection interactions and impacts on human health, 1st ed.,
pp. 333–355. Boca Raton, FL: CRC Press,Taylor & Francis Group.
Pierro A and Eaton S (2008) Metabolism and nutrition in the surgical neonate. Seminars
in Pediatric Surgery 17: 276–284.
Roberts SB and Rosenberg I (2006) Nutrition and aging: changes in the regulation of
energy metabolism with aging. Physiological Reviews 86: 651–667.
Schlein KM and Coulter SP (2014) Best practices for determining resting energy
expenditure in critically ill adults. Nutrition in Clinical Practice 29: 44–55.
Sisley S and Sandoval D (2011) Hypothalamic control of energy and glucose
metabolism. Reviews in Endocrine & Metabolic Disorders 12: 219–233.
Skillman HE and Mehta NM (2012) Nutrition therapy in the critically ill child. Current
Opinion in Critical Care 18: 192–198.
Wiskin AE and Davies JH (2011) Energy expenditure, nutrition and growth. Archives of
Disease in Childhood 96: 567–572.
Relevant Websites
http://www.fao.org/home/en/ – Food and Agriculture Organization of the United
Nations.
http://www.nal.usda.gov/fnic/DRI/Essential_Guide/DRIEssentialGuideNutReq.pdf –
Dietary Reference Intakes: The Essential Guide to Nutrient Requirements.
http://www.nature.com/scitable/topicpage/nutrient-utilization-in-humans-metabolism-
pathways-14234029 – Scitable by Nature Education: Nutrient Utilization in Humans:
Metabolism Pathways.
http://www.nutritionmd.org/health_care_providers/general_nutrition/macronutrients.
html – Nutrition MD.
http://www.who.int/nutrition/publications/nutrientrequirements/9251052123/en/ –
World Health Organization: Human energy requirements.
 Energy: Intake and Energy Requirements
DJ Millward, University of Surrey, Guildford, UK
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Energy intakes (EIs) that meet energy requirements (ERs) can
in principle be defined from physiological considerations of
energy balance. Thus, when EI from food and drink matches
total energy expenditure (TEE), there is energy balance with the
body neither gaining nor losing energy. This means that either
EI or TEE measured at energy balance could form the basis of a
reference EI or ER value. In practice, measurement of either EI
or TEE is not a simple task.
Energy Content of Foods
Within the International System of Units (SI), the unit of energy
is joule (J) and the energy content of food is normally in
kilojoule (kJ ¼ 103 J) and megajoule (MJ ¼ 106 J). The thermo-
chemical calorie is also still widely used especially in the United
States and is equivalent to 4.184 J (1 kcal ¼ 4.184 kJ).
Food energy content can be determined in terms of the
maximum amount of potentially available energy by measur-
ing the heat released after its complete combustion to carbon
dioxide and water in a bomb calorimeter. This is the gross
chemical energy content (GE) of all organic food constituents
of which the macronutrients carbohydrate, fat, protein, and
alcohol account for the main components. However, not all
GE from food is available for human metabolism because of
losses during food utilization at the level of digestion, metab-
olism in the intestine, especially in the colon, and post-
absorption metabolism. This means that the available energy
to the organism, defined as metabolizable energy (ME), is the
difference between GE intake and all losses in feces, in urine, a
small amount in sweat, and in postprandial increases in
metabolism. Energy losses in feces largely represent incomplete
digestion and the energy content of bacteria produced in the
colon, whereas energy losses in urine and sweat are due pri-
marily to the urea content, which represents the incomplete
catabolism of protein. The value quoted as the energy content
of foods on food labels and in the food composition tables is
the ME content. Historically, the ME content of food was
calculated from the amounts of protein, fat, carbohydrate,
and alcohol in the food (determined by chemical analysis)
using specific energy conversion factors called the Atwater
factors, which are estimates of the energy content of each
macronutrient and alcohol, numerically rounded for practical
purposes. These energy values are 17 kJ g1 (4.0 kcal g1) for
protein, 37 kJ g1 (9.0 kcal g1) for fat, 17 kJ g1 (4.0 kcal g1)
for carbohydrates, and either a rounded value of 29 kJ g1
(7.0 kcal g1) or an unrounded value of 6.9 kcal g1 for alco-
hol. However, there are several issues that complicate the cal-
culation of ME and that can result in differences between
agencies in how ME is calculated.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00250-6
For fat, the GE content will vary slightly according to the
chain length and degree of saturation of constituent fatty acids
in dietary triglycerides. Also, the ME will be less than the GE
because of some loss as fecal fat. In practice, because dietary fat
incorporates a mixture of different fatty acids, these differences,
which could influence the GE, are ignored, and it is usually
assumed that the digestibility is 95%. As a result, ME content
of dietary fat is assumed to be 95% of GE, that is, 37 kJ
(9.0 kcal g1), the general Atwater factor.
For carbohydrates, there are two complicating factors.
Firstly, the GE of carbohydrates in food varies according to
their molecular form, that is, glucose, disaccharides, and
starch. The GE per unit weight is 15 kJ (3.6 kcal g1), 16 kJ
(3.8 kcal g1), and 17 kJ (4.0 kcal g1), respectively. FAO has
recommended that a conversion factor of 16 kJ g1
(3.8 kcal g1) should be used when carbohydrate is expressed
as monosaccharide equivalents and 17 kJ g1 (4.0 kcal g1)
when determined by direct chemical analysis of carbohydrate,
that is, the general Atwater factor, and in the United Kingdom,
this method is used. However, in the United States, carbohy-
drate is calculated from total dry weight of food minus protein,
fat, and ash, and this will include unavailable carbohydrate
(i.e., dietary fiber) and other noncarbohydrate components
(e.g., lignin, organic acids, tannins, waxes, and Maillard prod-
ucts) in the calculated value. As a result, for a mixed diet, total
EIs can be 12% higher and carbohydrate intake 14% higher
when carbohydrate by difference is used in dietary analysis
compared with available carbohydrate analyzed directly.
Thus, estimates of the extent of underreporting in dietary sur-
veys in the United States are often lower than those in the
United Kingdom.
Secondly, carbohydrates include nonstarch polysaccharides
(NSPs) and resistant starch; these may also provide energy
through fermentation in the colon to form short-chain fatty
acids. Some of these products are absorbed into the blood-
stream or used locally by colonocytes as energy, and a conver-
sion factor of 8 kJ g1 (1.9 kcal g1) has been suggested by FAO.
However, in the United Kingdom, the food composition tables
ignore energy from NSPs and resistant starch and assume all
carbohydrates are completely absorbed so that the ME is calcu-
lated using the general Atwater factor of 17 kJ g1 (4.0 kcal g1),
which is carbohydrate expressed as monosaccharide.
For protein, the GE can vary according to the amino acid
content and the ME according to its digestibility and the amino
acid content, which determines its nitrogen content and the
amount of urea produced and excreted per gram of protein.
These differences especially the digestibility can be large so that
highly digestible animal proteins like in egg may yield 40% more
ME than less digestible plant proteins. In practice, the ME con-
tent of dietary protein is assumed to be 17 kJ (4.0 kcal g1), the
general Atwater factor, on the basis of an assumed loss of urinary
energy as urea. This will be a relatively accurate value because
dietary protein usually represents a mixture of several animal and
511
512 Energy: Intake and Energy Requirements
plant protein sources. Because the immediate metabolic fate of
amino acids absorbed into the body includes deamination and
other reactions such as gluconeogenesis that contribute to the
postprandial increase in heat production (the thermic effect of
food (TEF)), it has been suggested that this should also be
accounted for in a value defined as the net metabolizable energy
(NME). However, this value is not generally used and the TEF is
usually assumed to be part of energy expenditure.
For alcohol, the ME is assumed to be 29 kJ (6.9 kcal)/g
consumed, the unrounded general Atwater factor. Alcohol is
oxidized completely and does not directly add to body energy
stores. There is a component of postprandial thermogenesis
following alcohol consumption with reported values ranging
between 9% and 28%, but this is not included in the ME.
Energy Intakes
The amount of food energy consumed is assessed as part of
national nutrition surveillance in many countries and in the
course of many studies of diet and health interactions. However,
it is well known that accurate measurement of dietary EI is diffi-
cult. Moreover, the extent of the unreliability of the various
methods is not as widely appreciated as it should be. There are a
number of ways in which food intakes can be measured, and the
likelihood that the various methods are accurate can be tested to
some extent by modeling intake values with predicted values of
TEE to see if the values are physiologically sensible. This compar-
ison is a valid test because on a daily basis, differences between EI
and TEE are likely to be small and not usually measureable.
At the national population level, the FAO compiles data on
food balance sheets, converted to per capita overall food
energy supply. Clearly, this measure is unlikely to be useful
in identifying variation in intakes between subpopulations or
influences of age, gender, and lifestyle. Furthermore, food
wastage at all points in the food supply chain will result in an
overestimate of per capita overall food energy supply. This is
most likely to have increased over recent decades through
wastage, especially at the prehousehold level. An analysis of
these data in the United Kingdom shows little change between
1961 and 1990 (at about 13.4 MJ/day/per capita); thereafter,
however, this intake increases by 5% to the year 2000, and by a
further 2% to 2009, eventually to reach 14.3 MJ/per capita/
day. This value is higher than would be expected and modeling
shows it to be an overestimate of EIs by about 40%.
At the household level, records of household food pur-
chases adjusted for number and ages of household members
enable a better estimate of per capita EI. In the United
Kingdom, such records have been collected annually on a
complete cross section of the British population from 1950.
This is now the Expenditure and Food Survey, a 2-week diary
record of all expenditure by each household member over the
age of 7 years in about 5000 UK households. Such records are
subject to both underestimation through underreporting of
purchases and overestimation through food wastage in the
home, although attempts are made to minimize the former
and account for the latter error. Currently, reported values are
9.58 MJ/per person/day, which are 20–30% lower than values
in 1955. This value is slightly lower than the lower range (25th
centile) of the predicted current UK all-adult population TEE.
At an individual level, food EIs can be measured directly
from questionnaires of food intakes recorded as the frequency
of consumption of lists of individual food types, i.e., food
frequency questionnaires; recorded as a recall of intakes over
the previous 24 h, as dietary records collected over a number of
days (e.g., 4–7 days), or most accurately as records of weighed
food intakes over periods of up to 7 days. In national surveys
such as NHANES (the US National Health and Nutrition
Examination Survey) and NDNS (the UK National Diet and
Nutrition survey), a balance is struck between likely accuracy
and the numbers of subjects who can be studied. Currently, for
the UK NDNS, 4-day dietary records are used with annual
measurements in a rolling program collecting anthropometric
and biochemical measures of nutritional status as well as food
intakes. There has been a downward trend in the mean
reported daily intakes for total energy for adult men and
women from 1987, with the most recent value indicating
7.86 MJ day1. In fact, this value is much lower (and unphy-
siological) than would be predicted by simple modeling of
likely TEE, almost certainly due to underreporting.
Energy Expenditure as the Basis for ERs
From the earlier, it is quite clear that measurement of EI is
exceedingly difficult and unlikely to provide information that
can be reliably transformed into dietary reference values (DRVs)
for energy. In any case from what is known about the regulation
of energy balance, it is clearly more physiologically appropriate
to base the derivation of energy reference values on TEE than on
EI. This is because, in humans, it appears that EI is finely regu-
lated through the appetite mechanism to enable EI to match TEE,
rather than the other way round. TEE comprises the basal meta-
bolic rate (BMR), energy expended at rest in the postabsorptive
state and at thermoneutrality, energy expended in physical activ-
ity (physical activity energy expenditure (PAEE)), and other
components such as postprandial thermogenesis (TEF) and ther-
mogenesis associated with growth. TEE does not appear to
include a significant component of regulatory EE responsive to
EI, that is, dietary-induced thermogenesis, apart from the heat
increment of feeding that is associated in a near stoichiometric
way with postprandial metabolism, mainly amino acid oxida-
tion. The diet-related thermogenesis associated with uncoupled
fat oxidation in brown adipose tissue mitochondria, which is a
feature of rodent metabolism, is not believed to be quantitatively
important in humans after the first months of life apart from a
small, recently identified component of cold-induced non-
shivering thermogenesis. Thus, it is TEE, mainly a function of
size (BMR) and lifestyle (PAEE), that can be identified as the
physiological determinant of the ER: that is, the ER can be pre-
dicted specifically as the rate of TEE plus any additional needs for
growth, pregnancy, and lactation.
ERs for What Purpose and for Whom?
Given the context of target populations exposed to an obeso-
genic food supply and built and social environment, to which
it is genetically susceptible, and that have become overweight,
all recent definitions of the ER refer to healthy body weights.

For example, in the United Kingdom, the ER is defined as “the
amount of food energy needed to balance energy expenditure
in order to maintain body size, body composition and a level
of necessary and desirable physical activity consistent with
long-term good health. This includes the energy needed for
the optimal growth and development of children, for the
deposition of tissues during pregnancy, and for the production
of milk during lactation consistent with the good health of
mother and child.” This specifically ‘prescriptive’ approach
involves identifying ER values for infants, children, and adults
in relation to best estimates of healthy body weights: for chil-
dren, growth standards and reference weights that are below
current UK values and, for adults, a weight associated with a
minimum risk of mortality, that is, W equivalent to a BMI of
22.5 kg m2 at actual heights. This is below the current average
UK population BMI of 27 kg m2. Application of ER values
derived in this way to under- or overweight groups will tend to
mediate weight change toward the desirable range. The ques-
tion of including within a prescriptive ER, sufficient EI to
balance a ‘desirable’ level of PAEE, is also an important con-
sideration given that much of the population is thought to be
sedentary. Achieving this in practice, however, is much more
difficult since any advice to overweight populations to con-
sume more energy carries obvious risk. In practice, advice on
desirable physical activity is usually given separately including
information on the additional energy needed for various
amounts of additional PAEE.
In the public health context, ER values can be defined as
DRVs for food energy that provide a best estimate of the food
energy needs of a population and its subgroups and represent
criteria against which to judge the adequacy of their food EIs.
For all nutrients other than energy, the DRV is identified as the
reference nutrient intake (RNI), a value at the upper bound of
the reference ranges, that is, the estimated average reference
value (EAR) þ two standard deviations, ensuring low risk of
deficiency for any individual. However, for dietary energy, the
DRV is defined differently: that is, as the EAR, itself; this is
because an RNI defined as earlier would represent an excess EI
for the majority of the population, leading to weight gain and
obesity in the long term. In contrast, the EAR represents an
intake associated with similar probabilities of excessive and
insufficient EIs for any individual within the population.
Because EIs and the requirement are highly correlated by virtue
of the appetite mechanism’s adjusting EI to match ER, such
probabilities are much lower than would be expected, that is,
<50%. Thus, in adults, the DRV for energy is the EAR, that is,
the intake associated with a healthy body weight at existing
height and levels of physical activity. During infancy and
childhood, the requirement also has to meet the needs for
healthy growth and development, whereas during pregnancy
and lactation, the requirement must meet the needs for devel-
opment of a healthy baby, supporting adequate lactation while
minimizing postpregnancy weight gain.
Utilizing Doubly Labeled Water-Derived Measurements
of Energy Expenditure to Derive ERs
Measurement of TEE can be achieved by several methods. Short-
term measurements under highly defined conditions can be
Energy: Intake and Energy Requirements 513
made by calorimetry with direct calorimetry, measurement of
heat loss from the subject in a calorimeter, the most accurate
method. Indirect calorimetry, the most commonly used
approach, measures oxygen consumption and/or carbon dioxide
production from which TEE is calculated using standard
formulas. The doubly labeled water (DLW) method is generally
recognized as the most accurate measure of free-living TEE cur-
rently available. The DLW method is a minimally invasive stable
isotopic technique of measuring carbon dioxide (CO2) produc-
tion in free-living subjects over a period of several weeks. After a
measured dose of DLW (i.e., water containing the stable isotopes
of hydrogen, 2H, and oxygen, 18O2), samples of saliva or urine
are taken over 2 weeks, and the rate of dilution of the two
isotopes in these samples is measured. As water is lost from the
body, the two isotopes are lost and the enrichment of both 2H
and 18O2 falls. However, 18O2 in water exchanges with the oxy-
gen in CO2 because of the carbonic anhydrase reaction such that
there is an additional loss of 18O2 as C18O2. As a result, 18O2 is
lost from the body faster than 2H with the difference providing a
measure of the CO2 production rate. TEE is then estimated from
this rate after assigning an energy value to CO2 calculated from
the assumed average respiratory quotient value. This in turn is
estimated from the macronutrient composition of the dietary
intake. The method provides more accurate measures of TEE
than other noncalorimetric methods such as heart rate monitor-
ing and has dramatically improved our understanding of human
energy expenditure.
However, making use of DLW-derived data is not straightfor-
ward with either of two main approaches that can be employed.
The first involves a definition of the ER in which TEE is expressed
in a factorial way. TEE can be considered as comprising three
components, BMR, TEF, and PAEE. With TEF assumed to be
about 10% of EI, then for subjects at energy balance (TEE¼ EI),
TEE ¼ BMR þ0.1  TEE þ PAEE. However, PAEE will vary not
only with the extent of physical activity but also as a result of
body weight. Thus, an alternative way of expressing TEE is to
normalize it for the BMR assuming this captures influences of
weight age and gender by expressing it in terms of multiples of
the BMR as the physical activity level (PAL), that is,
ERTEE ¼ PAL  BMR
BMR is predictable with adequate accuracy from anthropo-
metric variables (weight, age, height, and gender). PAL is
assumed to include just PAEE although any TEF will also be
included. In the past, PAL has been derived from time-
allocated lists of activities for which energy costs are known
and PAL values or ranges have been identified that equate to
lifestyles. Thus, in free-living populations, PAL can range from
<1.3 in immobile subjects to between 3 and 4.7 for limited
periods of time in soldiers on field exercises or elite endurance
athletes, for example. Within the general population, however,
the overall range of PAL values for individuals in energy bal-
ance and leading sustainable lifestyles is between 1.38 for the
most sedentary and 2.5 for the most active. TEE can be pre-
dicted from BMR and a lifestyle-related value of PAL. DLW data
can identify PAL values in population groups. In this case, BMR
can be measured separately or predicted so that the values for
TEE determined by the DLW method can be expressed in terms
of PAL. This has allowed a much better understanding of the
range and variation in PAL as a function of lifestyle.
514 Energy: Intake and Energy Requirements
The second approach models the DLW-derived TEE values
against anthropometric characteristics of the reference popula-
tion in regression equations, allowing TEE and ER values to be
predicted for population groups: for example, children or
adults. However, regression modeling does not easily allow
for inter- and intraindividual variation in PAEE. Because of
this, committees using this approach have had to make provi-
sion for the likely variation in PAEE within the population
group by introducing multilevel ordinal variables derived
from PAL data in the regression model to modify the total
height and weight contribution to TEE.
Limitations of the Factorial Model of TEE
The increasing amount of reported DLW measurements of TEE
has in fact allowed a detailed analysis of the various factorial
models, which have all been shown to be unsatisfactory in
terms of being able to predict TEE of population groups accord-
ing to their lifestyles. This is because values predicted for
individuals or groups according to their lifestyles do not corre-
spond well with measured, DLW-based values. In fact, most
DLW or whole body calorimeter studies show considerable
variation in TEE between individuals involved in similar activ-
ities. A likely explanation for this is indicated by 24 h calorim-
eter and DLW studies showing a wide variation in PAL values
(from 1.15 to 1.7) in subjects with restricted activity. Further-
more, these 24 h values correlate with free-living DLW-
measured PALs (1.35–2.15). Such observations have led to
the development of the concept of spontaneous physical activ-
ity (SPA). SPA is body movements associated with activities of
daily living, change of posture, fidgeting, and ‘a propensity for
locomotion.’ SPA is a highly reproducible, within-individual
familial trait; it accounts for a considerable fraction of between-
individual variation in TEE and displays an inverse relationship
with future weight gain. Nonexercise activity thermogenesis is
another term used to describe a somewhat extended definition
of this phenomenon. Other individuals have been identified
who exhibit marked reductions in activity, that is, to levels
approaching the BMR, after periods of intense activity. The
extent and variation in SPA, currently not quantifiable within
population groups, calls into question the predictability of PAL
from lifestyle information as currently collected, especially with
the precision previously assumed.
A New Approach to the Factorial Model
The recent UK report on energy DRVs adopted an alternative
approach to the use of PAL values in the definition of TEE and
the ER of target population groups. In this report, it was con-
cluded that when there are sufficient DLW measurements of
TEE available for a particular population group to enable it to
serve as a reference, the most useful information is the distri-
bution of PAL values within that reference population. The
assumption is made that the reference population groups are
sufficiently demographically similar to the target population
and that the DLW data are extensive enough and representative
of the reference population. In this case, the distribution of
PAL values observed in the reference population in terms of
25th, median, and 75th centiles can be used to predict TEE and
hence the ER as a function of BMR for the less active, average,
and more active members of the target population group. The
principle is shown in Figure 1. The restriction of the range of
ERs to just three values is clearly a major departure from
previous approaches for adults. However, in most cases only
the median population value will be used. It is in principle
similar to the method adopted by FAO in their recommenda-
tions for children. However, it is an approach that recognizes
the reality that prediction of rates of TEE for individuals or
population groups is inherently uncertain and that, in practice,
most health professionals use a very restricted range of ER
values.
Identifying Suitable PAL and EAR Reference Values for Adults
Identifying reference, DLW-derived PAL values is difficult
because most published studies contain only small numbers
of subjects recruited in various ways for various reasons, that is,
not necessarily representative. For example, in a recent meta-
analysis of DLW data of adults, 4971 individuals were identi-
fied. However, these subjects derived from 183 cohorts with a
median cohort size of only 14 and with only 10% having >70
subjects. If few of the individual cohorts are representative of
the general population, then it cannot be assumed that com-
bined data sets will be representative either. In contrast, DLW
studies of energy expenditure have been reported for two large,
randomly selected, urban-population cohorts, with a com-
bined population of 929 men and women aged 30–69 years.
The two study populations were quite similar, both being
recruited from the Washington, DC, metropolitan area. They
were judged to be a reasonable match to the UK population in
terms of the extent of overweight and obesity (mean BMI
values of 27.2 kg m2: current UK values 27 kg m2) and eth-
nicity. PAL was evaluated for the whole cohort and the median
and distribution of PAL values within each cohort did not
differ and were not influenced by gender. For the combined
cohort (n ¼ 929), the distribution, shown in Figure 1, was
skewed, with subjects clustered at the lower end of the range.
PAL values of 1.49, 1.63, and 1.78 were identified for the 25th,
median, and 75th centiles, corresponding to sedentary, low,
and moderate activity. Neither median value nor distribution
of PAL values for BMI categories differed significantly, and the
regression of PAL on BMI was nonsignificant (p ¼ 0.64 for
slope: R2 < 0.1%). This suggests that with increasing BMI, the
decrease in extent of PAEE is matched by its increasing energy
cost. PAL fell slightly with age, although age explained only
0.4% of the variance, that is, falling from PAL values of 1.69 at
30 years to 1.63 at 70 years. Although the minimum age was
30, PAL values of the youngest subjects aged 35 years did not
differ from those aged >35 years suggesting that the lack of
18–30 years of age is unlikely to represent a significant source
of bias when the data set is used to represent PAL values for all
adults. Derived EAR values for adult men and women are
shown in Table 1.
Identifying Suitable PAL and EAR Reference Values for
Infants, Children, and Adolescents
For infants, most recent committees have calculated energy
reference values from the energy deposited in new tissue plus
TEE. The energy costs of tissue deposition have been calculated
 Energy: Intake and Energy Requirements 515

Lifestyle/phenotype/activity: overall range

Frequency sedentary extremely active

18
within
population

Effect of additional activity on PAL

150 min/week moderate intensity activity,

PAL = +0.15

300 min/week sport/strenuous leisure activity

PAL = +0.3

Competitive training
PAL = +0.6
0
1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Physical activity level
1.49 1.63 1.78
less population more
active (median) active
(25th (75th
centile) centile)

Figure 1 Schematic representation of energy expenditure and consequent energy requirements of the adult population as a function of the physical activity
(PAL) value. From the UK report Dietary Reference Values for Energy 2011, Scientific Advisory Committee on Nutrition London TSO 2012. The
distribution of PAL values is that of the reference population identified as appropriate to define the UK dietary reference values for energy. The likely increases
in PAL (and consequent energy needs) shown for various activities include the current UK recommendations that adults participate in at least 150 min
of moderate intensity activity per week: an increase in PAL of 0.15. These increases in activity are shown as they would affect initial PAL values at the 25th
centile and median values, with the increases indicated by the length of the arrows. Although for those with PAL values at the 25th centile, the
recommendations for increased activity will only increase PAL to the median level, this can still be expected to have health benefits. The increases shown
are only approximate. They will vary to a small extent with the initial PAL, that is, with the intensity of the activity replaced, and will be slightly less
than the values shown, especially for subjects with high initial PAL values. The equivalent values in terms of energy are shown in Table 1.

Table 1 Estimated average energy requirements for adults (MJ day1)

EAR values (MJ day1)

Height (cm) Weight (kg) BMR (MJ day1) Less active Population More active

Men 175 69.2 6.7 10.0 10.9 11.9

þ30 min moderate intensity activity, 5 days per week: þPAL ¼ 0.15 þ1
þ60 min active sport five times per week: þPAL ¼ 0.30 þ2
þintense aerobic exercise in competitive training: þPAL ¼ 0.60 þ4
Women 162 58.7 5.4 8.0 8.7 9.5
þ30 min moderate intensity activity, 5 days per week: þPAL ¼ 0.15 þ0.8
þ60 min active sport five times per week: þPAL ¼ 0.30 þ1.6
þintense aerobic exercise in competitive training: þPAL ¼ 0.60 þ3.2

Values shown for illustration are prescriptive aimed for a healthy body weight equivalent to a BMI of 22.5 kg m2 at mean adult heights for the UK population. BMR is calculated
from the Henry prediction equations (Henry, C. J. (2005) Basal metabolic rate studies in humans: measurement and development of new equations. Public Health Nutrition
8:1133–1152), based on weight, age, sex, and height. EAR values calculated as BMR  PAL where PAL ¼ 1.49 (less active), 1.63 (population), and 1.78 (more active). Also shown are
additional daily energy needs associated with the selective amounts of increased activity shown in Figure 1.
Source: Values derived from the UK report, Dietary Recommendations for Energy 2011, Scientific Advisory Committee on Nutrition London TSO 2012.

from an analysis of the body composition of healthy infants
during normal growth. Longitudinal DLW studies of TEE over
the first 2 years of life have been performed in healthy, well-
nourished, nonstunted infants, born at full term with adequate
birth weight, who have been either breast-fed or formula-fed.
These measurements have allowed the development of simple
prediction equations for TEE as a function of weight for breast-
fed, formula-fed, and mixed-fed infants, in the latter case by
predicting TEE values intermediate between the breast-fed and
formula-fed values. EAR values for energy have then been
calculated from the sum of TEE and energy deposition for
breast-fed, formula-fed, or mixed-fed infants. For the United
Kingdom, EAR values are calculated from weights of children
growing along the trajectory of the UK-WHO Growth Stan-
dards. Although it is recognized that exclusive breast-feeding
is highly unlikely to continue beyond the recommended first 6
months of life, in most recent reports, EAR values have been
calculated by this model for the first 12 months of life for each
mode of feeding (adopted during the first 6 months) and
tabulated for boys and girls at monthly intervals. This is
because after 12 months, a different calculation of the EAR is
used as described later. For boy infants, EAR values increase
516 Energy: Intake and Energy Requirements
from 2.03, 2.36, and 2.17 MJ day1 for breast-fed, formula-fed,
and mixed-fed infants at 1 month to 3.2, 3.3, and 3.25 MJ day-
1
at 12 months, respectively. For girl infants, EAR values
increase from 1.82, 2.16, and 1.97 MJ day1 for breast-fed,
formula-fed, and mixed-fed infants at 1 month to 2.91, 3.05,
and 2.98 MJ day1 at 12 months, respectively.
For children and adolescents, the same principle used to
derive EAR values for adults is used based on TEE derived from
PAL  BMR. However, because of potential changes in age in
both body composition and behavior that may influence TEE,
the starting point for derivation of EAR values is an analysis of
changes in PAL with age for boys and girls. In fact, while there
are a large number of published DLW studies of infants,
children, and adolescents, not all of them allow individual
PAL values to be calculated, and most of the studies involve
small numbers (average n ¼ 20). Thus, only a limited analysis
of the distribution of PAL values at each age and each gender
can be performed. In fact, PAL values were obtained for 170
studies (3502 individual measurements, 2082 females, and
1420 males), with reported values for every age from 1.5 to
18 years allowing mean study values for boys and girls to be
examined as a function of age, as shown in Figure 2. PAL
values were not influenced by gender, but did increase with
age; this effect is driven by low values for preschool children
and some groups of preadolescents. As a result, all studies were
grouped into three age categories 3, >3 to < 10, and 10–18
years. Whether this age increase in PAL is a physiologically or
behaviorally real phenomenon once children enter schooling
is not clear since several studies of children aged 5–10.5 years
show individual PAL values that vary markedly (e.g., from 1.19
to 2.34). It is possible therefore that schoolchildren from a very
2.4
Female
Male
2.2
2.0
1.8
PAL
1.6
1.4
1.2
group1 group 2
1.39 1.57
1.0
0 2 4 6
Figure 2 PAL values for energy (MJ day1) for infants, children, and adolescents. From the UK report Dietary Reference Values for Energy 2011,
Scientific Advisory Committee on Nutrition London TSO 2012. Median values for the indicated age ranges are shown. Each point represents a single age
group reported in a single publication or in publications, which list mean values for each of several age groups. Boys and girls are shown separately.
early age exhibit a similar very wide range of activities and PAL
values as observed in adults.
EAR values for children also include the cost of energy
deposition during growth, which is substantial in the first
year of life, tapering off thereafter. Thus, for preschool chil-
dren, schoolchildren, and adolescents, growth costs are much
less, and the energy deposited can be accounted for by a simple
þ1% adjustment of PAL (PAL  1.01), which results in accept-
ably low levels of error. This resulted in adjusted median PAL
values for the three age groups of 1.40, 1.58, and 1.75. These
values were used to define the population EAR values of chil-
dren as a function of BMR predicted for age, weight, height,
and gender. Population EAR values for energy as a function of
age are shown in Figure 3. The gender differences reflect the
slightly higher BMR values for boys due to differences in weight
and body composition.
Energy Reference Values for Pregnancy
It is customary to calculate the ERs for pregnancy and lactation
as increments to be added to the mother’s EAR. Ideally, women
should begin pregnancy at a healthy body weight, and this
raises the question as to whether ER values for pregnant
women should be calculated as for nonpregnant women on
their healthy body weight (i.e., a BMI of 22.5 kg m2) or on
their actual body weights. It is the case that women who are
overweight or underweight at the beginning of pregnancy are
at risk of poor maternal and fetal outcomes, although this is
not completely understood. Because of this uncertainty, it is
usual practice to adopt a precautionary approach and not
to make ER recommendations aimed at weight loss for
group 3
1.73
8 10 12 14 16 18 20
Age

median PAL values used
14 1.40 1.58 1.75
12
Boys
10
EAR values: MJ/d
Girls
8
6 measurements of energy expenditure and the amounts and
4 from tissue stores. On the basis of a milk energy output of
2
0 need for an increment of 330 kcal day1 (or 1.38 MJ day1) for
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Age (years)
Figure 3 EAR values for energy (MJ day1) for infants, children, and
adolescents. Values derived from the UK report Dietary Reference Values
for Energy 2010, Scientific Advisory Committee on Nutrition London
TSO 2012. Values are calculated from BMR  PAL values shown for the
various age groups and are prescriptive values calculated for healthy
weights at each age. PAL values are median values for the published DLW
data. BMR values are calculated from the Henry equations (reproduced
from Henry, C. J. (2005). Basal metabolic rate studies in humans:
measurement and development of new equations. Public Health Nutrition
8, 1133–1152), using weights and heights indicated by the 50th centiles
of the UK-WHO Growth Standards (ages 1–4 years) and the UK 1990
reference for children and adolescents (Royal College of Paediatrics and
Child Health (2011) UK-WHO Growth Charts: early years. Available at
http://www.rcpch.ac.uk/growthcharts).
overweight pregnant women. For this reason, the recent UK
report identified EARs for pregnancy and lactation as estimates
of the incremental EIs likely to be associated with healthy
outcomes for mother and child that should be added to EAR
values calculated at actual preconceptional body weights.
As to the amount of extra food energy required for pregnant
women, the key issue is that of identifying the extent to which
the demands for gestational weight gain (GWG) due to growth
of the products of conception and of maternal weight can be
met by adaptive physiological and behavioral changes of the
mother as opposed to increased dietary EIs. A balance needs to
be struck between allowing for optimal fetal growth and avoid-
ing the excessive weight gain likely to occur with overestimates
of energy needs. In this context, the recent UK report con-
cluded that, in general, it is unlikely that women require extra
energy in the first trimester of pregnancy and compensatory
reductions in PAEE during the second and third trimesters are
likely to reduce the demand for extra EI at this time. Further-
more, although careful studies of energy expenditure and
deposition indicate an apparent need for additional energy,
such calculations include maternal fat deposition, which is not
a determinant of birth weight. Indeed, on the one hand, lon-
gitudinal body composition studies have indicated that some
fat deposition remains 6 months postpartum and may contrib-
ute to weight gain in women through successive pregnancies.
This is undoubtedly undesirable. On the other hand, there is
strong evidence that inadequate GWG is associated with
decreased birth weight and fetal growth (small for gestational
age). Recommendations for constraining weight gain may also
be inappropriate for pregnancies in vulnerable groups such as
Energy: Intake and Energy Requirements 517
teenagers. On this basis, it was concluded that the EAR for
pregnancy should be limited to an additional intake of
0.8 MJ day1 (191 kcal day1) during the last trimester.
Energy Reference Values for Lactation
The energy needs for lactation can be calculated from DLW
composition of breast milk and estimated energy mobilization
500 kcal day1 and an average weight loss of 0.8 kg month1,
which is equivalent to 170 kcal day1, this would imply the
the first 6 months during which time exclusive breast-feeding is
recommended.
After the first 6 months, the energy cost of lactation will
depend on the amount of breast milk consumed by the infant,
which is likely to be diminishing. The need for additional
maternal EI will also be modified by maternal body composi-
tion, so recommendations for additional intakes are generally
not identified for this period of the infant’s life.
Conclusion
Historically, reference values for dietary food energy needs
developed from crude observations of food EIs to an assumed
detailed and extensive understanding of the energy costs of
human activities. However, the advent of DLW studies of
actual energy expenditure has revealed that the assumed ability
to predict accurately the energy needs of individuals or popu-
lation groups with certainty was overly optimistic. Because of
this, the most recent national report has markedly simplified
the estimation of energy needs identifying only three activity
levels for any population group. These are a population EAR
value based on a PAL value of 1.63 and BMR at weights
equivalent to a BMI of 22.5 kg m2 at individual heights.
Given that for UK men and women, mean heights are 175
and 162 cm, respectively; this equates to 10.9 (2605) and 8.7
(2079) MJ day1 (kcal day1), respectively, as shown in
Table 1. For the chronically ill or for immobile elderly men
and women, a lower PAL value is appropriate with the 25th
centile PAL value (PAL ¼ 1.49), corresponding to ER values of
10.0 (2393) and 8.0 (1915) MJ day1 (kcal day1). For men or
women responding to advice to generally increase activity, the
75th centile PAL value (PAL ¼ 1.78) is more appropriate
and corresponds to ER values of 11.9 (2848) and 9.5 (2274)
MJ day1 (kcal day1).
In each case, these values are prescriptive in terms of sup-
porting a healthy body weight. Thus, for the overweight or
obese, that is, the majority of the UK population, the popula-
tion ER values are less than weight-maintaining EIs and should
allow for some degree of weight loss. However, the recommen-
dations are not prescriptive in terms of physical activity
although the extent to which PAL values will be higher for
those specifically engaged in additional physical activity can
be predicted as shown in Figure 1, with daily energy incre-
ments shown in Table 1. Thus, for those engaged in 30 min of
moderate, intensity activity for 5 or more days of the week,
60 min of active sport five times per week, or intense aerobic
518 Energy: Intake and Energy Requirements
exercise associated with training for competitive sport, the ER
will increase by an amount equivalent to 0.15, 0.3, or 0.6 PAL
units, respectively. Importantly, the principle of a reference
population distribution of PAL values as the basis for EAR
values allows for simple updates as appropriate DLW data
accumulate.
See also: Dietary References: US; Dietary Surveys: National Food
Intake; Energy Metabolism; Food Composition Databases.
Further Reading
FAO (2003) Food energy – methods of analysis and conversion factors. Rome: FAO
Report of a Technical Workshop no. 77.
FAO (2004) Human energy requirements. Rome: FAO Report of a Joint FAO/WHO/UNU
Expert Consultation no. 1.
Institute of Medicine (2005) Dietary reference intakes for energy, carbohydrate, fiber, fat,
fatty acids, cholesterol, protein, and amino acids. Washington, DC: National
Academy Press.
Millward DJ (2013) Energy balance and obesity: a UK perspective on the gluttony v.
sloth debate. Nutrition Research Reviews 26: 89–109. http://dx.doi.org/10.1017/
S095442241300005X.
Millward DJ (2012) A new approach to establishing dietary energy reference values.
Current Opinion Clinical Nutrition and Metabolic Care 15(5): 413–417.
Scientific Advisory Committee on Nutrition (2010) Dietary reference values for energy.
https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/
339317/SACN_Dietary_Reference_Values_for_Energy.pdf (accessed on
02.12.2014).
Relevant Websites
http://www.cdc.gov/nchs/nhanes.htm – NHANES, National Health and Nutrition
Examination Survey.
http://www.efsa.europa.eu/ – EFSA European Food Safety Authority.
http://www.food.gov.uk/the-website-of-the-food-standards-agency – Food Standards
Agency.
https://www.gov.uk/government/statistics/national-diet-and-nutrition-survey-results-
from-years-1-to-4-combined-of-the-rolling-programme-for-2008-and-2009-to-
2011-and-2012 – NDNS, National Diet and Nutrition survey.
 Enteral Feeding
DL Waitzberg and RS Torrinhas, University of São Paulo, São Paulo, Brazil
ã 2016 Elsevier Ltd. All rights reserved.
Definition
Enteral nutrition (EN) can be defined as a set of therapeutic
procedures applied to solely or partially replace or supplement
oral feeding for maintenance or recovery of nutritional status
of malnourished or eutrophic patients in hospital, ambulatory
or home settings, through the enteral (gastrointestinal tract)
administration of commercial or noncommercial diets com-
posed by controlled predefined or estimated amount of iso-
lated or combined nutrients and specially formulated and
developed for use by enteral probes or oral intake, aiming at
the synthesis or maintenance of tissues, organs, or systems.
Relevance
EN is part of specialized nutritional therapy, which also
includes parenteral nutrition (PN) (endovenous nutritional
supply). Its main relevance is to restore or maintain the nutri-
tional status of patients in order to prevent malnutrition. It has
long been known that malnutrition is associated with
increased morbidity and mortality in hospitalized patients.
Both EN and PN allow achieving the protein-calorie
requirements and the minimum daily requirement of vitamins
and minerals. When the gastrointestinal tract is structurally
and functionally intact, it is preferred to use EN. When you
cannot reach 60% of caloric needs by EN, we must consider the
associated use of PN.
There are metabolic, safety, cost/benefit, and mainly phys-
iological benefits when providing EN. The supply of nutrients
through the digestive system maintains intestinal anatomy and
its natural microbiota. These benefits allow the improvement
of the intestinal immune system and the decrease of infectious
complication risk in surgical patients.
Indications
EN should be indicated in situations in which the digestive
tract is fully or partially functional and when oral intake is
insufficient to reach two-thirds to three-quarters of the daily
nutritional needs, mainly in malnutrition conditions. The
main indications for EN use are listed in Table 1. In the other
hand, EN is relative or temporarily contraindicated in some
conditions, such as gastrointestinal tract obstruction, diarrhea
and vomiting refractory to drug therapy, short bowel syndrome
with severe malabsorption, severe paralytic ileus, high-output
intestinal fistulas, massive bleeding from the gastrointes-
tinal tract, severe malabsorption syndrome, and inability to
access gastrointestinal tract.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00255-5
Planning
Route of Feeding
After the indication of the route of feeding, the time by which
EN will be required should be estimated to then proceed with
the best access choice. Particularly for surgical patients, the
estimate must comprise the time of pre- and postoperative or
perioperative (both) nutrition. At least 10 days of preoperative
EN are associated with improved nutritional status in moder-
ate malnourished patients. However, patients with severe mal-
nutrition associated with other clinical comorbidities may
require longer EN treatment, particularly those with benign
diseases.
Short-term EN (less than 6 weeks) is achieved using a naso-
gastric feeding tube (in gastric, duodenal, or jejunal position).
Insertion of nasogastric feeding tubes in gastric or postpyloric
position can be done manually or with endoscopic or fluoro-
scopic assistance and at the bedside in most cases. Although
rare, complications can occur during the passage of a nasogastric
feeding tube, such as pneumothorax, empyema, mediastite,
esophageal perforation, bleeding and gastric perforation, injury
to teeth, tracheobronchial injury, and arrhythmias.
For long-term EN (more than 6 weeks) gastric or jejunal
nutrition ostomies are preferred. Gastrostomy and jejunost-
omy nutrition can be performed through open or percutane-
ous surgery using endoscopic, radiological, or laparoscopic
techniques. Planning for the nomination and selection of the
EN access is provided in Scheme 1.
After the choice of a nasogastric feeding tube or ostomy, it
should be decided where the distal end of the feeding tube will
be placed in gastric or intestinal position. Gastric access can be
obtained with nasogastric or gastrostomy and the postpyloric
access through nasojejunal feeding tube, gastrojejunostomy, or
jejunostomy. Some criteria applied to determine the placement
of a nasogastric feeding tube include the speed of gastric emp-
tying, gastroparesis, use of drugs inhibiting gastric and diges-
tive motility, and risk of pulmonary aspiration.
Formulation
The EN can be administered intermittently or continuously.
Selection of pathway for EN administration and the type of
infusion to be adopted will influence its formulation design.
This also involves determining the total period for diet admin-
istration, the volume to be infused, infusion rate, if gravity drip
will be used, and in which form it will be provided (infusion
pump or by bolus). Table 2 outlines the programming of EN
according to feeding tube positioning in pre- or postpyloric
location.
Enteral formulations should be nutritionally complete
when used as exclusive nutrition or as a supplement to patients
with normal oral ingestion; or nutritionally incomplete when
519
520 Enteral Feeding

Table 1 Enteral nutrition indication for adult patients

Clinical setting Recommendation

Cancer Less than 60% of energy needs intake for 5 consecutive days
Dysphagia
Elderly Severe neurologic dysphagia
Emotional depression at hyporexia phase
Contraindicative for terminal dementia
Surgery High nutritional risk (5–7 preoperative days)
Head and neck cancer (early – 24 h postoperative)
Gastrointestinal cancer (early – 24 h postoperative)
Severe trauma (early – 24 h postoperative)
Malnutrition at the surgery period (early – 24 h postoperative)
Insufficient oral intake to achieve basal energy expenditure for more than 10 postoperative days (early – 24 h
postoperative)
Heart disease Loss of body weight (6%) in 6 months (heart cachexia)
Crohn disease or ulcerative Nutritional supplementation higher than 600 kcal day
1 (consider)
colitis

Adequate gastrointestinal function?

No Yes

Parenteral Nutrition Enteral Nutrition (3 - 4 weeks)

Nasogastric tube feeding Enterostomy

Pulmonary aspiration risk? Pulmonary aspiration risk?

No Nasogastric tube No
Gastrostomy
feeding

Yes Nasoduodenal or Yes

nasojejunal tube Jejunostomy
feeding

Scheme 1 Planning for indication and pathway selection of enteral nutrition.

used only as a supplement nutrition. The evaluation of the
digestive and absorptive capacity of the patient should be
performed for better enteral formula selection (Scheme 2).
Several enteral formulations are based on fresh food, pro-
cessed food, or both fresh and processed food. Therefore,
nutrients comprising EN are generally the same constituents
of a normal diet, consumed by the oral route, including car-
bohydrate (40–60% total energy needs), protein (14–20%
total energy needs), fat (15–30% energy needs), and fiber
(40–20 g l
1). Different factors should be considered to facili-
tate the choice of the most appropriate enteral formulation for
patients with EN indication, such as caloric density, osmolarity
and osmolality, administration pathway, source and complex-
ity of nutrients, and disease.
The EN caloric density (kcal ml
1) should be based on the
patient’s total calorie needs versus the volume of enteral diets
to be administered per day. Enteral diets with higher energy
density have a lower amount of water, which can range from
690 to 860 ml l
1 diet. The categorization of enteral formulas,
according to its energy density, is shown in Table 3.
Vitamin and mineral supply varies according to the specific
needs of the patients and their disease. In the specific nutri-
tional needs, you should evaluate the indication of additional
micronutrient supplementation, even when the formulation,
per se, achieves those values recommended by the Recom-
mended Dietary Allowance (RDA). Clinical nutritional patient
evaluation should include objective and/or subjective indica-
tors to identify, as early as possible, any risk of specific
 Enteral Feeding 521

Table 2 Programming of EN according to feeding tube positioning

Tube
feeding
position Volume Osmolality Fractionation Administration time

Stomach Allows high-volume Hyperosmolar solutions are Depends on the total volume/day About 120 drops per min (or
supply tolerated, but the higher solution and patient tolerance. Lower time (min) ¼ total volume (ml)/
osmolality the slower stomach fractionation (four to six times 6) from the beginning of
emptying per day) and higher volume in therapy
each supply may be used
Postpyloric During intermittent Better tolerance for formulations Continuous or intermittent Initial phase: 60 drops/min (or
supply, the volume with less than 550 mOsm l
1; fractionation, generally between time (min) ¼ total volume (ml)/
should not exceed dripping of hyperosmolar six and eight supplies per day in 3); ‘adapted’ phase: 120 drops
300 ml h
1 in solutions should be strictly each 3 h per min (or time (min) ¼ total
adapted patients controlled by using infusion volume (ml)/6)
pump

Patient can achieve 60% of their nutritional needs by oral pathway?

Yes No

Oral Nutrition Enteral Nutrition

Normal conscience? Metabolic dysturb?

Yes Yes No

General diet Specialized enteral diet Conventional enteral diet

No

Light or liquid diet with Can ingest / absorb intact Can ingest / absorb intact
supplements nutrients? nutrients?

Yes Yes

Specialized diet may be Polymeric conventional

polymeric diet

No No

Specialized diet with Specialized diet with

hydrolyzed, or elemental,
hydrolyzed, or elemental, or
or formulation with
formulation with modules modules

Scheme 2 Planning for selection of enteral diets.

micronutrient deficiency for it to be immediately corrected vitamin and mineral supply. Therefore, EN dietary planning
and/or prevented. attends to the need for supplementation or not of these micro-
Some specialized and very specific formulations to particu- nutrients. For the long-term use of incomplete enteral feeding,
lar clinical situation (e.g., renal failure) are insufficient in some the supplemental vitamins and minerals should be indicated.
522 Enteral Feeding
Table 3 Categorization of enteral formulas according to its energy
density
Energy density Value (kcal ml
1) Formula
Very low <0.6 Sharply hypocaloric
Low 0.6–0.8 Hypocaloric
Standard 0.9–1.2 Normocaloric
High 1.3–1.5 Hypercaloric
Very high >1.5 Sharply hypercaloric
In patients with malabsorption syndromes, investigate the
possible fat soluble vitamins (A, D, E, and K) deficiency to
correct it shortly. There is a lack of specific vitamin and mineral
recommendations for critically ill patients. However, in such a
condition, the needs of antioxidant nutrients are increased due
the oxidative stress, and it is recommended to supplement
vitamins A, C, and E, zinc, and selenium.
EN osmolality (mmol l
1 solution) and osmolality
(mOsm kg
1 water) are associated with its digestive tolerance.
Although the stomach tolerates diets with higher osmolality,
more distal portions of the gastrointestinal tract respond better
to isosmolares formulations. Therefore, hyperosmolar diets
infused by gastrostomy or nasogastric feeding tube have better
digestive tolerance when compared with administration by
postpyloric or jejunal probes.
The nutrients that most affect the osmolality of a solution are
simple carbohydrates (mono- and disaccharides), which have
greater osmotic effect than the higher molecular weight carbo-
hydrates (starch); minerals and electrolytes, due the property of
its dissociation into smaller particles (e.g., sodium, potassium,
and chloride); hydrolyzed proteins; crystalline amino acids; as
well as medium-chain triglycerides, because they are more sol-
uble than long-chain triglycerides. The more hydrolysates com-
ponents contains the formulation, the higher its osmolality.
Enteral diets should not exceed the value of the renal solute
load tolerated by the kidneys (800–1200 mOsm, in normal
situation). Renal solute load can be calculated by adding
1 mOsm for each mEq of sodium/potassium/chloride, and
5.7 mOsm (adults) or 4 mOsm (children) for each gram of
protein from its formula. Special attention should be given to
critical clinical situations, such as sepsis, postoperative,
polytrauma, and severe burn, where the urine becomes very
dense, with high osmolality (around 500–1000 mOsm kg
1),
even under appropriate hydration.
Importantly, the influence of the medication osmolality is
usually neglected. The mean osmolality of liquid medications
administered orally or by feeding tube ranges from 450 to
10 950 mOsm kg
1 water. Certain manifestations of gastroin-
testinal intolerance may be related to the medication, although
it is often attributed to enteral formulation.
In specific clinical situations, there may be demands for
change in the types of nutrients used; the quantity and/or
form these should be presented. In such cases, nutritional
therapy becomes more specialized. These adaptations involve
changes from simple source of nutrients used until its
physicochemical and structural modifications. Thus, special-
ized formulations for enteral use may provide different sources
of vitamins, minerals, carbohydrates, lipids, and proteins, and
these nutrients may be presented in their entirety or hydro-
lyzed (wholly or partly) structure.
Some specialized EN formulations are part of immunonutri-
tion. The immunonutrition is a nutritional intervention that
explores the particular activity of various nutrients in alleviating
inflammation and modulating the immune system, in which are
included the omega-3 fatty acids, arginine, glutamine, nucleotides,
and antioxidants. There is a current consensus that perioperative
immunonutrition can beneficiate elective surgical patients, espe-
cially those malnourished patients submitted to major gastroin-
testinal surgery. In these patients, administration of enteral diets
containing n-3 PUFA, nucleotides and arginine contributes to
decrease postoperative infectious and noninfectious complica-
tions and must be initiated 5–7 days preop (500–1000 ml day
1)
and maintained in the postoperative period.
Although the benefit of using this enteral formula combin-
ing different nutrients with immunomodulatory functions is
well established in surgical patients, data are lacking to confirm
or guide the effective and safe use of enteral diets containing
isolated immunonutrients in different clinical populations,
including arginine and glutamine. In hemodynamically stable
condition, arginine may offer immunologic and metabolic
benefits, but its participation in the synthesis of nitric oxide
may constitute a potential risk for septic patients. Enteral glu-
tamine should be considered to treat burn patients and trauma
victims, but there is not sufficient evidence for its use in criti-
cally ill patients with failure of multiple systems.
Other nutrients that may compose specialized EN formula-
tions include the branched chain amino acids (BCAAs). BCAAs
provide primary fuel for skeletal muscle during stress and
sepsis. Therefore, leucine, isoleucine, and valine may be
added to specialized EN formulas as supplemental metabolic
sources to attend the metabolic needs of skeletal muscle during
hypermetabolic conditions.
Administration
EN should be initiated as soon as possible to prevent intestinal
atrophy and its associated complications (mainly infection)
due the lack of nutrients, a concept known as early EN. In
critically ill patients, EN should be started within the first
24–48 h after admission. Under the metabolic point of view,
the use of early EN can prevent excessive secretion of catabolic
hormones by inhibiting the increase in serum cortisol and
glucagon and maintain nutritional status to prevent loss of
body weight and muscle mass and negative nitrogen balance.
However, the benefits of early EN do not extend to all
patients, and its efficient use is restricted by the need for
hemodynamic stability and adequate tissue perfusion and vis-
ceral oxygenation. Therefore, some clinical conditions, such as
paralytic ileus, abdominal distention, nausea, and vomiting,
may hinder the selection of potential candidates to benefit
from early EN. The points that limit the indication of early
EN remain to be clearly defined, with consensus and contro-
versy points, which can give rise to different clinical behaviors.
When the nasogastric feeding tube is placed in the stomach,
the concern about the dose and infusion rate is not so relevant
due the local adaptation mechanisms. Intermittent gastric
administration (30–60 min each one) can be initiated with
the volume of 60 ml in its total concentration and progress
up to 250 ml each four hours, respecting tolerance and nutri-
tional purpose; whereas continuous infusion can be initiated

with the volume of 10–40 ml h
1 in its total concentration and
progress from 10 to 20 ml every 8–12 h, as tolerated.
For postpyloric (duodenum or jejunum) access, attention
must be increased because fast dripping can cause colic and
diarrhea, with decreased caloric use and patient injury. With
the technique of continuous duodenal infusion, the dose and
rate to be employed correspond to those described for the
intragastric placement, with the difference that the concentra-
tion of the diet should be iso- or hypotonic.
Monitoring
Nasogastric or by ostomy feeding is associated with mechani-
cal, gastrointestinal, infectious, pulmonary, and otorhinolar-
yngologic complications, which is prevented or treated by
monitoring patients appropriately. Aspiratory pneumonia is
considered the most serious complication in EN. It can occur
due to excessive diet supply, delayed gastric emptying (with
potential risk for neurological disability of the reflex mecha-
nisms for vomiting protection) and paralytic ileus.
Aspiration of gastric residue may be useful to evaluate its
emptying and to avoid the risk of regurgitation and aspiration,
despite a lack of consensus for its routine use. For gastric residues
larger than 200 ml (nasogastric feeding tube) or than 100 ml
(gastrostomy) associated with abdominal discomfort and
distension, the EN must be must interrupted and the patient
clinically and radiologically investigated. When not associated
with digestive symptoms, it is recommended to delay the diet for
an hour and reassess the gastric residual volume. Some drugs,
such as erythromycin, metoclopramide, bromopride and don-
peridona can be used to accelerate gastric motility.
Among the EN-associated complications, diarrhea diag-
nosed by three or more liquid bowel movements per day also
takes a prominent position. Specialized anamnesis can be
useful for the differential diagnosis of diarrhea during EN to
exclude other potential etiologies, such as infectious and/or
inflammatory gastroenterocolites.
Conclusion
As part of specialized nutritional therapy, EN is not free of
complications, but its use ensures the maintenance of the intes-
tinal anatomical integrity, tropism, and natural microbiota that
would be impaired when providing PN. Moreover, considering
that the gastrointestinal tract and liver process the nutrient
before its reaching the systemic circulation, EN is very effective
for maintaining the homeostasis of amino acids pool, as well as
the muscle mass. Currently, EN is preferred to PN because ‘when
the gut is functioning and can be used, it should be used,’ and it
is extensively applied around the world to effectively maintain
or reestablish patient nutritional status. Furthermore, EN-related
complications may be avoided or minimized through the ade-
quate nutritional planning (including suitable choice of enteral
access and placement, administration form, and formulation
design) and strict patient monitoring.
Enteral Feeding 523
See also: Amino Acids: Determination; Amino Acids: Metabolism;
Carbohydrate: Digestion, Absorption and Metabolism; Diarrheal
Diseases; Dietary Fiber: Determination; Dietary Practices; Dietary
References: US; Energy: Intake and Energy Requirements; Energy
Metabolism; Fatty Acids: Determination and Requirements; Fatty Acids:
Essential Fatty Acids; Fatty Acids: Fatty Acids; Fatty Acids: Metabolism;
Fish Oils: Composition and Health Effects; Malnutrition: Concept,
Classification and Magnitude; Malnutrition: Prevention and
Management; Nutrition and Infection; Protein: Digestion, Absorption
and Metabolism; Protein Quality and Amino Acids in Maternal and
Child Nutrition and Health; Protein: Requirements; Proteins: Chemistry,
Characterization, and Quality; Storage Stability: Mechanisms of
Degradation; Storage Stability: Shelf Life Testing; Vitamins: Overview.
Further Reading
Bartlett Ellis R and Fuehne J (2015) Examination of accuracy in the assessment of
gastric residual volume: a simulated, controlled study. Journal of Parenteral and
Enteral Nutrition 39(4): 434–440.
Baskin W (2006) Acute complications associated with bedside placement of feeding
tubes. Nutrition in Clinical Practice 21(1): 40–55.
Baxter Y, Waitzberg D, Gama-Rodrigues J, and Pinotti H (2014) Critérios de decisão na
seleção de dietas enterais. In: Waitzberg D (ed.) Nutrição enteral e parenteral na
prática clı́nica, 1st ed, pp. 659–676. São Paulo: Atheneu.
Braga M, Wischmeyer P, Drover J, and Heyland D (2013) Clinical evidence for
pharmaconutrition in major elective surgery. Journal of Parenteral and Enteral
Nutrition 37(5 Suppl.): 66S–72S.
Heyland D, Dhaliwal R, Suchner U, and Berger M (2004) Antioxidant nutrients: a
systematic review of trace elements and vitamins in the critically ill patient. Intensive
Care Med 31(3): 327–337.
Howard P, Jonkers-Schuitema C, Furniss L, et al. (2006) Managing the patient journey
through enteral nutritional care. Clinical Nutrition 25(2): 187–195.
Kreymann KG, Berger MM, Deutz NEP, et al. (2006) ESPEN guidelines on enteral
nutrition: intensive care. Clinical Nutrition 25(2): 210–223.
McClave SA, Kozar R, Martindale RG, et al. (2013) Summary points and consensus
recommendations from the North American Surgical Nutrition Summit. Journal of
Parenteral and Enteral Nutrition 37(5 Suppl.): 99S–105S.
Pearce C (2002) Enteral feeding. Nasogastric, nasojejunal, percutaneous endoscopic
gastrostomy, or jejunostomy: its indications and limitations. Postgraduate Medical
Journal 78(918): 198–204.
Prittie J and Barton L (2004) Route of nutrient delivery. Clinical Techniques in Small
Animal Practice 19(1): 6–8.
Sanderson I and Croft N (2005) The anti-inflammatory effects of enteral nutrition.
Journal of Parenteral and Enteral Nutrition 29(4 Suppl.): S134–S140.
Schröder O, Hoepffner N, and Stein J (2004) Enteral nutrition by endoscopic means; I.
Techniques, indications, types of enteral feed. Zeitschrift Gastroenterologie 42(12):
1385–1392.
Waitzberg D, Plopper C, and Terra R (2000) Access routes for nutritional therapy. World
Journal of Surgery 24(12): 1468–1476.
Zhang Y, Gu Y, Guo T, Li Y, and Cai H (2012) Perioperative immunonutrition for
gastrointestinal cancer: a systematic review of randomized controlled trials. Surgical
Oncology 21(2): e87–e95.
Relevant Websites
http://www.criticalcarenutrition.com – Critical Care Nutrition.
http://www.espen.org/ – ESPEN - The European Society for Clinical Nutrition and
Metabolism.
https://www.nutritioncare.org/– ASPEN - The American Society for Parenteral and
Enteral Nutrition.
 Enzymes: Analysis and Food Processing
T Haertlé, FIP, BIA, INRA BP, Nantes, France
ã 2016 Elsevier Ltd. All rights reserved.
Proteins with catalytic properties are called enzymes. They are
the key molecules responsible for the correct functioning of
living organisms. Because of their catalytic properties, enzymes
have long been used as crucial tools in many food technolo-
gies. Their deliberate use started in Neolithic times, at the dawn
of human food processing, when they were employed in the
first intentional fermentation processes (1). Since then,
humans have used starters or rennets containing beneficial
enzymes.
As with all proteins, enzymes are composed of 20 amino
acids linked by peptide bonds, which, from a chemical point of
view, are a subtype of the amide bond. The character of the side
chains of amino acids (polar, apolar, negatively or positively
charged, H-bond donor or acceptor) defines most of the intrin-
sic and extrinsic interactions of the enzyme molecule and its
interactions with the surrounding medium. Thus, the character
and spatial arrangement of the amino-acid side chains define
the structural and catalytic properties and specificities of
enzymes. The classification of enzymes, as proposed by Inter-
national Union of Biochemistry and Molecular Biology, orga-
nizes enzymes according to the reactions they catalyze (3). In
this way, enzymes are classified into six main categories:
• EC 1: Oxidoreductases
• EC 2: Transferases
• EC 3: Hydrolases
• EC 4: Lyases
• EC 5: Isomerases
• EC 6: Ligases
According to a study carried out by the Freedonia Group Inc. in
2003 (9), the global enzyme industry was worth $5.8 billion
US dollars in 2010, and it is forecast to rise 6.8% annually to $8
billion US dollars in 2015. The Freedonia study covers the total
sales of enzymes, including carbohydrases, proteases, polymer-
ases, nucleases, and lipases, for use in various markets, such
as biotechnology, food, and pharmaceuticals. It was pointed
out in Freedonia study that three important factors drive the
growth of the world enzyme market:
• Novel advances made in production biochemistry, fermen-
tation processes, and recovery methods have resulted in the
affordable large-scale production of enzymes.
• The development of various applications employing
enzymes has greatly increased market demand.
• Enzymes are capable of catalyzing many different reactions,
enhancing their collective commercial usefulness.
Despite increased growth in world demand for enzymes, only
approximately 200 of the 4000 types of enzymes are commer-
cially available on the global market.
Of these 200 enzymes, about 20 are produced in industrial
quantities. The various difficulties impeding the industrializa-
tion of enzymes are as follows: (i) it is currently difficult to find
enzymes with high yield, high activity, and high stability;
524 Encyclopedia of Food and Health
(ii) enzymes used for drug and food purposes must pass
through multiple phases of clinical trials and government
approval, such as those conducted by the U.S. Food and Drug
Administration (FDA) and the European Food Safety Authority
(EFSA), which is both time-consuming and costly; and (iii)
enzymes for other purposes, such as detergents, require strict
testing and approval by government sectors dealing with envi-
ronmental safety. Not all the classes of enzymes are used in
food processing with equal intensity. Some of the key food
enzymes are described below, grouped according to function
(e.g., processing carbohydrates or proteins).
Processing Carbohydrates
a-Amylase, EC 3.2.1.1
Synonyms: 1,4-alpha-D-glucan glucanohydrolase.
a-Amylase catalyzes the endohydrolysis of 1,4-a-D-
glucosidic linkages in polysaccharides containing three or
more 1,4-a-linked D-glucose units.
Uses and applications: a-amylases of different origins (4, 5)
serve as processing aids in the production of alcoholic bever-
ages, starch hydrolysis, brewing, bread- and sugar-making, and
fine bakery.
Origins: Bacillus amyloliquefaciens, Bacillus licheniformis,
Aspergillus oryzae, barley malt, porcine pancreas.
Amylases are used extensively in the food, textile, detergent,
paper, and pharmaceutical industries. a-Amylases are also used
as sugarcane or sugar beet processing aids, solving the viscosity
and raw sugar filtration problems typical to starches. The spe-
cific ability to hydrolyze the internal a-(1,4) links in amylose
and amylopectin, yielding shorter, less viscous polysaccharide
chains, has made a-amylases attractive for use in sugarcane and
sugar beet processing. Although different sources of a-amylase
are available, enzymes of microbial origin are generally pre-
ferred because of their advantages, such as cost-effectiveness,
efficient production, and consistency. Most commercial
a-amylases are produced from Bacillus species. These amylases
are thermophilic, thermostable, and acidic enzymes isolated
from Bacillus strains, and they are widely used in starch
processing.
Their unique activities, efficiencies, and stabilities are typi-
cally specific to the Bacillus species from which the enzymes
originated, unless they were genetically modified or produced.
Overall, a-amylase performance is affected by several variables:
pH range, calcium requirements, temperature stability, hydro-
lytic activity, Rt, and resistance to osmotic conditions, that is
Brix tolerance. Because of the diverse functionality of commer-
cial a-amylases, their optimal application in the sugarcane
industry requires a full understanding of their activities and
potential to hydrolyze different forms of starch under various
industrial conditions.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00257-9

a-Amylases [a-1, 4-glucan glucohydrolase] are among the
most important enzymes used for the direct conversion of
starch to glucose, which can be further fermented into ethanol.
It breaks successive bonds from the nonreducing end of the
straight chains producing glucose. The production of ethanol
from starch requires two steps, enzymatic hydrolysis followed
by fermentation, which are performed by two different organ-
isms (bacteria and yeast).
The quantitative increase in glucoamylase secretion and
ethanol production requires strain improvement, because the
quantities produced by the wild strains are often too low.
Strain improvement in industrial applications is
generally achieved by mutation with agents such as
methylnitronitrosoguanidine(MNNG), ethidium bromide,
and ultra violet light. Production strains that are mostly
used in industrial fermentations are developed by repeating
random mutations followed by selection.
A common substrate for a-amylase production by solid-
state fermentation (SSF) is wheat bran. Enzyme extracted
from these substrates contains various impurities, including a
significant amount of melanine-like product-coloring matter,
which can interfere in the juice clarification process, if the
enzymes are used in crude form. Usually, a-amylases are puri-
fied from these colored extracts by different purification steps.
The purification of the targeted enzymes should be such that
the number of steps are minimized in order to be cost-effective,
because the yields and the number of operational steps are
inversely proportional to each other. Often a-amylases are
partially purified by activated charcoal in a single step.
a-Amylases are calcium-containing enzymes, binding at
least one calcium ion per monomeric unit. The calcium ions
convey the resistance to pH, temperature, proteolysis, and
denaturation by different agents and by heat. The thermosta-
bility of a-amylase is increased greatly by bound calcium ions.
Recently, many calcium-fortified fruit juices have been pro-
posed as healthy and nonfat alternative foods, especially for
children. Therefore, many fruit juices are clarified with use of
a-amylase in presence of calcium chloride.
a-Amylase has been found to be a possible causative aller-
gen for protein contact dermatitis (PCD), however. Main activ-
ity assay: One unit will liberate 1 mg of maltose from starch in
3 min at pH 6.9 and 20
C. The a-amylase assay uses
ethylidene-pNP-G7 as the substrate. Once the substrate has
been specifically cleaved by a-amylase, the smaller fragments
produced can be acted upon by a-glucosidase, which causes
the ultimate release of the chromophore, which can then be
measured at 405 nm.
b-Amylase EC 3.2.1.2
Synonyms: 1,4-a-D-glucan maltohydrolase. b-Amylase cataly-
zes the exohydrolysis of 1,4-a-D-glucosidic linkages in
polysaccharides, resulting in the successive liberation of malt-
ose units from the nonreducing ends of the chains.
Uses and applications: Partial delactosylation of milk and
other dairy products. Β-Amylase is also employed in the starch
and alcohol industries.
Sources: Kluyveromyces lactis, barley, wheat grains.
b-Amylase is a member of family 14, which comprises
glycosyl hydrolases, and it catalyzes the release of successive
Enzymes: Analysis and Food Processing 525
b-maltose from the nonreducing ends of a-1,4-linked oligo-
and polyglucans.
b-Amylase is a major component of so-called diastatic
power (i.e., the combined a-amylase, b-amylase, debranching
enzyme, and a-glucosidase activities) of malt. Exclusive malt-
ose production is utilized by the industry for the production of
maltose-rich syrups and the nondigestible sweetener maltitol.
b-Amylases have been found not only in various plants,
such as sweet potato, barley, wheat, and soybean, but also in
some bacteria. b-Amylase, alone or the combination with
transglucosidase treatment, can also be used to enhance the
slow digestion properties of starch. For amylopectin with a
higher proportion of short chains, the amylopectin structure
itself slows enzymatic digestion, due to its higher branch den-
sity and short chain length, which are more difficult for the
amylolytic enzymes to digest. Starch is the major reserve poly-
saccharide of higher green plants, and it is the second most
abundant natural biopolymer on earth after cellulose. Starch is
also the main energy-providing material in cereal- and tuber-
based food products for the majority of the world population.
In human nutrition, the proper rate of glucose release and
absorption from digesting starch plays an important role in
nutrition, helping to maintain adequate energy levels. Accord-
ing to the rate and extent of starch digestibility, starch is gen-
erally classified as rapidly digestible starch (RDS), slowly
digestible starch (SDS), or resistant starch (RS). RDS induces
a fast increase in blood glucose and insulin levels, whereas SDS
is slowly digested throughout the small intestine, with a low
glycemic response. Recent studies have shown that obesity and
related metabolic diseases, such as diabetes mellitus and car-
diovascular diseases, may be associated with the long-term
consumption of foods with high RDS contents, which is pos-
itively correlated to a high glycemic index and will cause a
substantial fluctuation of glucose homeostasis regulatory hor-
mones and high stress to the regulatory system. Therefore,
improving food quality with higher amounts of SDS is becom-
ing an area of interest for academia and industry. Generally,
starches differ in digestion rates. b-Amylase hydrolysis is
important for making sugar syrup and brewing beer and liquor
derived from starch. Thanks to the application of b-amylolysis,
the amount of SDS can be more than doubled.
b-Glucanase from Talaromyces emersonii belongs to the fam-
ily of hydrolytic enzymes. This food enzyme catalyzes the
hydrolysis of 1,3 linkages or the b-1,4 glucan of the cereals
used in the production of beer, and it helps reduce the viscosity
of musts caused by barley glucans.
Main activity test: According to Bernfeld (1955), the rate at
which maltose is released from starch is measured by its ability
to reduce 3,5-dinitrosalicylic acid. One unit releases one micro-
mole of b-maltose per min at 25
C and pH 4.8.
Xylanase EC 3.2.1.8
Synonyms: endo-1,4-b-xylanase.
Uses and applications: bread making, special bread, the
production of maize starch and alcohol through fermentation.
Origins: Aspergillus oryzae, Bacillus subtilis, Bacillus subtilis,
and Trichoderma longibrachiatum.
Xylanase hydrolyzes the b-1,4 linkages-D-xylosidic inside of
chains of xylans and arabinoxylans releasing reducing sugars.
526 Enzymes: Analysis and Food Processing
Hemicelluloses are hydrolyzed by the cooperative action of a
group of enzymes, and the main enzyme involved in the
depolymerization of xylan is endo-b-1,4-xylanase. The b-1,4-
endoxylanases (1,4-b-D-xylan xylohydrolase, EC 3.2.1.8),
which are produced by algae, protozoa, molluscs, crustaceans,
insects, seeds of terrestrial plants, bacteria, and many fungal
species belonging to the genus Aspergillus, are commonly
found in nature. However, the xylanase enzymes of filamen-
tous fungi are potentially the most interesting for industrial
uses, because they are produced in greater amounts compared
with those of yeast and bacteria. These enzymes can be used in
monogastric feeds in order to hydrolyze nonstarch polysaccha-
rides such as b-glucan and arabinoxylan that are found in
vegetables, thus increasing feed conversion, body weight gain,
and the yield of production (4, 5). To date their use was
broadened to many processing industries, such as pulp,
paper, food, and textile. Of the many reported applications
for xylanase, its use as a food supplement has played an impor-
tant role for monogastric animals, because it can improve the
utilization of nutrients.
Xylanases are used in the baking industry to stabilize dough,
to make dough flexible, and to improve gluten strength. Xylan is
the principal hemicellulose, which is the major plant cell wall
polysaccharide component. Arabinoxylans are important in the
formation of gluten and other networks in dough, and it has
been reported that the nonwater-extractable arabinoxylans exert
a negative effect on bakery products, whereas the water-
extractable forms of arabinoxylans, with medium to high molec-
ular weights, have a beneficial effect.
Xylanases have been added to dough to accelerate the pro-
cess of bread making by helping to breakdown polysaccharides
in the dough. Enzyme activity is effective in dough by cleaving
arabinoxylan chains and thus modifying their functions. More-
over, xylanases contribute to the decrease of dryness and stiff-
ness of the dough with increasing elasticity, extensibility, and
coherency, as well as increases in volume and decreases in
bread density. Xylanase treatments also result in higher mois-
ture retention and the improvement of sensory perception of
bread. Dough characteristics and bread quality improve signif-
icantly in response to xylanase treatments during tempering, as
compared to application during mixing.
Main activity test: Xylanase activity is tested using an
assay based on the release of dyed fragments from 4-O-methyl
glucuronoxylan dyed with Remazol Brilliant Blue dye or
p-pydroxybenzoic acid hydrazide measured at 410 nm.
Glucose Oxidase EC 1.1.3.4
Synonyms: b-D-glucose:oxygen 1-oxidoreductase
Uses and applications: gluten strengthening, gelation of the
soluble arabinoxylans in the flour.
Origins: Aspergillus niger.
Glucose oxidase can be used for strengthening the dough.
Glucose oxidase catalyzes the oxidation of b-D-glucose to form
D-glucono-1,5-lactone and hydrogen peroxide. Glucose oxidase
has a gluten-strengthening effect by inducing the formation of
protein–protein bonds that strengthen the protein network and
thereby strengthen and stabilize the dough. Glucose oxidase
catalyzes the oxidation of glucose into gluconolactone, with the
concomitant reduction of the oxygen to hydrogen peroxidase.
Five mechanisms are known, concerning the effects of the sugar
oxidase enzymes, as follows: (i) generation of disulfide bridges
between the proteins, (ii) formation of di tyrosines, (iii) block-
ing the effect of glutathione, (iv) gelation of the soluble arabi-
noxylans in the flour, and (v) alteration of the equilibrium
between the different enzyme systems present in the dough.
Main activity assay: Glucose oxidase can be utilized for the
enzymatic determination of D-glucose in solution. As glucose
oxidase oxidizes b-D-glucose to D-gluconolactate and hydrogen
peroxide, horseradish peroxidase is often used as the coupling
enzyme in glucose determinations. Although glucose oxidase is
specific for b-D-glucose, solutions of D-glucose can be quanti-
fied, because a-D-glucose will mutorotate to b-D-glucose as the
b-D-glucose is consumed by the enzymatic reaction. Thus, this
enzyme is also used in biosensors that measure the amount of
glucose in blood.
Processing Proteins
Rennet/Chymosin EC 3.4.23.4
Synonyms: chymosin, rennin.
Uses and applications: Rennet or chymosin is used for
cheese making and has highly specific milk-clotting activity
relative to its proteolytic activity.
Origins: Fourth stomach of the suckling calf and fungi such
as Mucor miehei, Mucor pusillus, and Endothia parasitica.
Aspartic proteases (EC 3.4.23.X) are endopeptidases having
two aspartic acid residues that are critical for catalytic activity
within their active sites. They are an important class of pro-
teases, which are widely used as milk-coagulating agents. Chy-
mosin (EC 3.4.23.4), the principal aspartic protease used for
cheese making, has highly specific milk-clotting activity rela-
tive to its proteolytic activity.
Chymosin has broad specificity, similar to that of pepsin A,
and it clots milk through the cleavage of a single Ser-Phe105-
Met-Ala bond in k-casein. By predominant cleavage of the
Phe105–Met106 bond of bovine k-casein, chymosin has high
specificity for splitting the caseinomacropeptide from the
casein micelles, and therefore, it induces milk clotting.
Because they are responsible for the hydrolysis of the k-casein
chain in casein micelles of milk, these enzymes are used as milk
coagulants in cheese preparation. Because cheese consumption
is growing all over the world, the production of different
cheese varieties has been increased. Consequently, the avail-
ability of calf rennet is limited, and now, calf rennet only
covers 20–30% of the world demand for milk coagulant.
Calf rennet obtained from the fourth stomach of a suckling
calf is used all over the world, except in India, for the manu-
facture of almost all varieties of cheeses. However, due to the
acute shortage of calf rennet, a substitute for calf rennet has
been developed from fungal sources such as Rhizomucor miehei,
which produces acid protease for the manufacture of goat and
sheep cheeses. Substitute proteases are also derived from plants
such as wild thistle (Cynara cardunculus), and recombinant
rennet-chymosin is used for milk coagulation. Chymosins
can be obtained from various sources, such as animals, plants,
and fungi. The properties of different chymosins have been
reviewed by Kumar et al. Chymosins, the major component

of rennet (milk clotting enzyme), are the acid proteases pro-
duced in the fourth stomach of milk-fed ruminants.
Although the cost has been reduced due to the biotechno-
logical preparation of recombinant chymosins, natural alter-
natives are still preferable. However, the unavailability of calf
stomachs and ethical problems associated with animal slaugh-
ter have forced a search for other alternatives to calf chymosin.
In this regard, various microbial alternatives are used for chy-
mosin production, but not all of these sources are suitable for
the production of quality cheese, because they often generate a
bitter taste. Recombinant chymosin production has been
achieved in different microorganisms in response to the con-
sumer demand for vegetarian and vegan cheeses. Buffalo,
sheep, goat, and rabbit are believed to be good sources of
rennet. Bovine (Bos tarus), buffalo (Bubalos bubalis), and lamb
(Ovis platyurea) chymosins have been characterized extensively.
Substitutes from microbial sources would be more
interesting, considering the stable availability of these
sources and the low cost due to the possibility of using cheap
substrates for fermentation. It has been reported that microbial
coagulants represent 53% of the demand for milk-clotting
preparations. Chymosins from microbial origins have found
well-defined applications in cheese manufacturing. Some
microbial chymosins from fungi such as Mucor miehei, Mucor
pusillus, and Endothia parasitica have been purified, and their
enzymatic properties have been investigated in detail. These
fungal enzymes have been used in large-scale cheese produc-
tion since the 1960s. Apart from low cost and stable character-
istics, chymosins from fungi are also better accepted by people
whose eating habits and religious beliefs exclude the use of
animal enzymes or recombinant rennets.
Main activity test: Skimmed milk solution is incubated at
35
C for 10 min. Then 0.5 ml of rennet is added to the solu-
tion and mixed immediately. The time from adding rennet to
milk coagulation is recorded.
Transglutaminase EC 2.3.2.13
Synonyms: Transglutaminase, factor XIIIa, fibrinoligase,
fibrin-stabilizing factor, glutaminylpeptide g-glutamyltransfer-
ase, polyamine transglutaminase, tissue transglutaminase,
R-glutaminyl-peptide:amine g-glutamyl transferase, (TGase;
protein-glutamine gamma-glutamyltransferase), fibrinoligase,
glutaminylpeptide gamma-glutamyltransferase, polyamine
transglutaminase.
Uses and applications: Stabilization of the gluten network
in dough, reconstitution of matrix meat products.
Origins: Blood coagulation factor XIII, pig/bovine liver,
Streptoverticillium moboarense.
Transglutaminases are the enzymes used for the stabiliza-
tion of the gluten network in dough, allowing manufacturers
to overcome the lack of emulsifiers and produce good bakery
products from frozen dough. It is also used for the reconstitu-
tion of meat products.
Transglutaminase is an efficient and promising food enzyme
because it catalyzes acyl transfer reactions, deamidation, and
crosslinking (polymerization) between protein intra- or inter-
chain glutamine (acyl donor) and lysine (acyl acceptor) peptide
residues. The reactions promoted by this food enzyme create
profound changes in the proteins composing food matrices,
Enzymes: Analysis and Food Processing 527
leading to improved texture and stability in terms of tempera-
ture, syneresis, emulsifying properties, gelation, and increased
water-binding capacity, without changing the pH, color, flavor,
or nutritional quality of food.
Protein cross-linking may be carried out with help of trans-
glutaminase (TG) either prior to or simultaneously with the
addition of rennet. Enzymatic cross-linking affects both the pri-
mary and the secondary stages of rennet coagulation. The impact
of protein cross-linking on the primary enzymatic phase is
mainly related to increased coagulation times due to the inhibi-
tion of caseinomacropeptide release. Consequently, a higher
extent of protein cross-linking may lead to reduced gel firmness.
The simultaneous reaction of TG and rennet results in signifi-
cantly increased yield due to the enhanced lactoserum binding of
the gel network stabilized by additional covalent bonds resulting
from transglutaminase action. In conclusion, cross-linking has a
strong impact on the entire coagulation process, leading to ren-
net gels with modified functional properties.
Transglutaminases may even render food protein more
nutritious because of their potential use for the incorporation
of essential amino acids in proteic matrices. Microbial trans-
glutaminase (MTGase), an extracellular enzyme from the class
of transferases, is produced commercially via traditional fer-
mentation by the microorganism Streptoverticillium moboarense.
This food enzyme, which acts in a wide range of pH and
temperature (pH 5.0–8.0, optimum temperature of 50
C,
but with activity between 40 and 70
C), is Ca2þ independent,
and its activation does not require special cofactors. This
enzyme was also granted generally recognized as safe (GRAS)
status by the FDA in 1998, as well as approval from European
(French and Danish) food control agencies, making MTGase
very attractive for the food industry. According to existing
reports, native and MTGase-modified proteins differ only in
the number of bonds between glutamine and lysine residues.
Several studies have tested a variety of food proteins as sub-
strates for MTGase: milk and whey proteins, soy globulins,
myofibrillar proteins, albumins, and others. One of the main
industrial applications of MTGase is in the restructuring and
consolidation of meat products. Through the cross-links
MTGase catalyzes, the transglutaminase increases stability,
improves texture, and decreases losses during cooking, giving
the restructured meat product an appearance resembling that of
intact muscle. In the dairy industry, transglutaminase has been
used in the production of yogurt, in which it has been largely
used for the prevention of syneresis during whey separation,
because of its capacity to increase the gel water-holding capac-
ity. Despite transglutaminase’s innovative potential in the for-
mulation of various food products, it remains underexploited
for industrial uses, particularly in products of plant origin.
Considering the food industry’s significant interest in using
food-grade transglutaminases because of their specificity, mild
reaction conditions, and very low risk of toxin formation,
understanding how transglutaminases modify the functional
properties of food proteins is the most important step in promot-
ing their industrial use in novel, reconstructed food products.
MTGases belong to the class of transferases widely known to
modify the functional properties of proteins in food systems.
The main mechanisms of their action are polymerizations that
result in many functional and biophysical changes, including
changes in protein hydrophobicity. MTGases decrease protein
528 Enzymes: Analysis and Food Processing
solubility, inducing protein gelation. They also affect emulsifi-
cation, foaming, viscosity, and water-holding capacity, all of
which depend on radical decreases in cross-linked protein solu-
bilities. Because MTGase promotes the polymerization of pro-
teins via decarboxylation of free amino acids, it could be used to
suppress the formation of biogenic amines in foods containing
high amounts of free amino acid, such as the meat of certain fish
(e.g., mackerel, tuna, etc.).
Given that the majority of plastics are nonbiodegradable
and their excessive use has led to serious environmental prob-
lems, industry is seeking alternative natural sources for those
materials. Therefore, reticulated fish skin and scale gelatines, in
combination with soya proteins, could be used to produce
edible and biodegradable films, improving food product qual-
ity and reducing waste.
Among the materials used to prepare edible films, gelatine
has been extensively utilized because of its relative abundance
and excellent film-forming ability. Recently, edible films based
on fish gelatin have received increasing attention due to
concerns about mad cow disease and foot-and-mouth disease.
Transparent films with good mechanical properties can be
prepared from the fish scale gelatin extracted at pH 5. How-
ever, gelatin films have poor water resistance properties,
because of the highly hygroscopic property of gelatin. Addi-
tionally, the mechanical properties of gelatin films decrease
when in contact with the surfaces of foods with high moisture
contents. These properties restrict the application of edible
films prepared from unmodified native gelatin only.
It was reported that the physical properties of protein films
could be improved by adding transglutaminase, which is con-
sidered to be a safe and effective cross-linking agent. The
addition of soy protein isolate (SPI) and its coreticulation
with TG could also greatly improve the swelling property of
gelatin films.
Main activity assay: Most tests rely on measuring the absor-
bency caused by OPA-reactive Lys on the casein during the
transglutaminase action.
Papain EC 3.4.22.2
Synonyms: Papainase, papaya peptidase I.
Uses and applications: Papain is a processing aid for the
production of protein hydrolyzates as aromatic bases, aswell as
plant protein hydrolyzates. Papain is also used in the brewery
and in meat tenderization.
Origins: Carica papaya (papaya), Ananas comosus
(pineapple).
Papain is a cysteine protease with wide specificity, cleaving
the peptide bonds of basic amino acids, such as leucine and
glycine. It also hydrolyzes esters and amides. Papain will digest
most protein substrates more extensively than the pancreatic
proteases do.
The ability of papain to improve beef meat tenderness is
attributed to the increased degradation of myofibril proteins
and the disruption of muscle fiber structures, because of its
proteolytic action. The main components of meat, myofibrils,
mainly consist of actin, myosin, and various accessory proteins
and connective tissue, which consists of various collagen sub-
types and elastin. The relative proportions of these compo-
nents influence meat structure and texture and, thus, the
relative tenderness of a meat. It is known that the disruption
of these components of meat structure in various ways, includ-
ing the partial hydrolysis of meat proteins, contributes to ten-
derness. The potential of exogenous proteases, such as papain,
to tenderize meat has been exploited for a long time.
Main activity assay: A titrimetric determination of the acid
produced during the hydrolysis of benzoyl-L-arginine ethyl
ester (BAEE) serves to measure papain activity. One unit
hydrolyzes one micromole of benzoyl-L-arginine ethyl ester
per min at 25
C and pH 6.2, under the specified conditions.
Asparaginase EC 3.5.1.1
Synonyms: Colaspase.
Uses and applications: Asparaginase is used in the deami-
dation of food preparations containing L-asparagine and
carbohydrates, which are cooked at temperatures above
120
C, such as bread and other cereal products, as well as
fried products based on potatoes, generating polyacrylamide.
Asparaginase is an enzyme that catalyzes the hydrolysis of
asparagine to aspartic acid.
Origins: Aspergillus oryzae.
Main activity assay: Asparaginase hydrolyzes asparagine to
generate aspartic acid, which can be detected colorimetrically
(l ¼ 570 nm) or fluorescently (Ex/Em ¼ 535/590 nm) using a
coupled enzymatic reaction.
Processing Lipids
Lipases EC 3.1.1.X
Uses and applications: Cheese production, oil and egg indus-
tries, bakeries.
Origins: Pancreas, Fusarium venenatum, Fusarium oxysporum,
hybrid between Thermomyces lanuginosus and Fusarium
oxysporum.
The importance of lipases (triacylglycerol acylhydrolases, E)
is undeniable, and the impact of this group of enzymes is
clearly seen in their widespread application in many industries,
including organic synthesis, paper manufacturing, oleochem-
istry, dairy, cosmetics, perfume, biosensors, and detergents.
Lipases catalyze the hydrolysis of fats into fatty acids and
glycerol at the water–lipid boundary, as well as the synthesis of
esters from fatty acids and glycerol at the water-insoluble substrate
boundary. The unique capability of lipases to react only at the
interface between aqueous and nonaqueous phases distinguishes
them from esterases. Lipases hydrolyze the ester bonds in
triglycerides, yielding diglycerides, monoglycerides, and fatty
acids, and they catalyze the hydrolysis of ester bonds in sn-1 diacyl
phospholipids to form 2-acyl-1-lysophospholipid and fatty acids.
This improves the properties of dough and the quality of bread,
leading to a finer and more homogeneous crumb structure.
Phospholipases are also used in cheese production, as well
as the oil and egg industries. Engineered phospholipase C may
be used for the efficient degumming of edible vegetable oils;
the enhancement of the natural emulsifying properties and
heat stability of yolk and/or whole egg; the enzymatic hydro-
lysis of vegetable lyso lecithins into lecithins in order to
improve their emulsifying properties; and the degumming of
vegetable oils during refining, which eliminates phospholipids

from the oil during the purification step, in order to obtain
satisfactory taste and quality, provide better storage stability,
and facilitate the rest of the manufacturing process.
Lipases often have high chemo-, region-, and enantioselec-
tivity, so that they are considered to be one of the most important
industrially utilized biocatalysts. The current trend in lipase
research is to focus on microbial lipases rather than lipases
derived from animals and plants, because lipases from microor-
ganisms have several advantages, including the ability to catalyze
diverse reactions, produce high yields, and reduce production
costs. In addition, microorganisms have the advantage of relative
ease of genetic manipulation. Other major advantages of micro-
bial lipases include (i) structural stability in organic solvents, (ii)
independence from cofactors, (iii) catalyze reactions utilizing a
wide range of substrates, and (iv) high enantioselectivity. A
detailed description of bacterial and fungal lipases, as well as a
brief account of lipase purification techniques and screening
methods, can be found in a recent review by Nagarajan.
Omega-3 polyunsaturated fatty acids (o3 PUFA) from fish
oils promote well-established health and anti-aging benefits
that justify their use as functional ingredients in dietary sup-
plements, healthy foods, and nutraceutical products. Among
them, eicosapentaenoic (EPA, C20:5) and docosahexaenoic
acids (DHA, C22:6) continue to receive particular attention
because of their numerous biological properties and positive
effects on human health. In addition to recognized benefits for
the prevention of cardiovascular diseases, EPA and DHA were
reported to protect from inflammation-mediated disorders,
including obesity and diabetes, as well as Alzheimer’s and
related neurodegenerative diseases.
Human metabolism exhibits a limited ability to synthesize
o3 PUFAs. The dietary supply of preformed compounds there-
fore appears to be an essential alternative. However, the prac-
tical use of such lipids as food ingredients is often limited by
their high susceptibility to oxidation, which is responsible for
the undesirable off-flavor and odor of rancid oils associated
with a loss of nutritional value. Various solutions can be
implemented to minimize these degradation pathways, and
the pathway most commonly used by manufacturers is the
addition of antioxidants. Intensive research has been pursued
on natural phenolic antioxidants issued from plants. Many
studies reported that such antioxidants are highly effective at
protecting o3-enriched food products from oxidation. In addi-
tion, some works pointed out the interest of formulations
mixing o3 PUFAs and phenolic compounds for the prevention
of Alzheimer’s disease and the treatment of obesity through
lipid-lowering effects. Another approach involves bringing
together o3 lipids and phenolic compounds in a single entity.
Such an association was shown to improve the stability of
highly oxidizable fatty acids, while facilitating the solubiliza-
tion of phenolic compounds in lipid phases. Additional effects
could include the increased bioavailability of phenols, as well
as cumulative and even synergistic biological activities. In
addition, PUFA phenolic esters were reported to exhibit higher
antimicrobial, antioxidant, and antiinflammatory activities
than native phenols do.
Fatty-acid phenolic esters are preferentially produced by
enzymatic bioprocesses based on the use of lipases that exhibit
high selectivity towards polyfunctional substrates and mild
reaction conditions, as compared to chemical synthesis
Enzymes: Analysis and Food Processing 529
pathways. Most often, esterification reactions were carried out
in dry organic solvent, but few studies reported the possibility
of processing without any solvent.
The synthesis of biodiesel by lipase-catalyzed reactions has
also become a matter of great commercial interest due to its
environmental friendliness over petrodiesel fuel.
The main advantages of using biodiesel are that it is biode-
gradable, can be used without modifying existing engines, and
produces less harmful gas emissions, such as sulfur oxide.
Chemically, biodiesel is a mixture of alkyl esters with long-
chain fatty acids, and it is typically made from nontoxic, bio-
logical resources such as vegetable oils, animal fats, or even
used cooking oils. In this sense, due to the low miscibility of
the substrates, the application of ultrasound irradiation to this
reaction system may lead to high reaction rates. Although the
rates of triglyceride conversion to fatty-acid alkyl esters are
known to be slower than the alkali-catalyzed routes,
enzymatic catalysis has attracted much attention, because it is
more ecofriendly and has the potential for industrial
implementation, due to the simplicity of the product-refining
process and the use of lower reaction temperatures. In fact, the
key advantage of an enzymatic biodiesel process is that
triglycerides, as well as free fatty acids, can be efficiently trans-
formed into biodiesel under the same mild conditions, and the
byproduct, glycerol, can be easily recovered as part of a high-
quality, high-value stream, without any complex process. The
high cost of enzyme production remains the major obstacle to
the commercialization of enzyme-catalyzed processes, but con-
siderable progress has been made to address it, including the
use of solvent-tolerant commercial and noncommercial immo-
bilized lipases and new immobilization technologies, making
catalyst reutilization possible.
From Fusarium venenatum, Fusarium oxysporum, hybrid
between Thermomyces lanuginosus and Fusarium oxysporum
Main activity assay: Lipase hydrolyzes a triglyceride substrate
to form glycerol, which may be quantified enzymatically by
monitoring a linked change in the OxiRed probe absorbance at
570 nm. Other spectrophotometric methods for lipase activity
determination use synthetic lipase substrates transformed
via enzyme-catalyzed hydrolysis into spectrophotemetrically
detectable products. The predominant substrates are
p-nitrophenyl and naphthyl esters of the long chain fatty
acids, as well as thioesters. The lipolysis of the p-nitrophenyl
esters (e.g., laurates, palmitates, oleates) gives rise to yellow-
colored p-nitrophenol measured at 405–410 nm.
Other Food Enzymes
Phytases EC 3.1.3.8 X
Uses and applications: Degradation of antinutritional factors
(phytates).
Origins: Bacillus spp., Aspergillus ficum, Aspergillus niger,
plants.
Phytases catalyze the hydrolysis of phytate molecules,
known as antinutrients, which complex certain essential
minerals, and it is used in bread making to reduce the content
of the bread phytates.
530 Enzymes: Analysis and Food Processing
Phytates (myo-inositol-1,2,3,4,5,6-hexakisphosphates) are
widely thought to play a significant role in decreasing the
bioavailability of minerals by chelating cations and forming
complexes, which may be insoluble or otherwise unavailable
under physiological conditions. A number of studies, includ-
ing animal experiments and human trials, have been done on
the negative effects of phytates, and these studies clarify that
dietary phytate has a negative impact on the bioavailability of
mineral ions (i.e., Zn2þ, Fe2þ/3þ, Ca2þ, Mg2þ, Mn2þ, and
Cu2þ). Additionally, phytate has also been reported to form
complexes with proteins at both low and high pH values, and
the complex formations may alter the protein structure, which
induces changes in the physicochemical and functional prop-
erties, enzymatic activity, and proteolytic digestibility of pro-
teins (or enzymes). Thus, it is necessary to partially, or even
completely, eliminate the antinutritional effect of phytates
when phytate-rich products are produced or used. There are
several processing techniques, such as soaking, germination,
malting, fermentation, ultrafiltration, and ion exchange
columns, which can be used to decrease the inhibitory effect
of phytate on mineral absorption. In addition, phytase
(myo-inositol hexakisphosphate phosphohydrolase), which
catalyzes the conversion of phytate to inositoland inorganic
phosphate, can also be used to reduce phytate in food proces-
sing. Some research studies describe the application of phytase
in food processing to reduce phytate in products such as white
wheat rolls, cereal porridges, wheat bran flour, glandless cotton
flour, bread, corn wet milling, and rice bran protein isolate. It
was reported that 85% of phytates in soybean meal and 67% of
phytates in cottonseed meal could be hydrolyzed by Aspergillus
ficuum phytase at 50
C and pH 4.0–5.5.
Phytic acid (PA) can be degraded by phytase enzymes,
leading to an increase in the bioavailability of zinc and
other minerals. PA degradation was reported to occur during
the fermentation of cereals such as wheat, which contain
endogenous phytases, and on the addition of exogenous
phytases during the preparation of cereal flours for comple-
mentary feeding. Both processes require long preparation in
slurry form, however, with adjustments to pH and tempera-
ture for efficient dephytinization. A simpler procedure, suit-
able for in-home fortification mixtures, would be to add the
phytase to the cereal porridge at the time of consumption so
that PA degradation could take place during the digestive
process. This would then have a positive effect on zinc and
iron adsorption.
Phytate can be hydrolyzed in the gastrointestinal tract
(GIT), at least to some degree, by the intrinsic phytases present
in feeds originating from plants, endogenous phytases, or pos-
sibly by microbial phytase from the gut microbiota present in
the GIT. More recently, however, phytases have been
commercially produced from microbial sources and are often
included in pig and poultry diets to improve the availability of
minerals, such as Ca and P, as well as other nutrients, ostensi-
bly through greater phytate hydrolysis. Several studies have
been published examining the efficacy of dietary microbial
phytase supplementation on Ca and P availability in pigs, but
these studies largely focus on disappearance across the total
tract, while largely overlooking apparent ileal mineral absorp-
tion. There is a lot of information about the fate of dietary
phytate P and total P and Ca throughout the GIT of pigs,
particularly in response to the presence of dietary microbial
phytase and in relation to corn–soybean meal diets.
Sources: Aspergillus niger, plants.
Main activity assay: The assay is based on the enzymatic
hydrolysis of sodium phytate under controlled conditions via
the measurement of the amount of orthophosphate released.
The phytase activity is expressed in phytase units (FTU). One
phytase unit (FTU) is defined as the amount of enzyme that
liberates 1 mmol of inorganic phosphorus per minute from
0.0051 mol/l sodium phytate at 37 and pH 5.50, under the
conditions of the test.
Legislation Concerning the Safe Use of Food Enzymes
With very rare exceptions, largely limited to their allergenic
properties, the consumption of most food enzymes does not
present a particular danger. However, some of these enzymes
in active form, such as certain proteases and lipases, may have
detrimental effects on the cell membranes of human skin or
the GIT. Thus, safe practice requires that all enzymes used in
food formulations should be either inactivated, denatured, or
eliminated before the end-product stage. Because the great
majority of food enzymes are the mixtures of various compo-
nents present in their production media including other
enzymes, present sometimes in even greater amounts that a
desired enzymatic product, and an array of other compounds,
the safety of the product should conform to current security
and health standards, assuring safety for consumers.
The first legislation addressing security verifications for
food enzymes was voted on and enforced in Denmark and in
France. These laws, in turn, inspired European legislation for
securing the safety of foods created with help of industrially
produced enzymes (12, 13). In most cases, if there is no con-
siderable history of safe use, as in the case of rennet, the new
enzymatic processing aid should fulfill many sanitary
requirements, such as microbiological, toxicologic, mutagenic,
carcinogenic, and allergenic safety.
See also: Biscuits, Cookies, and Crackers: Chemistry and
Manufacture; Bread: Breadmaking Processes; Bread: Chemistry of
Baking; Cheese: Chemistry and Microbiology; Enzymes: Functions and
Characteristics; Fermented Foods: Fermented Milks; Whey and Whey
Powders: Fermentation of Whey; Wines: Champagne and Sparkling
Wines – Production and Effervescence; Wines: Wine Production.
Further Reading
CEF Panel (2009) Guidance on the submission of a dossier on food enzymes for safety
evaluation. The EFSA Journal 1305: 1–26.
CEF Panel. (2014). Explanatory note for the guidance of the scientific panel of food
contact materials, enzymes, flavourings and processing aids (cef) on the
submission of a dossier on food enzymes. EFSA Supporting Publication:EN-689.
Chang DS and Tsujisaka Y (1976) Amylase components and crystallization of
a-amylase of Aspergillus niger on wheat bran. Journal of Fermentation Technology
54(1976): 264–266.
Danley DE and Geoghegan KF (1988) Structure and mechanism of formation of
recombinant-derived chymosin C. Journal of Biological Chemistry 263(20):
9785–9789.

Fischer EH and Stein EA (1960) a-Amylases. In: Boyer PD, Lardy H, and Myrbäck K
(eds.) The enzymes, vol. 4, pp. 313–343. New York: Academic Press.
Gaspar AL and de Góes-Favoni SP (2015) Action of microbial transglutaminase
(MTGase) in the modification of food proteins: a review. Food Chemistry
171C: 315–322. http://dx.doi.org/10.1016/j.foodchem.2014.09.019.
Kindstedt P (2012) Cheese and culture: a history of cheese and its place in western
civilization. White River Junction, USA: Chelsea Green Publ978-1-60358-412-8.
Kumar A, Grover S, Sharma J, and Batish VK (2010) Chymosin and other milk
coagulants: sources and biotechnological interventions. Critical Reviews in
Biotechnology 30(4): 243–258. http://dx.doi.org/10.3109/07388551.2010.483459.
Nagarajan S (2012) New tools for exploring “old friends – microbial lipases” Applied
Biochemistry and Biotechnology 168: 1163–1196.
Purr A (1935) The activation phenomena of papain and cathepsin. Biochemical Journal
29(1): 13–20.
The Freedonia Group Inc (2014) World enzymes to 2015 – demand and sales forecasts,
market share, market size, market leaders. OH, USA: The Freedonia Group Inc,
pp. 1–338.
Enzymes: Analysis and Food Processing 531
Relevant Websites
http://www.amfep.org/content/first-eu-positive-list-food-enzymes – Association of
Manufacturers and Formulators of Enzyme Products (AMFEP).
http://www.ancient.eu/article/223/.
http://www.chem.qmul.ac.uk/iubmb/enzyme/ – Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology.
http://www.efsa.europa.eu/en/topics/topic/foodenzymes.htm – European Food Safety
Authority (EFSA).
http://www.foodnavigator.com/Policy/EFSA-issues-food-enzyme-guidance – European
Food Safety Authority (EFSA).
http://www.freedoniagroup.com/World-Enzymes.html – Freedonia group.
 Enzymes: Functions and Characteristics
D Talens-Perales, J Marı́n-Navarro, and J Polaina, Instituto de Agroquı́mica y Tecnologı́a de Alimentos, Valencia, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Enzymes: Concept and History
Enzymes are biological macromolecules that act as facilitators
(catalysts) of every metabolic reaction that takes place in or is
produced by the action of living organisms. With the exception
of a small group of catalytic RNA molecules, all enzymes are
proteins, and their activity depends on their native protein
conformation. The function of enzymes is to increase the
speed (rate) of biological reactions so that they occur in a
time scale useful for organisms. An example of enzyme action
can be taken from how bread supplies us with energy. The
essential component of bread is starch, a polymer of glucose.
When we eat bread, digestive enzymes splice starch down to
free glucose molecules, which then undergo a chain of enzy-
matic reactions to be finally converted into CO2 and H2O. The
overall balance of these reactions is highly exergonic, and the
energy liberated sustains our physical and mental activity.
However, if we keep a piece of bread in a container, it can
remain for years without any noticeable change. The same
reactions that occur in our organism are thermodynamically
possible and may occur in the container, but in the absence of
enzymes they take place at an extraordinarily low speed that
makes them useless to sustain life. Enzymes are therefore
agents whose role is to reduce the time of chemical reactions.
Although virtually all enzymes are proteins, there are many
cases in which the protein needs another chemical component
(cofactor) to perform its catalytic activity. This cofactor may be
an inorganic ion, such as Fe2þ, Mg2þ, Mn2þ, or Zn2þ, or a more
complex organic molecule, named coenzyme, such as coen-
zyme A. When the coenzyme is tightly bound to the protein, it
is called a prosthetic group, such as the heme group. The
protein part is called apoenzyme or apoprotein, and the com-
plete, catalytically active enzyme with both components is
called holoenzyme.
The use of enzymes by humans dates to the earliest times of
civilization, far before the time when the concept of enzyme
could be conceived. Ancient enzyme uses were associated with
the production of certain types of foods and beverages. The use
could be indirect, as in the production of foodstuffs resulting
from microbial fermentation, such as bread, wine, beer, or
vinegar. It could also be direct as in cheese production from
milk, using renin obtained from the stomachs of ruminants. Of
course, early civilizations were not aware of the nature or
function of the substances that they used in the elaboration
of enzyme products, which was based on empirical observa-
tions and folklore.
It was during the nineteenth century that the development
of biochemistry provided an emerging picture of the nature
and properties of enzymes. In 1833 Anselme Payen and Jean-
François Peroz described the isolation of an amylolytic sub-
stance from germinated barley that they called diastase. Two
years later, the chemist Jöns Jacob Berzelius coined the term
catalysis (1835), referring to the property of certain substances
532 Encyclopedia of Food and Health
to accelerate chemical reactions. One year later Theodor
Schwan discovered the digestive enzyme peptidase. However,
it was not until 1876 that Wilhelm Kühne purposed the term
‘enzyme’ derived from the Greek word ‘zymé,’ which means
ferment. In 1894 Emil Fischer proposed the lock-and-key
theory to explain the mechanism of enzyme action. In 1897,
the brothers Hans and Eduard Buchner demonstrated that the
transformation of glucose in ethanol can be carried out by
chemical substances (enzymes) present in cell free extracts.
During the first half of the twentieth century, the chemical
nature of enzymes and their mechanism of function were
elucidated. In 1903, Victor Henri published the first mathemat-
ical model describing the process of enzyme action (enzyme
kinetics). Ten years later, in 1913, Leonor Michaelis and Maud
Menten published their very well-known mathematical model
of enzyme kinetics relating the velocity at which an enzymatic
reaction proceeds with the concentration of substrate. In 1926
James B. Summer purified and crystallized the enzyme urease
isolated from jack beans. His work provided first evidence that
enzymes are proteins. This was confirmed in the next few years
by the work of John Northrop with digestive enzymes pepsin,
trypsin, and chymotrypsin. Around 1950, two key contribu-
tions were determinant for understanding the structure and
function of proteins in general, and enzymes in particular.
Frederick Sanger’s work with insulin showed that proteins are
composed by chains of amino acids linked by peptidic bonds.
Based on Sanger’s discovery, Kaj Linderstrøm-Lang made an
accurate description of the shape and organization of the archi-
tectural elements that compose protein molecules. Already in
the second half of the twentieth century, around 1960, John
Kendrew and Max Perutz using X-ray crystallography resolved
the first three-dimensional structure of proteins. Although they
worked with globular, noncatalytic proteins, myoglobin and
hemoglobin, their work pioneered subsequent studies linking
protein structure and enzymatic function. Enzyme studies were
fostered by the onset and development of genetic engineering
techniques in the second half of the twentieth century. As a
result of this development, it is possible nowadays to obtain in
large amount and pure form almost any enzyme from any
organism.
One-Gene–One-Enzyme
In 1941 George Beadle and Edward Tatum published a highly
influential article in which they proposed the so-called one-
gene–one-enzyme hypothesis. At that time, the principles of
Mendelian inheritance were understood, and it was known
that enzymes were proteins, but the chemical nature of
the genetic material remained a mystery. Beadle and Tatum
worked with the bread fungus Neurospora crassa, looking for
genetic mutations causing metabolic defects. The selected
mutants required the addition of a specific supplement,
http://dx.doi.org/10.1016/B978-0-12-384947-2.00256-7

typically an amino acid (e.g., arginine or leucine) to restore
their capability to grow. These, called auxotrophic mutants,
were therefore impaired in the metabolic process – pathway –
leading to the biosynthesis of the required compound. The
genetic analysis of a group of mutants requiring the same
supplement showed that they were affected in different genes
controlling successive steps – chemical reactions – of the same
pathway. From this, Beadle and Tatum concluded that each
gene determines the existence of an enzyme that catalyzes a
specific biochemical reaction.
Later discoveries rendered this theory an oversimplification.
Presently, we know that there are many genes that do not
encode enzymes as they have a regulatory function; other
genes encode nonenzymatic proteins. There are also enzymes
that are composed by more than one polypeptide and are
therefore encoded by more than one gene. Nevertheless, with
many exceptions, Beadle and Tatum’s insight was essentially
true and established an early, first connection between the
genome and the thousands of biochemical reactions that take
place in a living organism.
Structure and Function of Enzymes
The working functionality of a molecule resides in its chemical
composition and spatial conformation – structure. Enzymes
are molecular machines able to catalyze chemical reactions.
They are built by at least one polypeptide chain, and their
structure derives from the corresponding particular sequence
of amino acids. The general features of protein structure were
first proposed by Linderstrøm-Lang and later confirmed by
crystallographic analysis of Kendrew, Perutz, and the work of
Linus Pauling. The structure of a protein results from a hierar-
chical organization of the molecule at four levels. The basic
level – primary structure – is determined by the specific linear
sequence of the amino acids that compose the polypeptide
chain. Stretches of amino acids bound in a polypeptide
chain, depending on their composition, can take an apparently
random conformation – coil or loop – or adopt particular
patterns. These may have the shape of a helix – a-helix – or a
zigzagged extended conformation of chain segments (b-strands)
interconnected laterally with the shape of a sheet – b-sheet.
These patterns constitute the following – secondary structure –
level. Every polypeptide chain, with its distinct secondary struc-
ture elements, adopts a specific three-dimensional arrangement
that is referred to as tertiary structure. Often, proteins are made
of more than one polypeptide chain – monomer – which can be
identical or different. The specific association of two or more
monomers in proteins constitutes its quaternary structure
(Figure 1). Although the amino acids of a polypeptide chain
are linked by covalent union between the amino group of
one amino acid and the carboxyl group of the next – peptide
bond, the secondary, tertiary, and quaternary structures are
maintained by numerous, usually noncovalent, forces such as
hydrogen bonds, salt bridges (electrostatic), and hydrophobic
interactions. Proteins contain secondary structure (a-helix and
b-chain) elements connected by coil regions of different length.
Generally, the secondary structure elements cluster at the inter-
nal hydrophobic core of the protein, whereas the connecting
Enzymes: Functions and Characteristics 533
coils are at the surface. A relatively small three-dimensional
pocket within the overall structure of an enzyme constitutes
the catalytic site, which is formed by the amino acid residues
directly involved in catalysis and those that participate in bind-
ing the substrate. In a way, it could be considered that the
secondary structure elements, which are evolutionary conserved
among similar, phylogenetically related proteins – homologues,
are mostly involved in maintaining the protein framework,
whereas some of the loops build the catalytic pocket, providing
substrate specificity and enough flexibility for the conforma-
tional changes occurring in the enzyme during the catalytic
process.
Presently, two methods are used for protein structure deter-
mination, X-ray crystallography, and nuclear magnetic reso-
nance (NMR). Analysis of X-ray diffraction patterns provides
a very accurate picture of protein structures with a resolution
up to 1 Å. A drawback of this technique is that it requires
the protein to be in crystalline form. Protein crystals are
often difficult to grow, and sometimes their quality is not
good enough to obtain the three-dimensional structure with
sufficient accuracy. Around 1970, NMR was applied to the
resolution of protein structures thanks to the work of Kurth
Wüthrich. NMR analysis is carried out using protein solution
samples, much easier to prepare than crystals; however, the
practical use of this technique is still limited to proteins of
relatively small size. Progress made during the last decades
has yielded a huge number of protein structures, most of
them enzymes, available at the Protein Data Bank (http://
pdb.org/). A highly useful complement to the experimental
techniques are bioinformatics tools, such as SWISS-MODEL
(http://swissmodel.expasy.org/) or I-TASSER (http://zhan-
glab.ccmb.med.umich.edu/I-TASSER/), which allow predict-
ing the three-dimensional structure of a protein from its
amino acid sequence, on the basis of existing homology with
other proteins of known structure.
Name, Types, and Classification of Enzymes
Enzymes are named by adding the suffix ‘-ase’ to a term
describing or referring to their activity. For instance: citrate
synthase, peptidase, lactase, etc. Exceptions to this rule reflect
the circumstance that the enzyme was named before the
specific reaction that catalyzes was known. This was the case
for enzymes such as pepsin or lysozyme. Before a systematic
way for naming enzymes was adopted, the same enzyme
could be named differently according to the context where
it was studied. Conversely, different enzymes could receive
the same name. This created misunderstanding and made
difficult the interpretation of published results. The situation
became unbearable when the rapid growth of biochemical
research increased enormously the number of enzymes that
were identified and characterized. In 1956, the International
Union of Biochemistry (IUB) appointed a commission, the
International Commission on Enzymes, or simply the
’Enzyme Commission’ (abbreviated EC), whose mission was
to establish a rational methodology for the classification and
nomenclature of enzymes. The adopted classification was
based on chemical activity (function). Each enzyme was
 534
Enzymes: Functions and Characteristics
Asn lle
lle Ala Asp
lle Gly
Val Asn

Cys
Asn
Gly
Tyr Glu
Thr Glu Val
(a)

Figure 1 (a) Primary structure fragment of the enzyme b-glucosidase A (BglA) from Paenibacillus polymyxa. The amino acidic residues are identified with a three-letter code. (b) Two different secondary
structure elements from BglA: a parallel b-sheet (left panel) and an a-helix (right panel). Hydrogen bonds connecting residues of different b-strands in the b-sheet are indicated by dashed lines. (c) Tertiary
structure of a BglA polypeptide (monomer). The structure represented on the left is an a/b-barrel, made of an inner cylindrical (barrel-shaped) b-sheet, surrounded by a-helixes. On the right side, a ‘surface’
representation of the monomer shows the contour of amino acid residues that are fully accessible to the solvent. Catalytic residues are colored in red. A molecule of the enzyme’s substrate, the
disaccharide cellobiose, is represented in the center of the figure. (d) Quaternary structure of BglA. This enzyme is an octamer of identical monomers (PDB ID: 1BGA).

assigned a classification number (EC number) and a system-
atic name to identify the reaction that it catalyzes. In addi-
tion, for traditional or practical reasons, enzymes often have a
common or trivial name. The classification proposed by the
EC established three hierarchical levels: classes, subclasses,
and sub-subclasses. Six classes of enzymes were considered:
oxidoreductases, transferases, hydrolases, lyases, isomerases,
and ligases. Within each class, subclasses, and sub-subclasses
were established attending specific characteristics of the reac-
tion being catalyzed, such as the nature of the substrate and
the chemical groups involved in the reaction. EC numbers are
composed of four set of digits, separated by dots. The first,
second, and third sets of digits refer to the class, subclass, and
sub-subclass. Finally, the fourth digit identifies individual
enzymes within a given sub-subclass. As an example, lactase
is the common name of the enzyme that breaks the disaccha-
ride lactose, the milk sugar, into its two monosaccharide
components, glucose and galactose. The systematic name of
the enzyme is b-D-galactoside galactohydrolase and its EC
number is EC 3.2.1.23. The number 3 specifies the class
name (hydrolase). The second number – 2 – the subclass
(glycosylase). The third number – 1 – indicates the enzyme’s
property of hydrolyzing O- and S-glycosides. The fourth digit –
23 – identifies this particular enzyme as b-galactosidase.
Nowadays, around 6500 enzymes, virtually the full set of
enzymes involved in the metabolism of living organisms, are
known. An updated view on enzyme names and classification is
given by the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (http://www.
chem.qmul.ac.uk/iubmb/enzyme/).
Proteins are classified on the basis of their structural sim-
ilarity. At the level of tertiary structure, that is, the three-
dimensional appearance of the protein, natural polypeptides
tend to adopt characteristic shapes that arise from different
combinations of secondary structure elements. These shapes
can be classified at different levels into a defined number of
classes (http://scop2.mrc-lmb.cam.ac.uk/). Enzymes are pro-
tein molecules whose functions – catalytic properties – are
determined by their structure, which in turn depends on their
sequence. On the other hand, resemblance at the primary
structure level (amino acid sequence) reflects obvious evolu-
tionary relatedness. Therefore, systematic classification of
enzymes on the basis of their structure affords information
about both their phylogenetic relationship and their function.
A particular case of structural classifications of enzymes is the
Carbohydrate Active Enzymes Database (http://www.cazy.
org/), which provides comprehensive information about all
sorts of enzymes related to carbohydrate metabolism.
How Enzymes Work
Most biochemical reactions are energetically favorable. How-
ever, they would take place extremely slowly without the exis-
tence of a catalyst. This is because the chemical conversion of a
substrate into a product undergoes an intermediate state (tran-
sition state), which has a higher energy than the substrate. The
term activation energy (Ea) is used to describe the energy required
to reach this transition state and is related to the reaction rate
(the higher the Ea, the lower the reaction rate). It may also be
Enzymes: Functions and Characteristics 535
described as a kinetic barrier that needs to be surpassed for the
reaction to proceed. The role of enzymes is to supply an envi-
ronment that greatly increases the chances for the reaction to
occur, by decreasing the energy of the transition state and there-
fore the Ea. The environment for the catalysis is provided by the
active site. The interaction between specific amino acid residues
of the enzyme, in a particular three-dimensional configuration,
and substrate is mediated by multiple weak interactions and
allows that the substrate is recognized unmistakably among
other similar molecules. Once the substrate is bound to the
enzyme, catalytic amino acid residues transiently react with the
substrate, assisting its transformation into the final product
through a more energetically favorable transition state than
that occurring without an enzyme. It is important to highlight
that the enzyme, as any other catalyst, is not consumed during
the reaction and recovers its original form when the reaction is
completed. Enzymes act as molecular machines able to break,
ensemble, or transform molecules. They have evolved as highly
specialized tools, which explains the existence of as many dif-
ferent enzymes as metabolic reactions.
Enzyme Kinetics
Enzyme kinetics is the discipline that studies how enzymatic
reactions take place, the rate at which they occur, and the
influence of environmental conditions in the reaction process.
The study of the rate of the reaction and its connection with the
other most critical factor, the available amount of the substrate,
is essential to understand enzyme action. In 1913 Michaelis and
Menten published, in German, a paper whose title translated to
English is ’The Kinetic of Invertase Action.’ In this paper, which
became a milestone of biochemistry, the authors established the
relationship between the velocity of an enzymatic reaction and
the concentration of the substrate according eqn [1]:
V0 ¼ Vmax ½S=Km þ ½S [1]
In this equation, V0 (initial velocity) is the rate at which the
reaction proceeds at a given substrate concentration, Vmax is the
maximal speed that the enzyme achieves (at saturating sub-
strate concentrations), [S] is the substrate concentration, and
Km is the Michaelis–Menten constant, which reflects the affin-
ity of the enzyme for the substrate (the lower the Km, the
higher the affinity). Its value corresponds to the concentration
of substrate at which the reaction rate is half of the maximum.
Michaelis and Menten deduced their equation from the study
of the enzyme invertase that splits the disaccharide sucrose into
its two constituent monosaccharides, glucose and fructose.
This reaction was a convenient model system to study the
behavior of an enzyme because the conversion of substrate
into products was easily monitored by physical techniques.
When a beam of polarized light traverses a sugar solution, the
polarization plane is rotated, either to the right – dextro
rotation – or to the left – levo rotation. This optical activity
can be measured with a polarimeter, and the measured value
correlates with the concentration of the sugar in the solution.
Sucrose and glucose are dextrorotatory, whereas fructose is
levorotatory. Because optical activity of fructose is higher
than that of glucose, the enzymatic reaction can be followed
536 Enzymes: Functions and Characteristics
by measuring the rotation of light from right to left. Using this
procedure, Michaelis and Menten determined the rate at which
sucrose was transformed by the action of the invertase. The
correlation that they found between the speed of reaction and
sucrose concentration fitted a hyperbolic curve. The reaction
rate increases as the substrate increases with an asymptotic
tendency to a maximum (Vmax; Figure 2(a)) according to
eqn [1]. The study of many other enzymes in the following
years confirmed the general value of the Michaelis–Menten
model, even though there are enzymes that show different
kinetics. This is the case, for instance, of allosteric enzymes,
in which the interaction between enzyme and substrate is
affected by another molecule – effector – that regulates the
activity of the enzyme. In 1934, Hans Lineweaver and Dean
Burk described a transformation of the Michaelis–Menten
equation (eqn [2]). In the graphical representation of the
transformation, the function of 1/V0 versus 1/[S] is a straight
line (Figure 2(b)), where the intercept with the x axis is 1/Vmax
and the slope is Km/Vmax.
1=V0 ¼ ðKm =Vmax Þ1=½S þ 1=Vmax
Linear regression analysis of experimental data by means of the
Lineweaver–Burk, or double reciprocal, plot soon became a
useful, albeit inaccurate, tool to easily calculate enzyme kinetic
parameters (Km and Vmax) from experimental determinations.
Currently, the availability of computational tools has facili-
tated nonlinear regression analysis, using directly eqn [1],
and this is the preferred method to obtain more accurate values
of Km and Vmax. However, the Lineweaver–Burk plot is still
useful to corroborate the Michaelis–Menten behavior of an
enzyme or to study the effect of different types of enzyme
inhibitors.
The Effect of Physical and Chemical Agents on
Enzyme Activity
Like most chemical reactions, enzyme-catalyzed reactions pro-
ceed faster when the temperature increases. This happens
Vmax
V0
V max
2
(a) Km [S]
Figure 2 Michaelis–Menten kinetics. (a) Variation of the initial rate of the reaction (V0) with the substrate concentration ([S]). The asymptote
corresponding to maximum velocity (Vmax), achieved at saturating substrate concentrations, is indicated with a dashed line. The Michaelis constant (Km)
value, which is indicative of the affinity of the enzyme for the substrate, corresponds to the substrate concentration for which the enzyme initial
rate is half of Vmax. (b) Lineweaver–Burk or double reciprocal plot of Michaelis–Menten kinetics.
because an increase of the system’s energy accelerates the mol-
ecules in the reaction, increasing the probability of interaction
between the chemical components. However, in the case of
enzymatic reactions the boosting effect of temperature is lim-
ited by the stability of the enzyme. Each enzyme has an opti-
mal temperature at which it shows maximal activity. This is
because enzymes structure is maintained by multiple
noncovalent weak interactions such as hydrogen bonds,
electrostatic, and hydrophobic interactions, which are influ-
enced by physicochemical conditions, such as temperature.
High temperature disrupts the network of interactions that
holds the molecular architecture of enzymes. If the change is
drastic, the protein structure disassembles, and the enzyme
losses its activity irreversibly. This process is called heat dena-
turation. Enzymes show different degrees of sensitivity to tem-
perature. In general terms, as a result of evolution, they are
adapted to the temperature conditions in the natural habitat of
the producing organism. For instance, human enzymes work at
a temperature of around 37  C. Enzymes from hyperthermo-
philic archaea function at temperatures of around 100  C at
[2]
which virtually all human enzymes are denatured. On the
other hand, enzymes from psychrophilic organisms work opti-
mally at temperatures close to 0  C at which human enzymes
are structurally stable but inactive. Another critical parameter
for enzyme activity is pH, which affects mainly electrostatic
interactions holding the enzyme structure. As happens with
temperature, there is a pH at which the enzymes work opti-
mally and a working range at which they remain active. If the
pH increases or decreases outside the working range, the
enzyme structure can be affected irreversibly.
Chemical compounds can affect enzyme activity. As men-
tioned earlier, some inorganic ions may be required as cofac-
tors for the activity of some enzymes. Some compounds may
bind reversibly or irreversibly to critical sites in the protein
structure (within or outside the catalytic pocket), reducing or
abolishing enzyme activity. Other compounds can react with a
functional group of the enzyme leading to chemical modifica-
tions (e.g., oxidation, carbonylation) that may cause (revers-
ible or irreversible) inactivation. A special case of inhibitors are
those called ’suicide inactivators.’ These are compounds of
1/V0
−1/Km 1/ Vmax
(b) 1/[S]

chemical structure similar to the enzyme’s substrate, which
bind to the active site and are converted into a reactive
molecule, which interacts irreversibly with one of the catalytic
residues. These kinds of inhibitors are very useful research tools
as they help to explain the mechanism of catalysis, can facili-
tate the crystallization and structural analysis of enzymes, and
in some instances can be used as drugs in therapeutic
treatments.
Enzyme Regulation
A simple living organism such as the bacterium Escherichia coli
contains around 4000 genes that direct the synthesis of a fairly
equivalent amount of proteins, most of which are enzymes. In
humans, the number of protein-encoding genes is around
35 000. Not all proteins encoded in a genome are needed at
the same time or in the same quantity. Because protein synthe-
sis is a highly costly process for living cells, living organisms
have developed regulatory mechanisms that supply the cells
with the proteins that are required at a certain moment and in a
convenient amount. Regulation is exerted at different levels in
the flux of information, which goes from DNA to mRNA and
from mRNA to protein. Genes can be classified into two cate-
gories, according to their expression rate. There are genes
required at all times, for example those encoding enzymes
acting in central metabolic pathways. These are called house-
keeping genes, and they are expressed in a constitutive –
continuous – way. For other genes, which are required just at
certain stages, the first level of regulation is transcription. The
synthesis of a regulated mRNA may be increased in the pres-
ence of a signal molecule called inducer. The consequent effect
of increasing the encoded enzyme is called induction. An
alternative mechanism is gene repression, in which the tran-
scription of a gene is decreased or suppressed by the presence
of a molecule called a repressor. As consequence of these two
mechanisms the presence or absence of individual enzymes
and even the levels at which they are present in the cells is
controlled. Another level of regulation takes place on the life
span of the mRNA molecule. In some cases, mRNAs translation
responds to environmental signals through the interaction of
specific sequences (UTRs, for untranslated regions) with pro-
tein factors. In other cases, as long as an mRNA is present in the
cytoplasm, it continues to be translated by the ribosomes into
the encoded protein. Different mRNA species differ in their
intrinsic stability. As a general rule, mRNAs from highly regu-
lated genes are more unstable than mRNAs from housekeeping
genes and tend to disappear quickly. Even though an mRNA is
eliminated, the enzyme resulting from its translation will
remain present in the cytoplasm for a certain time, depending
on its intrinsic stability. If its action is no longer required, its
activity is generally suppressed by a mechanism of inhibition
that can be exerted in different ways. One type of competitive
inhibition is caused by a molecule that competes with the
substrate for the active site of the enzyme. Often the inhibitor
is the product of the enzymatic reaction. The action of the
inhibitor depends on its concentration and on the affinity
that the enzyme has for it. The effect is reversible because an
increase on the concentration of the enzyme’s substrate will
make difficult the access of the inhibitor to the active site.
Enzymes: Functions and Characteristics 537
Another type of reversible inhibition is noncompetitive or
allosteric. In this case, the inhibitor binds to a region of the
enzyme other than the active site, called the allosteric site.
Industrial Enzymes
Since early times of the development of biochemistry,
researchers realized the industrial potential of enzymes. In
1907, the German company Röhm and Haas commercialized
for the first time an enzyme preparation. The product, mar-
keted with the name OROPON, represented a revolutionary
innovation for leather bating, as an alternative to traditional
procedures, which used animal excrements in the process. The
innovation resulted from the work of Otto Röhm, who had
studied the properties of enzymes like trypsin, isolated from
pig pancreas, and its digestive effect on skins and furs. A few
years later, in 1914, the Röhm and Haas firm launched
BURNUS, the first enzyme containing detergent for laundry.
One century after these precursory accomplishments, enzymes
have innumerable applications in the food, chemical, and
health industrial sectors. Enzymes are becoming an advanta-
geous alternative to an increasing number of industrial chem-
ical processes. Enzymes are natural, nontoxic, biodegradable,
efficient, and highly specific catalysts that operate under mild
conditions. Therefore, the use of enzymes generally represents
an eco-friendly – green – alternative to conventional proce-
dures. Current global production of industrial enzymes can be
estimated to represent a value between 4 and 5 USD billion.
Danish company Novozymes dominates world market with a
share that approaches one-half of the total. Currently, other
important European players are DSM in The Netherlands and
BASF in Germany. In the United States, DuPont has achieved
an important role in the enzyme business after the acquisition
of Danisco and Genencor. The main area of enzyme applica-
tion is the food and feed sector, which consumes over one-
third of enzyme’s production, closely followed by detergents
and cleaners. Biofuel (ethanol), pulp and paper, textile,
leather, and pharmaceutical industries also make extensive
consumption of enzymes. Starch processing represents a
major field for enzyme application. Simple sugars, monosac-
charides, resulting from the enzymatic hydrolysis of starch
carried out by a-amylase and glucoamylase, are used as sweet-
eners in different types of foods and beverages and are also
used by biofuel industries, as fermentable material to produce
ethanol. Traditional food industries like bakery and brewery
also make extensive use of enzymes. In bakery, amylolytic
enzymes are used to increase the amount of fermentable
sugars, increasing loaf volume. Xylanases help dough handling
and improve crumb structure. Proteases digest the gluten,
improving dough texture. Lipases are used to obtain an emul-
sifying effect that reduces bread staling. In brewing, amylases
are used to increase fermentable sugars. Glucan hydrolyzing
enzymes (b-glucasases) have an important application in
reducing the viscosity of beer, making for easier filtration. Pro-
teases prevent turbidity and chill-haze formation in beer.
ALDC (a-acetolactate-decarboxylase) prevents the formation
of diacetyl, a compound that is formed as a natural product
of yeast metabolism but has undesirable off-flavor characteris-
tics. The detergent industry is the largest single market for
538 Enzymes: Functions and Characteristics
enzymes. Proteases and lipases are included in detergent for-
mulations to eliminate stains of different chemical nature.
Cellulases help to remove substances attached to cotton fibers.
Enzymes improve different operations in the pulp and paper
industry such as bleaching, pitch control, and deinking. Xyla-
nases, lipases, cellulases, and laccases are used for this purpose.
Enzymes have numerous applications in the pharmaceutical
and health care industries. An example is the use of glucose
oxidase in sensor strips to monitor glucose level in serum.
Most industrial enzymes are produced by microorganisms.
Producer strains of fungi and bacteria are grown under well-
defined conditions in either submerged or solid state fermen-
tation. Genetic engineering techniques are routinely used to
achieve homologous or heterologous expression of genes
encoding enzymes of interest in suitable host strains and to
enhance the performance of the producing strains. Production
procedures are different for intracellular and secreted enzymes.
In the first case, the cells of the producing microorganism need
to be disrupted to release the enzyme, which may require
expensive purification methodologies such as preparative chro-
matography. Secreted enzymes are recovered from the culture
medium by filtration. The use of enzymes for food applications
requires GRAS (generally recognized as safe) status granted by
the U.S. Food and Drug Administration (FDA), the European
Food Safety Authority (EFSA), or the views of qualified experts.
Extremozymes
Life thrives in extreme conditions. Living organisms can find
niches at temperatures higher than 50  C (thermophilic), or
even higher than 100  C (hyperthermophilic), below 20  C
(psychrophilic), extreme acid or alkaline conditions at pH
lower than 3 (acidophilic) or higher than 9 (alkaliphilic), or
under extreme salinity (halophilic). Organisms capable of liv-
ing in such environments have enzymes that must endure
extreme conditions. These ’extremozymes’ have unique prop-
erties that could be used for many purposes. Thermophylic and
hyperthermophylic enzymes show an increased number of
interactions (hydrophobic, salt bridges, disulfide bonds) to
maintain the protein structure at high temperatures. They
are valuable tools for the degradation of complex
polysaccharides (such as starch or lignocellulosic materials)
or substrates whose solubility is greatly increased at high tem-
peratures. In contrast, psychrophilic enzymes have weaker pro-
tein interactions to ensure sufficient flexibility to sustain
enzymatic activity at low temperatures. Psychrophilic enzymes
are used in detergents and other textile-related applications.
Acidophilic or alkaliphilic enzymes usually fall in the thermo-
philic category and can be used for similar purposes. Produc-
tion of extremozymes at industrial scale is still a challenge
because generally extremophilic organisms are not easily
culturable, and often the genes coding for their enzymes are
not well expressed in conventional hosts. Nonetheless, this is
an active research field because extremozymes may find count-
less applications in various industrial fields.
See also: Enzymes: Analysis and Food Processing.
Further Reading
Berg JM, Tymoczko JL, and Stryer L (2010) Biochemistry, 7th ed. New York: W. H.
Freeman.
Branden C and Tooze J (1999) Introduction to protein structure. New York: Garland
Science.
Fersht A (1998) Structure and mechanism in protein science. A guide to enzyme
catalysis and protein folding. New York: W. H. Freeman.
Kessel A and Ben-Tal N (2010) Introduction to proteins: structure, function and motion.
Boca Raton, FL: CRC Press.
Nelson DL and Cox MM (2012) Lehninger principles of biochemistry, 6th ed. New York:
W.H. Freeman and Company.
Polaina J and MacCabe AP (eds.) (2007) Industrial enzymes – structure, function and
applications Berlin: Springer.
Schulz GE and Schirmer RH (2013) Principles of protein structure. New York: Springer.
Relevant Websites
http://www.cazy.org/ – Carbohydrate Active Enzymes Database.
http://www.chem.qmul.ac.uk/iubmb/enzyme/ – Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology.
http://pdb.org/ – Protein Data Bank.
http://scop2.mrc-lmb.cam.ac.uk/ – SCOP.
http://swissmodel.expasy.org/ – Swiss Model.
http://zhanglab.ccmb.med.umich.edu/I-TASSER/ – I-TASSER.
 Escherichia coli and Other Enterobacteriaceae: Food Poisoning
and Health Effects
JL Smith and PM Fratamico, Eastern Regional Research Center, Wyndmoor, PA, USA
Published by Elsevier Ltd.
Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation
or endorsement by the US Department of Agriculture.
Introduction
The family Enterobacteriaceae consists of a heterogeneous group
of rod-shaped, gram-negative bacteria, varying in their ecological
niche, host range, and pathogenic potential for humans, animals,
insects, and plants. Members of the family are aerobes or facul-
tative anaerobes, are motile by peritrichous flagella or nonmotile,
do not form endospores or microcysts, and are chemoorgano-
trophic with both respiratory and fermentative metabolisms, and
most grow well at 22–35  C. Foodborne pathogens in the family
Enterobacteriaceae generally do not demonstrate an atypical heat
resistance, and they are inactivated at temperatures lower than
the pasteurization temperature for milk (72  C for 15 s). The
family Enterobacteriaceae contains more than 50 genera and a
large number of species, and there are several human pathogens
within this family. Important foodborne pathogens in the family
Enterobacteriaceae include Cronobacter spp. (primarily C. sakaza-
kii), Escherichia coli, Salmonella enterica, Shigella (boydii, flexneri,
sonnei, and dysenteriae), and Yersinia (enterocolitica and pseudotu-
berculosis). In the United States,  3.6 million cases of foodborne
illness are caused each year by bacterial pathogens, and  1.5
million of domestically (the United States) acquired foodborne
cases are due to diarrheagenic E. coli, Salmonella, Shigella, and
Yersinia. Thus, four genera within the family Enterobacteriaceae
cause  40% of bacterial foodborne illness in the United States.
The 2011 data from the European Union indicate that the path-
ogens Salmonella, Campylobacter, and pathogenic E. coli account
for 48% of the outbreaks and 27% of the cases of the foodborne
disease. This chapter will focus primarily on pathogenic E. coli
categories, with some discussion also on other foodborne path-
ogens in the family Enterobacteriaceae.
Intestinal Pathogenic E. coli
E. coli is a commensal organism that makes up part of the
normal flora of the intestinal tract of humans and animals. E.
coli belongs to the family Enterobacteriaceae and is a gram-
negative, facultatively anaerobic, non-spore-forming, rod-
shaped bacterium. Not all strains of E. coli are harmless, and
some strains have acquired virulence mechanisms, making
them pathogens. Some of these strains cause diarrhea and
other intestinal diseases, while other E. coli pathogens induce
disease at nonintestinal sites.
Enterohemorrhagic E. coli (Also Known as Shiga
Toxin-Producing E. coli)
In 1977, Konowalchuk and coworkers in Canada discovered
that culture filtrates from some E. coli strains produced a
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00260-9
cytopathic effect on Vero cells (African green monkey kidney
cell line). Since most of the strains producing this effect were
from infants with diarrhea, they speculated that the toxin
produced by these strains, which they referred to as verotoxin,
had a role in the genesis of diarrhea. The E. coli that produced
verotoxins were referred to as verotoxigenic E. coli or VTEC. In
1983, O’Brien and LaVeck purified the toxin from
Konowalchuk’s prototype strain, E. coli O26:H11 H30, and
showed that it was structurally and antigenically similar to
Shiga toxin produced by Shigella dysenteriae, and they named
the toxin, Shiga-like toxin. In 1983, there were two outbreaks
of hemorrhagic colitis (HC) in the United States, and it was
reported by Riley and coworkers that they were linked to eating
undercooked hamburgers contaminated with E. coli O157:H7,
a rare E. coli serotype that was later shown to produce vero-
toxin. Subsequently, a link was established between VTEC
infection and development of hemolytic uremic syndrome
(HUS). The term enterohemorrhagic E. coli (EHEC) was coined
by Myron Levine at the University of Maryland and refers to
Shiga toxin-producing E. coli (STEC)/VTEC strains that have
the same clinical, epidemiological, and pathogenic features
associated with the prototype EHEC organism E. coli O157:H7.
STEC/EHEC O157:H7 and certain non-O157 STEC ser-
ogroups are major foodborne pathogens that cause diarrhea,
HC, and HUS, and occasionally, death results in infected indi-
viduals. A conspicuous feature of STEC O157:H7 and non-
O157 STEC intestinal pathology is the intestinal attaching
and effacing (A/E) lesion, which is dependent on a type 3
secretion system (T3SS). The secretion system is encoded on a
pathogenicity island, the locus of enterocyte effacement (LEE).
The T3SS structure forms a pore complex that spans from the
inner bacterial membrane to the host cellular membrane and
enables the bacteria to inject bacterial effector proteins directly
into the eukaryotic host cells following attachment. The effec-
tor proteins subvert eukaryotic cell processes and enable the
pathogen to modulate the host environment to induce bacte-
rial infection and disease symptoms. Among the non-O157
STEC, there are both LEE-positive and LEE-negative ser-
ogroups. For example, STEC serogroups O26, O45, O103,
O111, O121, and O145 are generally LEE-positive and T3SS-
dependent, whereas serogroups O1, O2, O5, O8, O48, O73,
O76, O87, O91, O113, O118, O123, O128, O139, and O174
may be LEE-negative and T3SS-independent. However, only a
few of the several hundred non-O157 serotypes have been
tested to determine their LEE and T3SS status.
The eae (E. coli attaching and effacing) gene located on the
LEE encodes the production of intimin at the bacterial outer
membrane. Tir (transmembrane intimin receptor), also encoded
by eae, is translocated by T3SS to the host cell membrane; Tir
allows attachment of the bacterial intimin protein to the host cell
539
540 Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects
membrane leading to an intimate attachment of the bacterium
with the host cell. The intimate attachment is characterized by
the A/E lesion with localized loss of the microvilli (effacement)
and an accumulation of polymerized actin pedestals beneath and
surrounding the adherent bacterial cell. The bacteria remain
extracellular during infection. There are non-O157 STEC that
are LEE-negative and do not produce A/E lesions; however,
these strains may produce severe diarrhea, HC, and even HUS,
but their virulence mechanisms have been poorly studied. Other
genes, such as the Saa (STEC agglutinating adhesion) and Iha
(IrgA homologue adhesion), are also carried by STEC serogroups
that may play a role in attachment; however, additional research
is needed to determine the function of these genes.
The most critical virulence factor in STEC is a
bacteriophage-encoded cytotoxin similar to the Shiga toxin
produced by Shigella dysenteriae. In E. coli, the Shigella Shiga
toxin was initially called Shiga-like toxin but was renamed to
Shiga toxin 1 and Shiga toxin 2. STEC produce two Shiga
toxins: Stx1 and Stx2. Stx2 has 60% sequence homology to
Stx1. There are a number of genetic variants for stx1, including
stx1a, stx1c, and stx1d, and for stx2, there are seven variants,
stx2a to stx2g. Stx2a is a more potent toxin than Stx1; the LD50
(in mice) of Stx1 is >1000 ng, whereas the LD50 of Stx2a is
6.5 ng. Stx1 and Stx2 are AB5-type toxins; the B-pentamer of
the holotoxin binds to globotriaosylceramide (Gb3) present on
host microvascular endothelial cell surfaces (the kidney, intes-
tine, and brain) followed by endocytosis of the holotoxin. A
subunit of the A portion of the toxin is an N-glycosidase that
acts on the 28S RNA of the 60S ribosomal subunit leading to
the inhibition of protein synthesis and apoptosis of endothe-
lial cells, particularly those of the kidneys with resultant dam-
age of the renal endothelial cells. There are swelling of the renal
cells and detachment of the cells from the basement
membrane and formation of fibrin thrombi in the kidney
capillaries. Narrowing of the capillary lumens leads to a
reduced blood supply to glomeruli with a loss of kidney func-
tion resulting in HUS. HUS is characterized by hemolytic ane-
mia (destruction of red blood cells), acute kidney failure
(uremia), and low platelet count (thrombocytopenia).
Early symptoms of STEC infection include watery diarrhea
and abdominal cramps. Approximately 90% of confirmed
STEC infections result in HC, and 5–15% of the HC cases
progress to HUS. The mortality rate for HUS ranges from 5%
to 10%. A few patients suffer long-term consequences affecting
the gastrointestinal tract, liver, cardiovascular system, or cen-
tral nervous system (CNS); however, most patients recover
without major consequences. Very young and elderly patients
show the most susceptibility to complications and death due to
HUS. Antibiotics are not recommended in the treatment of
STEC-infected patients and can increase the risk for develop-
ment of HUS due to toxin release from lysed STEC cells. Fluid
hydration may be necessary in diarrheic patients. Patients with
severe bloody diarrhea may need blood transfusions, and in
cases of HUS, dialysis may be necessary.
The main reservoir for STEC is the intestinal tract of cattle.
However, other ruminants, including sheep, goat, buffalo,
guanaco, deer, and elk, and nonruminants, including cats,
dogs, pigs, horses, rabbits, and chickens, also can carry STEC.
Contact with cattle, the cattle environment, and food products
originating from cattle provides major risk factors for STEC
infection to humans. Recreational, drinking, and irrigation
waters contaminated with cattle feces can be sources of STEC.
Infected individuals can transmit STEC person-to-person, and
food may be contaminated by cross contamination or by an
infected food preparer. In the United States, STEC O157:H7
was declared an adulterant in beef in 1994 after a large out-
break linked to contaminated ground beef occurred, and
recently, STEC serogroups O26, O45, O103, O111, O121,
and O145 were declared adulterants, since these serogroups
cause the majority of the non-O157 STEC infections and illness
similar to that caused by STEC O157:H7. For the period 2009–
13 in the United States, O157:H7 STEC outbreaks were associ-
ated with foods, including spinach (1 outbreak), Romaine
lettuce (1), Lebanon bologna (1), beef (3), hazel nuts (1),
cheese (1), and prepackaged cookie dough (1). STEC ser-
ogroup O26 was associated with outbreaks linked to ground
beef and raw clover sprouts. An outbreak linked to STEC O121
involved frozen food products, and O145 was associated with
two outbreaks, one linked to Romaine lettuce and the other
due to an unknown source. Outbreaks are often associated
with STEC O157:H7, whereas non-O157 STEC are more
often associated with sporadic cases and less often with out-
breaks. However, it is now estimated that non-O157 strains
cause a greater number of illnesses than E. coli O157:H7.
Based on the Centers for Disease Control and Prevention’s
FoodNet program in the United States for the years 2000–10,
STEC O157:H7 was the cause of 5688 infections (74.0%),
whereas non-O157 STEC caused 2006 infections (26.1%). The
top six non-O157 STEC caused 1416 cases (18.4%). Serogroup
O26 was responsible for 5.8% of STEC cases, followed by
serogroups O103 (5.0%), O111 (4.2%), O121 (1.4%), O45
(1.2%), and O145 (0.8%). However, estimates of the total
STEC foodborne infections indicate that the non-O157 ser-
ogroups cause 64% of the STEC disease cases in the United
States. In the European Union, in 2011, nine STEC serogroups
were responsible for 4022 cases of disease. STEC O157:H7/H
was responsible for 54% of the STEC cases, and STEAEC (Shiga
toxin-producing enteroaggregative E. coli) O104 caused 26% of
the disease cases. Serogroup O26 accounted for 7% of the cases.
Serogroups O103, O91, O145, O128, O111, and O146 were
responsible for fewer cases in the descending order. In 2010,
nine serogroups (in the descending order, O157, O26, O103,
O145, O91, O63, O111, O128, and O146) accounted for 2110
cases; STEC O157:H7 caused 71% of the STEC illnesses followed
by 12% for serogroup O26. There was no report of STEAEC cases
in the European Union in 2010. Of 504 isolates of STEC isolated
for the period 2001–09 in Australia, the following serogroups
were the most common: O157 (58%), O111 (13.7%), O26
(11.1%), O113 (3.6%), O55 (1.3%), and O86 (1.0%). The
information on STEC isolates from patients in the United States,
the European Union, and Australia indicate that serogroup
O157 was the most common cause of STEC illness.
Strict hygiene during animal slaughtering, vegetable and fruit
harvesting, product handling, shipping, and food preparation is
critical in controlling and preventing STEC-induced disease.
Enteropathogenic E. coli
Enteropathogenic E. coli (EPEC) are an important cause of
persistent and potentially fatal diarrhea in infants and young
 Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects
children in developing countries. EPEC possess the LEE path-
ogenicity island similar to the Shiga toxin-producing E. coli but
do not produce Stx. The formation of A/E lesions is character-
istic of EPEC and is dependent on LEE island genes encoding
T3SS, which acts in a similar way to the secretory system in
STEC and allows intimate attachment of the bacteria to the
host cell. LEE genes encode for effector proteins that subvert
host cellular processes and induce gastrointestinal symptoms.
Non-LEE-encoded genes produce effector proteins that inhibit
phagocytosis, activate innate immune responses, and have a
role in colonization and pathogenic responses. The E. coli
adherence factor plasmid (pEAF) has the bfp operon, which
encodes the bundle-forming pilus. The EPEC strains that carry
the eae and bfpA genes are termed typical EPEC, whereas the
atypical EPEC strains have the eae gene but lack bfpA. The
typical EPEC strains are a major cause of infantile diarrhea in
developing countries but are rare in the developed world.
Diarrhea manifests as a severe watery stool with a large amount
of mucus but is rarely bloody. The atypical EPEC induce a less
severe illness. Treatment consists of prevention of dehydration
induced by diarrhea. Humans appear to be the only reservoir
for typical EPEC, whereas both animals and humans are reser-
voirs for atypical EPEC. Contaminated food and water have
been associated with EPEC diarrhea. EPEC infections are gen-
erally sporadic, and food vehicles are often unknown. Raw
chicken and beef, cold pork, meat pie, and other foods, as
well as water, have been implicated in a few outbreaks. How-
ever, any foods exposed to the feces of an infected human may
induce diarrhea.
Enteroinvasive E. coli
Enteroinvasive E. coli (EIEC) induce an invasive inflammatory
colitis with copious watery diarrhea. EIEC are closely related to
Shigella species but do not produce Shiga toxin. A number of
EIEC strains contain the sen gene, which encodes for Shigella
enterotoxin-2 (ShET-2). ShET-2 is translocated to the host cell
via T3SS and may participate in the inflammation of the intes-
tinal epithelial cells. The virulence effector genes, the sen gene,
and T3SS genes are located on a large plasmid, pInv. EIEC does
not carry the LEE pathogenicity island. When ingested, EIEC
reach the colon, gain access to the colonic submucosa through
the M cells, and are then taken up by macrophages. The bacte-
rial cells escape from the macrophages and invade and repli-
cate in the colonocytes. Bacterial virulence effectors are
secreted via T3SS into the host cells, which enable the bacteria
to evade the immune system and to promote cell-to-cell dis-
semination. The role of T3SS in EIEC is internalization of the
bacteria into the host cell; A/E lesions are not seen in EIEC
infection. EIEC-contaminated food and water are associated
with the illness. EIEC infections caused by EIEC are rarely
detected in the United States; however, a large outbreak
occurred in Texas in 1992 in which 370 people became ill
after consuming guacamole. Foods are most likely contami-
nated by infected food handlers, and EIEC infections may also
occur via person-to-person transmission. Outbreaks associated
with EIEC in several countries have been reported with water,
imported cheese, vegetables, and potato salad as the vehicles of
infection. Rehydration therapy and antibiotics are recom-
mended as treatments for EIEC infection. Since humans are
541
the only known reservoir of EIEC, it is probable that most EIEC
infections are due to food prepared by an ill individual.
Enterotoxigenic E. coli
Enterotoxigenic E. coli (ETEC) infection induces a watery diar-
rhea, which may be a mild, self-limiting disease or a severe
purging cholera-like disease. The infection may attack children
in developing countries or other areas with poor hygiene and
unclean water. In addition, ETEC is a major cause of traveler’s
diarrhea. The organism colonizes the surface of the small
bowel and produces enterotoxins, which induce the diarrhea.
ETEC elaborate two types of plasmid-encoded enterotoxins:
heat-labile enterotoxins (LT-1 and LT-2) and heat-stable
enterotoxins (STa and STb). LT-1 is an AB5 toxin closely related
to cholera toxin. LT intoxication of host intestinal cells leads to
permanent activation of adenylate cyclase with increased levels
of cyclic AMP (adenosine monophosphate). Cyclic AMP acti-
vates protein kinase A, leading to phosphorylation of ion
channels and secretion of electrolytes into the intestinal
lumen. The increased gut osmolarity leads to a watery diarrhea.
STa is a small, single-peptide toxin, which binds to the host cell
guanylate cyclase, leading to increased cyclic GMP (guanosine
monophosphate) levels. Chloride secretion is increased, and
NaCl absorption is inhibited. The increased osmolarity in the
intestinal lumen results in diarrhea. ETEC strains may produce
LT, ST, or both. Rehydration therapy and antibiotics are recom-
mended as treatments for ETEC infection. Humans are the
reservoir of ETEC, and ingestion of food or water contaminated
by an infected person is often the likely cause of illness. Foods
implicated in infections include Brie cheese, curried turkey,
mayonnaise, crab meat, deli foods, and salads.
Enteroaggregative E. coli
Enteroaggregative E. coli (EAEC) include a heterogeneous pop-
ulation of E. coli strains that do not produce LT or ST and that
adhere in a stacked brick pattern to HEp-2 cells and to each
other. These strains cause a watery diarrhea in both industrial-
ized and third-world countries. Diarrhea can be of relatively
long duration ( 14 days) in some cases. EAEC infects children
and immunocompromised individuals, and these pathogens
are an important cause of traveler’s diarrhea. The major
features of EAEC pathogenesis are colonization of the intesti-
nal mucosa with mucoid biofilm formation, elaboration of
enterotoxins, and mucosal inflammation. The plasmid
(pAA)-encoded AAF (aggregative adherence fimbriae) and
AggR (regulator) are required for colonization; however, AAF
is not present in all EAEC strains, and therefore, other adhesins
are likely involved in EAEC colonization. Adhesion is followed
by secretion of Pet (plasmid-encoded toxin) and EAST1 toxins
(plasmid-encoded EAEC heat-stable toxin 1). EAST1 is a small
polypeptide enterotoxin that induces diarrhea by secreting
cyclic GMP. The pet gene is located on pAA and encodes Pet,
a heat-labile protein toxin belonging to the serine protease
autotransporter family. Pet is an enterotoxin inducing cytoskel-
etal changes and epithelial cell rounding. In addition, the
enterotoxin ShET-1 may be found in some EAEC. However,
the role of enterotoxins in EAEC-induced diarrhea is not clear.
EAEC strains that show the stacked brick phenotype and that
542 Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects
contain pAA are termed typical EAEC, whereas those strains
that have the stacked brick phenotype but lack pAA are termed
atypical EAEC. Both atypical and typical strains induce diar-
rhea. Rehydration therapy and antibiotics are recommended as
treatments for EAEC infection. Ingestion of food has been
associated with EAEC-induced diarrhea; however, the reservoir
of the organism is unknown. Salsa in Mexico, unpasteurized
cheeses in Italy, and school lunches in Japan have caused out-
breaks of EAEC-induced illness. It is probable that infected
food handlers are the source of the organism.
Diffusely Adherent E. coli
Diffuse adherence is characterized by uniform attachment of
bacteria to HeLa or HEp-2 cells, whereas localized adherence is
when bacteria attach in groups to only a few sites on the
cultured cell surface. Diffusely adherent E. coli (DAEC) are a
poorly characterized heterogeneous population of E. coli show-
ing diffuse attachment to cell surfaces and are believed to be a
cause of diarrhea. The DAEC pathotype colonizes the small
bowel and may be responsible for watery diarrhea in children.
Diffuse adherence is mediated by both fimbrial (Dr and
F1845) and afimbrial (Afa) adhesions. A secreted autotran-
sporter toxin (SAT) has been demonstrated in DAEC strains
associated with diarrhea. SAT induces lesions on tight junc-
tions of epithelial cells causing increased permeability and may
be a virulence mechanism in DAEC. However, the mechanism
of diarrhea induction in DAEC strains is not certain.
Shiga Toxin-Producing Enteroaggregative E. coli
The Shiga toxin-producing enteroaggregative E. coli (STEAEC)
may be a newly emerging pathotype of E. coli. The serotype
O104:H4 caused a large outbreak associated with close to 4000
cases of illness, over 800 cases of HUS, and over 50 deaths in
Europe in 2011. This pathotype is unusual in that the organism
carried the pAA-virulence plasmid (and its virulence genes) of
EAEC strains and a stx2-harboring prophage. Thus, this
STEAEC strain presents the stacked brick phenotype of EAEC
and production of Shiga toxin. The STEAEC strain is not a
pathogenic E. coli with new virulence factors but is rather a
pathogen that has combined the virulence properties of the
EAEC with the production of Shiga toxin of the STEC patho-
type. The strong morbidity and mortality induced by this strain
may be due to its strong adherence to the host cell leading to
higher levels of Stx transfer. The serotype O111:H2 with both
Stx2 and EAgg (AggR) was reported in 1997 as a cause of a case
of HUS, and in 2012, an outbreak induced by serotype O111:
H21 positive for Stx2c and aggR occurred in Ireland. Therefore,
it is probable that the transfer of the Shiga toxin gene into other
EAEC serotypes will produce STEAEC serotypes that may cause
serious outbreaks of Shiga toxin-induced HC and HUS in the
future.
Recent Outbreaks in the United States due to E. coli
Pathotypes
For the years 2009–10, the Centers for Disease Control and
Prevention reported that there were 58 outbreaks due to STEC
serotypes (53 due to STEC O157:H7 and 5 due to non-O157
STEC) with 651 total cases. In 2009, there were three outbreaks
due to ETEC with 85 cases, and in 2010, EPEC caused one
outbreak with 7 cases. The vehicles of infection were not
provided. There were no reported outbreaks caused by EIEC,
EAEC, or DAEC for 2009–10.
Nonintestinal Pathogenic E. coli
The nonintestinal pathogenic E. coli, which are a cause of
illness outside of the gut, are termed extraintestinal pathogenic
E. coli (ExPEC). The intestinal pathogenic E. coli (IPEC) are
obligate pathogens, whereas the ExPEC are facultative patho-
gens that reside in the gut of a fraction of individuals and
behave as harmless commensal organisms. In general, the
IPEC have well-defined virulence traits, whereas ExPEC strains
are very diverse and have few common virulence factors among
them. Thus, it is difficult to define ExPEC by virulence proper-
ties. At the present time, ExPEC and nonpathogenic E. coli
cannot be differentiated by molecular epidemiological
techniques, and therefore, there is a limited understanding of
the biology of ExPEC.
Urinary tract infections (UTIs) are common bacterial infec-
tions, particularly in women, and extract extremely high health
costs in terms of morbidity, treatment, and loss of productivity.
Uropathogenic E. coli (UPEC) are ExPEC that cause  80% of
community-acquired UTIs and  30% of nosocomial UTIs.
The UPEC reside in the gastrointestinal tract and can be trans-
ferred to the urinary tract. Infection of the urethra is termed
urethritis, that of the bladder is termed cystitis, and that of the
kidneys is termed pyelonephritis. Individuals at risk for acquir-
ing UTIs include the very young, elderly, females, pregnant
women, and individuals who are immunocompromised or
who are undergoing urinary catheterization. The UPEC possess
a number of virulence traits that make them efficient patho-
gens of the urinary tract such as specific adhesins; several toxins
including hemolysin, cytotoxic necrotizing factor type 1
(CNF1), and a protease toxin (SAT); and iron acquisition
systems. The primary reservoir for UPEC is the human intesti-
nal tract; however, the original source (e.g., possibly avian,
food, or environmental source) of UPEC is not clear.
The pathology leading to UTIs by UPEC includes adherence
and colonization, evasion of host defenses, and damage to
host cells and tissues. The UPEC are transferred from the
rectum to the periurethral area and ascend the urethra into
the bladder. Type 1 fimbriated UPEC attach to the bladder
epithelial cells and trigger apoptosis and exfoliation, as well
as invasion and multiplication of UPEC in the bladder cells. If
the UTI is not treated, the organisms may ascend the ureters
and induce pyelonephritis. Pyelonephritis can cause irrevers-
ible kidney damage, kidney failure, and death. Some individ-
uals may have high bacterial counts in the urine but are
asymptomatic. Treatment of UTIs is becoming more compli-
cated due to increased resistance of UPEC to antimicrobial
compounds.
Neonatal meningitis E. coli (NMEC) strains are ExPEC that
represent the most frequent cause of gram-negative meningitis
in the newborn. Bacterial meningitis is an inflammation of the
meninges with high morbidity and mortality. Severe neurolog-
ical sequelae occur in 30–50% of infants who survive
 Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects
meningitis. The neonate (neonatal period is the first 28 days
after birth) acquires NMEC during birth or from the environ-
ment, and the organisms invade and multiply in the blood-
stream. The NMEC pathogen then crosses the blood–brain
barrier (BBB). The BBB is a structural and functional barrier
made up of the brain microvascular endothelial cells (BMECs),
which separates the CNS from the vascular component of the
body. The BBB regulates the transition of macromolecules into
the CNS and protects the CNS against entry of microbes and
toxins that may be present in the blood.
There are several stages of NMEC pathogenesis: bacteremia,
binding of bacteria to the surface of the BMEC, and invasion of
the BMEC followed by invasion of the meninges and the CNS.
Meningitis is associated with bacteremia counts
of >103 CFU ml 1 blood. Outer membrane protein A
(OmpA), K1 capsule (carried by the predominant strains of E.
coli that cause neonatal meningitis), and O-lipopolysaccharide
are necessary for survival and multiplication of NMEC in the
circulatory system. OmpA and FimB (fimbriae) of NMEC bind
to BMEC, and the organism invades the BMEC via a trans-
cellular traversal mechanism in which the bacteria penetrate
the cells without disrupting them. OmpA, Ibe proteins, and
CNF1 induce actin cytoskeletal rearrangement and formation
of microvillus-like protrusions, which facilitate entry of the
organisms into the BMEC. NMEC leave the BMEC and invade
the meninges and CNS, and then, they multiply and induce the
release of proinflammatory compounds. In addition, there are
brain edema and increased intracranial pressure with menin-
gitis and neuronal injury. Neonatal meningitis is treated with
appropriate antibiotics.
Avian pathogenic E. coli (APEC) are responsible for a num-
ber of economically important poultry diseases. Interestingly,
some human ExPEC and some APEC strains have similar phy-
logenetic backgrounds and share some of the same virulence
genes. This relationship between ExPEC and APEC suggests
that APEC may be zoonotic pathogens that can lead to
human diseases such as UTIs or neonatal meningitis through
the ingestion of poultry meat. Recent studies support the role
of chicken meat as a potential source of organisms inducing
UTIs to humans; however, food has not been shown to be
related to neonatal meningitis.
Other Enterobacteriaceae
Cronobacter sakazakii
The genus Cronobacter consists of seven species, namely, C.
sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjen-
sii, C. condimenti, and C. universalis. Cronobacter species
are gram-negative, motile by peritrichous flagella, non-spore-
forming, facultatively anaerobic, rod-shaped bacteria. Crono-
bacter species are more thermotolerant when compared with
other genera in the Enterobacteriaceae family, and they can
grow in temperatures over a range of 6–47  C. The reservoir of
Cronobacter is unknown; however, plants are a possible source,
and the organisms have been isolated from a wide range of
food products. Most Cronobacter species have been associated
with human illness; however, the majority of the infections in
infants and neonates have been associated with C. sakazakii,
and this species has been the most studied. Infants that ingest
543
rehydrated powdered milk formula contaminated with
C. sakazakii suffer bacteremia, meningitis, and necrotizing
enterocolitis; mortality is greater than 50%. Infections among
immunocompromised adults, particularly the elderly, have
been reported. The outer membrane protein, OmpA, is an
important virulence factor in the development of meningitis
in newborn rats. OmpA-positive Cronobacter cross the intesti-
nal barrier, multiply in the blood, and invade the brain endo-
thelial cells. The pathogen crosses the BBB and eventually
causes meningitis. Cronobacter lacking the ompA gene do not
bind to intestinal epithelial cells, do not cross the intestinal
barrier, and do not lead to pathology. Other potential viru-
lence factors include biofilm formation and membrane-
associated efflux pumps that expel a broad range of inhibitory
compounds. However, it is clear that knowledge of the viru-
lence mechanisms of the Cronobacter species is limited.
Shigella
The Shigella species are small, gram-negative, nonmotile, fac-
ultatively anaerobic rods, which do not produce spores or
capsules. The optimum growth temperature is 37  C. The
organisms are the causative agents of bacillary dysentery (shig-
ellosis). All of the species except Shigella sonnei have several
serotypes. Humans are the natural host of the Shigella species.
The organisms are transmitted via the fecal–oral route by
infected individuals and contaminated food and water. Shig-
ellosis is an invasive infection of the colon that ranges from
short-term watery diarrhea to inflammatory bowel disease. In
inflammatory bowel disease, watery diarrhea proceeds to
bloody mucoid stools, abdominal cramps, and tenesmus. In
healthy individuals, shigellosis is self-limited and resolves
without sequelae. In malnourished infants and young children
in developing countries, shigellosis can result in acute,
life-threatening complications. HIV-infected and immuno-
compromised individuals may suffer persistent diarrhea and
malnutrition if infected by Shigella. Cellular invasion and
spread of infection involve invasion of the enterocytes, intra-
cellular multiplication, intra- and intercellular spread, killing
of the host cells, and tissue damage. Shigella dysenteriae, but not
other Shigella species, produce Shiga toxin. Shiga toxin is
encoded chromosomally in Shigella dysenteriae, whereas it is
phage-encoded in STEC. Similar to STEC, infection by Shigella
dysenteriae can induce HUS. The virulence of Shigella is depen-
dent on a virulence plasmid encoding T3SS components. T3SS
is necessary for invasion and secretion of virulence factors into
intestinal epithelial cells leading to inflammatory destruction
of the epithelial lining of the intestine. Rehydration therapy
and antibiotics are used to treat shigellosis.
Salmonella enterica
Salmonella spp. are rod-shaped, gram-negative, non-spore-
forming, predominantly motile with peritrichous flagella, che-
moorganotrophic, and facultatively anaerobic. The range of
temperature for growth is 2–54  C, and the optimum is 35–
37  C. There are more than 2000 different serovars based on
flagellar, carbohydrate, and lipopolysaccharide characteristics.
Salmonella causes disease in both animals and humans. Serovars
Typhi, Paratyphi, and Sendai cause enteric fever, whereas most
544 Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects
serovars cause enterocolitis and diarrhea. The elderly, infants,
and immunocompromised individuals are at the highest risk
for Salmonella infections. Ingestion of food or water contami-
nated with nontyphoidal Salmonella is followed by crampy,
abdominal pain, diarrhea, nausea, and vomiting. Enterocolitis
in children is marked by increased severity of inflammation,
bloody diarrhea, long duration, and increased risk of complica-
tions. Salmonellosis is self-limited in immunocompetent hosts.
A small number of infected individuals develop a sequela known
as reactive arthritis. Important virulence genes are carried on the
Salmonella pathogenicity islands (SPI-1 through SPI-5). Other
virulence traits include the virulence plasmid (pSLT), adhesins,
flagella, and proteins involved in biofilm formation. Antibiotics
are recommended in severely ill patients; however, it is important
to realize that antibiotic resistance is common in the salmonel-
lae. Hydration therapy is necessary in cases of severe diarrhea.
Yersinia
Yersiniosis is a self-limiting gastrointestinal disease in immu-
nocompetent individuals; however, in young children and
immunocompromised individuals, the disease is more severe
and may result in death. Sequelae such as reactive arthritis may
occur after infection. The organism is rod-shaped, gram-
negative, psychrotrophic (can grow at 4  C), and facultatively
anaerobic. Yersiniosis is a foodborne disease caused mainly
by Y. enterocolitica and Y. pseudotuberculosis, and the flea-
transmitted Y. pestis rarely causes foodborne illness. Pigs are
the major reservoir of Y. enterocolitica; pig tonsils are a common
site of Yersinia colonization. The absence of the virulence plas-
mid (pYV) is associated with lack of pathogenicity in Y. enter-
ocolitica. pYV encodes the Yersinia adhesin YadA, which
mediates mucus and epithelial cell attachment and host cell
invasion. A T3SS is also encoded by pYV and is involved in the
injection of virulence-related effector proteins, the Yersinia
outer membrane proteins (Yops), into the host cell. The Yop
proteins interfere with host signal induction (evasion of
immune responses), disrupt host actin cytoskeleton, and
induce apoptosis in host cells. Chromosomal genes encoding
the invasion protein (Inv) and the attachment-invasion locus
(Ail) have a role in cell binding and invasion by Yersinia. The
most common vehicle for yersiniosis is contaminated food,
including meat, oysters, fish, raw milk, and cheese. Antibiotics
are used to treat yersiniosis.
See also: Diarrheal Diseases; Emerging Foodborne Enteric Bacterial
Pathogens; Escherichia coli and Other Enterobacteriaceae: Occurrence
and Detection; Food Poisoning: Classification; Food Poisoning:
Epidemiology; Food Poisoning: Tracing Origins and Testing;
Salmonella: Detection; Salmonella: Properties and Occurrence;
Salmonella: Salmonellosis; Shigella; Yersinia enterocolitica: Properties
and Occurrence; Yersinia enterocolitica: Detection and Treatment.
Further Reading
CDC (2013) Surveillance for foodborne disease outbreaks – United States, 2009–2010.
Morbidity and Mortality Weekly Report (MMWR) 62: 41–47.
Clements A, Young JC, Constantinou N, and Frankel G (2012) Infection strategies of
enteric pathogenic Escherichia coli. Gut Microbes 3: 71–87.
Coburn B, Grassl GA, and Finley BB (2007) Salmonella, the host and disease: a brief
review. Immunology and Cell Biology 85: 112–118.
Croxen MA and Finlay BB (2010) Molecular mechanisms of Escherichia coli
pathogenicity. Nature Reviews Microbiology 8: 26–38.
Drummond N, Murphy BP, Ringwood T, Prentice MB, Buckley JF, and Fanning S
(2012) Yersinia enterocolitica: a brief review of the issues relating to the zoonotic
pathogen, public health challenges, and the pork production chain. Foodborne
Pathogens and Disease 9: 179–189.
European Food Safety Authority (2013) EU summary report on trends and sources of
zoonoses, zoonotic agents, and food-borne outbreaks in 2011. EFSA Journal 11(4):
3129.
Fàbrega A and Vila J (2013) Salmonella enterica serovar Typhimurium skills to succeed
in the host: virulence and regulation. Clinical Microbiology Reviews 26: 308–341.
Farfan MJ and Torres AG (2012) Molecular mechanisms that mediate colonization of
Shiga toxin-producing Escherichia coli strains. Infection and Immunity
80: 902–913.
Healy B, Cooney S, O’Brien S, et al. (2010) Cronobacter (Enterobacter sakazakii): an
opportunistic foodborne pathogen. Foodborne Pathogens and Disease 7: 339–350.
Kaper JB, Nataro JP, and Mobley HL (2004) Pathogenic Escherichia coli. Nature
Reviews Microbiology 2: 123–140.
Mellata M (2013) Human and avian extraintestinal pathogenic Escherichia coli:
infections, zoonotic risks, and antibiotic resistance trends. Foodborne Pathogens
and Disease 10: 916–932.
Melton-Celsa A, Mohawk K, Teel L, and O’Brien A (2012) Pathogenesis of Shiga toxin-
producing Escherichia coli. Current Topics in Microbiology and Immunology
357: 67–103.
Scallan E, Hoekstra RM, Angulo FJ, et al. (2011) Foodborne illness acquired in the
United Statesdmajor pathogens. Emerging Infectious Diseases 17: 7–15.
Smith JL, Fratamico PM, and Gunther NW (2007) Extraintestinal pathogenic Escherichia
coli. Foodborne Pathogens and Disease 4: 134–163.
Wang F, Yang Q, Kase JA, Meng J, Clotilde LM, Lin A, and Ge B (2013) Current trends
in detecting non-O157 Shiga toxin-producing Escherichia coli in food. Foodborne
Pathogens and Disease 10: 665–677.
Relevant Websites
http://www.cdc.gov/cronobacter/technical.html – Centers for Disease Control and
Prevention.
http://www.cdc.gov/nczved/divisions/dfbmd/diseases/shigellosis/ – Centers for
Disease Control and Prevention.
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/yersinia_g.htm – Centers for Disease
Control and Prevention.
http://www.cdc.gov/ecoli/ – Centers for Disease Control and Prevention.
http://www.cdc.gov/salmonella/general/ – Centers for Disease Control and Prevention.
http://www.fda.gov/Food/FoodborneIllnessContaminants/
CausesOfIllnessBadBugBook/ – U.S. Food and Drug Administration.
 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection
S Fanning, L Rogers, K Power, and P Ó Gaora, University College Dublin, Dublin, Ireland
ã 2016 Elsevier Ltd. All rights reserved.
Enterobacteriaceae
All members of the Enterobacteriaceae possess some common
bacteriological features. They are gram-negative, rod-shaped
facultative anaerobes. Bacterial isolates are classified as being
either motile by means of peritrichous flagella or nonmotile,
being devoid of any flagella. Members of this family are gen-
erally 0.3–1.0  0.6–6.0 mm in size and are non-spore-forming.
They can ferment glucose and are catalase-positive (except for
Shigella dysenteriae) and oxidase-negative, and they reduce
nitrate to nitrite. Table 1 provides a summary of the general
characteristics of some of the members of this family.
Enterobacteriaceae family members are associated with a
wide range of disease (Figure 1). Illnesses associated with
members of the Enterobacteriaceae include wound infections,
urinary tract infections (UTI), gastroenteritis, meningitis,
pneumonia, septicemia, and hemolytic uremic syndrome
(HUS). While some of these species are true pathogens, others
are regarded as opportunistic.
Members of this bacterial family can grow readily on a
variety of microbiological culture media. They are also widely
dispersed throughout the environment being found in soil,
water, plants, and the gastrointestinal tract of animals and
humans.
The Genus Escherichia
The genus Escherichia was described originally by Theodore
von Escherich who first discovered Escherichia coli in 1885.
This bacterial genus contains five species with E. coli being
the best known. Other species include E. albertii, E. fergusonii,
E. hermannii, and E. vulneris. A number of species are
pathogenic; however, the majority are commensals or oppor-
tunistic pathogens.
Features associated with isolates of the E. coli genus
Escherichia coli cells are 0.5 mm in length and 2.0 mm in diam-
eter. Optimal growth occurs at a temperature of 37  C, though
some strains can grow at temperatures up to 49  C. The opti-
mal range of pH is between 6.0 and 7.0, with few being able to
grow at a pH < 4 or > 9. E. coli grown on MacConkey agar
forms small, red/pink, smooth, and circular colonies. When
further assessed, these are motile by means of peritrichous
flagella (Table 1).
Environments known to contain E. coli
These bacteria can be found in the gastrointestinal tract of
warm-blooded animals in which it colonizes within hours of
birth. Strains can also be found throughout the environment,
in water, food, and soil. Most E. coli isolates are harmless and
play an important role as constituents of the normal biota or
microbiome found in a healthy human intestinal tract. They
aid digestion in the gut, produce vitamin K2, and prevent
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00259-2
pathogenic bacteria from thriving in the environment of the
intestine.
Relevance of E. coli to public health
Pathogenic strains of E. coli cause a variety of illnesses such as
food poisoning with severe stomach cramps, watery or bloody
diarrhea, vomiting, and nausea. Severe complications include
kidney failure, seizures, and stroke. The main route of trans-
mission is through fecal–oral contamination, due to poor
hygienic conditions.
E. coli has a very fluid genome that enables the uptake of
many different mobile genetic elements. This genomic fluidity
has had a large impact on the evolution of pathogenic serovars
with phages, plasmids, and pathogenicity islands playing a role
in their diversity and evolution. This has resulted in the emer-
gence of pathogenic strains that are associated with their own
virulence factors and disease symptoms. Pathogenic strains are
classified into a number of pathotypes as follows:
• Verotoxigenic E. coli (VTEC)
• Enterotoxigenic E. coli (ETEC)
• Enteropathogenic E. coli (EPEC)
• Enteroaggregative E. coli (EAEC)
• Enteroinvasive E. coli (EIEC)
• Diffusely adherent E. coli (DAEC)
Pathotypes of Escherichia coli Important to Human Health
Verotoxigenic E. coli
VTEC strains are defined by the production of cytotoxins that
disrupt protein synthesis within host cells. These toxins are
referred to as either verotoxins (vt) due to their activity against
Vero cells in vitro or Shiga-like toxin 1 (stx1) and Shiga-like
toxin 2 (stx2) due to their similarity to the Shiga toxin pro-
duced by Shigella dysenteriae.
Dissemination of these strains occurs via the fecal–oral route
arising from substandard hygienic conditions. An infectious
dose can be as low as 10–100 cells. Ruminant animals are an
important reservoir for VTEC. Infections can occur through
contact with an infected individual, directly from an animal
shedding the organism or its fecal matter, via the consumption
of a contaminated food containing the bacterium or through
the consumption of contaminated water.
Symptoms of a VTEC infection can range from mild diarrh-
ea to severe bloody diarrhea. Potentially fatal complications
arising from VTEC infections include HUS and thrombotic
thrombocytopenic purpura.
Enterohemorrhagic E. coli (EHEC) is a subset of VTEC that
is classified as pathogenic to humans. Usually, strains are
classified as EHEC when they contain a verotoxin-encoding
gene in combination with an eae gene that encodes intimin,
an outer membrane protein that is involved in attachment to
epithelial cells. Some eae-negative strains may also be EHEC.
545
546 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection

Table 1 Typical characteristics of Enterobacteriaceae

Escherichia
Features coli Salmonella Yersinia Cronobacter Enterobacter Citrobacter

Size (mm) 0.5  2.0 3.4  0.7 0.8  1–2 31 0.6  2–3 1  2–6
Motility Motile Motile Nonmotile (between 25 and 35  C) Motile Motile Motile
Optimal
Temperature or range ( C) 37 30–37 28 6–45 30–37 37
pH range 6–7 6.5–9 4–10 5–10 4.4–9 5–8
Flagella Peritrichous Peritrichous Peritrichous Peritrichous Peritrichous Peritrichous
Encapsulated Yes Yes Yes Yes Yes No
Pathogen category Major Major Major Opportunistic Opportunistic Opportunistic

Enterobacteriaceae

Major food-borne pathogens Opportunistic food-borne pathogens

Escherichia Salmonella Yersinia Cronobacter Enterobacter Citrobacter Klebsiella

coli species species species species species species

VTEC S. Typhimurium Y. pestis C. sakazakii E. cloacae C. freundii K. pneumoniae

ETEC S. Enteritidis E. aerogenes C. koseri
Y. enterocolitica
EHEC Y. pseudotuberculosis
EPEC
EIEC
DAEC
Figure 1 Enterobacteriaceae of importance to public health.

They produce highly potent toxins and can form attaching and
effacing lesions on epithelial cells, a characteristic typically
associated with EPEC strains. In terms of public health, E. coli
O157 is one of the most commonly detected EHEC serotypes,
although other serotypes are also being recognized.
Outbreaks of EPEC infection have been linked to the con-
sumption of contaminated drinking water as well as some
meat products. In the case of tEPEC, humans are thought to
be the only reservoir while both animals and humans are
reservoirs for aEPEC.

Enteropathogenic E. coli Enterotoxigenic E. coli

EPEC strains are noted for their ability to produce attaching
and effacing (A/E) lesions. In this case, bacteria attach to the
host epithelial cell membrane disrupting the cell surface result-
ing in the effacement of microvilli at the site of adherence.
EPEC contains a 35 kbp pathogenicity island called the locus of
enterocyte effacement, which encodes genes responsible for
the attaching and effacing lesions.
These strains are further classified into typical enteropatho-
genic E. coli (tEPEC) and atypical enteropathogenic E. coli
(aEPEC) based on the presence and absence of the E. coli adher-
ence factor plasmid that is only found in tEPEC strains. tEPEC is a
well-documented cause of gastroenteritis in infants. aEPEC is
more prevalent than tEPEC in both developed and developing
countries and is an important risk factor in juvenile endemic
diarrhea and diarrheal outbreaks.
ETEC strains are characterized by their expression of entero-
toxins and the presence of host-specific fimbriae required
for attachment to intestinal cells. Two types of toxins, heat-
stable (ST) and heat-labile (LT) toxins, are produced by ETEC.
Different strains can harbor either one or both of these
toxin-encoding loci. The toxins act to stimulate the intestinal
lining leading to an increase in fluid secretion resulting in
diarrhea.
These pathogenic E. coli are the leading cause of traveler’s
diarrhea. Symptoms range from mild to severe cholera-like
illness characterized by watery stool(s), vomiting, and stomach
pains lasting up to 3 days. The infectious dose is estimated at
108 cells but is lower in immunocompromised patients.
ETEC is mainly transmitted through food and water con-
taminated with human and animal feces. In humans, ETEC
 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection
infections are widespread though rarely life-threatening and
usually self-limiting. It is a major pathogen in animals and
can even cause death.
Enteroinvasive E. coli
These bacteria are characterized by their ability to invade epi-
thelial cells and disseminate from cell to cell while replicating
within. EIEC differ from typical E. coli in that they are non-
motile and cannot decarboxylate lysine nor ferment lactose.
EIEC strains possess an invasion plasmid that encodes the
virulence genes conferring EIEC invasive capacity.
EIEC strains are highly related to Shigella species at the
genetic, biochemical, and pathogenic levels. They cause a syn-
drome identical to bacillary dysentery more often linked to
Shigella species, with profuse diarrhea and high fever. The
infectious dose of EIEC is estimated at 106 cells whereas that
of Shigella is similar to EHEC, in the range of ten to several
hundred cells.
There are no known animal reservoirs of EIEC. Transmis-
sion usually occurs through contact with infected humans
through the fecal–oral route. EIEC infections can also occur
through ingestion of contaminated food or water.
Enteroaggregative E. coli
EAEC strains are characterized by their ability to adhere to
epithelial cells in an aggregative manner resulting in a stacked-
brick pattern. They do not secrete LT or ST enterotoxins. EAEC
pathogenesis involves adherence to the intestinal mucosa
resulting in increased production and deposition of a mucus
biofilm leading to mucosal toxicity due to inflammation and
cytokine release. EAEC strains can cause persistent infection by
evading the immune system of the host.
These organisms are associated with acute or persistent
diarrhea with little or no fever and no vomiting. They are
especially prevalent among children in developing countries.
EAEC strains are considered emerging pathogens, as VTEC
O111:H2 strains isolated from a HUS outbreak in France also
possessed genetic markers of EAEC E. coli. EAEC outbreaks
have been linked to infant food including formula, milk, and
water.
In the summer of 2011, a novel strain of E. coli O104:H4
emerged and caused a serious food-borne outbreak in Lower
Saxony, Germany. This outbreak was associated with serious
illness such as bloody diarrhea with a high incidence of HUS.
Epidemiological investigations reported 3816 cases with 845
instances of HUS and 54 deaths. At first, E. coli O104:H4 was
thought to be an EHEC strain, but it was later identified as an
EAEC that had acquired the ability to produce Shiga toxin.
Diffusely adherent E. coli
DAEC strains are characterized by their adherence to Hep-2
cells in a diffuse pattern. Approximately 75% of these bacteria
express the Afa/Dr family of adhesins, but not the typical
virulence genes of other E. coli pathotypes.
DAEC are a major cause of UTI and can cause watery diarrh-
ea typically without blood but with vomiting. Infection is
characterized by the growth of fingerlike cellular projections
on the surface of infected epithelial cells that envelop the
bacteria. Sources associated with DAEC infections include
547
contaminated food such as undercooked beef, contaminated
water, and contact with infected animals.
Enterobacteriaceae of Importance to Human Health
The genus Salmonella
This bacterial genus was named after Daniel Elmer Salmon
following the discovery of the type strain Salmonella enterica
in 1885. The Salmonella genus contains three species, S. bongori,
S. enterica (including six subspecies), and S. subterranea. The
genus is subdivided into more than 2500 serovars based on
antigenic features. Salmonella enterica and its serovars are most
commonly associated with human disease.
Features associated with isolates of the Salmonella genus
Salmonella cells are 2–3.4 mm long and 0.7–9.0 mm in diame-
ter, with rounded ends. Most have peritrichous flagella, though
S. subterranea has one lateral flagellum. Optimal growth occurs
within a pH range from 6.5 to 9.0 and across a temperature
range of 30–37  C. Salmonella species do not grow at pHs <3.5
and >9.5 nor at temperatures <10  C and >42  C. Salmonellae
are acid-tolerant and can grow at lower pH limits of 3.7–4.4.
Salmonella species can produce hydrogen sulfide from thiosul-
fate. The motility of Salmonella isolates is strain-dependent and
specific to the host environment.
Environments known to contain Salmonella
Salmonella species are isolated from aquatic and sedimentary
environments where they can multiply though these are
regarded as transitional environments between host infections.
Some strains such as S. subterranea are specifically adapted for
living in soil. Salmonellae possess the ability to survive in low-
moisture conditions and are associated with many food-borne
outbreaks especially in the ready to eat food market.
Relevance of Salmonella to public health
Salmonella species cause illnesses including typhoid fever, para-
typhoid fever, and food-borne illnesses. These bacteria are
zoonotic pathogens, being present in (food-producing) ani-
mals from which they can transfer to humans. S. enterica
subspecies enterica serovars Typhimurium and Enteritidis are
commonly reported members of this genus. These bacteria can
exhibit resistance to one or more antibiotics, compromising
treatment in some infected individuals.
Nontyphoidal salmonellosis is food-borne, and
consequently, the food chain is an important route of infection
for humans. Salmonella typhimurium is one of the serovars
most often associated with cases of food-borne infections.
Clinical signs associated with salmonellosis include diarrhea,
dehydration, fever, and abdominal pain that can last for up to
7 days. There is a greater risk of infection in the young, the
elderly, and immunocompromised individuals.
The genus Yersinia
Yersinia pestis (the ‘plague bacillus’) was first identified by
Alexandre Yersin in 1894 and classified as Pasteurella pestis
soon thereafter. Von Logham reclassified Pasteurella pestis and
Pasteurella rodentium into the new genus of Yersinia in 1944.
The name was accepted as an official genus in 1980. The genus
Yersinia contains 17 species, of which only 3 are known to be
548 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection
pathogenic to humans. These include Y. pestis, Y. enterocolitica,
and Y. pseudotuberculosis.
Features associated with isolates of the Yersinia genus
Yersinia species form gray-white translucent colonies after 24 h
of growth on blood agar plates. After 48 h on MacConkey agar,
they form clear colonies. Yersinia strains are nonmotile at
temperatures between 25 and 35  C (Table 1). Y. enterocolitica
and Y. pseudotuberculosis are psychotropic, being capable of
growth at temperatures of 4  C or even lower.
Environments known to contain Yersinia
Rodents are the natural reservoirs of Yersinia species, although
other mammals can occasionally serve as hosts. Yersinia pestis
can be transmitted to humans subcutaneously through flea
bites and can become airborne during pandemics. Due to the
ability of enteropathogenic Yersinia species to grow at low
temperatures, refrigerated foodstuffs can become contami-
nated with these organisms. Swine and wild animals are com-
mon reservoirs.
Relevance of Yersinia to public health
The pathogenic species of Yersinia have been linked with a wide
range of illnesses in humans including Crohn’s disease, yersi-
niosis, mesenteric lymphadenitis pseudoappendicitis, and sys-
temic infectious disease, commonly known as plague.
Y. pestis is the causative agent of the plague, which has been
responsible for three human pandemics throughout history.
These are the Justinian plagues, recorded from the sixth to
eighth centuries, the Black Death from the fourteenth to
nineteenth centuries, and the modern plague from the nine-
teenth century to the present time.
Y. enterocolitica is primarily a food-borne pathogen found in
some food-producing animals such as pigs and other mam-
mals. After ingestion of contaminated water or food, Y. enter-
ocolitica colonizes the intestine causing yersiniosis, an acute
gastrointestinal condition. Symptoms include fever, abdomi-
nal pain, vomiting, and diarrhea. Treatment usually consists of
an aggressive course of antimicrobial chemotherapy.
Y. pseudotuberculosis is the least common of the three Yersi-
nia pathogenic strains. It causes an illness characterized by
fever and acute abdominal pain arising from mesenteric
lymphadenitis, an inflammation of the lymph nodes.
The genus Cronobacter
Originally, members of this bacterial genus were described as
yellow-pigmented Enterobacter cloacae being subsequently
reclassified as Enterobacter sakazakii in 1980. A further and
more recent reclassification was reported in 2007 with the
acceptance of the new genus name, Cronobacter, based on a
polyphasic study of a large collection of these isolates.
Currently, the genus Cronobacter contains seven species,
namely, C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii,
C. dublinensis, C. condimenti, and C. universalis.
Features associated with isolates of the Cronobacter genus
Cronobacter are 3 mm long and 1 mm in diameter. Cronobacter
species produce two colony morphotypes, one being described
as glossy and the other having a matte texture. Some 80% of
strains identified produce a yellow pigment on tryptone soya
agar at 25  C. These strains can grow over a wide range of
temperatures from 5  C to 44–47  C depending on the iso-
late. Optimal growth occurs in pH ranging from five to ten
with some isolates being more susceptible to acid exposure.
Cronobacter have a notable tolerance to desiccation, being able
to withstand dry conditions for up to 2 years in infant formula.
Cronobacter strains are motile by means of peritrichous flagella.
Environments known to contain Cronobacter
Cronobacter has been isolated from a variety of food and envi-
ronmental sources such as dairy products, dried meats, pow-
dered infant formula (PIF), households, livestock facilities,
food manufacturers, and PIF production facilities.
Relevance of Cronobacter to public health
Cronobacter species are regarded as opportunistic pathogens
linked with life-threatening infections in neonates. Clinical
presentation can include necrotizing enterocolitis, bacteremia,
and meningitis. These organisms can also infect older adults,
although these cases tend not to be life-threatening.
The genus Enterobacter
The genus Enterobacter was accepted in 1960 with Enterobacter
cloacae being designated as the type species. With the applica-
tion of modern typing techniques, nine species are currently
recognized in this genus. These are E. cancerogenus, E. aerogenes,
E. mori, E. ludwigii, E. kobei, E. soli, E. asburiae, E. cloacae, and
E. hormaechei.
Features associated with isolates of the Enterobacter genus
Enterobacter species are motile by four to six peritrichous fla-
gella. Some are encapsulated (Table 1). Generally, these bacte-
ria are 0.6–1.0  2–3 mm in size. Enterobacter species produce
round, iridescent, flat, nonpigmented, irregular-edged colo-
nies, when grown on nutritive agar. The optimum growth
temperatures are between 30 and 37  C, though they can
grow in temperatures up to 44  C.
Environments known to contain Enterobacter
Enterobacter can be found on human skin, plants, soil, water,
sewage, intestinal tracts of animals, including humans, dairy
products; and clinical specimens such as feces, urine, blood,
sputum, and wound exudates.
Relevance of Enterobacter to public health
Enterobacter species are considered opportunistic pathogens,
rarely causing disease in healthy individuals. The species
E. cloacae and E. aerogenes are the main pathogens in the
genus. These organisms are associated with bacteremia, lower
respiratory tract infections, skin and soft tissue infections,
UTIs, endocarditis, osteomyelitis, arthritis, and ophthalmic
infections. Outbreaks involving E. cloacae have been reported
in the ICUs of hospitals.
E. cancerous is rarely associated with human infections and
is considered a plant pathogen.
The genus Citrobacter
The genus Citrobacter was first proposed in 1932. Citrobacter
contains 11 species: C. amalonaticus, C. farmeri, C. braakii,
 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection
C. freundii, C. gillenii, C. murliniae, C. sedlakii, C. werkmanii, C.
youngae, C. koseri, and C. rodentium.
Features associated with isolates of the Citrobacter genus
Citrobacter species are 1.0  2.0–6.0 mm in size. They are found
either singly or in pairs, are devoid of a capsule, and are motile.
C. freundii form small, circular, convex dark pink colonies on
MacConkey agar. Rough or mucoid forms have also been
reported. Citrobacter species grow optimally at a temperature
of 37  C.
Environments known to contain Citrobacter
Citrobacter are found in a variety of environmental sources,
including soil and water, and in the human intestines. They
are rarely the primary source of illness, though some strains
can cause infections of the urinary tract, sepsis, and infant
meningitis.
Relevance of Citrobacter to public health
Citrobacter species are not regarded as significant etiological
agents in human disease. C. freundii and C. koseri have mainly
been isolated from urinary and respiratory tract infections.
Citrobacter can cause septicemia in patients that display a num-
ber of predisposing factors. Citrobacter have also been found to
cause meningitis, septicemia, and pulmonary infections in
neonates and young children, and some of these cases have
been linked to contaminated batches of PIF. Citrobacter is
considered an opportunistic pathogen.
Detection of Members of the Enterobacteriaceae Family Using
Conventional and Molecular Methods
Conventional microbiology
Conventional microbiological-based techniques make use of
morphological and biochemical differences between genera
and bacterial isolates recovered from different sources. Selec-
tive and differential bacterial culture media for enrichment and
isolation of strains have been developed.
Examples of detection using bacterial morphology and reactions
on specific culture media
Macroscopic morphology can aid with the identification of
features visible to the naked eye. It describes colony appearance
in terms of its shape, texture, pigment, speed of growth when
incubated, and the growth pattern. Microscopic morphology
allows for an examination of individual bacterial cells for their
shape, size, Gram stain, and acid-fast reactions to aid identifi-
cation. The goal is to achieve an initial presumptive identifica-
tion of the bacterium on the culture plate.
The application of specially designed culture media is a
method commonly used to aid the identification of key path-
ogens of significance to public health. As an example, E. coli
O157 must be identified quickly due to its association with low
infectious doses. Its impact on human health makes it the
focus of numerous commercial diagnostic companies. Many
of these exploit the inability of VTEC O157:H7 strains to
ferment the carbon source contained in sorbitol MacConkey
agar. When grown on this media, VTEC O157:H7 produces a
colorless colony distinguishing it from other organisms on the
same plate. However, more recently, some E. coli O157 have
549
been identified with the ability to ferment sorbitol so other
means for confirmatory identification must be developed and
applied. Non-O157 strains may sometimes be differentiated
through the use of Chromocult and Rainbow agar on the basis
of their colony color. However, currently, there are no suitable
commercial agars available for this purpose.
Similarly, Salmonella species are cultured from food (after a
period of resuscitation) and from clinical samples directly onto
selective agar media, usually xylose–lysine–deoxycholate agar,
and incubated at 37  C for 24 h. Colonies can be preliminarily
classified based on their morphology.
Examples of diagnostic biochemical tests
Commercially available biochemical galleries exploit the
expression or otherwise of different phenotypic characteristics
to identify bacteria. Some of these tests exploit the biochemical
capacity of an isolate in terms of its ability to ferment a range of
sugars, susceptibility to various antibacterial agents including
drugs, and the capacity to metabolize complex polymers. In
addition, some strategies will allow for the assessment of the
motile nature of an isolate (see Table 1 in the preceding text).
Molecular-based diagnostic microbiology
Polymerase chain reaction
The polymerase chain reaction (PCR) is an in vitro version of
the natural DNA replication process that occurs within all
living cells. It can be applied to rapidly amplify a discrete
segment of genomic DNA. Characteristically, double-stranded
DNA is denatured, separating it into two discrete strands.
Primers, short oligonucleotide sequences of synthetic DNA,
hybridize to opposite DNA strands, and, following the addi-
tion of a thermostable DNA polymerase and the four dNTPs, a
round of synthesis is initiated, extending the DNA from the
synthetic primer on each strand. Up to 30 reiterated cycles of
denaturation, annealing, and extension are completed within a
typical PCR reaction, amplifying the original DNA target
exponentially.
In a more recent development of the conventional PCR
approach, quantitative real-time PCR (qPCR) emerged. This
strategy enables quantification of target-specific amplicons. In
this approach, a housekeeping gene is chosen to act as a nor-
malized comparator against which a variable DNA target is
measured, resulting in quantification of the latter. qPCR can
be applied for the quantification of individual genes and bac-
teria of importance to human health.
As an example, VTEC organisms can be detected by target-
ing their unique virulence genes, including stx and eae (as
described earlier). Real-time PCR assays have been developed
that allow the detection of the major VTEC-associated genes
enabling the differentiation of strains in real time. These targets
can be combined into multiplex assay platforms that can facil-
itate the detection of several different VTEC serogroups
simultaneously.
Various PCR-based methods for the detection of Cronobac-
ter strains exist. These include a conventional 1,6-a-
glucosidase-based PCR and a dnaG-based qPCR that can detect
all seven of the Cronobacter species. When used in combination
with a species level PCR assay, individual species of the genus
can be identified. This PCR method used the rpoB gene for
which specific primers have been designed for each species.
550 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) makes use of suitable
restriction enzymes to digest bacterial DNA at a limited num-
ber of sites within the genome, producing large or macro-DNA
fragments that can be separated based on their size. The careful
separation of these DNA fragments is achieved through the
application of a periodical alternating electric field to a gel
matrix. Comparison of these restriction profiles can indicate
whether or not isolates are epidemiologically linked. PFGE
has been used to aid with the tracking of many food-borne
outbreaks and is regarded as the gold standard in molecular
subtyping. PFGE has been standardized between different labo-
ratories allowing for the comparison of PFGE profiles. PulseNet
is an initiative that was originally developed at the CDC that
facilitates the global tracking of food-borne pathogens.
Multilocus sequence typing
Multilocus sequence typing is a genome-based protocol used
for the characterization of bacterial species by sequencing
internal regions of typically seven housekeeping genes. To
enable accurate sequencing, fragments of between 450 and
500 base pairs are used. Bacterial species display sufficient
polymorphism within the chosen genes to assign an identifier
to the alleles detected. By combining the sequences of (usually
up to) seven loci, a species-specific allelic profile can be gener-
ated for each bacterial isolate.
Optical mapping
Optical mapping is a recently developed technique that gener-
ates high-resolution, ordered, whole-genome restriction maps
from single DNA molecules independently of available
sequence information. Purified DNA is isolated from a bacte-
rium of interest and digested with a suitable restriction endo-
nuclease. The DNA is then stained, scanned, accurately
measured, and subsequently assembled into a complete
genome restriction map. These maps can be compared to
discover genetic differences, such as insertions or deletions
between strains. Optical mapping can be used to distinguish
between different species and isolates.
Optical mapping was applied to distinct E. coli O104:H4
German outbreak isolates in order to characterize differences.
In this case, regions of differences contributing to the pathoge-
nicity of this unusual strain were identified.
Whole-genome sequencing
Genome sequencing involves the decoding of a bacterium’s
complete genetic code, more specifically the determination of
nucleotide order, within the chromosome. The technology
underpinning this approach has evolved following a number
of advancements over the years, and it is the most powerful
analytic approach to molecular determination.
Next-generation sequencing
Next-generation sequencing (NGS) or high-throughput sequen-
cing describes a number of sequencing technologies in common
use. These approaches generate large volumes of sequence data
at relatively low cost, thereby facilitating the rapid sequencing
of DNA and RNA. This technology is significantly quicker and
cheaper than the traditional Sanger-based technology.
A number of NGS technologies now exist for this purpose,
such as
• Illumina (Solexa) sequencing,
• Roche 454 sequencing,
• Pacific Biosciences single-molecule real-time sequencing,
• SOLiD sequencing,
• Life Technologies Ion Torrent sequencing.
Despite the differences in the chemistries used, all the methods
in the preceding text share some common features such as the
random fragmentation of the template DNA, amplification of
the target to be sequenced on a solid surface such as a bead or
glass slide, and direct step-by-step detection of each nucleotide,
following incorporation.
Sequencing technologies provide for the determination of
the base order from thousands of small fragments that are
subsequently assembled to generate a consensus genome
sequence. Once assembled, this draft sequence must then be
checked to confirm that correct assembly has taken place,
followed by careful gene annotation and importantly the dis-
covery of features of interest, including the identification of
resistance and virulence genes along with other phenotypic
data on a molecular basis.
NGS is a high-resolution and therefore accurate and reliable
means of bacterial characterization.
In the 2011 German outbreak mentioned earlier, NGS tech-
nology facilitated the whole-genome characterization at an
early stage of this incident. An E. coli O104:H4 outbreak isolate
and a historic E. coli O104:H4 HUS isolate from 2001 were
characterized using Ion Torrent’s NGS in combination with
optical mapping. Within 62 h, draft genomes were available
enabling the genes to be examined. It was found that the
strains carried genes associated with both EAEC and EHEC.
Hence, it was proposed that the outbreak strain was a highly
pathogenic hybrid of EAEC and EHEC strains.
See also: Emerging Foodborne Enteric Bacterial Pathogens;
Escherichia coli and Other Enterobacteriaceae: Food Poisoning and
Health Effects; Salmonella: Detection; Salmonella: Properties and
Occurrence; Salmonella: Salmonellosis; Shigella.
Further Reading
Bolton DJ, O’Sullivan J, and Duffy G, et al. (eds.) (2007) Methods for detection and
molecular characterisation of pathogenic Escherichia coli. Ireland: Pathogenic
Escherichia coli Network. http://www.antimicrobialresistance.dk/data/images/
protocols/e%20coli%20methods.pdf.
EFSA Panel and on Biological Hazards (BIOHAZ) (2013) Scientific opinion on VTEC-
seropathotype and scientific criteria regarding pathogenicity assessment. EFSA
Journal 11(4).
EFSA Panel and on Biological Hazards (BIOHAZ) (2014) Scientific opinion on the
evaluation of molecular typing methods for major food-borne microbiological
hazards and their use for attribution modelling, outbreak investigation and scanning
surveillance: part 2 (surveillance and data management activities). EFSA Journal
12(7).
Ekiri AB, Landblom D, Doetkott D, Olet S, Shelver WL, and Khaitsa ML (2014) Isolation
and characterization of shiga toxin-producing escherichia coli serogroups O26,
O45, O103, O111, O113, O121, O145, and O157 shed from range and feedlot cattle
from postweaning to slaughter. Journal of Food Protection 77(7): 1052–1061.
Fricke WF and Rasko D (2014) Bacterial genome sequencing in the clinic: bioinformatic
challenges and solutions. Nature Reviews Genetics 15(1): 49–55.
 Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection
Kaper JB, Nataro JP, and Mobley HLT (2004) Pathogenic Escherichia coli. Nature
Reviews Microbiology 2: 123–140.
Latreille P, Norton S, Goldman BS, Henkhaus J, Miller N, Barbazuk B, Bode HB,
Darby C, Du Z, Forst S, Gaudriault S, Goodner B, Goodrich-Blair H, and Slater S
(2007) Optical mapping as a routine tool for bacterial genome sequence finishing.
BMC Genomics 8: 321.
Leekitcharoenphon P, Nielsen EM, Kaas RS, Lund O, and Aarestrup FM (2014)
Evaluation of whole genome sequencing for outbreak detection of Salmonella
enterica. PloS One 9(2), p.e87991.
Mellmann A, Harmsen D, Cummings C, et al. (2011) Prospective genomic
characterization of the German enterohemorrhagic Escherichia coli O104:H4
outbreak by rapid next generation sequencing technology. PloS One 6(7) p.e22751.
Metzker ML (2010) Sequencing technologies – the next generation. Nature Reviews
Genetics 11(1): 31–46.
551
Pennington TH (2014) E. coli O157 outbreaks in the United Kingdom: past, present, and
future. Infection and Drug Resistance 7: 211–222.
Quinn PJ and Markey BK (2003a) Enterobacteriaceae 1. In: Concise review of veterinary
microbiology, pp. 38–42. Oxford: Blackwell Publishing.
Quinn PJ and Markey BK (2003b) Concise review of veterinary microbiology. Oxford:
Blackwell Publishing.
Robinson ER, Walker TM, and Pallen MJ (2013) Genomics and outbreak investigation:
from sequence to consequence. Genome Medicine 5(4): 36.
Relevant Websites
http://www.cdc.gov/ecoli/ – CDC Escherichia coli.
www.cdc.gov/pulsenet – CDC PulseNet.
 Essential Oils: Isolation, Production and Uses
CM Cook, Hellenic Agricultural Organization-Demeter, Thermi, Greece
T Lanaras, Aristotle University of Thessaloniki, Thessaloniki, Greece
ã 2016 Elsevier Ltd. All rights reserved.
Background
Essential oils are volatile, aromatic components of herbs and
spices and have been used since ancient times in flavoring,
food preservation, medicine, and perfumery. Some well-
known aromatic and spice plants from which essential oils
are produced commercially are shown in Table 1. The plant
families Lamiaceae and Apiaceae are particularly rich in aro-
matic plants. The essential oil can be found in the flowers,
leaves, roots, rhizomes, fruits, seeds, wood, and resins of
these plants. On the surface of leaves and calyces, the essential
oil is located in specialized cells called glandular trichomes.
Essential oils are synthesized by plants, as secondary metab-
olites, and are bioactive compounds with biological properties.
They are considered to play a role in plant protection against
herbivores making them unpalatable; in the attraction of
pollinators; in plant protection against fungal, viral, and bac-
terial infections; in communication and signaling between
other plants; and in allopathy by reducing the competition of
neighboring plant species, among others. The essential oil
content of plant material can vary with season, bioclimatic
zone, growing conditions, and stress.
Essential oils can easily be isolated from plant material by
hydrodistillation and are liquids at room temperature. They are
a complex mixture of terpenes and terpenoids, which make up
the characteristic aroma of each plant, and it is the composite
effect of all the constituents present in it. They can be present
from a mere trace up to around 95% of the essential oil. Most
often, main components are precursors, intermediates, and
end products of a particular biosynthetic pathway, and their
relative proportions may depend on the time of year or the
developmental stage of the plant. Terpenes are chemical com-
pounds of low molecular weight, which are made up of iso-
prenoid units consisting of 5 carbon atoms, and these can join
together to form units of C10 mono-, C15 sesqui-, C20
diterpenes. Oxygenated terpenes or terpenoids can be alde-
hydes, alcohols, ketones, acids, esters, ethers, and others.
Natural biodiversity is reflected by plants and their essential
oils. It is possible to have several different species producing an
essential oil with the same composition and major component
(same biosynthetic pathway), for example, carvacrol in ‘oreg-
ano’ plants: Origanum vulgare subsp. hirtum (‘Greek’), O. onites
(‘Turkish’), and Thymbra capitata (‘Spanish’ oregano). On the
other hand plants of the same species, for example, Mentha
spicata can have completely different aromas: ‘peppermint’
(menthol), ‘spearmint’ (carvone), ‘wild mint’ (piperitone and
piperitenone oxide), and ‘lavender’ (linalool and linalyl ace-
tate). These are known as chemotypes, plants of the same species
having terpenoids arising from different biosynthetic pathways.
A return to the use of natural products in everyday life has
stimulated interest in different aspects of essential oils and
prospective uses in medicine, cosmetics, and the food industry.
This interest is reflected in the marked increase in research
552 Encyclopedia of Food and Health
publications on essential oils over the last twenty years
(Figure 1) focused primarily on not only essential oil compo-
sition and biological properties but also isolation methodolo-
gies, species variation, chemotaxonomy, and others.
Isolation
The volatile nature, general insolubility in water, and solubility
in organic solvents are properties of essential oils that can be
exploited when designing isolation methods. The nature of the
plant material and the lability of essential oil components
should also be considered. Traditional and commercially most
commonly used methods are distillation using water and/or
steam, cohobation, maceration, enfleurage, and cold pressing.
However, new technologies are continually being developed in
order to decrease extraction times and energy costs.
Traditional Technology
Hydrosteam distillation
Distillation using water and/or steam is the most widely used
and cost-effective method for the production of the majority of
essential oils worldwide. It involves the application of heat and
moisture to vaporize and release the essential oils from the plant
material followed by the cooling of the vapor mixture and the
separation of the oil from the water. Although the boiling point
of most essential oil components generally ranges from 150 to
300
C, they can be evaporated with steam/boiling water at
100
C, as the combined vapor pressures of two immiscible
liquids will be equal to atmospheric pressure at a lower temper-
ature than that at which the essential oil will vaporize.
Hydrodistillation is the most simple of processes and has
been used by man for centuries for the extraction of essential
oils and is used mostly by small-scale producers. The plant
material is almost completely covered by water in the chamber.
The water is boiled using an external heat source, and the
essential oils together with the steam are condensed and sepa-
rated. Essential oil content of aromatic plants is determined
using a Clevenger-type distillation apparatus, according to the
European Pharmacopoeia. Disadvantages of hydrodistillation
are that the process is slow and requires energy for heating,
distillation rates may vary if the heat source is not controlled,
and the direct heat source may cause charring of plant material
in the base of the chamber. Prolonged contact of essential oils
with the hot water can cause the hydrolysis of some essential
oil constituents, for example, esters can hydrolyze to acids and
alcohols. However, advantages of hydrodistillation are high
yields of essential oil and good recovery of constituents. It is
simpler and faster than steam distillation.
In steam and water distillation, direct contact with the boiling
water is avoided by supporting the plant material on a grid.
Advantages are that the process is faster and requires less fuel
http://dx.doi.org/10.1016/B978-0-12-384947-2.00261-0
 Essential Oils: Isolation, Production and Uses 553

Table 1 Some well-known aromatic plants and spices used
commercially for their essential oil
Plant family Common (and scientific name)
Anacardiaceae Mastic (Pistacia lentiscus var. chia)
Annonaceae Ylang-ylang (Cananga odorata)
Apiaceae Dill (Anethum graveolens), parsley (Petroselinum
crispum), anise (Pimpinella anisum), coriander
(Coriandrum sativum), cumin (Cuminum
cyminum), caraway (Carum carvi)
Asteraceae Chamomile (Chamaemelum nobile and Matricaria
chamomilla), tarragon (Artemisia dracunculus),
yarrow (Achillea millefolium)
Burseraceae Frankincense (Boswellia sp.)
Caprifoliaceae Valerian (Valeriana officinalis)
Lamiaceae ‘Greek’ oregano (Origanum vulgare subsp. hirtum),
peppermint (Mentha x piperita), spearmint (Mentha
spicata), sage (Salvia officinalis), lavender
(Lavandula sp.), rosemary (Rosmarinus officinalis),
basil (Ocimum basilicum), marjoram (Origanum
majorana), lemon balm (Melissa officinalis),
pennyroyal (Mentha pulegium), thyme (Thymus
sp.), patchouli (Pogostemon cablin)
Lauraceae Bay (Laurus nobilis), cinnamon (Cinnamomum sp.)
Myristicaceae Nutmeg (Myristica fragrans)
Myrtaceae Clove (Syzygium aromaticum), eucalyptus
(Eucalyptus sp.), wintergreen (Gaultheria sp.)
Oleaceae Jasmine (Jasminum sp.)
Piperaceae Black pepper (Piper nigrum)
Poaceae Citronella grass (Cymbopogon nardus, C.
winterianus)
Rosaceae Damask rose (Rosa x damescena and R. centifolia)
Rutaceae Lemon (Citrus limon), bergamot orange (C.
bergamia)
Santalaceae Sandalwood (Santalum sp.)
Schisandraceae Star anise (Illicium verum)
Zingiberaceae Cardamom (Amomum sp. and Elettaria sp.), ginger
(Zingiber officinale), turmeric (Curcuma longa)
than hydrodistillation while giving high reproducible yields. The
method is used in developing countries as portable field distilla-
tion units, combining simple construction, ease of operation,
and low cost with capacities of 100–2000 kg plant material.
In direct steam distillation, steam is generated in a boiler
(external source) that is then passed through the plant
material, which is supported on a grid in the chamber. Steam
at atmospheric pressure has a temperature of 100
C; however,
the use of high-pressure steam correspondingly increases the
temperature enabling a more rapid and complete distillation of
essential oil. The advantages are reduced fuel costs due to
higher thermal efficiency and suitability for large-scale produc-
tion (1–3 ton plant material). However, setup costs are quite
high and stills should be of stainless steel.
Distillation with cohobation: most essential oils have very
low solubility in water; however, the solubility of rose oil, for
example, is quite high. To avoid the loss in the condensing
water, this water is returned to the still when using water and
steam distillation for redistillation.
Solvent extraction
In the perfume industry, essential oil production is carried out
using volatile solvents such as petroleum ether and hexane at
50
C. This temperature is lower than that of distillation reduc-
ing the possibility of degradation of thermally labile
compounds, and thus, essential oils are considered to have a
more natural odor. After filtration, the essential oil is recovered
by reducing the volume of solvent under vacuum evaporation;
however, this can lead to severe losses of essential oil volatile
components. Solvent extraction is advantageous when plant
material is very limited (e.g., single leaf analysis) or where the
major essential oil constituents are of interest rather than the
essential oil content. Disadvantages are that nonvolatile com-
pounds may be coextracted and that toxic solvent residues
remain in the extract and can then contaminate food and
fragrances. The process is also more expensive than distillation
and creates waste disposal problems for the used solvents.

Enfleurage
Enfleurage is the extraction of essential oils using cold fat and
has been used traditionally in the production of perfumes from
fresh flower petals with low essential oil content, for example,
3000
jasmine and violets. The process has limited application on a
large scale as it is very labor-intensive and has been superseded
Number of publications

by modern processes such as extraction with volatile solvents.

Dry flower petals are placed on glass plates coated with a thin
2000 layer of a high-quality odorless fat. Emitted essential oils are
absorbed by the fat, and after extraction (in the order of 24 h or
more hours), the petals are removed (defleurage) and replaced
by fresh petals. This is an accumulative process that is repeated
1000
until the end of the flowering season. The essential oil that is
absorbed in the fat is subsequently extracted with alcohol.

Cold pressing (expression)

2000 2005 2010 This mechanical method is almost exclusively used in the
Year production of citrus fruit (e.g., lemon, orange, and bergamot)
Figure 1 Trends in essential oil publications: 1995–2014. Web of essential oils. Pressure is used to crush the peel (pericarp) by
Science (all databases), Thomson Reuters (2015) Thomson Reuters Web rolling the fruit against sharp objects. Essential oil glands
of Science. Available online: http://apps.webofknowledge.com (2015) located in pits on the pericarp are punctured, releasing the
(Accessed 30 January 2015). essential oil, which is washed away with water. The essential
554 Essential Oils: Isolation, Production and Uses

oil and water are separated by centrifugation. The process is
carried out at low temperature so there is no thermal degrada-
tion of the essential oil components. Essential oil can be
extracted before or after the extraction of the juice. Tradition-
ally, expression was carried out by hand.
New ‘Green’ Technology
In recent years, research has centered on designing more effi-
cient extraction procedures, which give higher yields (process
intensification) with lower energy consumption and lower
ambient operational temperatures. Conventional steam distil-
lation and solvent extraction procedures although being gen-
erally simple have disadvantages of requiring long extraction
times and large volumes of solvents. Industries are moving
towards the application of greener nonconventional technolo-
gies with an emphasis on safer, sustainable, environmentally
friendly, and more economical protocols. These non-
conventional technologies include supercritical fluid extrac-
tion (SFE), solid-phase microextraction (SPME), ultrasound-
assisted extraction (UAE), and microwave-assisted extraction
(MAE), which are increasingly finding applications at a pilot
and industrial level. It is interesting to see whether the trends in
essential oil publications with respect to methods of isolation
(MAE, solvent extraction, SPME, SFE, and hydrodistillation)
for the period 1995–2014 (Figure 2) follow the trends in the
development of new nonconventional technologies. Hydro-
distillation is by far the primary isolation method reported in
publications that would appear to be a result of the simplicity
and low cost of the method. However, rising trends are also
observed for SFE and SPME as might be expected.
Supercritical fluid extraction (SFE)
Carbon dioxide is a supercritical fluid when it is subjected to
temperatures and pressures above its critical temperature
(304.25 K) and pressure (7.39 MPa). It then expands like a
gas but has the density of a liquid. Supercritical CO2 is an
important industrial solvent, which is odorless, colorless, non-
flammable, and of low toxicity and environmental impact,
compared to other organic solvents used in essential oil extrac-
tion, such as hexane and acetone, which are toxic and flamma-
ble. Other advantages of supercritical CO2 extraction are
relatively low temperature, shorter extraction times, selectivity,
solvating power that varies with temperature and pressure
enabling fractional extraction, absence of toxic residues that
remain in extracts, absence of oxidation of essential oil com-
pounds, ease of isolation of analytes from the supercritical CO2
by pressure reduction (evaporation), and CO2 recycling by
condensation.
For essential oil extraction by SFE fractionation, supercriti-
cal CO2 at temperatures of 40–50
C and a pressure of about
11 MPa is passed into a heated extraction column packed with
plant material. Usually, the plant material is incubated for a
period of time (static mode) followed by a flow of new fluid
(dynamic mode). The supercritical CO2 with dissolved com-
pounds is subjected to pressure reduction to remove the CO2.
Isolated essential oils may contain small amounts of other
coextracted compounds such as cuticular waxes.
Solid-phase microextraction (SPME)
SPME is a rapid, solvent-free sample extraction technique. The
SPME apparatus consists of a fiber holder and assembly con-
taining a 1–2 cm thin, fused-silica retractable fiber coated with
a thin polymeric coating such as polydimethylsiloxane (sta-
tionary phase). The sample is placed in a vial sealed with a
septum. The fiber is extended through the needle into the
headspace above the sample. The volatile analytes establish
an equilibrium between the sample matrix, the gas phase
above the sample, and the polymeric coating on the fused-
silica fiber to which the analytes adsorb. After a given extrac-
tion time, the fiber is withdrawn and can be inserted directly
into the injection port of a capillary GC column where the
volatile analytes are rapidly, thermally desorbed. As no solvent
is injected, there is no solvent front to mask component peaks

microwave extraction
solvent extraction
solid phase microextraction
supercritical fluid extraction
1500 hydrodistillation
Number of publications

1000

500

0
1995–1999 2000–2004 2005–2009 2010–2014
Year
Figure 2 Trends in essential oil publications with respect to methods of isolation (microwave-assisted extraction, solvent extraction, solid-phase
microextraction, supercritical fluid extraction, and hydrodistillation) for 5 year periods from 1995 to 2014. Web of Science (all databases), Thomson
Reuters (2015) Thomson Reuters Web of Science. Available online: http://apps.webofknowledge.com (2015) (Accessed 30 January 2015).

eluting early from the column improving detection limits and
resolution. SPME is used in the analysis of flavor and fragrance
components in foods and beverages and is easily automated. It
can be applied to very small samples and also to living systems.
Ultrasound-assisted extraction (UAE)
Ultrasound (US) has been used traditionally in biochemistry as
a procedure for the production of plant extracts, as it causes the
rupture of cell membranes and the release cell contents into the
medium. The main factors that cause cell breakage are the
water jets and shock waves caused by the collapse of cavitation
bubbles generated by US. Part of the US pressure wave is
dissipated as heat. The application of US to essential oil isola-
tion enhances the homogenization of the plant material and
thereby decreases the extraction time and the total energy
consumed in the process. Analytes are more concentrated in
the extract. The process is clean with no residues left in the
extract. Combined with other extraction procedures, it can
improve yield, selectivity, and product quality.
Recently, it has been shown that UAE using large-scale
multiple transducer flow reactors operating at high power
density (700 W l1) promotes process intensification in the
isolation of essential oils as compared to classical maceration
in hydroalcoholic extracts. US has also been used to improve
hydrodistillation extraction (sono-Clevenger) by greatly reduc-
ing extraction time without reducing yield or essential oil
quality. Combined US and hydrodistillation extractions have
been implemented on an industrial scale.
Microwave-assisted solvent extraction (MAE)
MAE is an extraction technique that combines microwave radi-
ation technology with traditional solvent extraction. Micro-
wave radiation is used to heat the solvent and plant material.
Advantages of MAE are shorter extraction times (minutes rather
than many hours), the use of less solvent, higher extraction
yield, higher-quality products with lower cost, and lower
energy consumption and CO2 emissions when compared to
Soxhlet or solvent extraction with stirring. Apparatus is simpler
and less expensive than other methods such as SFE. Under
optimized conditions (solvent volume, power, and heating
time), the quality of essential oil and yield obtained are similar
to those obtained by hydrodistillation.
Solvent-free microwave extraction (SFME)
Solvent-free microwave extraction (SFME) combines micro-
wave heating and ‘dry’ distillation (no added solvent or
water) under conditions of atmospheric pressure and 100
C.
Isolation and concentration of volatile compounds are per-
formed in a single step. The internal heating of water within
the plant material swells the plant cells and leads to rupture of
the essential oil glands. Essential oil is evaporated by the in situ
water of the plant material, and the distillate is passed through
a cooling system outside the microwave. Excess water is
returned to the extraction vessel to restore the in situ water of
the plant tissue. SFME significantly reduces the time, energy,
and plant material required for extraction. The reduction in
extraction time and small amounts of water utilized decrease
the likelihood of the hydrolysis, esterification, and/or oxida-
tion of essential oil compounds.
Essential Oils: Isolation, Production and Uses 555
Production
Production technology should be optimized and standardized
in order to improve the overall yield and quality of the essential
oil product. Raw plant materials, extraction yield and extraction
time, and quality (composition, color, and texture) are impor-
tant factors for industrial production, which looks for efficient,
fast, and economic processes. Subsequently, storage conditions
and containers should be optimized for maximum shelf life.
The quality of the raw plant material and an adequate
supply for industry can be guaranteed by sustainable agricul-
tural practices. Indiscriminate collection from the wild can lead
to the reduction of wild resources, loss of biodiversity, and
plant material of inconsistent quality. Cultivation of selected
plants with high essential oil and biomass yields and of desired
chemical composition (quality) is preferable. Harvesting
times and postharvest drying and processing conditions must
also be optimized and standardized so as to minimize any
deterioration in quality and yield (appearance and loss of
essential oil).
Extraction procedures should be carried out in stills and
vats made of noncorrosive materials (e.g., stainless steel and
glass) of optimal design and under optimal standardized con-
ditions (temperature and time of extraction) for reproducible
results. Plant materials such as seeds, roots, and wood may
need to be crushed, ground, or soaked prior to extraction.
Distilled essential oils with a high moisture content must be
clarified using desiccants or on an industrial scale by high
speed centrifugation. Moisture, oxygen, high temperature,
and light adversely affect the storage life of the essential oil.
Essential oil composition (quality) can be analyzed using
gas chromatography (separation of compounds) coupled with
detection methods such as mass spectrometry (MS). For exam-
ple, the gas chromatographic analysis of oregano (Origanum
vulgare subsp. hirtum) essential oil (Figure 3) indicates many
component peaks and the presence of four main components
p-cymene (5), g-terpinene (6), thymol (9), and the major
component carvacrol (10). Some characteristics of these
components are given in Table 2.
The standardization and quality control of essential oils
ensure product quality and safety in worldwide markets. Stan-
dards are important and aid in the detection of adulterated and
synthetic essential oils (addition of synthetic compounds and
mixing with cheaper lower-quality oils, or solvents, to increase
bulk). Standardization is an important reference for industry
providing the properties of the natural components of products.
The International Organization for Standardization technical
committee ISO/TC 54, Essential oils, has developed 1. specific
monographs for quality standardization of all essential oils; 2.
standardized analytic methods to control the quality of
international standards of analytical methods and specifications;
3. requirements for transport, labeling, and marking; and 4.
nomenclature and botanical names, with the aim of expanding
global trade in essential oils and ensuring their quality and safety.
ISO/TC 54 aims to 1. facilitate the global trade in essential oils, 2.
enhance the quality of essential oils on the market, 3. protect the
health of essential oil consumers, and 4. enhance the safety of
essential oils. Many essential oils (or components) demonstrate
toxicity and/or are hazardous in pregnancy and for persons with
asthma or epilepsy and thus should be treated as medicines. The
556 Essential Oils: Isolation, Production and Uses

TRACE GC-Channel 1

10
Pk #
3000 OH 3000

2000 2000
OH
mAU

mAU
1000 1000

9
6
4 5

11

12
3
1
2

8
0 0

0 10 20 30 40 50 60 70
Time (min)

Figure 3 Gas chromatograph of Origanum vulgare subsp. hirtum (oregano) essential oil. Experimental details: the oil was analyzed by GC-FID using
a ThermoQuest Trace GC with an HP-5MS capillary column (5% diphenyl-, 95% dimethyl- polysiloxane, non-polar, 30m  0.25 mm ID  0.25 mm film
thickness) coupled with a flame ionization detector. Carrier gas: helium. Flow rate: 1.0 ml min1. Column temperature was 60
C for 5 min, increased
2
C min1 until 150
C and then increased 6
C min1 until 280
C. The injector and detector temperatures were 240
C. Kovats indexes were calculated
by comparison of the relative retention times of the separated essential oil components with those of an homologous series of C8-C26 n-alkanes standards.

Table 2 Some characteristics of the four main compounds of Food Industry

oregano essential oil analyzed by gas chromatography (see Figure 3)
About 60 % of essential oil production is used in the food
Chemical industry primarily for flavoring and seasoning of food prod-
Peak Compound Kovats Molecular Formula abstract service ucts such as cured meats, alcoholic and soft drinks, ice cream,
number name index formula weight number and confectionary, among others. Essential oils also represent a
source of natural antimicrobials for food preservation. Carva-
5 p-Cymene 1024 C10H14 134 99-87-6
crol, a major component of oregano essential oil, has many
6 g-Terpinene 1059 C10H16 136 99-85-4
beneficial bioactivities in the preservation of food, which
9 Thymol 1290 C10H14O 150 89-83-8
10 Carvacrol 1299 C10H14O 150 499-75-2 include antioxidative properties in food (fats and oils); the
inhibition of foodborne human pathogenic bacteria, fungi,
Compiled from data given in Adams, R. P. (2007). Identification of essential oil and parasites in human foods (eggs, meat and poultry prod-
components by gas chromatography/mass spectrometry (4th ed). Allured Publishing ucts, milk, and leafy vegetables) and animal feed; and also the
Corporation: Carol Stream, IL inhibition of microbial and fungal toxin production. The many
health promoting effects of carvacrol create a potential for its
use as a multifunctional food in pure and encapsulated form
committee has also participated in the revision of and new and in edible films.
European Pharmacopoeia monographs for essential oils.
Pharmaceutical Industry
Essential oils are generally not recommended for internal use
Uses undiluted due to their toxicity, nor for direct application to the
skin, with the risk of dermatitis, allergic reaction, or skin photo-
Essential oils contain many bioactive compounds (e.g., terpe- sensitization. However, topical application of some essential oils
noids), which, apart from their fragrance and flavor, also show such as lavender and oregano when diluted in plant oils aids in
a wide range of biological activities and properties. For exam- the healing of wounds and burns. Essential oils are used as anal-
ple, some of these properties are antibacterial, antifungal, gesics, expectorants, decongestants (menthol and eucalyptus),
antioxidant, insecticidal, insect repellent, acaricidal, larvicidal, antiseptics, flavors to neutralize unpleasant tastes, and others.
anthelmintic, anti-inflammatory, cytotoxic, antibiotic, anticar- Recently, some essential oil constituents such as geraniol have
cinogenic, analgesic, and local anesthetic. As such, these shown potential antitumor effects, inducing growth inhibition
natural products from more than 300 essential oils are widely and apoptosis in cancer cells. Mice fed with diets supplemented
used in the food and beverage industry; in the pharmaceutical with geraniol (25–75 mmol kg1) showed a dose- and time-
industry and medicine; in perfumery, cosmetics, and health- dependent growth inhibition of tumor growth in vivo together
care products (toiletries); and in veterinary science and with an induction of apoptosis. It also reduced cholesterogenesis.
alternative medicine (aromatherapy). As the uses are many, it The doses of geraniol were nontoxic to the mice; the mevalonate
is useful to examine them on a general level and then focus on biosynthetic pathway was inhibited. As such, geraniol could be a
some recent advances relevant to food and health. potential candidate for cancer chemotherapy.

Animal Feed Supplements and Animal Health
Essential oils have been evaluated for their effects on growth
performance and meat quality of animals raised for meat pro-
duction (e.g., poultry, pigs, and rabbits) as a safe and natural
feed supplement alternative to the overuse of nontherapeutic
antibiotics (growth promoters) and the subsequent develop-
ment of antibiotic resistance in bacterial pathogens of humans
and animals. For example, the supplementation of quail (Cotur-
nix coturnix) diets with juniper oil (100 and 150 mg kg1)
caused a significant increase in live weight, live weight gain,
and carcass yields. Further, the dietary juniper oil reduced thio-
barbituric acid levels in raw thigh meat samples in storage and
exhibited antioxidant activity, preventing lipid oxidation in
stored meat. Generally, essential oils as animal feed supple-
ments can protect against the colonization of pathogenic micro-
organisms in the gut, reduce the fermentation process and the
production of toxic metabolites, and have a positive effect on
gut microbiota and animal welfare.
Many essential oil constituents (e.g., trans-cinnamaldehyde,
thymol, carvacrol, citronellal, neral, linalool, and limonene) are
important insect repellents and are effective against mosquitoes,
fleas, ticks, flies, and lice, which can transmit infective diseases
such as malaria.
Acknowledgment
The authors thank Evaggelia Samara for the technical assis-
tance and acknowledge the financial support of the National
Strategic Reference Framework (NSRF) Greece, Research Fund-
ing Program, Action ARISTEIA II (NATURAL AROMA-4204),
for the essential oil analysis.
Essential Oils: Isolation, Production and Uses 557
See also: Essential Oils: Properties, Composition and Health Effects;
Flavor Enhancers: Characteristics and Uses; Spices and Flavoring
Crops: Fruits and Seeds.
Further Reading
Alexandru L, Cravotto G, Giordana L, Binello A, and Chemat F (2013) Ultrasound-
assisted extraction of clove buds using batch- and flow-reactors: a comparative
study on a pilot scale. Innovative Food Science and Emerging Technologies
20: 167–172.
Bordas A and Bermejo E (2012) Essential oils. A fresh look at the oldest known remedy
and beauty booster. ISO Focusþ July-August: 12–13.
Capuzzo A, Massimo ME, and Occhipinti A (2013) Supercritical fluid extraction of plant
flavors and fragrances. Molecules 18: 7194–7238.
Chen H-C, Chi H-S, and Lin L-Y (2013) Headspace solid-phase microextraction
analysis of volatile components in Narcissus tazetta var. chinensis Roem. Molecules
18: 13723–13734.
Delazar A, Nahar L, Hamedeyazdan S, and Sarker SD (2012) Microwave-assisted
extraction in natural products isolation. Methods in Molecular Biology 864: 89–115.
Engelberth J (2010) Secondary metabolites and plant defense. In: Taiz L and Zeiger E
(eds.) Plant physiology, 5th ed., pp. 369–400. Massachusetts, USA: Sinauer
Associates Inc.
Filly A, Fernandez X, Minuti M, et al. (2014) Solvent-free microwave extraction of
essential oil from aromatic herbs: From laboratory to pilot and industrial scale. Food
Chemistry 150: 193–198.
Friedman M (2014) Chemistry and multibeneficial bioactivities of carvacrol
(4-isopropyl-2-methylphenol), a component of essential oils produced by aromatic
plants and spices. Journal of Agricultural and Food Chemistry 62: 7652–7670.
Galle M, Crespo R, Kladniew BR, et al. (2014) Suppression by geraniol of the growth of
A549 human lung adenocarcinoma cells and inhibition of the mevalonate pathway
in culture and in vivo: Potential use in cancer therapy. Nutrition and Cancer-An
International Journal 66: 888–895.
Hyldgaard M, Mygind T, and Meyer RK (2012) Essential oils in food preservation: Mode
of action, synergies, and interactions with food matrix components. Frontiers in
Microbiology 3: 1–24.
Yesilbag D, Cengiz SS, Cetin I, Meral Y, and Biricik H (2014) Influence of Juniper
(Juniperus communis) oil on growth performance and meat quality as a natural
antioxidant in quail diets. British Poultry Science 55: 495–500.
 Essential Oils: Properties, Composition and Health Effects
G Buchbauer and IM Wallner, University of Vienna, Vienna, Austria
ã 2016 Elsevier Ltd. All rights reserved.
Sources and Production
Essential oils are a group of plant secondary metabolites and
are obtained only by steam distillation (according to ISO rule
9235) from different plant organs especially from aromatic
plants. Those volatile compounds vary in odor and flavor.
The manner of this distillation method is important because
it determines the quality of the essential oil. Inappropriate
production can result in the loss of bioactivity. Essential oils
are concentrated liquids of complex mixtures and contain a lot
of bioactive compounds.
Patterns of Consumption
Essential oils are lipophilic compounds, which are able to cross
membranes very easily. Hence, essential oils are well absorbed
not only from the intestine but also through the skin and
the lungs. Because of their transdermal and pulmonary
absorption, they are used for many medical applications in
ointments, balms, and bath additives.
Availability, Absorption, and Metabolism
A high amount of essential oils is rapidly absorbed after dermal,
oral, or pulmonary administration and crosses the blood–brain
barrier. They are able to interact with the receptors in the central
nervous system (CNS) and to affect biological functions such as
relaxation, sleep, and digestion. Essential oil constituents can
be metabolized by the liver in the form of polar compounds
following limited phase I enzyme metabolism by conjunction
with glucuronate or sulfate. Furthermore, they can be exhaled
via the lungs as volatiles or as CO2. Sulfate and glucuronide
forms have been detected in urine and in plasma. Because of the
fast metabolism and short half-life of active compounds, a
minimum risk of accumulation in the body is suggested. The
respiratory tract exerts the most rapid way of entry, followed by
the dermal pathway. Essential oils and their metabolites can
also be absorbed and delivered to the body through oral
ingestion.
Health Effects
Essential oils and especially their constituents have a lot of
health effects such as antibacterial, antiviral, and antifungal
activities. Further fields of applications are anticancer therapy
and therapies for cardiovascular and nervous system disorders.
Additionally, they are used to reduce the level of cholesterol
and regulate the glucose level. Besides, they are useful in the
treatment of gynecological diseases. Essential oils are com-
monly used in the food and cosmetic industries.
558 Encyclopedia of Food and Health
Antioxidative Properties of Essential Oils
Nutritional properties of foodstuffs are affected by oxidation of
oils. Today, the tendency goes towards using natural com-
pounds such as essential oils for producing functional food.
A present study has tested Cinnamon zeylanicum and Zataria
multiflora Boiss as two natural preservatives. The use of this
essential oil prevents oxidation rate and reduces preliminary
and secondary oxidation products compared with butylated
hydroxyanisole (BHA). Foods, which contain fat and oils, can
be oxidized slowly during storage. As a result, different oxida-
tion products occur, which cause rancidity and reduction of the
sensory properties of foodstuffs. Lipid oxidation and fungal
growth reduce the shelf life of food products. To solve these
problems, manufacturers use antioxidants and preservatives. In
the majority of cases, the producers of foodstuff use synthetic
additives such as BHA and butylated hydroxytoluene as antiox-
idants, but the even low toxicity of those products has restricted
their use. Recently, aromatic plants and spices serve as a source
of biologically active substances such as antioxidants in food-
stuffs. Today, the focus is on natural antioxidants that can
replace synthetic additives that might be carcinogenic. Cinna-
mon, which belongs to the Lauraceae family, provides a variety
of oils with different aroma characteristics. This aromatic plant
contains the highest phenolic contents and strongest antioxi-
dant activity. The main components of cinnamon are cinna-
maldehyde and methyl eugenol. Studies have shown good
antioxidative and antifungal activities of Cinnamon zeylanicum
compared with control samples. The components that are
responsible for antiradical activity of cinnamon are eugenol,
cinnamaldehyde, cinnamic acid, and 1,8-cineole. The antifun-
gal nature of cinnamon belongs to the high phenolic contents
especially carvacrol, thymol, cinnamaldehyde, and eugenol.
Food manufacturers need antioxidants that resist at high tem-
perature during baking. Studies have been shown that concen-
trations of 500 ppm of Cinnamon zeylanicum essential oil can be
used instead of BHA. Foodstuff, which contains this oil, might
have nutritive and functional advantages compared to BHA.
Consumption of foodstuff, which includes natural additives,
can help us to prevent health disorders caused by oxidation, for
example, aging, atherosclerosis, and cancerogenesis.
Inhibition of Foodborne Pathogens by Essential Oils
Foodborne illness that is caused by microorganisms is a grow-
ing public health concern. The percentage of people that are
suffering from foodborne illness are up to 30%. Unsafe food is
responsible for illness and results in several deaths every year.
Nowadays, there are an increased concern of food safety and a
demand from customers of natural products, which are free
from synthetic additives. As a result, the use of natural antimi-
crobial compounds, such as essential oils, herbs, and spices for
food, is gaining interest. Essential oils offer a relatively safe
status and a potential for multipurpose functional use.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00262-2
 Essential Oils: Properties, Composition and Health Effects
Especially essential oils from the Lamiaceae and Apiaceae fam-
ilies exert bactericidal properties against microorganisms that
contaminate food products; therefore, they are considered as
natural food preservatives. Additionally, the widest spectrum
of action as well as the greatest antibacterial activity is shown
by phenolic compounds, for example, thymol and carvacrol.
Above all, Thymus vulgaris L., Origanum vulgare L., and Satureja
hortensis L. play an important role. The phenylpropanoid euge-
nol, which is a phenolic compound, is also known for its
antimicrobial properties, and eugenol occurs especially in the
essential oils of clove and of cinnamon leaves. All these essen-
tial oil compounds, especially as carvacrol, thymol, eugenol,
cinnamaldehyde, and cinnamic acid, are used in food to con-
trol natural spoilage process and to prevent growth of
microorganisms.
Moreover, essential oils could be used for the reduction of
biogenic amines and as a flavoring agent in Gouda cheese,
which has a positive health effect. Biogenic amines affect phys-
iological functions such as immune response, brain activity,
and gastric acid secretion. However, if biogenic amines occur in
high amounts in food, they can cause health damage. Different
concentrations of the essential oil from Z. multiflora Boiss
(Lamiaceae) were added to milk, to test the effect of this oil on
biogenic amines production such as tyramine and histamine
and microbial counts in Gouda cheese. Thus, the concentration
of tyramine and histamine was significantly reduced at the end
of the maturation period in comparison with the control group.
The higher the concentration of the essential oil, the higher was
the decrease in biogenic amines and microbial counts. Z. multi-
flora essential oil was most effective against yeasts, but there was
only a low reduction in Enterobacteriaceae counts.
Essential oils and spasmolytic activities
Present studies have shown promising spasmolytic activities
from Cuminum cyminum L. Generally, cumin is used as an
antioxidant and flavor compound. Besides these properties, it
also acts as an antiseptic, analgesic, anti-inflammatory, and
sedative and is used against stomach disorders, diarrhea, and
spasms. The antispasmodic activity of the hydrodistilled fruit
extract of Cuminum cyminum was tested on isolated of guinea
pig ileum. At the beginning, concentration-dependent
responses of acetylcholine – which induces such spasms – on
pig ileum were recorded and then a distinctive antispasmodic
activity was shown. The anticholinergic drug atropine was used
as the standard antispasmodic agent. These results proved that
acetylcholine alone causes contraction of ileum, but when
acetylcholine was given in combination with the hydro-
distilled fruit extract, a significant decrease of contraction was
noted. This high degree of spasmolytic activity was caused by
blocking the cholinergic receptors.
Essential Oils and Anticancer Activities
Cancer is a worldwide disease that can occur at any age and it is
the second cause of mortality particularly in low-income coun-
tries. Until 2030, the cancer mortality could increase by 50% to
reach 15 million worldwide. There is a relationship between
the production of reactive oxygen species and the origin of
oxidation and inflammatory, facts which can lead to cancer.
Oxidative stress acts as a DNA-damaging agent, which
559
furnishes an increase of the mutation rate within cells and
promotes oncogenetic transformation. ROS can activate signal-
ing pathways that contribute tumor development through the
regulation of cellular proliferation, angiogenesis, and metasta-
sis. It is worth to note that many cytotoxic molecules of plant
origin that possess a chemopreventive potential are used in
chemotherapy, for example, thymoquinone as an active ingre-
dient of Nigella sativa L. (Ranunculaceae). It should be consid-
ered that 30–40% of all kinds of cancer can be prevented with a
healthy lifestyle and dietary measures, and it is obvious that
nutrition has an impact on the cancer process. There are dietary
components, which are able to promote cancer progression,
and there are some that act as chemopreventive agents. N.
sativa exerts a positive effect as mediating inflammation and
cancerous cell growth especially to treat colon cancer. Nuclear
factor-kappa B activation was decreased by thymoquinone in a
dose-dependent manner. The chemotherapeutic effect of this
substance is comparable with the synthetic 5-fluorouracil. The
inhibiting effect of the essential oil from N. sativa on colon
carcinogenesis may be linked with the suppression of cell
proliferation in the colon mucosa. The essential oil of black
cumin seeds is known for its cytotoxic and apoptotic/necrotic
properties in a cancer cell line. An animal study has shown that
the addition of N. sativa seeds to the diet is able to protect
against oxidative stress, inflammatory response, and carcino-
genesis. Furthermore, this study reported that the daily intake
of these seeds (and thus of the volatile oil) results in the
reduction of chromosomal aberrations and damaged cells.
Additionally, animal models showed that thymoquinone
blocked angiogenesis in vitro and in vivo, thus preventing the
growth of cancerous cells. In conclusion, the anticancer and
anti-inflammatory properties of N. sativa and its essential oil
containing thymoquinone may be attributed partly to the sup-
pression of NF-kappa B activation pathway. This essential oil
can also be considered as a potential immunosuppressive
agent. Because of its nutritional quality, N. sativa is a potential
source of diet-based strategies, which may play a role in
improving human health.
Besides, monoterpenes that are components of nearly all
essential oils and especially of citrus fruits can be used to
prevent cancer. They show ideal chemopreventive properties,
such as antitumor activity, commercial availability, low cost,
oral bioavailability, and low toxicity. Because of this reason,
monoterpene studies are proceeding in human clinical trials
for chemotherapeutic activity. Several dietary monoterpenes
are known for their antitumor activity. It has been shown
that (þ)-limonene exerts chemopreventive activity against
rodent mammary, skin, liver, lung, and fore-stomach cancers.
(þ)-Limonene, the main constituent of orange oil, for exam-
ple, is widely used as a flavoring agent for fruit juices, soft
drinks, ice cream, and so on. The monoterpenes (þ)-limonene
and perillyl alcohol and their metabolism products showed a
high degree of oral bioavailability.
Essential oil constituents such as citral are also known for
their anticancer potential. Citral can be obtained from herbs
such as lemongrass, melissa, and verbena. Citral consists of
two isomeric acyclic monoterpene aldehydes, namely, geranial
(trans-citral, or citral A) and neral (cis-citral, or citral B). This
key component is commonly used as a food additive. Studies
showed that concentrations of 44.5 mM citral induced
560 Essential Oils: Properties, Composition and Health Effects
apoptosis in cancer cell lines, a concentration that is compara-
ble to the one of citral in a cup of tea prepared from 1 g of
lemongrass. Apoptosis is linked with DNA fragmentation
and induction of caspase-3 catalytic activity, whereby the ab-
unsaturated aldehyde group is responsible for this apoptotic
effect and might be a core structure for the design of pro-
apoptotic drugs.
Essential oils are also able to inhibit the formation of
N-nitrosodimethylamine in vegetables. Vegetables are rich in
nitrate that is a precursor of nitrite. Crops contain different
concentrations of nitrate, dependent on the species, genetic,
and environmental factors and cultivation conditions, whereas
the concentration of nitrite in plants is commonly very low. N-
Nitroso compounds such as nitrosamines that are formed by
N-nitrosation of secondary amines with nitrite in an acidic
environment act as strong carcinogens. Several foods contain
the precursors of this reaction. Meats, cured meat products, and
smoked fish are sources of secondary amines. Nitrite often
occurs in hams and sausages as an antimicrobial or coloring
substance. Essential oil constituents, such as carvone and lim-
onene, are able to activate the glutathione S-transferase that
reduces the risk of cancer. The terpene hydrocarbon (þ)-
limonene, which represents one of the major compounds of
citrus essential oils, exhibits anticarcinogenic properties against
mammary, lung, stomach, and skin cancer. It has been
reported that the essential oils of citrus show an inhibitory
effect on the formation of N-nitrosodimethylamine. Especially
Citrus junos Siebold ex Tanaka (Rutaceae), also known as yuzu,
which is a popular sour citrus fruit in Japan, shows an inhib-
itory effect on the formation of N-nitrosodimethylamine.
Because the Japanese diet is richer in vegetables than in other
countries, their intake of nitrate is very high, and thus, they
have a higher risk to suffer from stomach cancer. Bacteria in
saliva have an influence on the N-nitrosodimethylamine for-
mation, because they induce the reduction of nitrate. Although
the rate of formation of nitrosamines is proportional to the
square of nitrite concentration, it has been considered that
nitrite in vegetables especially cabbage, celery, spinach, and
mint is not mainly responsible for the formation of N-nitroso-
dimethylamine. In most cases, oral bacteria are responsible for
the reduction of nitrate into nitrite. Terpene hydrocarbons
such as myrcene, a-terpinene, and terpinolene show an inhib-
itory effect and cause the decrease of N-nitrosodimethylamine
formation. In conclusion, yuzu oil showed an inhibitory activ-
ity on the formation of N-nitrosodimethylamine in the pres-
ence of vegetables and saliva.
Essential Oils and Diabetes
Essential oils from different plants can also have a positive
effect on diabetes and hypertension. Nowadays, there is a
tendency to manage type-2 diabetes and hypertension with
natural sources. There are two major ways to handle this: the
scavenging of free radicals and the inhibition of key enzymes,
which are involved in starch digestion such as a-amylase and a-
glucosidase. By those enzymes, starch is converted into glu-
cose, thus increasing its concentration. Inhibition of these
enzymes leads to a delay of the absorption of glucose followed
by a moderate postprandial blood glucose elevation. There is
also a relationship between diabetes and an increased
generation of free radicals and a defective antioxidant defense
system. Additionally, oxidative stress is involved in the diabe-
togenic process. For that reason, antioxidant-rich foods have a
good dietary intervention in the management of this disease.
The use of the essential oil of black pepper seeds in relation
with diabetes and hypertension is known in folk medicine.
Aromatic spice plants such as black pepper show good antioxi-
dant properties, due to phenolic contents of the essential oil,
which possesses antimicrobial, antihypertensive, anticonvulsive,
and sedative activities. The essential oil is extracted from the
seeds and leaves from black pepper and contains mono- and
sesquiterpenes, besides phenolic compounds that are effective in
preventing diabetes in animal models. The essential oil of black
pepper is established for its radical scavenger abilities and its
ferric-reducing antioxidant activity attributed to the presence of
pinene and 1,8-cineole. Another therapeutic approach is the
inhibition of the starch-metabolizing enzymes. In this case,
the activity of a-glucosidase is more strongly inhibited by
the essential oil than the one of a-amylase. This circumstance
has a therapeutical purpose towards synthetic a-amylase and
a-glucosidase inhibitors. The use of those natural products is
important in preventing some side effects of the synthetic inhib-
itors. The essential oil of black pepper can inhibit also the
angiotensin-1-converting enzyme (ACE) activity. This fact is a
therapeutic approach in the treatment of hypertension, which is
one of many complications associated with type-2 diabetes.
In vitro, the ACE activity was inhibited in a concentration-
dependent manner by the essential oil, because it is able to
block the conversion of angiotensin-1 into the vasoconstrictor
angiotensin-2 that causes the hypertension.
Essential Oils and Mood Disorders
Furthermore, some essential oil constituents, such as (E)-
methyl isoeugenol (MIE), is often used as food flavor. This
essential oil from Pimenta pseudocaryophyllus (Gomes) L.R.
Landrum (Myrtaceae) leaf has calming properties. This ubiqui-
tous food additive appears attractive for the treatment of mood
disorders. Studies demonstrated the anxiolytic- and
antidepressant-like activities of MIE, suggesting the participa-
tion of serotonergic pathways. Among psychiatric diseases,
mood disorders are most common, that is, widespread with a
prevalence of up to 20% worldwide. Because of the low remis-
sion rate and the high rate of nonresponse to current treat-
ment, it is necessary to develop new therapeutic agents, such as
consumption of functional food. With this basic food nourish-
ment as well as health benefits are covered. Today, aromatic
plants have been largely explored as functional ingredients in
the pharmaceutical and food industries. The treatment of neu-
ral disorders with naturally occurring food flavors, such as MIE,
seems to be more acceptable than the use of pharmacother-
apies. Oral administration of MIE showed an increase in sleep
duration and a soothing activity of the CNS. An antiseizure
property of MIE was hypothesized too, because it acts similar
to diazepam, a CNS-calming compound. This hypothesis is
further supported by the antiseizure properties of aromatic
compounds with a similar chemical structure as MIE, such as
methyleugenol, eugenol, and 1-nitro-2-phenylethane.
Besides MIE, in diets, the widely consumed essential oil of
lemon is also known for its antidepressant-like effect and
 Essential Oils: Properties, Composition and Health Effects
therefore acts as functional food ingredient. Because of many
side effects, such as nausea and anorexia, by using conven-
tional medicaments against depression, there is a growing
interest in the search of new effective antidepressants, such as
functional foods that worldwide are used in the kitchen. Ani-
mal studies have been shown that the volatile oil from lemon
has increased the metabolic turnover of dopamine in the hip-
pocampus and of serotonin in the prefrontal cortex and stria-
tum. The oral administration of lemon essential oil does not
show any toxic effects or an influence on weight, blood, and
organs of rodents. Upon oral administration of 400 mg kg1
lemon essential oil, the dopaminergic activity in the striatum
and the hippocampus was significantly enhanced on account
of an increased concentration of dopamine and a decreased
turnover. It is known that depressed patients suffer from a
dysfunctional 5-hydroxytryptamine (5-HT) system; thus, selec-
tive 5-HT reuptake inhibitors enhance the activation of various
5-HT receptor subtypes. In conclusion, the essential oil of
lemon has a great potential to be used as an antidepressant
functional food ingredient.
Essential Oils and Osteoporosis
Furthermore, essential oils can also be useful in the treatment
of osteoporosis. This disease is a major health problem in aging
humans – especially women – when low bone mass leads to
osteoporotic fractures. The bone turnover is increasing and it
comes to a marked decrease in trabecular bone mineral density
(BMD) and bone mineral content. Osteoporotic fractures
reduce the quality of life of the patient and they are a burden
to health care. Hence, it is desirable from medical and
economical points of view to prevent the loss of bone mass.
A nutritional approach would be an inexpensive opportunity
to prevent low bone mass. Studies have shown that essential
oils from sage, rosemary, and thyme and essential oils from
pine, juniper, and eucalyptus inhibit or at least retard the
activity of osteoclasts. The essential oil of pine is the most
potent one and also the bitter orange-peel oil is very potent,
for which monoterpenes, such as thujone, eucalyptol, cam-
phor, borneol, menthol, and thymol, are responsible. Studies
have shown that pine oil reduces the loss of trabecular BMD in
animal models, and in vitro studies report that these mono-
terpenes directly inhibit the osteoclast activity. (1R,3R,4S)-()-
Menthol that is the most widely used monoterpenic alcohol in
human nutrition inhibits bone resorption in vivo and in vitro,
but the latter effect is weak. Also, the consumption of
monoterpene-rich fruits and vegetables is linked with a greater
BMD in humans. Animal studies have shown that vegetables,
salads, and herbs, as a part of human nutrition, are able to
inhibit bone resorption. Essential oils exert a positive effect on
the bone metabolism when they are added to food. Therefore,
essential oil-containing herbs are possible candidates for a
dietary approach to osteoporosis.
Essential Oils and Their Effect on the Gastrointestinal Tract
Essential oils are also famous for their positive effect on the
gastrointestinal tract. Mentha x piperita L., which belongs to the
Lamiaceae family, contains a volatile oil with the major com-
pound ()-menthol. This essential oil is used against irritable
561
bowel syndrome, because of its influence on gastric motility.
Peppermint tea is commonly used against acute and chronic
gastritis, enteritis, and disturbances of the GI tract by the anti-
spasmodic effect of menthol. Furthermore, peppermint tea is
commonly used as an antiemetic in pregnant women, besides
ginger and cannabis. This volatile oil shows the same effect
as 5-HT3 receptor antagonists, 5-HT4 receptor agonists, and
anticholinergics. Those receptors are also involved in the path-
ogenesis of irritable bowel syndrome. Peppermint oil and ()-
menthol are able to completely inhibit 5-HT3 receptor in a
concentration-dependent manner by relaxing the smooth mus-
cle and antagonizing the serotonin-induced stimulation. This
antagonistic effect on the 5-HT3 receptor channel influences in
a positive manner the disturbed motility during symptoms of
irritable bowel syndrome and emesis. The cationic influx
through 5-HT3 receptor channels was inhibited by peppermint
oil and its main constituent ()-menthol. The influence of
peppermint essential oil and ()-menthol leads to the reduc-
tion of serotonin-induced contraction in animal models. In
addition, they possess a direct relaxant effect.
Essential oils often are used as food additives to comple-
ment present therapies of gastritis and peptic ulcers. This dis-
ease is caused by an increased density of Helicobacter pylori in
the gastric mucosa. A nutritional approach can help people
with asymptomatic gastritis to manage the infection and to
decrease the development of the disease. Those essential oils
that exhibit the strongest bactericidal potential against H. pylori
P1 were also active against other Helicobacter strains. The most
effective single essential oils constituent against H. pylori are
carvacrol, isoeugenol, nerol, citral, and sabinene. The wide-
spread infection with H. pylori is well known as the major
etiologic factor in chronic active type B gastritis, gastric ulcers,
and gastric cancer. Nowadays, a triple therapy such as the
combination of proton pump inhibitor and two antibiotics is
common, but increasing antibiotic resistance is also responsi-
ble for an eradication treatment failure. Therefore, there is a
growing interest to find alternative treatments, for example, the
development of new nutritional approaches. Especially
patients with an asymptomatic gastritis profit from this nutri-
tional approach.
Essential Oils and Alzheimer’s Disease
Essential oils can also be used in the treatment of Alzheimer’s
disease, the most common cause of dementia in the aged pop-
ulation. It results in cognitive decline and mental deterioration
that is the result of massive and progressive loss of neurons
from different regions of the brain. Disorders of the CNS are
linked with neurotransmitter disturbances and insufficiencies
in cholinergic functions. Cholinesterase is responsible for the
hydrolyzation of choline, and if there is a high concentration of
this enzyme, then a lower concentration of choline in the
synaptic gap results. Investigations have shown that there is a
relation between increased levels of cholinesterase enzymes
and Alzheimer’s disease. This fact leads to the hypothesis that
this cognitive decline in patients is linked to the progressive
cholinergic degeneration. Hence, promising approaches for
the treatment of Alzheimer’s disease are cholinesterase inhibi-
tors that are able to enhance the level of cholinergic neurotrans-
mitters in the brain. Acetylcholinesterase (AChE) and
562 Essential Oils: Properties, Composition and Health Effects
butyrylcholinesterase (BChE) are the major cholinesterase
enzymes that are responsible for the decreasing choline levels.
Today, Alzheimer’s disease patients are treated with AChE
inhibitors, synthetic compounds that reveal some toxicity dur-
ing prolonged use. Hence, there is a great demand for alterna-
tive drugs to treat Alzheimer’s disease. It has been suggested that
dietary supplements with antioxidants and free radical scaven-
gers may decrease the mild cognitive impairment of
Alzheimer’s disease. A high amount of plants has been tradi-
tionally used for the enhancement of cognitive function and
alleviation of several symptoms linked with Alzheimer’s dis-
ease. Nowadays, essential oils of edible plants are considered to
inhibit AChE and BChE. Those volatile oils are of great interest,
because of their availability, few side effects, low toxicity, and
their biodegradability. Studies have shown that the essential
oils of Citrus aurantifolia (Christm.) Swingle (Rutaceae), Alpinia
galanga (L.) Willd. (Zingiberaceae), and Melissa officinalis L.
(Lamiaceae) possess the highest inhibitory activity against
AChE, whereas Melissa officinalis, Citrus aurantifolia, and Oci-
mum gratissimum L. (Lamiaceae) exhibited the strongest inhibi-
tory properties against BChE. Therefore, the leaf essential oil of
Citrus aurantifolia with the constituents (þ)-limonene, ()-
camphor, citronellol, cis-ocimene, and 1,8-cineole is a promise
for the prevention and treatment of Alzheimer’s disease,
because it inhibits both enzymes. 1,8-Cineole that is one of
the most potent AChE inhibitors also occurs in other plants
such as Rosmarinus officinalis L. (Lamiaceae).
Essential oils and atherosclerosis
The essential oil of sage could be used as food ingredient with
anti-inflammatory and antiatherogenic properties. Cholesterol
deposition in the intima of large- and medium-size arteries
together with a chronic inflammatory process leads to athero-
sclerosis. Oxidized low-density lipoproteins play a key role in
early inflammation. This type of LDL that is not recognized by
the LDL receptor is taken up by the scavenger receptors in
monocytes–macrophages and endothelial cells. Hence, it
comes to the accumulation of cholesterol in macrophages
and to the formation of foaming cells. Moreover, oxidized
LDL is able to induce the expression of proinflammatory cyto-
kines such as TNF-a, Il-1b, and IL-6 in macrophages and endo-
thelial cells. Essential oils from spices, which are added to food,
present various biological activities, such as antioxidant and
anti-inflammatory properties. As mentioned earlier, the oxida-
tion of LDL results in this atherosclerotic lesion and the initia-
tion of the inflammatory cascade. Therefore, oxidized LDL is
able to induce the expression of proinflammatory cytokines,
such as IL-1b, TNF-a, and IL-6 from monocytes and macro-
phages. IL-1b and TNF-a play an important role in the initial
amplification of the inflammatory response. Moreover, they are
able to induce the expression of adhesion molecules by endo-
thelial cells and promote secretion of different cytokines and
chemokines by monocytes. Furthermore, they are involved in
the process of foam cell formation, especially by means of
induction of growth factors. Activated macrophages, lympho-
cytes, fibroblasts, and vascular smooth muscle cells produce
during stimulation IL-1 and TNF-a. The inflammatory cytokine
IL-6 plays an important role in the development and progres-
sion of atherosclerosis. It has been reported that the essential oil
of Salvia officinalis L. (Lamiaceae) is able to inhibit COX-2, thus
exerting anti-inflammatory properties. Also, supercritical
extracts of Salvia officinalis are effective inhibitors of oxidized
LDL and induced proinflammatory cytokines. This sage extract
mainly consists of camphor, borneol, and 1,8-cineole that is a
strong inhibitor of TNF-a, IL-1b, and IL-6 production. Borneol
and camphor also exhibit excellent anti-inflammatory proper-
ties. Therefore, sage extract has a great potential to be used as an
anti-inflammatory agent to prevent atherosclerosis.
See also: Essential Oils: Isolation, Production and Uses; Herbs:
Composition and Dietary Importance.
Further Reading
Alimohammadi M, Es’haghi Gorji M, Jahed Khaniki G, Nabizadeh Nodehi R, Noori N,
and Rastkari N (2014) The evaluation of Zataria multiflora Boiss. Essential oil effect
on biogenic amines formation and microbiological profile in Gouda cheese. Letters
in Applied Microbiology 59(6): 621–630.
Arranza E, Jaimea L, Lopez de la Hazasa MC, Regleroa G, Santoyoa S, and Vicentea G
(2014) Supercritical sage extracts as anti-inflammatory food ingredients. Industrial
Crops and Products 54: 159–166.
Barzegar M, Kordsardouei H, and Sahari MA (2013) Application of Zataria multiflora
Boiss. and Cinnamon zeylanicum essential oils as two natural preservatives in cake.
Avicenna Journal of Phytomedicine 3(3): 238–247.
Benjakul S and Tongnuanchan P (2014) Essential oils: extraction, bioactivities, and
their uses for food preservation. Journal of Food Science 79(7): 1231–1249.
Catherine AA, Deepika H, and Negi PS (2012) Antibacterial activity of eugenol and
peppermint oil in model food systems. Journal of Essential Oil Research 24(5):
481–486.
Crowell PL (1999) Prevention and therapy of cancer by dietary monoterpenes. The
Journal of Nutrition 129(3): 775–778.
Edward K, Monika S, and Magorzata W (2012) Recent patents regarding essential oils
and the significance of their constituents in human health and treatment. Recent
Patents on Anti-Infective Drug Discovery 7(2): 133–140.
Fajemiroye JO, Galdino PM, Paula JAMD, Rocha FF, and Akanmu MA (2014) Anxiolytic
and antidepressant like effects of natural food flavour (E)-methyl isoeugenol. Food
& Function 5(8): 1819–1828.
Felix R, Lozano A, Mühlbauer RC, Palacio S, and Reinli A (2003) Common herbs,
essential oils, and monoterpenes potently modulate bone metabolism. Bone 32(4):
372–380.
Ferreira AR, Mata AT, Nogueira JMF, et al. (2007) Antioxidant and
antiacetylcholinesterase activities of five plants used as Portuguese food spices.
Food Chemistry 103(3): 778–786.
Hauk F, Heimes K, and Verspohl EJ (2011) Mode of action of peppermint oil and ()-
menthol with respect to 5-HT3 receptor subtypes: binding studies, cation uptake by
receptor channels and contraction of isolated rat ileum. Phytotherapy Research
25(5): 702–708.
Muhlbauer RC (2006) Are vegetables, salads, herbs, mushrooms, fruits and red wine
residue that inhibit bone resorption in the rat a promise of osteoporosis prevention?
Current Nutrition and Food Science 2(1): 69–78.
Nagori BP, Saini N, and Singh GK (2014) Physicochemical characterization and
spasmolytic activity of essential oil of cumin. International Journal of Biology,
Pharmacy and Allied Sciences 3(1): 78–87.
Sadiq BM and Tauseef SM (2010) Nigella sativa: reduces the risk of various maladies.
Critical Reviews in Food Science and Nutrition 50(7): 654–665.
Wallner, I. M. (2015). Essential oils: properties, composition and health effects. Master
Thesis, University of Vienna.
 Ethnic Foods
OI Bermudez, Tufts University School of Medicine, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Consumers of ethnic foods often have distinctive cultural char-
acteristics, including language, social norms, and behaviors
that provide them with a sense of connectedness. From a
cultural standpoint, ethnic foods serve as the familiar link
with the past and help those who are accustomed to them to
maintain and promote among peers their ethnic identity.
In countries like the United States, with a very diverse ethnic
population, the production, distribution, and consumption of
ethnic foods are an integral feature of the overall American
culture.
Native Americans are direct descendants from the original
population groups distributed throughout the country before
the European colonization. Since then, they have contributed
to the cultural dimensions of ‘ethnic foods’ with their food
production developed around the ‘three sisters’ (corn, beans,
and squash) system. Moreover, they developed a rich and
highly nutritive traditional cuisine with many similitudes to
those found in Central America. For example, in Guatemala,
the ‘three sisters’ system was termed as the ‘milpa.’
During and after the colonization, several waves of immi-
grants from Europe, Africa, and Asia brought from their coun-
tries of origin their ethnic foods and dishes. Then, their cuisine
evolved from the homeland traditional dishes to the modified
versions of ethnic cuisines that currently exist in these modern
times.
Similarly, more recent waves of immigrants from Central
and South America, the Caribbean, and the four corners of the
globe contributed to the rich diversity of the foods present
today in US households.
Several cooking techniques and eating styles from the orig-
inal traditions brought by original, earlier, and new American
settlers were exchanged and adopted by them. Traditional
dishes like tacos, tamales, pizza, and noodles and Chinese
dishes (e.g., lo mein) gave origin to new dishes, different
from their native, original versions. Those ethnic food trans-
formations are likely to continue as the American population
becomes more diverse and interrelated, with evolving sense of
taste, flavor, and smell for what they term as ‘ethnic foods.’
Ethnic Diversity of the American Population
The United States has a very diverse population. This diversity
was long in existence in the precolonial times with more than
300 tribal groups of Native Americans. Since then, the United
States had transformed into a ‘plurality nation.’ This pluralism
is reflected in the ancestry of the early settlers in the country,
which are those that contributed to the ethnic diversity of the
American cuisine. In the current times, those from German
(16%), Irish (11%), English (8%), and Italian (6%) descents
are the largest ethnic groups that contributed to the diversity of
the white population (see Figure 1). The Census Bureau
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00263-4
defines as white those having origins in any of the original
peoples of Europe, the Middle East, or North Africa.
Furthermore, it is the plurality of the more recent immi-
grants, along with that from the African-American population
that has given this country the ethnic diversity observed in the
early periods of the twenty-first century. According to the 2010
Census Bureau, the total population reached about 309 mil-
lion people. The racial and ethnic heterogeneity of this popu-
lation is represented in Table 1. As seen in this Table 1, whites
were the largest group (75%). Other ethnic groups include
black or African-Americans (14%), Asians (6%), American-
Indian and Alaska Natives (2%), and Native Hawaiian and
other Pacific Islanders (0.4%). Based on ethnicity, which is
reported by the Census Bureau as having or not Latino or
Hispanic background, 16% of the total US population is of
Latino or Hispanic origin.
This diversity of the major racial and ethnic groups in the
United States is reflected in the large number of ethnic groups
that, because of their geographic, socioeconomic, and cultural
origins, have contributed to the rich, mixed, and festive ethnic
foods that every day are present in the American households.
Ethnic Foods Present in the American Homes
Based on the geographic, climate, and, particularly, the plural-
ity of the American population, one could find an extraordi-
narily rich ethnic cuisine in most households across the
country. Most ethnic cuisines have been influenced by centu-
ries of food exchanges between indigenous groups and immi-
grants from all over the world. As result, the American cuisine
is a unique multiethnic blend. No single eating pattern has
prevailed over all the others, and no ethnic or cultural group
has emerged with an unchanged food pattern. However, some
ethnic cuisines are more popular than others, mainly due to
the high number of immigrants from the latitudes where those
ethnic foods and traditions originated.
In general, foods that are regarded as ethnic foods by a
cultural group vary over time and with the degree of accultur-
ation of the group consuming them. Even within major race
and ethnic subgroups, many variations in ethnic foods exist.
Some foods only bear a passing resemblance to the original
cuisine from which they were derived. However, all of them
fulfill important cultural roles for the diverse American popu-
lation. For example, in the United States, pizza, tacos, chili
with meat, and chicken chow mien are very different from
the Italian, Mexican, and Chinese dishes that inspired them.
Hispanic-Americans with a Mexican-American heritage differ
in their food preferences from Puerto Ricans, Colombians, and
Chileans, whose eating patterns involve a blend of Spanish,
African, Native Central/South American, and continental
North American influences. Similarly, Asian-Americans of Fil-
ipino, Japanese, or Chinese ancestry have very different food
choices and traditions. Among the over 300 different Native
563
564 Ethnic Foods

German, 15.5

Other Groups, 38.9 Irish, 11.2

English, 8.4

American, 6.5

Russian, 1.0

Scotch-Irish, 1.1
Swedish, 1.3 Italian, 5.6
Norwegian, 1.4 Polish, 3.1
Dutch, 1.5 Scottish, 1.8 French, 2.8
Figure 1 Ancestry of the American population, based on the 2010 US Census data. Reproduced from US Census Bureau. (2010). 2010 American
Community Survey. American Fact Finder: People Reporting Ancestry.

Table 1 Racial and ethnic population groups in the United States Table 2 Examples of ethnic cuisines and representatives dishes
commonly eaten by the American population
Race and Latino or Hispanic origin Number Percenta
Ethnic
Total population 308 745 538 100.0 cuisine Examples of popular dishes
Race categories
White 231 040 398 74.8 Puerto Rice with pigeon peas, tostones (fried green plantains),
Black or African-American 42 020 743 13.6 Rican habichuelas (stewed pink beans), pasteles (similar to
Asian 17 320 856 5.6 tamales), and rice pudding
American Indian and Alaska native 5 220 579 1.7 Mexican Tamales, tortillas (corn- or wheat-based), tacos,
Native Hawaiian and other Pacific Islander 1 225 195 0.4 enchiladas, guacamole, and mole poblano
Some other race 21 748 084 7.0 Italian Pizza, risotto, spaghetti, lasagna, ciabatta bread, cheeses,
Latino or Hispanic ethnicity (from any race 50 477 594 16.3 and wines
categories) Japanese Sashimi, sushi, soba, sukiyaki, yakitori, and miso soup

a
Percentages estimated from the total population.
Source: US Census Bureau. (2010). 2010 Census Briefs. Overview of Race and
Hispanic Origin: 2010.
American groups, the staples and other foods that they regard
as unique and distinctive to their cultures also vary.
The traditional ‘Mediterranean’ diet varies in terms of foods
and seasonings included in their cuisine. These differences are
reflecting the European and North African Mediterranean loca-
tions and cultural norms of the Mediterranean countries where
the Mediterranean diets originated. These traditional diets
include liberal amounts of fruits, vegetables, legumes, grains,
and fish. High amounts of monounsaturated compared with
saturated fats, low amounts of meats, and moderate consump-
tion of alcohol are also observed. Although there is a debate
about the extent to which such a regimen is exportable, some
‘Mediterranean’ ethnic foods have become very popular in
America. Examples of those foods include olives and olive
oil, breads, couscous, pastas, artichokes, arugula, grapes,
dates, figs, almonds, chickpeas, basil, bay leaf, and parsley.
The ethnically diverse cuisines found in the United States
include the contributions of most ethnic groups. Those groups
brought with them their native recipes and some of their
traditional food ingredients. And then, throughout the times,
they modified and adjusted those recipes to the availability and
cost of foods and seasonings in order to make them acceptable
by the general population. Table 2 contains a short list of
examples of ethnic cuisines present in the US households.
Germans, for example, brought and modified hot dogs and
hamburgers. Italians, with their basic ingredients wheat,
tomato sauce, and olive oil, also influenced the American
foodways. They introduced classic dishes such as pizza and
spaghetti, to the point that the Italian cuisine is the third
most popular in the country.
Ethnic cuisines of Asian origins, particularly represented by
Chinese and Japanese ethnic dishes, adapted to the American
palate. Chinese-American foods represent the second most
popular ethnic cuisine in the United States. Those ethnic
cuisines contributed with several unique ethnic dishes.
Noodles-based dishes and a large variety of vegetable dishes
(e.g., broccoli and tomatoes) are part of the incorporation of
non-Chinese foods added to satisfy the American preferences.
 Ethnic Foods 565

Table 3 Useful sources to consult on ethnic cuisines, ethnic foods, and food behaviors

Description of sources Electronic sites

Scientific journals
Journal of the Academy of Nutrition and Dietetics http://www.andjrnl.org/
Nutrition Today http://journals.lww.com/nutritiontodayonline/pages/default.aspx
American Journal of Clinical Nutrition http://ajcn.nutrition.org/
Journal of the American College of Nutrition http://americancollegeofnutrition.org/content/the-journal
Journal of Nutrition http://jn.nutrition.org/
Appetite http://www.journals.elsevier.com/appetite/
Public Health Nutrition http://journals.cambridge.org/action/displayJournal?jid¼PHN
Journal of Food Composition and Analysis http://www.journals.elsevier.com/journal-of-food-composition-and-analysis/
Ethnicity and Disease http://www.ishib.org/wordpress/?page_id¼1388
British Food Journal http://www.emeraldinsight.com/journal/bfj
Proceedings of the Nutrition Society http://journals.cambridge.org/action/displayJournal?jid¼PNS
Organizations, programs, and other resources
Dietary Guidelines for Americans http://www.health.gov/dietaryguidelines/
US Food Classification: MyPlate http://www.choosemyplate.gov/food-groups/
USDA Cultural and ethnic food and nutrition resources http://www.nal.usda.gov/fnic/pubs/ethnic.pdf
Society for the Study of Ingestive Behavior http://www.ssib.org/web/
American Institute of Wine & Food http://www.aiwf.org/
Center for Food Safety http://www.centerforfoodsafety.org/about-us
Center for Science in the Public Interest http://www.cspinet.org/
Oldways Preservation Trust http://oldwayspt.org/
Sally’s Place http://www.sallybernstein.com/
Slow Food USA http://www.slowfoodusa.org/
UN Food and Agriculture Organization (FAO) http://www.fao.org/home/en/
FAO food-based dietary guidelines http://www.fao.org/nutrition/nutrition-education/food-dietary-guidelines/en/

From Latin America, Mexicans contributed a large variety Table 4 Factors involved in the development of ethnically distinctive
of ethnic foods. An extensive list of Mexican foods, including cuisines
chili peppers, chocolate, black beans, tacos, ‘burritos,’ and
Factors Examples
tamales, is part of the rich offerings found across the country.
Currently, the Mexican cuisine is the most popular ethnic Ecological Geography, climate, range of native plants and unusual
food category in the country. Because the large Mexican factors species in area
population in the United States, along with their rich and Individual Philosophical, religious, and moral attitudes
varied traditional cuisine, the general American population factors
has incorporated many of those Mexican ethnic foods into Social factors Social structures, social relationships, social status,
their eating habits, diversifying the culinary landscape of migration patterns (both voluntary and involuntary)
‘American’ ethnic foods. Economic Trade, food prices, food distribution, imports, and
Ethnic food preferences and choices are important aspects factors exports
of the cultural heritage they represent. Some of the many
sources available on food-related aspects of culture are sum- Table 5 Theories on how ethnic food patterns developed
marized in Table 3. It includes the cultural anthropology liter-
ature on foodways, reports, and nutrition information Theory Comments
materials that describe foods, their uses, and related traditions.
Environmentalism Ethnic food traditions are determined primarily by
environmental and external influences
Cultural Humans mold their environments and diets quite
Ethnic Differences in Food Classification Systems determinism independently of their physical surroundings
Cultural ecology Both culture and environment are important
Ethnic groups vary in their foodways, social and physical con- Cultural history Historical influences on how foods have evolved
and developed over time
texts in which food preparation, serving, and eating occur.
Functionalism Use of foods to identify or please people socially
Table 4 describes the factors that are thought to be involved
and how foods are used in society
in the development of ethnically distinctive cuisines and ethnic
foods. Nutritional anthropologists subscribe to various theo-
ries on how ethnic food patterns have developed (see Table 5). medical views of foods as sources of nutrients that are based
No one theory appears to predominate over the other. on physiological and biochemical characteristics. They may
Individuals in different ethnic groups have different ways of also differ in their attitudes toward medical care and their
categorizing foods, which are sometimes at variance with health behaviors and in their beliefs about the therapeutic
566 Ethnic Foods

Table 6 Some ways in which foods are classified in different cultures
Food classification
system Descriptions
Food habits ‘Core,’ secondary core, and peripheral foods
Gastronomic Foods versus nonfoods, edible versus
nonedible
Religious/ Sacred versus profane, allowed versus
philosophical forbidden
Opposing categories Hot versus cold, yin versus yang, or other
or properties ways of ‘balancing food intakes to prevent or
cure disease’
Medicinal Foods for the prevention of diseases or as
treatment for certain diseases or
psychological states
Social Symbolic of a culture or belief system
Emotional Foods for the sick, party foods
significance
Nutritional Macro- and micronutrients
Hygienic Wholesomeness, cleanliness, freedom from
environmental contaminants,
microorganisms, or other undesirable
elements
Source: Kaufman-Kurzrock, D. L. (1989). Cultural aspects of nutrition. Topics in
Clinical Nutrition 4(2), 1–6.
and preventive properties of foods and the appropriate balance
between different foods. For example, there are systems in
various cultures that categorize foods into medicinal versus
nonmedicinal, hot versus cold, or yin versus yang. These are
classification schemes based on properties other than nutrient
content (see Table 6). Food classification systems may affect
nutritional status because they cause the exclusion of healthful
foods or food groups from the diet, encourage consumption of
harmful items, or produce a false sense of security among
eaters that all of their nutritional needs are met when in fact
they are not.
Dietary Assessment of Ethnic Food Consumption
Patterns
Familiarity with ethnic foods is important for dietary assess-
ment, particularly if they are imports from other countries. The
foods themselves, their names, composition, portion sizes, and
frequency of consumption vary. Accurate assessment of nutri-
ent intakes depends on a good knowledge of these factors.
Some ethnic foods may be especially rich sources of nutrients
or other food constituents, such as phytochemicals.
During the last 40 years, Americans experienced increases in
a series of nutritional and health threats associated with
unhealthy eating practices, including obesity, type 2 diabetes,
hypertension, cardiovascular diseases, and certain types of can-
cers. To halt those detrimental effects, people, in general, are
responding favorably to the new policies and recommenda-
tions to improve eating and health-related practices. For some
groups, those recommendations are being implemented with
the use of ethnic foods considered healthy alternatives. To
confirm those beliefs, food and nutrition experts need to ana-
lyze the growing number of ethnic foods that are being
incorporated into the already diversified, ethnic cuisine prac-
ticed by Americans. The application of tools and techniques for
dietary assessment may be needed in order to provide appro-
priate nutritional guidance for those groups adapting new
ethnic foods.
One challenge in evaluating diets that include many ethnic
foods is the appropriate choice of methods for dietary assess-
ment. Methods such as semiquantitative food frequency ques-
tionnaires are often designed to capture intakes of important
nutrients for those who conform to the predominant food
culture. These are often inappropriate for subgroups with very
different dietary patterns. Subgroup members may call the foods
by different names, consume items not included on the food
lists, or vary in amount and frequency of consumption. The use
of such instruments to assess intakes of individuals with uncon-
ventional dietary preferences can lead to highly erroneous con-
clusions. Subgroup-specific food frequency questionnaires that
take account of these factors are sometimes available, and when
they are, these should be used. For example, well-validated
ethnic-specific food frequency questionnaires have been devel-
oped for Japanese and Chinese-Americans living in Hawaii and
for Puerto Ricans living in the Northeast.
Another potential problem in the dietary assessment of
ethnic cuisines is that nutrient databases for converting dietary
intakes into nutrients for ethnic foods may not be available. In
research studies on individuals who use many ethnic foods,
nutrient databases that include entries for ethnic favorites are
needed to obtain complete information about intakes. When-
ever possible, food tables that are specific to the country or
ethnic group should be used. Country-specific tables of food
composition and nutrient databases are becoming increasingly
available, and these are the best option. For individuals who
eat largely ethnic cuisines in countries where the parent food
culture is different, special food composition tables for the
ethnic foods that are most popular in mainstream diets are
usually unavailable. Even for ethnic foods that are included in
nutrient databases, food composition information may be
incomplete, inappropriate, or lacking. Moreover, nutrient
values for foods that are rarely consumed outside of the ethnic
group may be absent, especially if the ethnic group is small.
This is a challenge with many local foods consumed by Alaskan
natives for which there is a gap in nutrient composition of
those foods.
The dietary assessment task is more difficult when no coun-
try or ethnic-specific food tables are available. Recipes made
from locally available ingredients or imported foods may differ
in their food composition from recipe values in food tables
used in Western countries. For example, a typical curry dinner
as served in India, Ghana, and England varies greatly in its
nutrient composition. For this reason, country-specific food
consumption tables are needed.
Ethnically Appropriate Dietary Planning and Interventions
The consumption of ethnic foods is usually interpreted as a
cultural phenomenon. However, it has physiological implica-
tions. Sometimes, alterations in eating habits are necessary to
achieve nutritional needs, but this is a difficult goal to achieve.
Food habits are among the oldest and most deeply entrenched
aspects of many cultures, and they cannot be easily changed.
 Ethnic Foods 567

Table 7 Examples of the importance of ethnically appropriate nutrition interventions

Resources for nutrition intervention Electronic sites

National Heart, Lung, and Blood Institute (NHLBI): Materials for American Indian/
Alaska Native Audiences
NHLBI: American Indian Health
NHLBI: Materials for Asian American/Native Hawaiian/Other Pacific Islander
NHLBI: Materials for Latino/Hispanic Americans
NHLBI: On the Move to Better Heart Health for African Americans
USDA: Cultural and ethnic food and nutrition resources
US Department of Health and Human Services. Office of Disease Prevention and
Health Promotion: Dietary Guidelines for Americans
US Department of Agriculture. US Food Classification: MyPlate
Oldways Preservation Trust: Inspiring good health through cultural food traditions
and lifestyles
Illinois State University: Healthy habits at the holidays
Mayo Clinic. Mediterranean diet: a heart-healthy eating plan
Winham, D.M., 2009. Culturally tailored foods and CVD prevention. American Journal
of Lifestyle Medicine 3, 64S
Lemacks, J. et al., 2013. Interventions for improving nutrition and physical activity
behaviors in adult African American populations. Preventing Chronic Disease 10,
120256
http://www.nhlbi.nih.gov/health/healthdisp/an.htm
http://americanindianhealth.nlm.nih.gov/index.html
http://www.nhlbi.nih.gov/health/healthdisp/aapi.htm
http://www.nhlbi.nih.gov/health/healthdisp/lat.htm
http://www.nhlbi.nih.gov/health/resources/heart/african-
american-index
http://www.nal.usda.gov/fnic/pubs/ethnic.pdf
http://www.health.gov/dietaryguidelines/
http://www.choosemyplate.gov/food-groups/
http://oldwayspt.org/
http://stories.illinoisstate.edu/student-affairs/health-
promotion-and-wellness/healthy-habits-holidays-2/
http://www.mayoclinic.org/healthy-living/nutrition-and-
healthy-eating/in-depth/mediterranean-diet/art-20047801
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782861/
http://www.cdc.gov/pcd/issues/2013/12_0256.htm

When they are changed, they often produce unexpected and
unwelcome reactions.
There is no simple formula for success, but ethnic specificity
and cultural sensitivity are important ingredients in crafting
successful nutrition planning and interventions. Table 7 pro-
vides some examples from the recent literature of ethnically
appropriate nutrition interventions.
The expertise of nutritional anthropologists who specialize
in cultural anthropology as it relates to food and foodways is
helpful. Some general principles that must be kept in mind are
discussed in the succeeding text.
Some foods are more important than others to eaters and
are the base of their food systems. Those foods constitute a
continuum or gradient that consists of the ‘core diet,’ which
includes universal, regular staple foods, the ‘secondary core’ of
foods that are used widely but not universally, and ‘peripheral’
foods that are used infrequently or during specific holidays or
celebrations. Such groupings are useful in planning nutritional
interventions since they often suggest opportunities for making
acceptable changes and possible pitfalls that need to be
avoided. Food habits are always difficult to change. The most
resistant habits of all involve foods in the core diet, especially
those that are also viewed by the eaters as ethnically appropri-
ate and vital to group cohesion.
The language used about foods and the names of favorite
foods themselves must be used in recruitment. Ethnic-specific
nutrition information, recommendations, dietary guidance,
and education are also vital if interventions are to be appropri-
ate to the dietary patterns and culture of intended recipients. In
many countries, ethnicity, socioeconomic status, educational
level, geographic location, and religious and ethical views are
often confounded. As these other factors vary, so will the
appropriateness of the intervention.
The importance of including ethnic foods in determining
the ultimate acceptance of nutrition interventions varies within
and between ethnic groups. In general, those who are least
acculturated to the larger mainstream society, those with lan-
guage barriers, the ill, the geographically isolated, the econom-
ically disadvantaged, traditionalists, and the religiously
conservative may regard ethnic foods as particularly important.
Those who are innovators and desire acceptance in the larger
culture may regard ethnic foods as less important. The degree
to which new foods are incorporated into eating styles varies
depending on contact between cultures, food availability,
trade, and social status of the group introducing the food and
philosophical, religious, and individual factors. Immigrants
differ in the extent to which they wish to remain unassimilated
in their foodways and true to their native cuisines. Ethnic
borrowing and mingling rather than rigid preservation of
unique eating traditions often occur.
Ethnic-specific food-based dietary guidelines and graphics
to guide food choices present nutritionally appropriate food
patterns in a culturally acceptable manner for many ethnic
groups. Many guidelines are currently available. They have
the advantage of including ethnic foods as a part of overall
diets. These guidelines are helpful as starting points for devel-
oping population-based nutrition education materials for
healthy persons. Graphics that depict appropriate food choices
have the additional advantage of being understandable even to
those who may be illiterate.
Special attention must be paid to the ethnic appropriate-
ness of nutritional counseling for those who are ill or in
especially vulnerable groups from the physiological stand-
point. Therapeutic diet manuals are sometimes available or
obtainable that can be helpful in designing diets that provide
medical nutrition therapy that is culturally appropriate.
568 Ethnic Foods
Additional useful techniques for crafting nutrition informa-
tion, education, and counseling interventions include the use
of ethnic and regional terms for foods and recipes. Interven-
tions should be adapted to be culturally appropriate. Food
preparation and cooking demonstrations can be used to
show how to incorporate recommendations into existing
food patterns. Nutrition education and counseling should
include attention to ethnic issues. Community social structures
in the ethnic group are helpful to make contacts with the target
ethnic groups.
Those who develop nutritional interventions that involve
food enrichment, fortification, or special supplements must
also pay attention to the role of ethnic foods and food patterns
in the diet. In many cultures, the target groups for such efforts
are those who are nutritionally vulnerable because of their age,
sex, and socioeconomic status. And they are likely to have
eating practices that are deeply embedded in their culture.
Nutrition counselors must remember that care of the sick is
heavily intertwined with food and health beliefs, social
relationships, and acculturation. In summary, nutrition
workers must avoid cultural biases and ethnocentricity in deal-
ing with their clients in both dietary assessment and planning.
See also: Amaranth; Authenticity of Food; Bananas and Plantains;
Cashew Nuts; Cassava: The Nature and Uses; Date Palm: A Wealth of
Healthy Food; Food–Herbal Medicine Interface; Legumes in the Diet;
Quinoa.
Further Reading
Administration on Aging and US Department of Health and Human Services, 2005.
Guidelines for Culturally and/or Linguistically Competent Agencies.
Backstrom A, Pirttila-Backman A-M, and Tuorila H (2003) Dimensions of novelty: a
social representation approach to new foods. Appetite 40(3): 299–307.
Bermudez OI and Tucker KL (2003) Trends in dietary patterns of Latin American
populations. Cadernos de Saúde Pública 19(Suppl. 1): S87–S99.
Bermudez OI and Tucker KL (2004) Cultural aspects of food choices in various
communities of elders. Generations (Journal of the American Society on Aging)
28(3): 22–27.
Broussard-Marin L and Hynak-Hankinson MT (1989) Ethnic food: the use of Cajun
cuisine as a model. Journal of the American Dietetic Association 89(8): 1117–1121.
Kaufman-Kurzrock DL (1989) Cultural aspects of nutrition. Topics in Clinical Nutrition
4(2): 1–6.
Khan LK and Martorell R (1997) Diet diversity in Mexican Americans, Cuban Americans
and Puerto Ricans. Ecology of Food and Nutrition 36: 401–415.
Koehler KM (1989) Core, secondary, and peripheral foods in the diets of Hispanic,
Navajo, and Jemez Indian children. Journal of the American Dietetic Association
89(4): 538–540.
Lee JH, Hwang J, and Mustapha A (2014) Popular ethnic foods in the United States: a
historical and safety perspective. Comprehensive Reviews in Food Science and
Food Safety 13(1): 2–17.
Lemacks J, Wells BA, Ilich JZ, and Ralston PA (2013) Interventions for improving
nutrition and physical activity behaviors in adult African American populations.
Preventing Chronic Disease 10: 120256.
Lin H, Bermudez OI, and Tucker KL (2003) Dietary patterns of Hispanic elders are
associated with acculturation and obesity. Journal of Nutrition 133(11): 3651–3657.
Nestle M (1995) Mediterranean diets: historical and research overview. American
Society for Nutrition 61: 135–205.
Sanjur D (1995) Hispanic foodways, nutrition, and health. Boston: Allyn and Bacon.
Relevant Websites
http://www.nal.usda.gov/fnic/pubs/ethnic.pdf – Food and Nutrition Information Center,
& USDA.
http://stories.illinoisstate.edu/student-affairs/health-promotion-and-wellness/healthy-
habits-holidays-2/ – Illinois State University.
http://www.mayoclinic.org/healthy-living/nutrition-and-healthy-eating/in-depth/
mediterranean-diet/art-20047801 – Mayo Clinic.
http://americanindianhealth.nlm.nih.gov/index.html – National Heart Lung and Blood
Institute.
http://www.nhlbi.nih.gov/health/healthdisp/an.htm – National Heart Lung and Blood
Institute.
http://www.nhlbi.nih.gov/health/healthdisp/aapi.htm – National Heart Lung and Blood
Institute.
http://www.nhlbi.nih.gov/health/healthdisp/lat.htm – National Heart Lung and Blood
Institute.
http://www.nhlbi.nih.gov/health/resources/heart/african-american-index – National
Heart Lung and Blood Institute.
http://oldwayspt.org/ – Oldways Preservation Trust.
http://factfinder.census.gov/faces/tableservices/jsf/pages/productview.xhtml?
pid¼ACS_10_1YR_B04006&prodType¼table – U.S. Census Bureau.
http://www.census.gov/prod/cen2010/briefs/c2010br-02.pdf – U.S. Census Bureau.
 Extrusion Cooking: Chemical and Nutritional Changes
JAG Arêas, CM Rocha-Olivieri, and MR Marques, Universidade de São Paulo, São Paulo, Brazil
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The Extrusion Cooking of Foods
Extrusion cooking has become one of the most prominent
technologies for thermal processing of food for a number of
reasons. It was adapted from the plastic industry and first used
for food processing in the 1920s to cook and model cereals in
several forms. It evolved in the 1970s to the high-shear, high-
pressure, high-temperature machines, which has become ever
since the major process used throughout the food industry to
generate expanded ready-to-eat products and textured vegeta-
ble protein. In the 1990s, modulated double-screw extruders
were developed, and more feed materials with distinct water
content could be processed. However, the most successful
configuration for extrusion cooking is still the one that through
a high shear at high temperature and pressure in a single screw
creates the final texture by orienting and cross-linking the food
macromolecules in a tridimensional stable structure, generat-
ing a wide variety of food products. In this extruder configura-
tion, the feed material, rich in carbohydrates and proteins and
with a low moisture content, is conveyed through an Archime-
des screw within a barrel forming a molten mass that is
expelled through a die. During this process, the molten mass
experiences elevated shear, temperature, and pressure, with
resident times at high temperatures of <5 s. Extrusion is thus
a high-temperature short-time (HTST) food process method
that inhibits most of the biological events but preserves most
of the nutritive value of the processed food. In addition, the
elevated pressure, temperature, and shear promote fragmenta-
tion of the feed material and orientation of the biopolymers
dissolved in the molten mass until the exit of the equipment.
The high pressure inside the equipment keeps the overheated
water, typically around 150
C, in the liquid state within the
molten mass. At the die exit, it then flashes off because of the
sudden drop of pressure, and the mass cools down. The bio-
polymers’ filaments that hitherto flowed into the molten mass
undergo an intense cross-linking, creating a supramolecular
network that stabilizes the final structure of the extruded prod-
uct. For this reason, extrusion has been widely used to cook
and model food in various shapes, generating a great number
of commercial convenient products. These extruded products
are usually stable, with a long shelf life, microbiologically safe,
and with an imparted texture, which increases their acceptabil-
ity. They can be ready-to-eat products or ground and becoming
ingredients that added to diverse products’ formulation.
Because the extrusion cooking process employs elevated
temperatures for short periods, usually below 5 s, several ben-
eficial effects are expected in the extruded food. Nonetheless,
some concern was also devoted to the potential deleterious
effects of this processing to the nutritive value of foods. The
reader should refer to the ‘Further Reading’ section to several
reviews, books, and websites on the extrusion cooking tech-
nology. This article will describe the most common chemical
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00266-X
changes throughout the extrusion cooking process and how
they affect the nutritive value of the extruded food.
The More Frequent Chemical Transformations
in Extrusion Cooking
Cross-Linking and Chemical Changes in Proteins
The diverse composition of the monomers that make up pro-
teins enables a wide spectrum of interactions between proteins
and other macromolecules. In general, the conditions of
extrusion cooking promote raw materials’ native protein disso-
ciation, denaturation, orientation, and cross-linking at the exit
of the equipment. Besides this macromolecular organization
and because of the high temperatures that a protein experiences
inside the equipment, extrusion cooking has been supposed to
produce some potentially toxic amino acid and peptide deriv-
atives, as it was described for other thermal processes. For
example, under alkaline conditions, amino acids may cross-
link during food thermal processing. As the extrusion cooking
process destabilizes the tertiary and quaternary structures of the
proteins by a combination of increased shear and temperature
within the extruder, protein–protein interactions and amino
acid cross-linking may occur. Proteins rich in alanine, lysine,
cysteine, and ornithine are more prone to protein–protein
interaction under specific conditions.
Thermal alkaline processes transform alanine into dehy-
droalanine, which may then react either with lysine to form
lysinoalanine, or with cysteine to form lanthionine, or with
ornithine to form ornithoalanine. Lysinoalanine is the most
common of these compounds, which interferes with growth
and induces changes in nucleus size and cytoplasm volume of
kidney tubules cells in rats. However, all studies on the extru-
sion cooking process showed that there is less lysinoalanine
formed in the extruded foods as compared with similar prod-
ucts prepared by traditional cooking methods. The majority of
reactions of protein cross-linking observed throughout the
extrusion process participate in the formation of the supra
molecular structure that stabilizes and texturize the extruded
products. Extrusion is safe as to the production of toxic com-
pounds derived from thermal decomposition of amino acids.
The intensity of these cross-links may also be dependent on
the other components from the food matrix and on the protein
functionality. The content and type of protein may produce
distinct structure resistance after extrusion as observed when
amaranth or chickpea flour are treated with sodium glutamate
and inosinate. The extrusion cooking of amaranth pasta mix-
tures may also cause a decrease of protein solubility in different
denaturant solvents in the presence of starch; and all this
knowledge may be used for technology improvements. How-
ever, none of these changes affect the nutritive value of the final
extrusion product nor produce any health threat.
569
570 Extrusion Cooking: Chemical and Nutritional Changes

Hopper
Breaker
plate
Die Barrel Feedthroat

Screw

Feedpipe
Screw drive
motor

Figure 1 Schematic representation of a typical single-screw extruder. Ambient pressure and temperature built up to the typical maxima of 5 MPa
and 150
C, respectively, from the feed hopper to the die end. The residence time of the feed material at these conditions is typically below 5 s. Public
domain picture. Author: Magnus Manske. http://commons.wikimedia.org/wiki/User:File_Upload_Bot_(Magnus_Manske).

(a) (b)

Figure 2 Examples of single-screw (a) and double-screw (b) extruders. From: Schweizer, J. P. (2011) The Right Extruder: Design Considerations -
http://www.foodmanufacturing.com/articles/2011/05/right-extruder-design-considerations.

Extrusion of bovine rumen protein with 3.8% of residual
lipid, for example, produced more structured extrudates with
higher content of disulfide linkages and noncovalent interac-
tions than those with less lipid content. Lung protein shows
similar results, indicating an important role of the lipid, espe-
cially phospholipid content. These findings are consistent with
some of the proteins becoming inaccessible upon thermal
treatment and with the reorganization of the macromolecules
at the die end favoring disulfide and noncovalent interactions.
Again, these changes only affect texture with no effect on the
nutritive value of the extruded food.
Chemical Changes in Carbohydrates
Extrusion cooking is a process where occur moderate carbohy-
drate hydrolysis and intensive starch gelatinization through
cleavage of intermolecular hydrogen bonds. For this reason,
extrusion causes an increase in water absorption until the
rupture of starch granules, which occurs during this process,
creating a viscous mass. The action of high pressure, high shear,
and high temperature, combined with the moisture content
and the amylose of the raw material, may subject the processed
material to significant changes.
One of the most important modifications undergone by the
material rich in carbohydrates and proteins, after extrusion, is
expansion. The rate of expansion is very important for an
extruded product, and for most of the cereal starches, such as
corn, wheat, and rice, the product has a maximum expansion
when subjected to temperatures between 150 and 200
C. The
gelatinized starch, the cellular protein, and the cellulose
encompass a complex that decisively influences the product’s
expansion ability after the mass passes through the die.
The extrusion cooking purpose is to transform starchy raw
materials, whose main feature in their original form is an
insoluble granular material, in a crunchy, fluffy, expanded
final product that easily breaks. Therefore, gelatinization rate

(a)
(b)
(c)
Figure 3 Examples of snacks and biscuits (a), breakfast cereals (b),
and pet food (c), produced by single-screw extrusion cooking.
Promotional photographs. Source: (a) http://www.kishanagrotech.com/;
(b) http://www.clextral.com; (c) http://www.diytrade.com.
measurements and the effects of extrusion are essential. Thus,
some physical tests are used, including viscosity, product
expansion in the die, shear strength, amylograph tests, consis-
tency index, and expansion ratio. There are also chemical tests,
such as gelatinization index, water absorption index (WAI),
water solubility index (WSI), and aqueous ethanol-soluble
carbonate and water-soluble carbohydrate.
During the extrusion cooking process at high temperatures
between 120 and 180
C and pressures from 3 to 16 MPa,
partial starch hydrolysis may occur. It appears that because of
thermoplastic treatment, starch flour has a variation in its
color; the milk-white flour at temperatures between 120 and
135
C turns dark brown at 150–180
C. At temperatures of
120–135
C, starch becomes completely gelatinized, but at this
point, hydrolysis does not occur yet. This will be only achieved
at higher temperatures and pressures. The consequence of this
Extrusion Cooking: Chemical and Nutritional Changes 571
high-temperature and high-pressure combination is the cleav-
age of 1,2-glycoside bonds of sucrose, 1,4-glycoside bonds of
raffinose, and 1,4-glycoside bonds of maltodextrins. The
higher the extrusion temperature and lower moisture content
of the raw material, the greater is the bond cleavage within the
molecules with high molecular weight.
For evaluation of the final extruded product, two factors are
quite important and shall be taken into account: the WSI and
the WAI. The conclusions found by many studies on these
properties were that the WAI increases for a wide variety of
starch products with the temperature rise in the extruder barrel.
The lower is the material initial moisture content used for
extrusion, the higher is the extrudates’ WSI.
The extrusion of starchy raw materials results in important
changes in product viscosity after dissolution in water. This
property is relevant for a technological point of view, but its
impact on nutritional properties is not clear. All processes that
increase starch solubility may increase its glycemic index (GI).
However, several cooking processes for amaranth showed that
the GI of starch of the extruded amaranth was not the highest
one. Even increasing starch solubility, the extrusion produces a
complex network made of starch and protein that may hinder
access of amylolytic enzymes to the food matrix. A more
detailed account of the impact of extrusion on the GI is in
Section Changes in GI of this article.
Chemical Changes in Lipids
Generally, the raw materials used throughout the extrusion
process contain low amounts of lipids. The friction necessary
for mechanical energy transfer to the extruded material is
essential for food extrusion. Large amounts of lipids cause
slippery at the barrel and impair the process.
Some lipid hydrolysis occurs in lipid extrusion by lipase
action. However, even high levels of free fatty acids in foods
have no toxicological effects on humans, although organolep-
tic and storage quality of the extruded foods may be impaired.
The proper combinations of humidity and heat are responsible
for reducing the lipase activity in the extrusion process,
decreasing the factors favoring free fatty acids’ production.
The inactivation of enzymes that occur during the extrusion
process promotes increased shelf life of the final product.
Fatty acids’ oxidation may also occur in a limited extent.
Some conditions in the extrusion cooking process that may
affect the rate of lipid oxidation, which are similar to the ones
found in any other process, are listed in the succeeding text:
(1) The rate of oxidation increases with the amount of oxygen
present and the surface area of the food exposed.
(2) Higher temperatures produce faster reaction rates.
(3) Oxidation rate is retarded at intermediate moisture con-
tents of water activity (Aw ¼ 0.3), but the rate is higher at
water activities below 0.1 and above 0.55.
(4) Light and metals act as prooxidants.
(5) The presence of antioxidants reduces the rate of oxidation
by terminating free radicals or by sequestering metals.
Chemically, the double bonds of unsaturated fatty acids can
easily react with free radicals and oxygen. Oxygen adds to a
carbon–carbon double bond in a chain of unsaturated fatty
572 Extrusion Cooking: Chemical and Nutritional Changes
acids forming hydroperoxides. These compounds break down
rapidly in the presence of some substances, factors, and process
conditions that affect the rate of lipid oxidation. Contents up
to  5% of total lipids improve expansion ratio and texture,
whereas the formation of trans fatty acids at this lipid concen-
tration is almost insignificant. As the lipid content of the raw
material for extrusion is typically below this content, extrusion
negligibly affects lipid oxidation.
Other Chemical Changes
During the extrusion cooking process, there is a strong
structural molecular rearrangement. The intense disturbance
considerably influences the shape and texture of the extruded
products. One such chemical change that occurs is the
amylose–lipid complex formation. The interest in amylose–
lipid complexes focused on their technological importance in
starchy food systems. A consequence of these chemical changes
is the low lipid extraction using solvents after extrusion.
At low hydration levels, the linear component of starch,
amylose [a 1–4]-glucose-linked polymer, behaves as a flexible
random coil with stretches of left-handed helical segments in
aqueous solution. Stable secondary conformations, known as
A-, B-, and V-amylose, are formed both in ionic solutions and
in solvents of lower polarity. In the presence of lipids, the
amylose chain undergoes a conformational change to V-amy-
lose, characterized by the interaction of the aliphatic lipid tails
with the hydrophobic core of the polysaccharide, forming a
very stable imperfect helix.
These amylose–lipid complexes turn up when the material
is between its melting and glass transition temperatures. The
barrel temperature and moisture within the feed material are
determinant in the process; the water acts as a plasticizer, and
the decrease of moisture content increases the temperature.
The presence of other components in the food matrix
increases the melting temperature of starch–lipid complexes,
which are also present at high barrel temperatures.
Moreover, the presence of triglycerides, diglycerides, and
different fatty acids’ saturation in the lipid fraction may result
in lower expansion, decreased degree of gelatinization of
starch, reduced dough viscosity, distinct dough flow properties,
and decrease of the hardness of the extrudate.
Nutritional Consequences of the Chemical Changes
Increasing Digestibility of Amino Acids
Comparable to other HTST processes, extrusion is the preferred
choice regarding nutrient retention, destruction of antinutritional
factors, microorganisms, and capability of producing different
textures. However, the intense high-pressure homogenization
can break down chemical bonds and promote new chemical
reactions that alter the protein digestibility. Mild heat treatment
of vegetable proteins generally improves digestibility, and
with increasing severity of temperatures, protein digestibility
decreases.
In some studies, the decrease of the true digestibility was
evident after extrusion. The process caused protein denatur-
ation with formation of insoluble compounds through new
protein–protein interactions that were responsible for the
stabilization of the structure. Intermolecular disulfide and
other hydrophobic bonds appear to be the major type of
linkages that occurs during extrusion and influence extrudate
characteristics. These results occurred due to previously hidden
amino acid residues becoming exposed and free to react with
other food components. The exposure of hydrophobic residues
reduces the solubility of extruded protein in aqueous systems,
decreasing digestibility.
However, most studies have demonstrated that the protein
digestibility improves because of the denaturation of protein,
which renders them more susceptible to digestion by proteo-
lytic enzymes. Extruded cowpea beans, a protein-rich legume,
showed an increase of 55.9% digestibility measured in vitro as
compared to the raw grain. Thus, extrusion enhances the
digestibility of proteins through denaturation and by inactiva-
tion of protease inhibitors.
Loss of essential amino acids
The amount of essential amino acids is an important indicator
of nutritional quality of extruded products. Process conditions
of extrusion influence the digestibility of the proteins and
affect the availability of certain amino acids. Thus, no doubt
that lysine is the most unstable amino acid during the extru-
sion process. Several chemical reactions, combined or not, are
responsible for amino acid losses, such as protein denatur-
ation, Maillard reaction, and protein cross-linking.
Increasing energy input and decreasing water cause signifi-
cant reduction in the availability of several amino acids. Loss of
unstable amino acids in the extrusion cooking process was
reported in the literature to be 30–40% lysine, 7–21% argi-
nine, 15% histidine, 8–21% cystine, 14% methionine and
tryptophan. During extrusion cooking of maize grits, below
14.5% water, there was also a decrease in cystine. Only minor
losses were found in other amino acids.
The nature of the extruded protein also influences the
extent of the loss of the available amino acids. In meat sources
of protein, bovine rumen, for example, extrusion did not affect
the concentration of very sensitive essential amino acids like
cystine and methionine, and the occurrence of any other lim-
iting amino acid in the extruded protein was not observed.
Maillard reaction
The Maillard reaction is one of many chemical reactions that
occur during the extrusion cooking process and has important
nutritional and functional consequences. Known as non-
enzymatic browning, Maillard reaction is actually a series of
reactions with a wide variety of compounds produced as a
result. This chemical reaction is characterized by the binding
of a reducing sugar (glucose, fructose, lactose, or maltose) and
a free amino group on an amino acid, usually the epsilon-
amino group of lysine.
Because of this, the main nutritional loss related to this
reaction is the quantity of available lysine. Data reported
throughout the literature indicated that small peptides from
the matrix can also be involved in this reaction. Another indi-
cation of Maillard reactions is the dark color of the extruded
material, due to the formation of melanoidins and other reac-
tion products; however, a darker color is not necessarily due to
the imperative presence of these materials.

Extrusion cooking appears to cause lysine losses that do not
exceed those for other methods of food processing (loss range
3–13%), but appears to depend on the raw material used. In
semolina pasta, for example, lysine is involved to loss of a
greater extent with respect to other amino acids in the range
of 43.9–62.0%. In food with lysine limitation, this loss is
important for the nutritional quality.
Extreme conditions of temperature (>180
C) and rotation
(>100 rpm) in combination with low moisture (<15%) pro-
mote the breakdown of poly- and oligosaccharides and release
reducing units of the molecule. On the other hand, an increase
in feed rate promotes less lysine loss due to a residence time
decrease and heat transfer to the mixture. Conditions of higher
pH, reducing sugar hydrolysis in the extrusion of mung bean
and cowpea flours, are also favorable for lysine retention.
For quality control purposes, the amount of available lysine
in an extrudate may be quantitatively measured through sev-
eral methods. The reaction of free lysine with fluorodinitro-
benzene is one of these. Total lysine may also be measured
after acid hydrolysis of the protein, and the amount of unavail-
able lysine can be calculated.
Although researchers are still unsure of the precise mecha-
nisms by which acrylamide is formed in foods, there is a strong
relationship between Maillard reaction and acrylamide forma-
tion on extruded products. Temperatures above 120
C, very
low moisture content of 5%, higher asparagine content, and
reducing sugars are favorable parameters. It has been suggested
that the butanedione, one of the several dicarbonyl com-
pounds formed during the Maillard reaction (Strecker degra-
dation), in the presence of free asparagine can produce high
amounts of acrylamide. On the contrary, glycine, L-lysine, and
L-cysteine added to formulation can reduce the formation of
acrylamide (95%, 91%, and 87%, respectively), and they can
be used as additives of the extrusion process.
Hydrolysis of Carbohydrates
Redistribution of soluble and insoluble fiber
Few studies have been performed about the effect of extrusion
cooking or other processes on dietary fiber. The degradation of
the components of dietary fibers may occur in processes that
involve shear. It is known that the glycosidic bonds of cellulose
can be broken during dry grinding in ball mill. It is also known
that degradation in the colon of the fiber is inversely propor-
tional to its particle size. Therefore, the disruption and homog-
enization of the particles due to the intense mechanical
working during extrusion could leave the dietary fiber more
available for fermentation in the colon.
The thermoplastic extrusion causes structural changes in bio-
polymers, with significant transformation in polysaccharides.
Because of this, the structure and composition of the fiber frac-
tion experience a shift in the molecular weight of constituent
polysaccharides. In general, one observes an increase between
10% and 15% in the content of the soluble dietary fiber (SDF)
fraction at the expense of the insoluble one. Enzymatic or acid
treatments can increase this proportion, being an additional tool
in achieving higher levels of soluble fiber.
A very important effect brought about by the extrusion
process is the degradation of phytic acid, in addition to rear-
ranging components of total dietary fiber (TDF), which would
Extrusion Cooking: Chemical and Nutritional Changes 573
improve the nutritional properties of food after this process.
The heat applied during the extrusion process modifies the
texture of raw foods, which can consequently alter the compo-
sition and the structure of these components.
In some foods, such as oat bran, the extrusion process
improves the functional properties of SDF. When one com-
pares the SDF raw oat bran with SDF-extruded oat bran, some
characteristics have been changed in the extruded product.
They are the increased aggregate formation, the increased gela-
tinization temperature, the increased solubility index, the
water absorption capacity, and water retention solvent. An
increase in the apparent viscosity and the consistency
coefficient is also observed, besides a decrease in the flow
behavior index, a decreased radial expansion, an increased
bulk density and breaking strength of extrudates, and an
improvement in foam ability.
Studies have also reported an increase in TDF of barley flour
after extrusion. This is due to the increase either in one or in
both fractions of fiber. These changes in the profile of dietary
fiber during the extrusion process can be explained by the
resistant starch formation that is determined as dietary fiber,
‘enzyme-resistant indigestible glucans,’ formed through trans-
glycosidation, and a structural transference from insoluble
to SDF.
Digestibility of starch
Many changes may be observed in the functional properties of
the starch due to the extrusion process. These changes can be
monitored by physical and chemical methods in the starch
granules and their components. They include the determination
of textural and rheological properties, which are related to the
digestibility of the starches and availability of glucose. Among
the many food processing methods, the extrusion cooking is the
one that mostly increases the rate of starch digestibility, due to a
decrease of particle size of the final product. The variation and
the digestibility rate will depend on some parameters, such as
amylose and amylopectin ratio, the feed material particle size,
the process temperature, the moisture of the raw material, and
the screw speed and geometry.
During extrusion, starch is exposed to high shear forces that
physically cleave glycosidic linkages, forming fragments of
lower molecular weight. Inside the extruder, the starch granule
is compressed and will slowly turn into a homogeneous and
molten mass where the disappearance of granular and crystal-
line structure is observed. However, the extent to which the
starch will break down depends upon the intensity of treat-
ment. The degree of digestibility of starch is an important
property of the extrudate, and it appears that the in vitro digest-
ibility of starches is significantly higher after the extrusion
process.
The structural integrity of the starch granules is lost due to
increased shear action and molding in the extruder barrel. This
makes the starch more available for enzymatic action. Never-
theless, low levels of starch digestibility observed in some
extruded foods with a high starch content can be attributed
to the formation of amylose–lipid complex, starch–protein,
and the limited availability of water, which impairs the starch
digestibility during the enzymatic hydrolysis. The decrease in
the grain size distribution results in increased surface area and
a higher degree of hydrolysis.
574 Extrusion Cooking: Chemical and Nutritional Changes
Extrusion potentially produces greater increase in starch
digestibility than any other processing methods. However,
some studies comparing four processing method – popping,
roasting, flaking, and extrusion – found that the rate of hydro-
lysis of starch is greater by popping, roasting, and peeling than
by extrusion.
Changes in GI
Values of the GI of several products and the respective glycemic
response can be influenced for many factors; and processing
plays a very important role among them. When the cellular
structure of the starch granule is preserved even after heat
processing, the final product keeps its GI, which is lower than
when the starch granule is broken. Nongelatinized starches are
less digested by humans, so heat processing is beneficial for a
complete starch digestion. After the heating and shearing dur-
ing the extrusion process, the starch granules are cooked and
broken down, and the crystalline structure of the pellet is
reduced, resulting in partial gelatinization and depolymeri-
zation. This will result in a viscous fluid of starch dispersion
in water. Extrusion processing conditions such as temperature,
moisture, and screw speed, among others, will affect the phys-
ical and chemical structures of starch and, subsequently, the
IGs of the final extruded products. Therefore, snack amylac-
eous products usually have higher GIs than the corresponding
raw materials.
Most of the extruded products are basically composed of
cereals. Thus, they have a high GI. Various studies have been
performed to increase the dietary fiber content after extrusion,
thereby lowering their glycemic response. They shed some light
on the mechanisms by which dietary fiber lowers the glycemic
response. In these studies, the fibers seem to coat the starch
granules, protecting them against the action of the amylolytic
enzymes and reducing the degradation of starch, eventually
producing a low glycemic response. There is also a physiolog-
ical effect of the soluble fiber fraction, increasing gut fluid
viscosity and reducing glucose absorption and hence the GI.
With increased dietary fiber content, it is possible to
enhance the amount of slowly digestible starch components
in processed food with reduced rate and degree of glycemic
response. The use of whole-wheat flour instead of the refined
flour in the extrudates reduces the amount of rapidly digestible
starch components and increases the quantity of slowly digest-
ible starch in extruded cereal-based breakfast. In the extrusion
process, starch is subjected to high shear forces that chemically
break glycosidic linkages forming fragments of lower molecu-
lar weight. Thus, this smaller branched starch forms new link-
ages, which are more resilient and, therefore, present lower GI.
These structures are considered resistant starch and greatly
contribute to the TDF content of the extruded food product.
Loss of Essential Fatty Acids and Vitamins
Extrusion of food can be a possible adverse effect with the
destruction of vitamins during processing. The loss or reten-
tion of vitamins in extruded foods can vary according to the
type of food, screw rpm, die diameter, moisture content, resi-
dence time at high temperatures, time and temperature of
processing, and throughput. In general, in lower temperatures,
the losses are minimal. In usual extrusion conditions at high
temperatures for a short time and with quick cooling of the
product during output, a relatively small loss of most of the
essential fatty acids and vitamins is observed. For example,
there are a 95% retention of thiamine and a small loss of
riboflavin, pyridoxine, niacin, and folic acid in cereals at tem-
peratures as high as 154
C.
Vitamins A and C are normally lost by oxidation and heat.
The retention of vitamin C is about 70% in extruded foods,
while the retention of vitamin A ranged from 50% to 140%
after extrusion. These differences are due to the intrinsic sus-
ceptibility of each vitamin and because vitamin A fortification
of extruded products is performed through a series of
b-carotene derivatives with distinct heat stability.
Other Nutritional Changes
Extrusion can maintain the levels of the lycopene in extruded
enriched snacks, if the source used to enrich is the tomato skin
powder.
The extrusion process can reduce the content of tannin that
sometimes is undesirable in food. The extrusion of flaxseed, for
example, showed 77% reduction of tannins.
A 13–35% reduction in phytate content occurs after the
extrusion of a wheat bran–starch–gluten mix. Phytate is known
to reduce absorption of iron and other essential minerals.
Fortification of extruded products must be performed care-
fully. The added minerals should have high bioavailability,
they should also be homogeneously mixed with food
ingredients, and these must be compatible with the processing.
Calcium salts, for example, reduce the expansion of snacks. The
mineral used may also be compatible with the postextrusion
storage. The bioavailability of the iron is generally not affected
by the extrusion process, and highly bioavailable iron is
observed after processing of leguminous seeds.
Overview of the Extrusion Cooking Effects on
Nutritional Value
In short, the extrusion process is widely used throughout the
food industry during recent years; and there are several nutri-
tional aspects of this thermal processing that should be taken
into account.
Among the positive aspects, the following can be featured:
(a) The possibility of developing a vast range of versatile prod-
ucts ready for consumption, with a long shelf life, since
various enzymes are inactivated, as lipases and lipoxidase.
(b) The destruction of antinutritional factors and microorgan-
isms is effective and the final product is safe with respect to
the production of toxic compounds derived from thermal
decomposition of amino acids.
(c) The transformation of starchy materials in a crunchy,
fluffy, expanded product that easily breaks with a final
pleasurable product with texture.
(d) The enhancement of the digestibility of proteins through
denaturation and by inactivation of protease inhibitors.

(e) The promotion of an increase of SDF fraction content and
degradation of phytic acid, in addition to rearranging
components of TDF.
(f) Extrusion process promotes a greater increase in starch
digestibility than any other processing methods.
The negative aspects of extrusion are minimal but should be
considered:
(a) Extruded snack products tend to have a higher glycemic
response in comparison with the raw materials, although
the increase in the dietary fiber content of the extruded
products can lower their glycemic response.
(b) Vitamins are destroyed during processing, with vitamin C
being the most affected, but in lower temperatures, the
losses are minimal.
(c) The availability of several amino acids is reduced; lysine
is the most unstable amino acid during the extrusion
process.
(d) There is a strong relationship between Maillard reaction
and acrylamide formation on extruded products.
See also: Amaranth; Browning: Non-enzymatic browning; Cooking:
Domestic Techniques; Extrusion Cooking: Principles and Practice;
Fatty Acids: Fatty Acids; Functional Foods; Oxidation of Food
Components; Proteins: Chemistry, Characterization, and Quality;
Quality Control in Food Processing; Starch: Modified Starches; Starch:
Sources and Processing.
Extrusion Cooking: Chemical and Nutritional Changes 575
Further Reading
Arêas JAG (1992) Extrusion of food proteins. Critical Reviews in Food Science and
Nutrition 32(4): 365–392.
Camire ME, Camire A, and Krumhar K (1990) Chemical and nutritional changes in foods
during extrusion. Critical Reviews in Food Science and Nutrition 29(1): 35–57.
Kokini JL, Ho CT, and Karwe MV (1991) Food extrusion science and technology.
New York: Marcel Dekker Inc.
Mitchell JR and Areâs JAG (1992) Structural changes in biopolymers during extrusion.
In: Kolkini JL, Ho CT, and Karwe MV (eds.) Food extrusion science and technology,
pp. 345–360. New York: Marcel Dekker Inc.
Mulla MZ, Bharadwaj VR, Annapure US, and Singhal RS (2011) Effect of formulation
and processing parameters on acrylamide formation: a case study on extrusion of
blends of potato flour and semolina. LWT - Food Science and Technology
44: 1643–1648.
Prudêncio-Ferreira SH and Arêas JAG (1993) Effect of phospholipid on protein-structure
and solubility in the extrusion of lung proteins. Food Chemistry 47(2): 111–119.
Thachil MT, Chouksey MK, and Gudipati V (2014) Amylose-lipid complex formation
during extrusion cooking: effect of added lipid type and amylose level on corn-based
puffed snacks. International Journal of Food Science and Technology 49(2): 309–316.
Zhang M, Bai X, and Zhang Z (2011) Extrusion process improves the functionality
of soluble dietary fiber in oat bran. Journal of Cereal Science 54(1): 98–103.
Relevant Websites
http://en.wikipedia.org/wiki/Food_extrusion – Wikipedia.
http://foodqualityandsafety.wfp.org/extrusion-cooking-for-fortified-blended-foods –
The World Food Programme (United Nations system).
https://www.google.com.br/search?q¼extrusionþcookingþofþfood&client¼firefox-
a&hs¼Jz7&rls¼org.mozilla:pt-BR:official&channel¼sb&tbm¼isch
&tbo¼u&source¼univ&sa¼X&ei¼jEkgVP33OPDjsASmvYGYAQ
&ved¼0CEIQsAQ&biw¼1920&bih¼945 – Google images of extrusion.
http://www.wisegeek.com/what-is-extrusion-cooking.htm – wiseGeek.
 Extrusion Cooking: Principles and Practice
L Moscicki, Lublin University of Life Sciences, Lublin, Poland
ã 2016 Elsevier Ltd. All rights reserved.
Extrusion-Cooking Technology
Extrusion technology, which is well-known in the plastics
industry can be modified for a wide variety of extrusion-cooking
applications in the food and agriculture industries, for which it
produces ‘engineered’ food and special feed. Generally speaking,
the extrusion cooking of raw vegetable materials involves the
extrusion of ground material under barothermal conditions,
with controlled moisture content, so that the shear energy,
which is exerted by the rotating screw and/or additional heating
of the barrel, heats the food material to its plasticizing or
melting point. In this changed rheological status, the food is
conveyed under high pressure through a die or a number of dies,
and the product expands to its final shape. As a result, the
extrudates have physical and chemical properties that are very
different from those of the raw materials. Food extruders, which
are often called extrusion cookers, belong to the family of high-
temperature short-time equipment, because they perform under
high pressure (up to 20 MPa) and high temperatures (up to
200
C) for only a short time (typically tens of seconds). This
form of processing restricts unwanted damage to nutrients such
as proteins, amino acids, vitamins, and enzymes. The process
conditions and raw materials used have a strong impact on the
food and feed properties during extrusion cooking, however,
and they can drastically influence the final product quality.
A modern food extruder is a process reactor that creates
different extrudates by varying screw layout, mixing elements,
clearances in the gaps, and the installed motor power and
barrel heating, in order to control the dough reaction. For
example, altering the process parameters and the combined
effects of heat and shear affects the degree of starch gelatiniza-
tion or denaturation of proteins in the presence of water.
Extrusion cooking is now used for the manufacturing of a
diverse array of foodstuffs (Figure 1), including the following:
Figure 1 Various extrusion-cooked foodstuffs and feedstuffs.
576 Encyclopedia of Food and Health
• Direct-extruded snacks, such as ready-to-eat (RTE) breakfast
cereals, differing in shape, color, taste, and texture
• Snack pellets: products destined for fried or hot
air–expanded snacks, precooked pasta
• Baby food, precooked flour, instant concentrates, functional
components
• Pet food, aquafeed, feed concentrates, and calf-milk
replacers
• Texturized vegetable protein or meat analogs (mainly from
soybeans)
• Crisp bread, bread crumbs, emulsions, and pastes
• Confectioneries: different kinds of sweets, gums, and jellies
• Barothermally processed products for the pharmaceutical,
chemical, paper, and brewing industries
Extrusion cooking can use raw materials that have not previ-
ously displayed great economic importance (e.g., legumes) or
have been regarded as waste. Of practical importance, the pro-
cess can also be implemented with relatively low effort, does not
require excessive capital investment, and uses equipment that
can be employed for multiple applications. The energy con-
sumption of food extruders is in the range 0.08–0.20 kWh kg1.
Thus, in many cases, food extruders provide a cost-competitive
alternative to other machines used for food and feed
manufacturing.
The direct extrusion of cereal snacks provides a good exam-
ple of extrusion-cooking technology. Figure 2 presents the
standard installation used in this process.
Preparation of Raw Material
Direct-extruded snacks are most often manufactured from pop-
ular cereal-based materials, such as corn, wheat, and rice.
Depending on its quality, the material must be properly ground
and weighed, according to the recipe, and mixed thoroughly
before being fed to the extruder. If preconditioning is required,
water and/or steam is added to the material before mixing.
Extrusion Cooking
Extrusion cooking is based on the concept that, after the mate-
rial leaves the die, the sudden decrease in pressure allows the
superheated water in the material to turn into steam, leading to
a rapid expansion of the material. This expansion is responsi-
ble for the porous structure of the extrudate, with the structure
shaped by bundles of molten protein fibers. The melt mass
leaving the extruder generally takes the shape of the extruder
dies. A rotary knife is installed outside the die, in a lengthwise
arrangement, and the appropriate setting of the knife speed
controls the product length. This allows for the production of
different, fancy shapes of extrudates, such as balls, stars, shells,
and rings, even when using a simple single-screw food
extruder. The technology for the production of each shape
requires an appropriate distribution of temperature, pressure,
http://dx.doi.org/10.1016/B978-0-12-384947-2.00265-8
 Extrusion Cooking: Principles and Practice 577

1
3
3 3

4 9
2 13

10

5 14
6 11

12
8
7
2

2
Figure 2 A diagram of a standard installation for the production of direct-extrusion snacks. 1, raw materials; 2, pneumatic conveyer; 3, collector; 4,
weight; 5, mixer; 6, preconditioner; 7, food extruder; 8, cutter; 9, dryer; 10, screen; 11, recycling of dust; 12, coating drum; 13, finished product;
14, packaging.

and moisture content in the material during processing. sorption, wet strength, dry strength, cooking loss, and viscosity
Therefore, the operator must be focused on flexibility and behavior after extrusion. A more detailed description of the
precise control, especially in regard to the thermal process. properties of starches and proteins also requires additional
Frequently, the process for the manufacturing of specific prod- chemical data, including dextrose equivalents, reaction rate
ucts must be developed empirically. constants, and data describing sensitivity to enzymatic degra-
dation. The food engineer and technologist must be able to
forecast the relationship between these properties and their
Drying, Coating, and Packing dependence on the extruder variables. Therefore, it is necessary
to provide a (semi)quantitative analysis of biopolymers in the
After extrusion, the extrudates are dried to a moisture content
extrusion-cooking process, which can be done by adopting an
of about 6–8% and cooled when necessary. Drying can be
engineering point of view, with the extruder considered to be a
performed with simple rotating drums installed with electric
processing reactor.
heaters, or it can use a gas-operated hot air installation working
at temperatures just above 100
C. Larger installations rely on
belt dryers, heated by gas-fired heat exchangers or steam, with
Food Extruders
hot air circulating through the unit sections. Cooling takes
place at the ambient temperature, as air flows through the
As previously mentioned, extrusion cooking involves machines
perforated belt of the cooler device. The flavor and vitamin-
in which the main operative body is one screw or a pair of screws
coating step is often integrated in drum dryers. Flavor
fitted in a barrel. During barothermal processing, the material is
additives, liquored or powdered, come in a wide variety of
mixed, compressed, melted, and plasticized in the end part of
forms, from smoked meat flavor to paprika aroma.
the machine (Figure 3). The range of physical and chemical
Popular direct-extrusion breakfast cereals include coated
changes in the processed material principally depends on the
cereal balls, rings, or shells in an icing sugar coating. The
parameters of the extrusion process and the construction of the
industrial production of this type of product requires the use
extruder.
of an additional high-performance drum dryer or belt dryer, so
There are many conventional methods for classifying food
that the application of the coating is a continuous process, thus
extruders, but the most practical accounts for the following
maintaining fixed product quality.
three factors.

Quality Parameters • The method of generating mechanical friction energy con-

The literature has described the influence of various extruder
variables, such as screw speed, die geometry, screw geometry,
and barrel temperature, on product quality. A number of mea-
suring methods are used to obtain the desired physical prop-
erties of extrudates. The most important are bulk density, water
verted during extrusion into heat: autogenic, isothermic
(heated), polytropic (mixed)
• The amount of mechanical energy generated: low-pressure
extruders producing relatively limited shear rate and
high-pressure extruders generating large amounts of
mechanical energy and shear
578 Extrusion Cooking: Principles and Practice

1 2 3 4 5 6 4 7 4 8 9 10

I II III

Figure 3 A cross section of a single-screw food extruder: 1, engine; 2, feeder; 3, cooling jacket; 4, thermocouple; 5, screw; 6, barrel; 7, heating jacket;
8, head; 9, dies; 10, cutter; I, transport zone; II, compression zone; III, melting and plasticizing zone.

• The construction of the plasticizing unit, with both the
barrel and the screw designed as a uniform, integrated
body or fixed with separate modules
In 1946, in the United States, single-screw extruder were devel-
oped to cook and expand corn and rice snacks. In combination
with an attractive flavoring, this product type is still popular,
and the use of single-screw extruder equipment to produce such
snacks is, in principle, is still the same. A wide variety of
extruder designs can be employed for this purpose. The old
method of cutting preshaped pieces of dough out of a sheet
with roller-cutters is also still in use, however, because the
complicated shapes of snacks require very expensive dies and
die heads for cooking and forming extruders. Although modern
techniques offer improved control of mass flow in food
extruders, new extruders with better mixing and a more steady
mass flow often show advantages over the traditional single-
screw equipment. In the mid-1970s, twin-screw extruders were
introduced for the combined process of cooking and forming
of food products, partly as an answer to the restrictions of
single-screw extruder equipment, because twin-screw extruders
provided a largely forced flow. Twin-screw extruders also tend-
ed to produce better results on scale-up from the laboratory
extruder types in use for product development.
During the processing of biopolymers, nearly all chemical
changes in food are irreversible. Continued treatment after such
an irreversible reaction in an extruder should be a process
controlled for temperature, time, and shear, leading to a series
of completely different functional properties of the produced
food. From a rheological point of view, two facts are the most
important. First, non-Newtonian flow behavior usually occurs
in food extruders. Second, chemical reactions occur during the
extrusion-cooking process (e.g., gelatinization of starch or
Maillard reactions), strongly influencing the viscosity function.
The rheological behavior of the product, which is relevant to
the modeling of the extrusion process, has to be defined directly
after the extruder screw tip, before expansion has occurred, in
order to determine the influence of water losses, the consis-
tency of the material, and temperature effects due to the flash-
ing process that occurs directly after leaving the die.
Today, the food extruder is integrated into the technological
line as a single- or twin-screw reactor, with the preheating step
performed in a specially built preconditioner. The forming task
of the extruder can also be separated from the heating and
shearing steps. In this case, the final shaping and forming are
done in a second, well-optimized post-die forming extruder,
which largely processes the food at a lower water level than is
present in the first reactor extruder. This method produces
cooked, preshaped, unexpanded food pellets, which can be
used for the production of RTE cereal flakes (e.g., popular
corn flakes) or as half product in the manufacturing of fried
snacks.
Single-Screw Food Extruders
The design of single-screw extruders is relatively simple. In
food extruders, sticking effects are prevented by the force of
friction between the material and the barrel wall, and this
friction is facilitated by the suitable grooving of the inside of
the barrel (longitudinal or spiral grooves).
These devices process relatively easy materials characterized
by a high friction coefficient, such as maize or rice grits (i.e.,
basic materials for the production of direct extrusion snacks or
breakfast cereals). For these materials, the use of the simplest
autogenic extruders is often sufficient, even at a low ratio of
screw length L to diameter D (L/D) ¼ 4–6. The main disadvan-
tages of single-screw extruders are poor mixing (as a result,
materials should be mixed before feeding) and limited effi-
ciency, especially when multicomponent mixtures of raw
materials are used. Smooth positive movement of the material
in a single-screw food extruder depends on the actual drag
flow, which is caused by the screw geometry and its rotation
minus the so-called back flow.
The operator must strictly follow the technological regime
during operation, which is determined experimentally or given
by expert technicians. A number of issues must be taken into
consideration in daily extruder operations:
• The moisture content and particle-size distribution of the
raw material mixtures must be properly chosen and
homogeneous. This prevents shooting or blocking in the
extruder and ensures the desired quality of the extrudates.
• Intensive cooling of the barrel (e.g., with cold water) con-
tributes to a lower processing temperature and increases the
friction inside the material. It must be correlated with the
quality requirements of the extrudate. A temperature drop
in the material raises its viscosity and positively contributes
to the extruder’s performance.
• The blocking of a few die holes results in a sudden increase
in pressure and leads to a powerful back flow, or even

lockout of the machine. In such a case, immediately
cleaning the blocked holes with a thin tool might correct
the problem. If this does not help, the operator must stop
the machine and dismantle the die. Postponing the disas-
sembly of the die with a plasticized material inside it may
lead to permanent damage to the equipment during the
next start-up.
• The smallest holes in the die cause a higher resistance
during the extrusion of the material, because smaller open-
ings increase pressure and reduce the extruder’s output,
given the higher incidence of back flow.
• Applying a plasticizing screw with a greater L/D ratio can
generate more extrusion pressure resulting from a longer
fully filled screw, which leads to improved plasticizing of
the material and reduced back flow.
• The loss of a defined clearance between the barrel surface
and the screw flights hampers the movement of the mate-
rial, reduces friction and pressure, and causes poor opera-
tion of the machine, which means poor product or no
product at all.
Twin-Screw Food Extruders
These machines are much more complex in terms of design but
much more universal in operation. However they are more
expensive. Nonetheless, twin-screw extruders have become
very popular with producers of extrusion-cooked foodstuff,
because of their ability to process a wider range of materials
(e.g., viscous and hard-to-break materials) and their lower
energy consumption, broadening the list of products the
machines can produce. Corotating food extruders are also
used to a greater extent because of their high productivity and
efficient material transportation, mixing, plasticizing, and
extrusion. The self-wiping and intermeshing flights of the
screws effectively force the material forward, and, as a result,
no material is locked in the space between the surface of the
barrel and the screw. Popular corotating twin-screw food
extruders most often produce RTE breakfast cereals, pet food,
and aquafeed.
The physical processes and heat exchange associated with
material transfer in twin-screw food extruders are more com-
plicated than they are in single-screw extruders. Readers who
wish to learn more about the engineering aspects of extrusion
can find detailed information in the literature. The extrusion-
cooking technique is fairly complicated, and its proper use
requires considerable expertise on the part of the operators.
Yet, when operators are properly trained, extruders can allow
food companies to vastly broaden their product offerings.
Counterrotating twin-screw food extruders are special-
purpose devices. In these machines, the screws rotate slowly,
but they can mix the material effectively, and their work resem-
bles a positive-displacement pump, generating high pressure
on the screws in the barrel’s closed C-shaped chamber. This
approach is especially suitable for high viscosity material. The
back flow of material in these extruders is very small because of
the tiny clearances between the screws and the barrel. They are
mainly used for the production of confectionery and chewing
gum, as well as the processing of fiber- and cellulose-rich
materials.
Extrusion Cooking: Principles and Practice 579
Figure 4 Modern multifunctional corotating twin-screw food extruder
with an axially open barrel.
Twin-screw food extruders now have a modular
construction, with the screws built from several different
elements mounted on the screw shaft. These elements handle
transporting, mixing, compressing, and melting the material
(Figure 4). By properly setting these elements, the operator can
control the behavior of material inside the extruder, thus
influencing the scope of the physical and chemical processes
during the extrusion-cooking process.
Extruders often produce a wide range of products, from
simple maize snacks to protein-based food. Snack production
requires high pressure and mechanical energy at a low L/D ratio.
Protein-based material needs long processing times, with many
intermediate stages applied. Modular screws allow for these
possibilities, either by the disassembly of the barrel units in
combination with the replacement of the screws or by changes
in the screw lay-out. Some food-extruder producers offer a
mobile feeder installed on the feed ports between the center
and the end of the barrel. In such cases, distance rings are placed
on the screw shafts ahead of the final set of screw elements.
During the operation of extruders, the engine load must be
maintained at an adequate level (maximum 80–85%). As a
result, the screw’s rotational speed, torque, and energy con-
sumption (expressed in kWh per 1 kg of product) must be
controlled. The proper choice of extruder machinery for the
type of production is also important for a successful operation.
Yet, no established and strict recommendations currently exists
regarding the range of temperature in different zones of the
plasticizing section of food extruders. Some temperature ranges
can be recommended during the processing of certain raw
materials, but, in the case of a particular machine, these ranges
should be verified empirically, or they should be based on the
manufacturer’s recommendations.
In the daily operation of food extruders, the start-up and
close-down of production causes difficulties. This is an impor-
tant job for the operator because it is relatively easy to block the
machinery. Cleaning the plasticizing unit, after it has stopped,
requires patience and time. If the stop procedure is incorrect,
permanent damage to the equipment can occur. On the other
hand, the start-up procedure is relatively uniform. After the
extruder heats up and reaches the desired temperature in indi-
vidual zones of the plasticizing section, the engine begins run-
ning at one-third of the nominal rpm level. Highly moisturized
material is then fed into the machine. Subsequently, the
580 Extrusion Cooking: Principles and Practice
machine performance is increased step by step, while the oper-
ator closely watches the outflow of material and the engine load.
After a few minutes, the machine reaches its nominal operation
parameters. The close-down must be done in the reverse order,
and finally, the operator completely cleans the machine after
taking down the die head, using some coarse material (e.g., oats)
for the final cleaning of the plasticizing section. The machine
will not restart when the working parts and the head are dirty.
Producers of extruded foodstuffs now prefer to fully control
the production process. In the case of high-volume production
rates, this level of control is indispensable, because only a
properly programmed control system is fast enough to govern
the production flow, responding immediately to failures of the
working equipment. Modern food extruders can easily replace
many conventional food-processing units, and these extruders
are capable of helping the industry develop new series of
products. The market expects new foodstuffs that are fancy in
shape, taste, and raw material composition, as well as attractive
from an economic point of view. As previously mentioned,
extrusion cooking can texturize proteins from vegetable
sources to create textures resembling those of meat, allowing
for healthier (low fat), palatable, and more sustainable prod-
ucts. Extrusion-cooking technology can meet these expecta-
tions, but food technologists need specialized knowledge.
See also: Acrylamide; Aerated Foods; Bread: Types of Bread; Cereals:
Types and Composition; Cooking: Domestic Techniques; Extrusion
Cooking: Chemical and Nutritional Changes; Functional Foods; Heat
Treatment: Principles and Techniques; Maize; Rheological Properties of
Food Materials; Snack Foods: Role in Diet; Snack Foods: Types and
Composition; Starch: Modified Starches; Starch: Sources and
Processing; Starch: Structure, Property, and Determination; Wheat:
Grain Structure of Wheat and Wheat-based Products.
Further Reading
Bouvier JM and Campanella OH (2014) Extrusion processing technology. Oxford:
Wiley.
Frame ND (ed.) (1994) The technology of extrusion cooking. Glasgow, England: Blackie
Academic & Professional.
Guy R (2001) Extrusion cooking, technologies and applications. Boca Raton, FL: CRC
Press Inc.
Harper JM (1981) Extrusion of foods. Florida: CRS Press.
Janssen LPBM (1978) Twin screw extrusion. Amsterdam: Elsevier Scientific Publishing
Company.
Janssen LPBM and Moscicki L (eds.) (2009) Thermoplastic starch. Weinheim: Wiley-
VCH Verlag GmbH & Co. KGaA.
Jowitt R (ed.) (1984) Extrusion cooking technology. Essex: Elsevier Applied Science
Publishers.
Mercier C, Linko P, and Harper JM (1998) Extrusion cooking. St. Paul, MN: American
Association of Cereal Chemists, Inc.
Moscicki L (2003) Effect of screw configuration on quality and SME value of corn
extrudate. Teka commission of motorization power industry in agriculture, vol. III,
182–186.
Moscicki L, Mitrus M, and Wojtowicz A (2007) Technika ekstruzji w przetworstwie
rolno-spozywczym (in Polish). Warsaw, Poland: PWRiL.
Moscicki L (ed.) (2011) Extrusion-cooking techniques. Weinheim: Wiley-VCH Verlag
GmbH &Co. KGaA.
Riaz MN (ed.) (2007) Extruders and expanders in pet food, aquatic and livestock feed.
Clenze: Agrimedia GmbH.
Van Zuilichem DJ (1992) Extrusion cooking. In: Craft or science?. The Netherlands:
Wageningen University PhD thesis.
Relevant Websites
http://www.bakerperkins.com/cereal.
http://www.bakerperkins.com/snack/products/snack-products.
http://www.buhlergroup.com/global/en/process-technologies/extrusion-dough-
preparation.htm#.U9j-qPl_vTp.
http://www.pavan.com/product.asp.
http://www.wenger.com/.
F
Famine, Hunger, and Undernourishment
R Milà-Villarroel, Nutrition without Borders and University of Vic-Central University of Catalonia, Barcelona, Spain
C Homs, Nutrition Without Borders, Spain
J Ngo, Nutrition Research Foundation, Barcelona, Spain; Nutrition Without Borders, Barcelona, Spain
J Martı́n, Nutrition Without Borders, Barcelona, Spain
M Vidal, Nutrition Without Borders, Barcelona, Spain
ã 2016 Elsevier Ltd. All rights reserved.

Introduction error to think that death from starvation or famine is homogenous;

At present, the worldwide population is estimated to be around
7 billion people. During the twentieth century, however, it is
estimated that more than 70 million died due to food crises.
Nowadays, at the height of the twenty-first century, the food
crises (famine) continue as a global humanitarian tragedy. These
crises are recognized and are mainly located in zones of the
African continent, such as in the Horn of Africa where an
endemic form persists. This does not mean, however, that
other regions of the world are free from the risk of food crises.
Generally, the risk persists primarily in developing countries
with deficient economic transformations combined with prob-
lems of political instability, natural disasters, wars, and/or social
conflicts. Moreover, these crises persist in concrete zones that are
poorly integrated into worldwide trade, as well as in urban and
rural areas whose public services lack the capacity to attend the
poorest and most vulnerable groups. However, although these
determinants can be found in different parts of the world and in
distinct moments, the current food crises concentrate mainly in
sub-Saharan Africa and some regions of South Asia.
Hunger, be it transient or endemic, is a historical reality
whose incidence continues being decisive at the present time.
Although the statistics on malnutrition and food insecurity
have greatly improved in the last decade, the most recent
reports of the Food and Agriculture Organization of the United
Nations (FAO) on the state of food insecurity in the world
indicate that much remains to be done. Hunger has fatal
repercussions in the life of millions of people; as part of the
process, it deprives its victims of health, causes massive migra-
tions to other countries as people seek sustenance, and aggra-
vates and/or causes illnesses such as malnutrition, AIDS,
tuberculosis, and malaria. In addition, it is both a source and
a consequence of instability, poverty, and underdevelopment
as attested to by the fact that the majority of armed internal
conflicts happen in countries with precarious food conditions.
One must ponder: Why, if it was possible to end famine
in hunger-prone countries such as Russia, China, India, and
Bangladesh during the twentieth century, due to the technical
advances in food production, do people still to continue dying of
hunger? Surely, this is because hunger is a phenomenon much
more complex than it seems on its face. In any case, it would be an
in fact, the causes and consequences of food insecurity greatly
differ between countries and even between different regions within
the same country. As such, a general theory explaining the periods
of hunger and famine cannot exist, and thus, each one of these
periods should be treated in a unique manner.
Definitions for the Discussion of Famine
To better understand the context of famine, it is necessary to
clarify some concepts associated to the term. Distinctions need
to be made among hunger, malnutrition, famine, and food
insecurity.
Hunger
Hunger is defined here as a condition resulting from an
individual’s inability to eat sufficient food to lead a healthy
and active life. It compromises a series of feelings, emotions,
and behavioral changes, not easily measurable in themselves
that result from disruption in an individual’s access to food.
Naturally, hunger is a recurring feature of food insecurity of
households and their members in developing countries.
Malnutrition
Undernutrition, by contrast, is defined as the measureable
nutrient deficiencies in a diet that can lead to illness (lack of
energy, retardation, and blindness) or death. The symptoms
themselves may not be recognized as indications of nutrient
deficiencies since interactions between undernutrition, care
behavior, and diseases are complex.
Famine
Famine is a widespread and extreme hunger that for individ-
uals results in a drastic loss of body weight and an increase in
morbidity. At the community level (as an interaction of these
two symptoms), a rise in the death rate and massive social
dysfunction and dislocation result. Famine is a process of socio-
economic crisis, relatively prolonged over time, and consists in
the progressive impoverishment of the most vulnerable groups

Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00269-5 581

582 Famine, Hunger, and Undernourishment
and the deterioration of their systems of sustenance, with
an increase in massive hunger. The process also implies popu-
lation movements, the propagation of epidemics, community
degradation, and, in severe cases, an increase in mortality that
results more from related epidemics than to starvation, itself.
Therefore, food crises (famine) are a catastrophic split of social,
economic, and institutional systems that provide food for pro-
duction, feeding, and consumption. For many years, these crises
have been considered as anomalies, crises that could be resolved
in a brief period of time and for which the previous situation
existing before the crisis could be reestablished. Nowadays, this
vision of a transient stage has been dispelled, as it is known that
food crises (famine) can destroy not only lives but also future
development of the community/region.
Food Insecurity
Food insecurity is a situation that exists when people lack secure
access to sufficient amounts of safe and nutritious food for
normal growth and development and an active and healthy
life. It may be caused by the unavailability of food, insufficient
purchasing power, inappropriate distribution, or inadequate use
of food at the household level. Food insecurity, poor conditions
of health and sanitation, and inappropriate care and feeding
practices are the major causes of poor nutritional status. Food
insecurity may be chronic, seasonal, or transitory.
Famine Mortality
The most serious manifestation of starvation is increased mor-
tality, although food crisis does not occur with the same inten-
sity or in the same way in all cases. Mortality data associated
with food crises also reveal that they do not affect all people
equally. From a biological and socioeconomic focus, the high-
est mortality rates are seen in vulnerable groups. First affected
are children under five and the elderly living in poor condi-
tions and in remote rural areas far from the cities. However,
data recorded in the great famines indicate a complex interac-
tion between biological vulnerability and socioeconomic vul-
nerability. The latter is closely associated with the cultural,
social, and economic contexts of each region or country.
The death toll in the severe famines is only approximate
estimates. The reason for not being able to set an exact number
of deaths is mainly due to the lack of official records of births
and deaths in some developing countries as well as basic
demographics of the population (population census and mor-
tality rates during nonfamine years). On the one hand, excess
mortality rates are estimated for population groups monitored
by the national population census, and the rates are compared
to the ‘normal’ population before the famine. On the other
hand, mortality is a parameter that does not assess the total
demographic impact of famine, as the falling birth rate may
have consequences on population size. This increased mortal-
ity in famines is due to two main factors. First, there is the effect
of starvation that brings about weight loss and the subsequent
undernutrition that puts individuals at increased risk for dis-
eases. Second, as a result of undernutrition, there is increased
susceptibility to infectious diseases as a result of weakened
immune systems. Furthermore, there is increased exposure to
epidemics unrelated to food (e.g., typhoid, cholera, measles,
and malaria) due to the lack of sanitation and hygienic
conditions caused by displacement and overcrowding in unsa-
nitary settings and the lack of potable water. Both circum-
stances imply that refugee camps are locations that generally
register the highest mortality rates.
Although the overall data indicate that famines throughout
history have produced a high number of deaths, in relation to
the national population of the countries where they occur, this
has rarely exceeded 2–3% of the total population. During
twentieth century, there were between 70 and 80 million
deaths. From the recorded data, a pattern of geographic and
seasonal appearance can be established. It seems that over the
decades, famines have been happening in the northern hemi-
sphere (Russia) and Asia (China, India, and Bangladesh), mov-
ing toward sub-Saharan Africa, where even today, it remains
endemic. In addition, this geographic shift has been associated
with a decrease in mortality. During the pre-1960 period, the
nine famines that occurred in Russia, China, and India led to a
total of 66 million deaths. However, the 16 famines that have
occurred since the 1960s in Africa have caused 10 million
deaths. This reduction is due to several advances made during
the twentieth century. In the first half of this period during the
colonial administration, vulnerability to famine in Asia, and to
a lesser extent in Africa, decreased because of improved com-
munications and transportation. Markets that were previously
isolated were linked with larger markets, and warning systems
were established and policies implemented to help those
affected by famine. In some colonized countries, decoloniza-
tion brought disparate effects. On the one hand, in countries
like India, decolonization produced greater government
involvement in the eradication of hunger through policies of
job creation or the delivery of humanitarian aid, both of which
led to excellent results. On the other hand, in many African
countries, decolonization was associated with the de novo
appearance of famines, induced by increases in political and
economic instability and civil wars. Moreover, in many cases,
drought and the lack of water for crops worsened the problems
of hunger in many of these countries. Thus, during the 1980s
and 1990s, some countries that had never been prone to fam-
ine such as Sierra Leone, Zaire, Angola, and Mozambique and
others that had previously suffered famine such as Sudan
and Ethiopia suffered major famines due to armed conflict
and/or drought. Today, in the Horn of Africa, the equation
drought þ war ¼ famine is still very much in vogue.
Current State of Global Food Insecurity
FAO’s latest estimates indicate that reducing global hunger
continues on the right track. It is estimated that approximately
805 million people were chronically undernourished during
2012–14. These figures represent a decline of more than 100
million in the last decade and of about 209 million over the
period 1990–92. However, one in nine people worldwide still
lacks sufficient food to lead healthy and active lives. The vast
majority of malnourished people live in developing countries
(791 million people), although it is in these same countries
where most of the improvements in the last two decades have
occurred (FAO). The decrease in the proportion of hungry peo-
ple has been approximately 7.4 percentage points from the
period 1990–92 to 2012–14, which has reduced the proportion
of people affected by hunger from 18.7% to 11.3% (Figure 1).
 Famine, Hunger, and Undernourishment 583

Prevalence (%) of undernutrition in developed and developing regions

25.00%
23.40%

20.00%
18.20%
18.70% 17.30%
14.50%
15.00%
14.90% 14.30% 13.50%
12.10%
10.00% 11.30%

5.00% 5.00% 5.00% 5.00% 5.00%

5.00%

0.00%
1990–92 2002–02 2005–07 2008–10 2012–14
World Developed regions Developing regions

Prevalence (%) of undernutrition in Africa

35.00%

30.00% 33.30%
29.80%
27.70% 26.50%
25.00%
25.20% 24.40% 23.80%
20.00% 22.60%
20.90% 20.50%
15.00%

10.00%
5.00% 5.00% 5.00% 5.00% 6.00%
5.00%

0.00%
1990–92 2002–02 2005–07 2008–10 2012–14
Africa Northern Africa Sub-Saharan Africa

Prevalence (%) of undernutrition in Western and Caucasian Asia

25.00% 23.70%

23.20%
20.00% 17.60% 17.40%

16.00% 14.10%
15.00% 12.70%
14.10% 15.30% 15.30%
11.30% 12.70%
10.00%
10.80%
9.50%
7.40%
5.00%

0.00%
1990–92 2002–02 2005–07 2008–10 2012–14
Asia Eastern Asia Caucasus and Central Asia

Figure 1 Trends in undernutrition prevalence rates.

Continued
584 Famine, Hunger, and Undernourishment

Prevalence (%) of undernutrition in Southern and Eastern Asia

35.00%

30.00% 30.70%

25.00% 23.20%
24.00% 22.30%
20.20%
20.00% 23.70% 18.50% 16.30%
18.30% 15.80%
17.60% 17.40% 14.10%
15.00% 12.70%
16.00% 15.30% 13.40%
10.00% 12.70% 10.80%
10.30%
5.00%

0.00%
1990–92 2002–02 2005–07 2008–10 2012–14
Asia Southern Asia Eastern Asia South-eastern Asia

Prevalence (%) of undernutrition in Latin America and Caribbean

30.00% 27.00%
24.40% 23.70%
25.00% 20.70% 20.10%
20.00%
14.40%
15.00% 10.70%
15.30% 7.70%
10.00% 6.10%
11.50% 5.10%
5.00% 8.70%
7.00% 6.10%
0.00%
1990–92 2002–02 2005–07 2008–10 2012–14

Latin America and Caribbean Latin America Caribbean

Prevalence (%) of undernutrition in Oceania

18.00%
16.00%
16.50%
14.00% 15.70% 15.40%
12.00% 13.50% 14.00%

10.00%
8.00%
6.00%
4.00%
2.00%
0.00%
1990–92 2002–02 2005–07 2008–10 2012–14
Oceania

Figure 1—Cont’d

This decline has been much more significant in developing
countries, where for the same period, the decrease was almost
10 percentage points (from 23.4% in 1990–92 to 13.5% in the
period 2012–14). Therefore, it seems that the hunger goal stated
in the Millennium Development Goals (MDGs) for 2015,
“reduce by half the proportion of undernourished people” is
within reach, but not the objective of the World Food Summit
(WFS) of “halving the number of chronically underfed people.”
According to estimates, it appears that by 2015, the proportion of
undernourished people would be 12.8%, a rate 1.1 percentage
points higher than that expected by the MDG (11.7%). However,
according to experts, increasing efforts in the most affected areas
(sub-Saharan Africa and South and West Asia) would make it
possible to accelerate the pace of reducing hunger. Nevertheless,
the estimate of people affected by hunger in 2015 is around
791 million, a far cry from the 500 million that constituted the
goal of 1996 World Food Summit.
Africa
In Africa, there are a total of 226.7 million undernourished
people (20.5% of undernourished people worldwide). The
data show that progress in reducing hunger in Africa, mainly
sub-Saharan area, has not been sufficient. Still, there have been
significant improvements from the period 1990–92 when the
prevalence of undernutrition in sub-Saharan area was 33.3%
and the prevalence for the period 2012–14 was 23.8%. In total,
it is estimated that in the period 2012–14, the number of
undernourished people in sub-Saharan area was 214.1 million
people. The lack of political stability and wars are the major
causes for less progress in sub-Saharan Africa compared to
other at-risk regions. A different situation is observed in
North Africa, where the prevalence of undernutrition was
observed to be less than 5% in all periods since 1990–92;
during the period 2012–14, there was an increase in undernu-
trition reaching 12.6%, for a total of 12.4 million undernour-
ished people. This increase rate can be attributed mainly to the
inclusion of Sudan into the region of North Africa.
Asia
Globally, Asia has a total of 525.6 million undernourished
people, representing 65% of the world’s undernourished pop-
ulation. Although globally, Asia is close to meeting the Millen-
nium Development Goals on hunger, it is the area with the
highest numbers of undernourished individuals, and there are
significant differences between the regions that make up this
area. The Caucasus and Central Asia, East Asia, and Southeast
Asia are the regions where most progress has been observed
over the last 20 years. The decreases in undernutrition rates
have been over 50% for West Asia (from 23.2% in the period
1990–92 to 10.8% in 2012–14). In Caucasus and Central Asia,
the decreases had been from 14.1% in 1990–92 to 7.1% in
2012–14, and a 66.5% decrease was observed in the case of
Southeast Asia (30.7% in 1990–92 and 10.3% in 2012–14).
Still and all, it is worthy to note that in the West Asia region,
there is a total of 161.2 million undernourished people.
Moreover, the worst scenarios are observed in West and
South Asia. In the region of South Asia, although progress
since 1990–92 has been seen, undernutrition rates of 15.8%
(with a total of 276.4 million people having food access
Famine, Hunger, and Undernourishment 585
problems) are still far from the Millennium Development
Goals. The situation is worse in West Asia where, albeit under-
nutrition rates are not as alarming, there is much concern about
the developments in recent periods where rates have gone from
6.3% in 1990–92 to 8.7% in 2012–14. This increased preva-
lence of undernutrition is also reflected in the number of under-
nourished people, increasing from 8 million in 1990–92 to 18.5
million in 2012–14, showing an increase of more than 10
million undernourished individuals. Deterioration of the econ-
omies and high political instability are the main causes of the
increase in rates in recent years in the West Asia region.
Latin America and the Caribbean
The Caribbean and especially Latin America presented the
most progress of all the developing regions in relation to
increased food security. This region has already met the Mil-
lennium Development Goals and is close to achieving the
target set by the WFS. In total, the region has in 2012–14 an
undernutrition rate of 6.1% (a total of 37 million undernour-
ished people), a decrease from the 68.5 million in 1990–92.
Latin America’s progress in improving food security has been
faster than in the Caribbean. In Latin America, the prevalence
of food insecurity for 2012–14 is 5.1%; in the last 20 years, the
total number of undernourished people has decreased from
60.3 million in 1990–92 to 29.5 million in 2012–14. In the
Caribbean, this reduction has been slower, declining from
27% in 1990–92 to 20.1% in 2012–14.
Oceania
Oceania is the region that has the least number of undernour-
ished people in the world (1.4 million representing 0.17% of
the world’s undernourished population). The prevalence of
undernutrition has varied little since 1990–92, showing only
a 1.7% decrease. The main concern in the area is that the rates
of undernutrition have been accompanied by a greater burden
of overweight and obesity. As such, the region suffers from a
considerable double burden of malnutrition.
Theories of Hunger
Hunger as a collective phenomenon has always existed, which
throughout history has involved different regions of the planet
and during given periods of time. It is a result of various factors:
poor harvests, wars, economic crises, and natural disasters.
The hunger of our time has the peculiarity that it is concentrated
in certain regions and countries; it now can be considered
as chronic and endemic, that is, not only due to natural disas-
ters or wars. For this reason, it is important to study the theo-
retical frameworks that have been developed to explain the
worldwide phenomenon. In recent decades, several different
explanatory paradigms have emerged, attempting to explain
the causes of famines. These can be grouped into five distinct
models:
Model 1. Demographic Theories (Malthusian and Neo-
Malthusian Theories)
The relationship between hunger, production and food
availability, and the population is the core of the reductionist
586 Famine, Hunger, and Undernourishment
Box 1 Criticisms of the Malthusian Theory
(a) Malthus wrote thinking of the English agricultural situation in the eigh-
teenth century when it was based on subsistence agriculture.
(b) Malthus failed to foresee the agricultural, transport, industrial revolution,
and demographic transition.
(c) Geographically marginal land is not necessarily less fertile, even if it is the
furthest away.
(d) Famine did not act as a ‘Malthusian leveler.’
theories of Malthus and the neo-Malthusians. The focus of
these theories was based on the fundamental consideration
that the amount of food production (production or food avail-
ability) and the number of people (population) are the only
two variables that determine the global food situation and
therefore the existence – or not – of hunger in the world.
From the relationship between both elements, the acceptance
of hunger as a fact that has to happen consistently was derived,
since the number of people grows geometrically, whereas the
amount of available food grows arithmetically. In this formula,
Malthus affirmed that in order to avoid hunger, a decrease in
the growth rates of the population was essential so as to
increase food availability. Currently, there are some authors
who keep the Malthusian theory alive, and they conceive hun-
ger as a result of the competitive relationship between the two
variables mentioned earlier: food production and population
growth and poverty. In other words, according to these princi-
ples, the causes of hunger are the lack of food and high rates of
population growth. Based on these theories, the balance
between food availability and population would be restored
through famines, epidemics, mass migration, and reduced fer-
tility. These theories were popular in the 1960s and 1970s
when the perception that the world was running out of food
resources was on the rise. Today, Malthusian theories have
evolved with new arguments that differ from the original the-
ory. New proposals differ from the original Malthusian argu-
ment because the constraint on population growth is
represented not only by arable land and food scarcity but
also by the availability of water, energy supply, and raw mate-
rials, as well as by land and air pollution. From this point of
view, the neo-Malthusian approach is strongly supported by
increasing the awareness of the need to protect the environ-
ment and its resources. The Malthusian approach is susceptible
to four criticisms outlined in Box 1.
Despite these critiques, these theories could still be consid-
ered quite current as they focus on the safety and availability of
natural resources and on nonpolluted air and water, thus
emphasizing the environmental and social constraints of our
planet.
Model 2. Climatic Theories (Based on the Decrease in
the Availability of Food or Food Availability Decline)
The food availability decline (FAD) approach assumes that
famines are caused by a sudden reduction of per capita food
supply. It is usually triggered by natural disasters (drought,
floods, pest infestation, etc.), wars, and epidemics that lead to
a contraction of the food supply. As a consequence, food prices
go up and people, who are not able to bear such an increase,
consume less calories and nutrients. Consequently, the most
vulnerable individuals start reducing their food consumption
because of rising prices. In the case of prolonged exposition to
a crisis, this may culminate in increased mortality due to
starvation and infectious diseases. This model points out the
insufficient production and availability of food as the main
causes of famines and starvation. This approach implicitly
assumes an equal division of the available food, but unfortu-
nately, such an assumption fails to reflect reality. The FAD
hypothesis implies that food security is essentially a matter of
expanding food availability. As a solution, it argues to increase
the supply and consequently the availability of food. This
solution has been strongly criticized by Amartya Sen. However,
he recognizes that famines can be caused by food availability
decline and he also assumes this hypothesis for its entitlement
approach.
Model 3. Food Intervention Decline
This new framework, the so-called food intervention decline
(FID), originates from the recent and growing awareness that
governments, and more generally political institutions, human-
itarian agencies, and nongovernmental organizations (NGOs),
have the responsibility to protect all citizens by promoting direct
public interventions. This model approach argues that people
starve because food policies and services fail to guarantee a
sufficient level of nutrition. When these policies and services
decline or disappear, persons start suffering and severe under-
nutrition may occur and consequently famine appears. This
approach identifies as main actors all those institutions that
are responsible for the production and implementation of mea-
sures to secure nutrition and healthy feeding.
The FID approach suggests a number of different policies
that may be broadly grouped into two categories, depending on
their target. One constitutes the policies established to prevent
undernutrition in at-risk zones. Examples are improving the
conditions of public health and the warning systems to prevent
the crisis, reducing social inequalities and social cohesion,
applying policies of price protection for basic foods, and forti-
fying the systems of education, health, and social services. The
other category includes policies establishing fast-responding
systems to deal with the appearance of food crises. Examples
consist of introducing free or price-reduced food consignments
into markets, establishing policies to control prices and subsi-
dize the food supply, creating food reserves (i.e., cereals), carry-
ing out direct interventions, and improving communication and
transport infrastructures.
All these interventions should be implemented in a
planned and carefully thought-out manner in accordance
with the nature of the problems and the underlying causes of
the famine. One of the main risks is to damage economies with
abundant food supplies, further depressing food prices affect-
ing agricultural incomes; the outcome could be further reduc-
ing the disadvantaged population’s purchasing power.
Model 4. Sen’s Food Entitlement Approach
As a response, mainly to the theories of Malthus and the neo-
Malthusians, some authors elaborated theories that deter-
mined that famines and hunger were not produced by a lack
of food in the market, but rather by the poverty of given social
sectors that deprived them of food access. These criticisms gave

place to a new approach, known as food entitlement, proposed
by the Indian economist Amartya Sen in his famous 1981 work
Poverty and Famines. This was decisive for the formulation of
the concept of household food security, which focused more
on families than on countries (the concept of national food
security was changed to household food security). The entitle-
ments are the capacities that a family has to access food by legal
means, be it by production, purchase, or receiving food in the
form of donations from the state or the community. Sen dem-
onstrated in diverse studies that famine was not frequently due
to a lack of supplies, but rather to a sudden loss of entitlements
in poor families, until reaching a point that food requirements
could not be met. He understood famine as a temporary con-
vulsion of the economic system. According to this theory, the
political answer to famines resided in the struggle against the
poverty. One of the advantages of Sen’s theory on famine is
that it is not a simple theory, but a model that is generalizable
and can explain different models of famine. In some way, Sen’s
model assumes as its own the models of FAD and of FID. In the
first case, it understands that the descent in food availability
can increase the price of foods and, as such, leads to impaired
food entitlements. FAD’s role is assumed to be one more factor
out of many that are related to entitlements. In the same way,
when Sen’s model suggests to adopt measures to improve food
purchases and food assistance, it assumes the hypotheses of the
food intervention decline (FID) model.
The fundamental criticism of Sen’s theory is that it assumes
that famine is like a sudden collapse in food consumption that
causes a particularly virulent form of starvation and involves
widespread death. That is to say, it would be considered as a
onetime and sporadic event, which is distinct from hunger and
whose appearance is characterized by an increase in mortality
due to starvation. In contrast, authors like Amrita Rangasami
and Alex de Waal have shown that famine is not so different
from hunger, but rather is a process that begins with hunger as
its starting point and, in the most severe cases, can lead to
massive death. In fact, the majority of famines nowadays are
characterized by a not so extreme starvation, thanks to the
palliative mechanisms of the family’s distinct coping strategies.
At present, it is not the increase in mortality that is the most
worrisome factor, but rather the misfortune and social pertur-
bation that is caused. As such, the victims perceive hunger not
so much as a life-threatening biological process but rather as a
socioeconomic process of impoverishment that threatens their
systems of livelihood. Therefore, it understands that Sen’s def-
inition of famine only refers to the last phase of the famine and
not to the entire process. In fact, authors like Purusottam
Nayak criticize Sen for limiting his explanation to the imme-
diate cause of famine, such as the loss of entitlements, but not
including background causes or dynamics once the process is
initiated. That is to say, Sen explains how famines are produced
but does not explain why or what causes them to happen.
Another critical vision of his theory addresses the exces-
sively economic vision of the problem, which centers only on
the loss food access and on subsequent starvation, but omits
other essential factors of famines like the impact of epidemics
on increased mortality, violence, wars, and population move-
ments or social destruction. Sen also contemplated famine
victims as passive subjects, ignoring their own capacities for
implementing coping strategies to mitigate the effects of fam-
ine. Sen himself indicated that his theory had some
Famine, Hunger, and Undernourishment 587
limitations, which could be summarized in two aspects: on
the one hand, a failure to recognize individuals as socially
embedded members of households, communities, and states
and, on the other hand, a failure to recognize that famines are
political crises as much as they are economic shocks or natural
disasters.
Sen’s theory, when centering on circumstantial economic
appearances, specifically on the relationship between markets
and people, tends to ignore the structural, historical, and polit-
ical factors. Therefore, his theory is soundly criticized for lim-
iting itself to measures of a technical nature, suggesting
solutions such as providing employment. Thus, resources are
generated to help those that have temporarily lost their enti-
tlements or both, but it does not contemplate structural trans-
formations that would eliminate the underlying structural
vulnerability.
Model 5. Complex Emergencies (Humanitarian and Political
Emergencies)
From the mid-1980s, certain authors posed a distinct vision of
the etiology of famines, based on political causes instead of
economic or demographic determinants. Authors such as Alex
de Waal, David Keen, and Mark Duffield are the maximum
exponents of this explanatory political model of famines. For
these authors, the victims of the famine are defined not by a
lack of economic but rather political power. Famines are seen
as political phenomena derived from the lack of government
initiatives and even of policy practices causing famines, such as
ethnic persecution, forced population displacement, and deny-
ing humanitarian aid.
Famine is a result of the failure of a ‘political contract’
between the country’s leaders and the population. This agree-
ment would impose obligations on the politicians, certain
obligations to guarantee the right to food. When this tacit
agreement exists, the apparition of famine can generate grave
political consequences, even including the downfall of govern-
ment or sanctions for its leaders; and as such, there is keen
interest in impeding the appearance of famines. In those coun-
tries that do not guarantee the civil rights of their citizens,
famines do not concern governing leaders, and therefore, the
fight against hunger is not a priority. This phenomenon has
mainly been observed in countries with authoritarian regimes
like Russia, China, or Ethiopia in the decades of the widespread
famines or in civil wars where such a political agreement does
not exist, as seen in distinct countries of the Horn of Africa.
Besides, these authors indicate that in any famine, there are
some sectors that become impoverished, but there are also
those that become wealthy, such as traders, landowners of
big agricultural farms, and soldiers taking advantage of the
hunger-weakened poor. Therefore, the analysis of famines
should also be done from the perspective of those that exert
political, economic, or military pressure, to benefit from the
transfer of resources.
When famine appears as a result of military conflicts, it may
be the case where hunger in this context is being used as a
weapon of war. Thus, war becomes the main cause of famines,
as it destroys crops and food production systems, generates
massive migrations, paralyzes health services, prevents or hin-
ders possible family coping strategies, and prevents the distri-
bution of humanitarian aid. From this perspective, famine is
588 Famine, Hunger, and Undernourishment
not so much the unavoidable consequence of a crisis, but
rather a deliberate aim caused and sustained by determined
social powers for taking ownership of resources from the most
vulnerable. This allows for the clear definition of political
responsibilities in famines and goes beyond the mere eco-
nomic factors proposed by Sen or the demographic changes
defended by Malthus. This new approach understands that the
famines are unleashed by a lack of political responsibility on
the part of governments and for their noncompliance with
fundamental human rights, especially the right to food.
Synthesis of the Famine Models
In short, none of these approaches are sufficient in and of
themselves to explain the nature of famines. On the one
hand, the Malthusian and neo-Malthusian explanations have
lost strength due to the fact that their suppositions are no
longer valid, although it is true that the concern for the envi-
ronment and resources of the planet is in the center of current
political debate.
On the other hand, the economic approach is for the time
being what dominates among the authors. The vision of fam-
ine is seen as a form of economic disaster heightened by some
natural catastrophe (drought, floods, etc.) and facilitated by
the lack of policies to protect markets, prevent hunger, and
intervene in states of emergency. By not considering the polit-
ical aspects in relation to wars or the related violence of armed
conflicts and by not respecting human rights, the vision of
famine is reduced to a technical problem that requires techni-
cal measures for improving the efficiency of food systems.
Finally, the political approach goes beyond mere economic
circumstances and deals with famine as a political problem of
not respecting fundamental human rights, of power conflicts
and wars, of repressing designated population groups, and of
the passivity of the international community in confronting
the issues. In these cases, the solution demands interventions
not only that are of an economic nature and addressing pro-
ductive market shares but also that focus on the international
level to respect human rights.
Conclusions
Nowadays, already well into the twenty-first century, many
people from different parts of the world continue suffering
from famine. The famine seen today is different from the
widespread famines of the past, as political, economic, and
social contexts have changed. Although the triggers and effects
are similar, the causes are much more extensive and much
more complex. Famine appears as a complex phenomenon of
factors, distinct in nature: economic, political, environmental,
and social. This multiplicity of factors confirms the high com-
plexity of the problem and therefore also hampers the election
of the best responses in each case. Therefore, the policies of
prevention and action against famine should take on a holistic
approach and consideration for all these aspects. For this rea-
son, although nowadays, famines are more predictable (as they
are processes that occur over months or even years, this allows
for early detection) and preventable (since they are a result of
economy or policies and, as such, are a product of human
factors that can be corrected), they are much more difficult to
resolve or counteract simply by means of developing structural
policies that guarantee the disappearance of hunger.
Further Reading
Cribb J (2010) The coming famine: the global food crisis and what we can do to avoid it.
Oakland: University of California Press.
de Waal A and Whiteside A (2003) New variant famine: AIDS and food crisis in southern
Africa. The Lancet 362(9391): 1234–1237.
Devereux S and Berge K (2000) Famine in the twentieth century, vol. 105. Brighton:
Institute of Development Studies.
Edkins J (2000) Whose hunger? Concepts of famine, practices of aid, vol. 17.
Minneapolis: University of Minnesota Press.
Keen D (1994) The benefits of famine: a political economy of famine and relief in
southwestern Sudan, 1983–1989. Princeton: Princeton University Press.
Rubin O (2011) Democracy and famine, vol. 37. London: Routledge.
Von Braun J, Teklu T, and Webb P (1999) Famine in Africa: causes, responses, and
prevention. Washington, DC: International Food Policy Research Institute.
Wang T, Hung CC, and Randall DJ (2006) The comparative physiology
of food deprivation: from feast to famine. Annual Review of Physiology
68: 223–251.
Watts MJ (2013) Silent violence: food, famine, and peasantry in northern Nigeria, vol.
15. Augusta: University of Georgia Press.
Watts MJ and Bohle HG (1993) Hunger, famine and the space of vulnerability.
GeoJournal 30(2): 117–125.
Relevant Websites
http://www.actionagainsthunger.org/blog/over-9-million-lives-transformed-dive-
details – Over 9 Million Lives Transformed: Dive into the Details | Action Against
Hunger.
http://econ.worldbank.org/WBSITE/EXTERNAL/EXTDEC/EXTDECPROSPECTS/0con
tentMDK:23112529pagePK:64165401piPK:64165026theSitePK:476883,00.
html – Prospects – Goal 1: Reducing Poverty and Hunger.
http://econ.worldbank.org/WBSITE/EXTERNAL/EXTDEC/EXTDECPROSPECTS/0.,con
tentMDK:23109795pagePK:64165401piPK:64165026theSitePK:476883,00.
html – Prospects – Goal 1: Eradicate Extreme Poverty and Hunger.
http://www.fao.org/hunger/en/ – Hunger | FAO | Food and Agriculture Organization of
the United Nations.
http://www.fao.org/post-2015-mdg/home/en/ – What – Home | FAO | Food and
Agriculture Organization of the United Nations.
http://www.fao.org/news/story/en/item/243839/icode/ – FAO.
http://www.fao.org/publications/sofi/en/ – The State of Food Insecurity in
the World (SOFI) 2014 | FAO | Food and Agriculture Organization of the United
Nations.
http://www.greenpeace.org/international/en/news/Blogs/makingwaves/fighting-world-
hunger-who-deserves-our-praise/blog/46999/ – Fighting world hunger: Who
deserves our praise? | Greenpeace International.
http://www.wfp.org/hunger/ – What is hunger? | WFP | United Nations World Food
Programme – Fighting Hunger Worldwide.
http://www.wfp.org/hunger/stats – Hunger Statistics | WFP | United Nations World Food
Programme – Fighting Hunger Worldwide.
http://www.wfp.org/hunger/who-are – Who are the hungry? | WFP | United Nations
World Food Programme – Fighting Hunger Worldwide.
http://www.wfp.org/hunger/causes – What causes hunger? | WFP | United Nations
World Food Programme - Fighting Hunger Worldwide.
http://www.wfp.org/zero-hunger – WFP and the Zero Hunger Challenge | WFP | United
Nations World Food Programme Fighting Hunger Worldwide.
http://www.worldbank.org/en/news/feature/2014/04/14/gafsp-improving-incomes-
reducing-hunger – GAFSP: Improving Incomes, Reducing Hunger.
http://www.worldhunger.org/articles/Learn/world%20hunger%20facts%202002.htm –
2015 World Hunger and Poverty Facts and Statistics by WHES. 2012:860.
 Fat Replacer
RS Chavan, National Institute of Food Technology and Entrepreneurship Management (NIFTEM), Kundli, India
CD Khedkar, College of Dairy Technology, Pusad, India
S Bhatt, Anand Agricultural University, India
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Although many nutrition recommendations remain controver-
sial, there is consensus among health and nutrition profes-
sionals in the developed world that the ratio of saturated fatty
acids to poly- and monounsaturated fatty acids in the diet
should be decreased, and intake of dietary cholesterol should
be limited. It is further agreed that consumption of calories
from fat should not be more than 30% of the daily total and
that consumption of cholesterol should be restricted to
<300 mg day
1. It is suggested that those with elevated Low-
density lipoprotein (LDL) cholesterol levels or cardiovascular
disease should restrict saturated fats to 7% of calories. Promi-
nence is also given to increasing the intake of fruits, vegetables,
and grains and modifying the type and amount of fat con-
sumed. The emphasis is on achieving an overall healthful
pattern of eating rather than on limiting the focus to merely
achieving goals for macronutrient composition.
In the food supply, 30% of the total fat comes from meat,
poultry, and fish, and 25% comes from baked goods and other
processed grain products. Milk products and fats/oils account
for 18% and 11% of the total fat, respectively. Collectively,
these food categories account for 84% of the fat in the food
supply. Although the type of fat in these contributors to fat
intake varies, the effort to reduce total fat and calorie intake has
focused largely on reducing the fat content of foods in these
categories. The effects of fat on cardiovascular disease are so
well documented that they have been recognized as major
factors in the etiology of cardiovascular disease. Dietary treat-
ment to prevent obesity and high blood cholesterol levels have
become the cornerstone of the National Cholesterol Education
Program, which has been designed to help prevent coronary
heart disease. Advocates of this program are boosted by reports
that coronary heart disease rates can be decreased by dietary
interventions and by a recent report that changes in lifestyle,
including diet, can bring about regression of severe coronary
atherosclerosis after a year without the use of lipid-lowering
drugs. Obesity is strongly associated with the development and
severity of both diabetes and hypertension, both of which are
major risk factors for cardiovascular disease. Due to this, the
consumer demand for low-fat foods has encouraged research
on reducing the fat content of foods. Problems of inferior
organoleptic and physical properties in these products sug-
gested the use of fat replacers (FRs) to provide the desirable
qualities.
Definition of FR
FR is defined as a carbohydrate-, protein-, or fat-based com-
pound that replaces one or more of the functions of fat to
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00271-3
reduce calories in foods. A broader scheme of FR is presented
in Table 1.
FR can be classified as synthetic fat substitutes (FS), emul-
sifiers or surface active agent, starch derivatives, maltodextrins,
hemicelluloses, b-glucans, bulking agents, microparticulates,
composites, and functional blends. FR can be further catego-
rized into the following groups:
FS: a synthetic compound designed to replace fat on a weight-
by-weight basis, usually having a similar chemical structure
to fat but resistant to hydrolysis against digestive enzymes.
Fat mimetics (FMs): a substance that can imitate physical
or organoleptic properties. FMs are also referred to as
‘texturizing agents.’ It requires a high water content to
achieve its functionality.
Fat analogs: are compounds with many of the characteristics of
fat but have an altered digestibility and altered nutritional
value.
Fat extenders: optimize the functionality of fat, thus allowing a
decrease in the usual amount of fat in the product.
Some of the applications of FR along with their usage levels are
given in Table 2.
Carbohydrate-Based FRs
Carbohydrate-based FMs incorporate water into a gel-type
structure, resulting in lubrication and flow properties similar
to that of fat. Carbohydrate-based FRs are categorized into
starch-derived, cellulose-based, fiber-based, gum-based, and
others.
Starch-derived FRs
Native starch is widely used in food and industrial applications
as a thickener, colloidal stabilizer, gelling agent, bulking agent,
and water-retention agent. Starches from different plant
sources such as wheat, maize, rice, and potato have received
extensive attention.
Resistant starch
Commercial resistant starches contain about 20–60% total
dietary fiber. Resistant starch is insoluble, having a low water-
holding capacity (WHC) and is most suitable for cereal-grain-
based products with lower levels of moisture content. Resistant
starch is generally used as an FR in baked goods, extruded
snacks, pasta, breakfast cereals, and beverages.
Maltodextrin
Maltodextrin is a nonsweet, nutritive saccharide polymer, with
a dextrose equivalent (DE) of < 20. Starches from botanical
sources like corn, potato, wheat, rice, tapioca, sago palm,
barley, and sorghum can be used as raw material. Maltodextrins
589
Table 1 Classification of fat replacers by nutrient source, functional properties, and use in food

Type of fat substitute Nutrient source (energy density) Functional properties Use in food

Derived from carbohydrate

Polydextrose Water-soluble polymer of dextrose (1 cal g
1) Bulking and retaining A wide range of foods, including
moisture baked goods, confections, and
frozen desserts
Modified food starch A variety of starch sources (1–4 cal g
1) Modifying texture, gelling, Processed meats, salad dressing,
thickening, and baked goods, frozen desserts, etc.
stabilizing
Dextrin and A variety of starch sources (4 cal g
1) Modifying texture and Baked goods, dairy products, salad
maltodextrins bulking dressing, sauces, and spreads
Gums and pectin Xanthan, guar, locust bean, carrageenan, alginates, Retaining moisture, Wide range of products, including
and fruit mouthfeel and modifying baked goods, sauces, and salad
texture dressings
Cellulose Various plant sources (virtually noncaloric) Modifying mouthfeel, Dairy products
texture, and pourability
b-Glucan Soluble fiber extracted from oats (1–4 cal g
1) Adding body and texture Baked goods and a variety of other
food products
Derived from protein
Microparticulated Denatured or microparticulated protein from egg/ Modifying mouthfeel Dairy products, spreads, and bakery
protein and milk (1–4 cal g
1) products
modified whey
Derived from fat
Olestra Sucrose polyester with triglycerides (noncaloric) Modifying texture and Savory snacks (stable for fried foods)
mouthfeel
Caprenin and salatrim Caprylic, capric, behenic acid, and glycerine, or Simulating properties of Confections, baked goods, and dairy
triglyceride of short- and long-chain fatty acids cocoa butter foods
(5 cal g
1)
Mono- or diglycerides Derived from vegetable oil and emulsified with water Adding moisture and Baked goods, vegetable dairy
(9 cal g
1) modifying texture and replacers
mouthfeel

Source: Judith, W. R. (2002). Fat substitutes and health an advisory from the Nutrition Committee of the American Heart Association. http://dx.doi.org/10.1161/01.
CIR.0000019402.35632.EB.

Table 2 Fat replacers and their food applications

Type of fat replacer Usage level (%) Food application

Modified starch (potato) 21 Bakery dough

Maltodextrin 40–45 Chocolate
Maltrin M040 20 Low-calorie pudding
Maltrin M100 10–28 Butter-like product
Polydextose 20 Peanut butter
HPMC 13 White chocolate bar
Pectin 0.18 Nonfat frozen dessert
Pectin (Slendid) and maltodextrin 0.5–3 þ 0–10 Fruit spread
Microcrystalline cellulose þ pectin 0.05–0.5 þ 0.1–10 Fruit spread
Microcrystalline cellulose þ k-carrageenan (93:7) 25 Yogurt and cottage cheese
Carrageenan gels 15 Frozen dessert
k-carrageenan 10–14 No-fat coffee creamer
l-carrageenan 0.2 Whipped toping
Carrageenan þ maltodextrin 1–3 þ 0–10 Fruit spread
Xanthan gum 0.18 Fat-free cookie
0.3 Low-fat spread
Pectin þ xanthan gum 0.63 þ 0.28 Fat-free fruit bar
Starch, pectin, guar gum, and gelatin mixture 1.29 Low-calorie yogurt
Simplesse 2–20 Fruit spread
Sucrose fatty acid polyester (Olestra) 10 Low-calorie cheese
Ethoxylated glucoside tetraoleate 13 Cake mix
10 Icing mix
56.25 Mayonnaise
50 Salad dressing
80 Margarine
Ethoxylated glucoside tetrastearate 25 Plastic shortening
13 Cake mix

Compiled from various sources: Roller (1996), Sharma and Ganeshkumar (1996), Akoh (1998), Sharma et al. (1998), Sandrou and Arvanitoyannis (2000), Chavan and Prajapati
(2009), Tiwari (2005).
 Fat Replacer 591

Table 3 Maltodextrin-based fat replacers

Usage
level pH Temperature
Trade name Type (%) stability stability ( C) Applications in food products Manufacturer

Paselli A2 Potato 20 3–7 25–212 Dressings, sauces, spreads, frozen desserts, Avehe American Inc.,
toppings, cakes, and butter NJ, USA
Stellar, Instant- Corn 20–25 4–6 25–200 Baked goods, frostings, fillings, gravies, A. E. Staley
Stellar sauces, and dairy products, confectionary Manufacturers, USA
Maltrin M040, Corn 20 3.5–7.5 300 Bakery and snack foods, beverages, Grain Processing
M100, M150 confectionary, dairy products, and salad Corporation, USA
dressings
Amalean I, Amalean Corn 1–8 3–7 25–200 Cheesecake, spoonable salad dressings, American Maize Product
II-Instant icings, cheese spreads, and desserts Co., USA
N-oil, Instant-N-oil Tapioca 25 3–8 25–225 Confectionary, soup, frozen desserts, spoon/ National Starch
pourable sauces, gravies, and puddings Manufacturers, USA
N-Lite LP, N-Lite B/ Maize 25–30 3–8 25–200 Salad dressings, soups sauces, bakery National Starch
L, N-Lite D, N-Lite products, dairy products, yogurt Manufacturers, USA
CL
Oatrim Oatflour 3–6 >3 Retort Dairy products, cereal, baked goods, and Rhone-Poulenc Food
meat products Ingredients, USA
Oat-fiber Oat 3–6 All All Processed meats, ice-cream, butter-coated Williamson Fiber
White Tan (770, fiber products and deep fried foods, and Products, County
780, 782) chocolate Cork, Ireland

Compiled from various sources: Roller (1996), Sharma and Ganeshkumar (1996), Akoh (1998), Sharma et al. (1998), Sandrou and Arvanitoyannis (2000), Chavan and Prajapati
(2009), Tiwari (2005).

are used as an FR in dairy products, confectionary, frozen
desserts, cereal baked goods, and meat products due to their
ability to form soft, spreadable, thermoreversible gels with
melt-in-the-mouth properties. Low-DE maltodextrin is usually
added at a level of 1–5% in liquid foods to impart a full-bodied
texture and mouth coating, whereas it can substitute about
25–35% of fat in cookies. Starches are usually hydrolyzed to a
DE’s value from 0 to 100, and as the DE increases, browning
reaction, freezing point depression, hygroscopitity, sweetness,
solubility, and osmality increase, whereas viscosity, cohesive-
ness, film-forming ability, and ability to prevent large crystal
formation decrease. Maltodextrin with low DE has similar
properties to those of native starch and can be used as an FR
(Table 3).
Maltodextrins based on potato are low DE (5), are cold
soluble, and provide low viscosity in the form of solution, but
at high concentration (>20% w/w) they are unstable and tend
to gels, which are plastic, and spreadable, which provides
shortening-like texture. Potato maltodextrin is produced by
solubilizing the slurry by jet cooking and treating it with
enzymes until the required degree of hydrolysis is achieved
(Paselli SA2, C* Pur 01906). Potato starch contains amylose
molecules with longer chains as compared to corn and wheat,
which retrograde less readily, thereby reducing the tendency to
cause turbidity and an undesirable texture in foods. Paselli SA2
is applied in products such as low-fat mayonnaise, sauces,
imitation cheese, and frozen desserts, whereas C* Pur 01906,
is used in baked goods. Preprepared gels consisting of one part
maltodextrin and three parts water provide 1 kcal g
1.
Corn maltodextrin is obtained from limited hydrolysis of
cornstarch. Maltrin M040, a product of Grain Processing
Corp., Muscatine, Iowa, is a spray-dried corn maltodextrin
(DE ¼ 5) and is able to partially or totally replace fat in
margarine, frozen desserts, salad dressings, and extruded
cereals and snacks. Tapioca maltodextrin is obtained by heat-
ing tapioca starch in the presence of hydrochloric acid, which
yields a gel and is characterized by a bland flavor, smooth
mouthfeel, and a texture similar to that of hydrogenated fat,
for example, N-oil, N-oil II. N-oil is used as a 20–35% aqueous
solution, contributes only 1 kcal g
1, and is used in products
like salad dressings, puddings, and margarine.
Oat maltodextrin is obtained by partial enzymatic hydroly-
sis of oat flour, oat fiber, or oat-bran. Oat maltodextrin (Otrim-
1, Otrim-5, Otrim-10) gels are useful for fat sparing in food
and food products like yogurt, baked products, and meat prod-
ucts. Oatrim-5, invented and patented by the USDA at the
National Center for Agricultural Research, is the only
carbohydrate-based FS and contains the unique combination
of b-glucan and low-DE maltodextrin. Wheat-based maltodex-
trin is produced by microfibrillation of wheat fibers and
maltodextrin, for example, Vitacel, produced by Rettenmaier,
is composed of 70% wheat fibers and 30% maltodextrin.
Vitacel can be used instead of hydrocolloids as a thickening
and binding agent for stabilization of emulsions, foams, and
liquid media.
Disadvantages associated with the usage of maltodextrins is
that the amylopectin present in it has a tendency to retrograde
slowly, giving rise to a phenomena of setback in low-fat spoon-
able salad dressings. Low-DE maltodextrins also suffer from
freeze–thaw stability and unreliable heat and acid stability.
Energy output of all the maltodextrin-based FMs is about
4 kcal g
1.
Polydextrose
Polydextrose was invented by Hans Rennhard at Pfizer Central
Research Laboratories, United States, in the mid-1970s.
592 Fat Replacer
Table 4 Microcrystalline cellulose and polydextrose-based fat replacers
Usage
level
Trade name Type (%)
Avicel RC/CL Cellulose gel 2–5.0
Tabulose Powdered MCC 0.5–20
Novagel RCN- MCC þ Guar gum 0.5–5.0
10; RCN-15
Polydextrose, Polymer of glucose with 5%
Litesse, sorbitol and citric acid
Litesse II (89:10:1)
Sta-Lite Polydextrose (fast – –
dissolving)
Compiled from various sources: Roller (1996), Sharma and Ganeshkumar (1996), Akoh (1998), Sharma et al. (1998), Sandrou and Arvanitoyannis (2000), Chavan and Prajapati
(2009), Tiwari (2005).
Polydextrose is composed of randomly cross-linked glucose
polymers with all types of glucosidic bonds, although 1–6
bonds are predominant (min. 90%), sorbitol end-groups
(max. 2%), and monoester bonds with citric acid. Polydextrose
exists in five forms viz., coarse powder, fine powder, type N,
type K, and type F. Polydextrose is used as a low-calorie bulking
agent that can replace all or part of the sugars and some of the
fats in foods while maintaining a pleasant texture and mouth-
feel. Polydextrose can also be used as a humectant, texturizer,
thickener, stabilizer, and cryoprotectant (Table 4). Litesse®, a
white- to cream-colored powder that has a pH (10% solution)
of 2.5–3.5 and melts above 130  C, is clear, bright, and
nonsticky; exhibits no crystallization; and supplies only 1 kcal -
g
1. According to the FDA, Litesse® may be used in frozen dairy
desserts, baked goods, confections, frostings, salad dressings,
gelatins, puddings, fillings, hard and soft candy, and chewing
gum. Litesse® II, specifically developed for light foods, provides
a higher level of sugar and fat replacement. The FDA has recog-
nized polydextrose as a ‘carbohydrate.’
Cellulose-based FRs
Cellulose is a polymer based on b-(1 ! 4)-linked D-glucose.
The most widely available cellulose-based FR are microcrystal-
line cellulose (MCC) and methylcellulose (MC) gums.
Microcrystalline cellulose
Developed in 1964, is a nonfibrous form of cellulose, ranging
in size from 25 to less than 1 mm. MCC is composed of anhy-
droglucose units linked through a b (1–4) glycosidic bond,
which is hydrophilic, linear, and high molecular weight, and
it accounts 75–95% by weight. The remaining 5–20% is solu-
ble in nature (Table 4).
MCC is produced using a-cellulose, which is composed of
paracrystalline and crystalline regions of microfibrils by
employing acid depolymerization and agglomeration, which
is followed by drying to give porous particles of MCC. In case
of colloidal grade, soluble hydrocolloids such as carboxy-
methyl cellulose, xanthan gum, or alginate are added to
pH
stability Applications in food products Manufacturer
>3.5 Salad dressings, dips, spreads, bakery, products, FMC Co., PA, USA
dairy products, meat products
– Mayonnaise, salad dressings, sauces, dietary Blanver
products, bakery products, imitation cheese Farmoquimica Ltd.,
products USA
– Bakery and snack foods, beverages, FMC Corporation, PA,
confectionary, dairy products, salad dressings USA
5–6 Pastry, confectionary products, dressings, Pfizer Inc., New York,
spreads, bakery fillings, toppings, chilled USA
desserts
Confectionary and bakery products A. E. Staley
Manufacturing
Company, IL, USA
prevent aggregation during drying. On proper dispersion, the
cellulose crystallites set up a three-dimensional network that is
highly thixotropic, is temperature stable, and adds body, while
imparting a clean mouthfeel. Functionality of MCC is affected
by different factors like adequate shear, order of addition, hard
water/electrolytes presence, and pH of the food. MCC, when
used in cakes at levels from 2% to 4%, improves the spongi-
ness, increases cake volume, provides better handling
properties, and creates improved product appearance. In
doughnuts, powdered grade MCC (1–3%) can reduce the fat
absorption. MCC is indigestible, and FDA regulations permit a
maximum addition of 1.5% (w/w) of finished ice cream, with
an exception in the case of fruit sherbets, wherein a maximum
of 0.5% (w/w) can be added.
MC gums
Hydroxypropylemethyl-cellulose (HPMC) and MC are gums
derived from chemical modification of cellulose derived from
wheat, maize, potato, and/or rice. The cellulose is made solu-
ble by adding sodium hydroxide and then treating with methyl
chloride to form MC and along with propylene oxide to give
HPMC, which is further dried using hot air, followed by grind-
ing and packaging. MC and HPMC are described in terms of
degree of substitution (DS) and molar substitutions (MSs). DS
is the amount of substituent groups on the anhydroglucose
units of cellulose, and MS is the average number of molecules
of substituent that have been substituted per anhydroglucose
unit. DS and MS affect physicochemical properties of MC gums
like water retention, sensitivity to electrolytes, dissolution tem-
peratures, and gelation characteristics. Methoxy substitution in
MC and HPMC corresponds to a DS range of 1.49–2.00 and
3.0–12.0%, respectively. In fried foods MC and HPMC cause
reduction in fat uptake, lower caloric value, improve cooking
economy, provide more moisture retention, and provide
improved yield. In cake and yeast-leavened doughnuts, oil
reduction of about 26–28% can be achieved along with
enhancement of air entrainment and promotion of uniform
and fine cell size in crumb structure. In liquid foods like sauces

and dressings, MC gums act as a stabilizer and provide pour-
ability, texture, and viscosity. In frozen dairy products, their
film-forming property, thickening capability, and lubricity
mimics the feel of fat. MC is labeled either as
‘methylcellulose’ or E-461 and HPMC as ‘hydroxpropyl
methylcellulose’ or E-464.
Dietary fiber-based FRs
Dietary fibers can be used to replace fat due to their wide range
of technological functionalities, and soluble fibers, such as
pectin and gums, possess a higher WHC of almost 20 times
their weight of water as compared to cellulosic fibers. WHC is
affected by concentration, temperature, presence of certain
ions, and pH of the food system. Fibers, such as pectin,
gums, b-glucans with mixed bonds, and polysaccharides
extracted from algae, form highly viscous solutions, whereas
that of inulin is minimal. The capacity of a fiber to bind fat
depends on the porosity, which is useful for avoiding excessive
absorption of frying fat in batters. Dietary fiber also acts as a
protective agent against cardiovascular diseases, diverticulosis,
constipation, irritable colon, colon cancer, and diabetes.
Pectin
Pectin is a complex mixture of polysaccharide composed of a
galacturonan backbone of which variable proportions can be
methyl-esterified and is usually obtained from citrus fruit and
apple. D-galacturonic acid units are linked by a-(1, 4)-
glucosidic bonds. Commercial pectins are divided according
to the degree of esterification into low methocxyl (LM) pectins
and high methoxyl (HM) pectins. HM pectins can also be
chemically amidated to obtain low methyl-esterified and ami-
dated (LMA) pectins. HM pectins are used mainly in sugar-acid
gels, whereas LM and LMA pectins are used in pectate gels.
Functionality of pectin is influenced mostly by the molecular
weight and the level and distribution of methyl-esters. Slen-
did®, manufactured by Copenhagen Pectin, Denmark, is pro-
duced by extraction from plant, purification, and isolation of
the pectin followed by a controlled desertification with the
help of either acid or base. Currently, it is used for fat sparing
in confectionary products, dairy products, and bakery fillings.
To mimic the physical and sensory characteristics of emulsified
fat, the particle size of 25–50 mm and usage level of 1.5–4.0% is
recommended. It is possible to reduce the fat content in a
frankfurter from 25% to 35% to 3–5% by using a wet prepara-
tion of 2% Slendid® 110. Pectin gives a trophic effect, increases
villus height and crypt depth in the small intestine, stimulates
intestinal cell proliferation and activity of brush border mem-
brane enzymes, and stimulates short-chain fatty acid produc-
tion in the cecum. Pectin gives a net energy of 9 kJ g
1 on
digestion. In the United States, pectin is given a Generally
Recognized As Safe (GRAS) status, whereas in the European
Union it is given a designation of E440 as a food additive.
Inulin
Inulin is an oligomer found in plants such as chicory and
Jerusalem artichoke and has a sweetening power of 30–65%
that of sucrose and a high degree of polymerization (DP) of
2–60. Inulin is produced by extracting inulin from chicory
roots by diffusing the roots in hot water followed by refining
and last spray-drying the concentrate. At a concentration of
Fat Replacer 593
40–45%, it forms a gel or crème having a fatty creamy feel with
characteristics like high water binding, stability against freeze
thaw, and inhibition of syneresis in mayonnaise and salad
dressings. Inulin with lower a DP of 25 is generally available
for high-performance fat replacement. Inulin crème has been
successfully applied in fat-reduced table spreads, frozen des-
serts, cheese products, meat products, fillings, sauces, and meat
replacers (Table 4).
b-glucan
b-glucan is a cell wall polysaccharide present in oat, barley, and
other grains, and recently yeast b-glucan has been used as a
FR in mayonnaise. b-glucan preparation is used to partially
substitute for vegetable oil in low-fat products such as salad
dressings, ice creams, yogurts, and cheese. b-glucans can also
be used for their therapeutic values, as they are found to be
immunomodulators, antitumorogenic, and antiviral agents for
treatment of hypercholesterolemia and stabilization of
glycemia.
Bacterial cellulose
It is produced by some Acetobacter, which is a high-value raw
material for producing dietetic and dessert foods due to its high
WHC. Instead of xanthan gum, bacterial cellulose at a concen-
tration of 3% can be used in ice creams. Bacterial cellulose
improves the quality of pastry products by reducing their
stickiness.
Z-trim
Z-trim refers to zero calories and can be used for replacing fat
and some of the glycemic materials (starches, sugars, and
syrups). It provides an aqueous gel fiber structure without
taste and imparts a smooth texture. Z-Trim gels also provide
insoluble fibers to be used separately or with other fibers such
as Oatrim. Z-Trim gel can be used for considerable reduction in
calories depending on the amount of fat and carbohydrates
replaced in food formulations.
Gum-based FRs
Gums are hydrocolloids, which are long-chain,
high-molecular-weight polymers that dissolve or disperse in
water. These gums are used along with a combination of
other ingredients viz. starches, bulking agents, emulsifiers,
and flavor to yield a finished product similar to a full-fat
system. Viscosifying and gelling property are affected by tem-
perature, pH, solvent quality, ionic strength, and presence of
specific ions. Hydrocolloids usually come from: (a) plant
materials such as seaweed, seeds, roots, and tree exudates; (b)
microbial biosynthesis; and (c) chemical modification of nat-
ural polysaccharides.
Locust bean gum
It is derived from carob seed, Ceratonia Siliqua and consists
of D-Mannopyronosyl backbone with attached D-
galactopyranosyl units existing in a ratio of 4:1. It is having
a limited solubility in cold water but on heating to 80  C
for 10 min it hydrates fully, resulting in a highly viscous pseu-
doplastic solution. Prolonged heating, high rate of shear, and
the time of heating can irreversibly degrade the locust bean
gum solution.
594 Fat Replacer
Guar gum
Guar gum or guran is the endosperm of the seed Cyampsis
tetragonolobus containing a backbone of (1 ! 4)-b-D-
mannopyranosyl units, with every second unit bearing a
(1 ! 6)-a-D-galactopyranosyl unit. Lower mannose:galactose
ratio (1.5–2) in guar gum indicates that there are few inter-
molecular interactions, and consequently, less heat is required
for solubilization (i.e., at 25–40  C). Guar gum is usually
utilized at a concentration < 1% and is stable between pH 3.5
and 9.0.
Protein-Based FRs
Protein-based FR is derived from proteins found in eggs, milk,
and other foods (Table 5). The concept of using proteins as FRs
is related to proteins with a particular spheroidal structure,
ranging from 0.1 to 2.0 mm in size and is usually produced
by microparticulation process.
Simplesse® (NutraSweet, Deerfield, IL) was the first FR
developed with protein and has received a GRAS status to be
used in frozen dessert. It contains 53% protein and is a micro-
particulated spray-dried powder that mimics emulsified fat by
forming a dispersed phase of particles that are free to move
independently. It is a multifunctional dairy ingredient made
from whey protein concentrate (WPC) that undergoes a
unique microparticulation process that results in uniform,
spherical, and deformable protein particles, much like fat
globules, which range in size from 0.1 to 3 mm. This process
helps to reduce the tendency to aggregate the spherical droplets
and form gel on heating. These droplets roll around on the
tongue, providing a smooth, creamy texture without chalkiness
or graininess. Simplesse increases the opacity of the product by
forming small spherical droplets of protein that allow more
scattering of light. Simplesse retains the biological property of
the protein used, so individuals who are allergic to egg or milk
proteins can experience allergic reactions to it.
Table 5 Protein-based fat replacers
Product name Type
SimplesseW Microparticulate whey protein
concentrate
Dairy-LoW Modified whey protein concentrate
K-BlazerW A blend of protein, food starch, gums,
and emulsifiers
LitaW Corn zein
Leancreme™ Microparticulated whey protein
Optipep™ Hydrolyzed whey protein
concentrates and isolates
N-Flate Nonfat milk, gums, emulsifiers, and
modified starch
ULTRA-BAKETM Starch, modified vegetable protein,
and xanthan gum
ULTRA- Egg-white, milk protein, corn syrup
FREEZETM solids, and modified starch
Compiled from various sources: Roller (1996), Sharma and Ganeshkumar (1996), Akoh (1998), Sharma et al. (1998), Sandrou and Arvanitoyannis (2000), Chavan and Prajapati
(2009), Tiwari (2005).
‘Dairy Lo,’ a thermally denatured WPC, interacts with water
and prevents iciness, provides opacity, controls viscosity, and
stabilizes air cells along with a fat-sparing effect in dairy prod-
uct when used at a level of 2–5%. The formulations supple-
mented with WPC showed greater elasticity, firmness,
chewiness, and gumminess compared to the control sample,
thus contributing to the texture of the fat-free dairy desserts.
Some of the other protein-based FRs available in the market are
K-Blazer®, Ultra-BakeTM, Ultra-FreezeTM, and Lita®.
Fat-Based FRs
Fat-like substances, which are resistant to hydrolysis by diges-
tive enzymes, comprise another major category of materials
being promoted as partial or full replacements for oils and
fats in bakery and other food products.
Structured lipids
A structured lipid is a triglyceride obtained by the hydrolysis
and random transesterification of medium-chain triglyceride
(MCTs) and long-chain triglyceride. Structured lipids provide
approximately half of the calories of the normal edible oil.
Caprenin, an MCT derived from coconut, palm kernel, and
canola oil, mimics the physical properties of cocoa butter or
confectionary fat. Another example of MCT is Neobee M-5,
derived from coconut oil, which prevents bloom formation
in chocolate and sticking of uncoated nutritional bars to pack-
aging material by migrating to the surface of these products.
Salatrim (short- and long-chain acyltriglyceride molecules) is a
‘family’ of triacylglycerols produced by the interesterification of
highly hydrogenated vegetable oils with triacylglycerols of
acetic and/or propionic and/or butyric acids. Salatrim prepa-
rations are available to emulate cocoa butter, as well as for use
in baked products and filled dairy products.
Applications in food products Manufacturer
Dairy products, spreads, bakery product, salad dressing, CP Kelco
dips, mayonnaise, frostings, sauces, soups
Milk/dairy products, baked goods (cheesecake), frostings, Cultor, New York, NY
salad dressing, mayonnaise-type products
Salad dressing, mayonnaise, frozen dessert, sour cream, Kraft Food Ingredients
baked goods, and dairy products Corp, Memphis, TN
Baked goods Opta Food Ingredients,
Inc., Cambridge, MA
Baked goods SPX-APV brand
Yogurts, ice cream, cheese, and drinking milk Carbery
Sports nutrition products and nutrition bars
Cake mixes, salad dressing, icings, glazes, desserts, ice
cream, and ground beef
Baked goods

Sugar polyesters
Olestra is a mixture of hexa-, hepta-, and octaesters of sucrose
prepared by esterifying sucrose with long-chain fatty acids
isolated from edible fats and oils. Olestra can be liquid or
solid at room temperature based on the fat source in the
sucrose polyester. Olestra has the organoleptic and thermal
properties of fat and thus it can serve as a zero-calorie replace-
ment for fat in a variety of foods like savory snacks such as
potato and corn chips.
Esterified propoxylated glycerol
Esterified propoxylated glycerol (EPG) is a family of propylene
oxide derivatives with a structure similar to that of natural fat.
It is been claimed that EPG is resistant to enzymatic hydrolysis
and that it can be substituted for fats and oils in products such
as table spreads, frozen desserts, salad dressings, and bakery
products.
Dailkyl dihexadecymalonate
A fatty alcohol ester of malonie and alkylmalonic acids, dailkyl
dihexadecymalonate (DDM) in combination with soybean oil
has been used to produce potato and tortilla chips. DDM has
also been tried in products like mayonnaise and margarine.
Trialkoxytricarballate
A tricarballylie acid, esterified with fatty alcohol, is being eval-
uated as an oil substitute to produce acceptable margarine and
mayonnaise-based products.
Safety of FRs
The use of FRs as a food additives to reduce the fat content in
food products also raises the concern of consumers’ safety. The
safety of the currently used FRs is ensured by the GRAS status
by the FDA. FRs could be used and consumed by the con-
sumers in large quantities in fat-less or reduced-fat products.
Many of the carbohydrate-based, protein-based, and fat-based
FRs have not shown major health concerns except for pol-
ydextrose and Olestra. Polydextrose can have a laxative effect,
which requires a labeling disclaimer when present at specified
levels. Olestra may cause leaky and fatty stools and loss of fat-
soluble vitamins. In general, there is limited evidence at the
present time on the long-term adverse consequences associated
with the consumption of these or any other reduced-fat foods
by adults. However, further research is required to study the
safe use of these products by children and adults and to eval-
uate fully their long-term health effects.
Conclusion
Due to the increasing demand for low-fat food products,
reduced-calorie and low-fat food markets are showing a
dynamic growth. As pointed out in the beginning of this
article, the FR can mimic one or more roles of fat in food
products. Classification of an FR usually is based on the nutri-
ent source. They provide the functional and sensory qualities of
fats in foods and are absorbed and metabolized normally. The
introduction of olestra, which is a nonabsorbable FS that can
affect nutrient absorption, raises questions about its potential
Fat Replacer 595
effect on overall health. Some research suggests that individ-
uals who consume a diet that is reduced in fat and calories and
includes use of fat-modified products have a better overall
nutrient profile than do individuals who do not use any fat-
modified products. The recent increase in the availability of
FRs in the market raises questions about the cumulative impact
of using them in multiple food products and the potential
interaction with medications and food ingredients. Within
the context of a healthy dietary pattern, FRs, when used judi-
ciously, may provide some flexibility in dietary planning,
although additional research with clinical trials is needed to
fully determine the longer-term health effects.
See also: Fats: Classification and Analysis; Fatty Acids: Determination
and Requirements; Fatty Acids: Fatty Acids; Food and Agriculture
Organization of the United Nations.
Further Reading
Akoh CC (1998) Fat replacers. Food Technology 52: 47–53.
Chavan RS and Prajapati PS (2009) Carbohydrate-based fat replacers – a review. Indian
Journal of Dairy Science 62: 1–14.
Cheskin LJ, Zorich N, Miday R, and Filloon T (1998) Gastrointestinal symptoms
following consumption of olestra or regular triglyceride potato chips: a controlled
comparison. JAMA 279(2): 150–152.
Clegg SM (1996) The use of hydrocolloid gums as fat mimetics. In: Roller S and
Jones SA (eds.) Handbook of fat replacers, pp. 191–212. Boca Raton, FL: CRC
Press, Chapter 9.
Huyghebaert A, Dewenttinck K, and deGrey W (1996) Fat replacers. IDF Bulletin
317: 10–15.
Judith WR (2002) Fat substitutes and health: an advisory from the Nutrition Committee
of the American Heart Association. Circulation 105: 2800–2804. http://dx.doi.org/
10.1161/01.CIR.0000019402.35632.EB.
Kaur L, Singh N, and Singh J (2004) Factors influencing the properties of
hydroxypropylated potato starches. Carbohydrate Polymers 55: 211–223.
Lucca PA and Tepper BJ (1994) Fat replacers and functionality of fats in foods. Trends
in Food Science and Technology 5: 12–19.
Mitchell HL (2004) Capture the opportunity: sustained fermentation with Litesse,
polydextrose and lactitol – the health benefits. Innovations in Food Technology
13: 24–26.
Ognean CF, Neli D, and Ognean M (2006) Fat replacers – review. Journal of
Agroalimentary Processes and Technologies XII: 433–442.
Osborn HT and Akoh CC (2002) Structured lipids: novel fats with medical,
neutraceutical, and food applications. Comprehensive Reviews in Food Science and
Food Safety 3: 110–120.
Roller S (1996) Starch-derived fat mimetics: maltodextrins. In: Roller S and Jones SA
(eds.) Handbook of fat replacers, pp. 99–118. Boca Raton, FL: CRC Press, Chapter
6A.
Sandrou DK and Arvanitoyannis IS (2000) Low-fat/calorie foods. Current status and
perspectives. Critical Reviews in Food Science and Nutrition 40: 427–447.
Sharma A and Ganeshkumar C (1996) Fat replacers as future dietary regimes: an
overview. Indian Dairyman 48: 27–35.
Stanton JM (1990) Fat substitute update. Food Australia 42: 472–475.
Relevant Websites
http://www2.ca.uky.edu/agc/pubs/fcs3/fcs3208/fcs3208.pdf.
http://www.circulationaha.org.
http://www.cfs.purdue.edu/fn/fn453/pdf_full/Fat_Replacers.pdf.
http://www.fda.gov/bbs/topics/ANSWERS/2003/ANS01245.html.
http://www.ift.org//media/Knowledge%20Center/Science%20Reports/Scientific%
20Statu%20Summaries/fatreplacers_0398.pdf.
http://journal-of-agroalimentary.ro/admin/articole/74033L66_FAT_REPLACERS_final.
pdf.
 Fats: Classification and Analysis
M Narváez-Rivas and M León-Camacho, Instituto de la Grasa (C.S.I.C.), Seville, Spain
ã 2016 Elsevier Ltd. All rights reserved.

Introduction – acetonitrile–ethanol–hexane,
– acetonitrile–tetrahydrofuran.
Fats are essential nutrients and concentrated sources of energy
They improve the solubility of triacylglycerols and have an
of the diet, supplying about 9 kcal g
1. They are soluble in
effect on the selectivity and efficiency of the separation. The
most organic solvents, such as hexane and ethyl ether, but
detector used and the nature of triacylglycerols may determine
basically insoluble in water.
the choice of mobile phase and the choice of an isocratic or a
By classical definition, fats are referred to total lipids, which
gradient system. For example, acetonitrile–acetone mixtures
can be classified into two different fractions depending on if
are not well suited to saturated components of higher molec-
they form or fail to form soaps when blended with sodium
ular weight, since they tend to precipitate out of solution.
hydroxide, considered as saponifiable and unsaponifiable frac-
For such purpose, the acetonitrile–dichloromethane combina-
tions, respectively. The different compounds that can be part of
tion may be the best available. Isocratic eluants are utilized
the saponifiable fraction are shown in the next scheme.
with detectors in which the change in the composition of the
mobile phase affects the obtained signal, as in the case of
The unsaponifiable matter contains terpenic (sterols, the refractive index (RI) detector. The gradient of elution can
4-methylsterols, 4,40 -dimethylsterols, carotenoids, tocoph- be used with detectors such as UV and evaporative light scat-
erols, and tocotrienols) and aliphatic compounds (saturated tering detectors (ELSDs).
and unsaturated hydrocarbons and fatty alcohols). The column temperature should be fixed and controlled to
The different ways of analysis of the fat compounds are achieve reproducible retention times. With low temperatures,
going to be described in the succeeding text. solubility effects may come into play, and the more saturated
molecular species may precipitate out of solution.
The detection systems for quantitative analysis of triacylgly-
Saponifiable Fraction
cerol species separated by HPLC have been RI, UV, evaporative
Triacylglycerols light scattering, and mass spectrometry detectors.
One of the most used for the mentioned purpose is the RI
Triacylglycerols consist of the trihydric alcohol glycerol esteri-
detector, in which the RI changes with the chain length and
fied with fatty acids. They represent the main lipid fraction of
with the unsaturation number. The response factor is consid-
foods. For the analysis of molecular species of triacylglycerols,
ered the same (1) for all triacylglycerols in the practice. This
chromatographic and spectrometric methods are available.
detector is affected by the temperature and composition of the
However, stereospecific determination of positional distribu-
mobile phase, and due to this, the analysis needs to be carried
tions of fatty acids in them remains a substantial technical
out with thermostated cells and isocratic elution. In some
challenge. Nowadays, the most used methods for their analysis
cases, flow gradients can be used. Another limitation of this
are HPLC and GC.
detector is its low sensitivity. However, RI detection gives good
results and is one of the most adequate for the quantitative
Reversed-phase HPLC
analysis of these compounds.
Good resolution of triacylglycerols by reversed-phase high-
It has been reported that UV detection can be used to get
performance liquid chromatography is possible. The retention
good quantitative data at wavelength between 200 and 220 nm,
times are dependent on the total number of carbon atoms of
where the ester bond absorbs. This detector shows the highest
the three fatty acids of the molecule, with each double bond
sensitivity. Nevertheless, the absorption of several solvents used
reducing the effective chain length of the species by the equiv-
in the mobile phase, like acetone, the unsaturations, and the
alent of about two carbon atoms.
conjugation of these unsaturations are a problem for the use of
The most frequently used columns have been those with
this detector in triacylglycerol analysis.
phases of the octadecylsilyl (C18, ODS, or ODS-2) type, since
An alternative to the detectors mentioned is the ELSD,
stationary phases similar in chain length to the fatty acyl chains
which is universal and simple. This shows the disadvantages
maximize the interactions and give a higher efficiency. Silver
that a calibration method must be taken into account and that
ion HPLC has been also used. The 250  4.6 mm column with
the linear range is between 50 and 250 mg. Below 50 mg, the
5 mm packing, the 100  3 mm column with a 3 mm packing,
sensitivity decreases, which is a problem for the analysis of the
and the 100  2.1 mm column with 1.7 mm packing have been
minor compounds. But this detector shows the advantages that
used, the last one being used in terms of saving time and
no solvent peak appears and the resolution of the peaks is good
solvent and with no loss of resolution.
and it is not affected by the changes in the solvents of the
Referring to the mobile phases, the most used are
mobile phase.
– acetonitrile–acetone, For both detecting and identifying molecular species of
– acetonitrile–isopropanol, triacylglycerols, mass spectrometry is used, showing a good
– acetonitrile–dichloromethane, sensitivity, although a careful calibration is also needed.

596 Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00274-9

 Fats: Classification and Analysis 597

The choice of injection solvent is another important practi- resolving triglyceride mixtures even better than nonpolar
cal point that should be mentioned. Samples should be phases, but they cannot reach temperatures over 300  C.
injected in a small volume of the mobile phase (5–10 ml). The polarizable phenyl methyl silicone phases are capable of
Hexane is not employed as injection solvent because a single enduring temperatures of about 360–370  C for a long time,
component can emerge as double peaks due to its properties, separating triacylglycerols by both chain length and the num-
which are so similar to those of the stationary phase that it ber of unsaturation. The fused silica capillary DB-17ht column
competes with this for the solute molecules. (30 m  0.32 mm i.d. and 0.15 mm film thickness) shows
a really good resolution for the different molecular species of
triacylglycerols.
High-temperature GC
Detection is carried out by using a flame ionization detector
Nowadays, the use of high-temperature GC with flame ioniza-
(FID), and the response factors need to be considered, which
tion detection for the separation and quantification of triacyl-
depend on a certain extent on the ratio of the carrier gas.
glycerols is a good alternative to HPLC, solving its problems.
The purity and nature of the carrier gas, flow rate, rate of
Triacylglycerols are not at all easy to volatilize for gas chro-
temperature change, and whether the carrier gas is maintained
matography purposes since their molecular weight is quite
at constant flow or constant pressure rate are other practical
high. The injection procedure is of great importance to avoid
factors that have to be considered.
any sample discrimination and achieve good quantification.
An example of triacylglycerol analysis by GC-FID is shown
Modern gas chromatographs usually come equipped to achieve
in Figure 1.
such purposes with cold on-column injection, split injection,
and/or programmed temperature vaporizer injection. The use
of cold on-column injection is one of the most desirable to
reduce or eliminate thermal decomposition (e.g., the pyrolysis Diacylglycerols and Monoacylglycerols
of the most unsaturated triacylglycerols), mass discrimination,
Diacylglycerols are esters of the trihydric alcohol glycerol in
and other undesirable effects associated with classical split/
which two of the hydroxyl groups are esterified with fatty acids,
splitless injection. Using this injection technique, the injector
and they are found as 1,2- and 1,3-isomers. They are generated
temperature should be lower than the solvent boiling point.
by acidic and enzymatic hydrolyses of triacylglycerols during
The new split/splitless injectors minimize mass discrimination
the transformation and storage of fats, and 1,3-isomers can be
loss and make possible to apply this technique to triacylglycerol
generated from 1,2-diacylglycerols due to an isomerization
analysis with similar results to those obtained by using cold
process, since the 1,3 species is more thermodynamically sta-
on-column injection.
ble. In tissues, 1,2-diacylglycerols can also be generated from
Another aspect that needs to be addressed is the nature of
phosphatidylinositol (PI) by the action of phospholipase C.
the stationary phase. The temperature stability of this is highly
The determination of molecular species of diacylglycerols in
important. The most stable to high temperatures is the non-
fats can be accomplished by a previous extraction and purifi-
polarity phases of the dimethylpolysiloxane type, which per-
cation by column chromatography or solid phase extraction
mit separation by chain length. However, high-polarity or
(SPE) and a subsequent chromatographic method, GC
polarizable phases allow remarkable separations by both car-
being the most appropriate for that purpose. An aliquot of
bon atom number and degree of unsaturation, but they are less
the fat in hexane and a suitable internal standard (e.g., 1,3-
stable to high temperatures. Columns coated with polar sta-
dipalmitoyl-glycerol and dinonadecanoine) are added prior to
tionary phases, such as cyanopropyl silicone, are capable of

mVolts

50 9

40

30
14

20 8 10 15

16
10 13
4
5
6 7 11 12 17
12 3
0

5 10 15 20 Time (Min)

Figure 1 Chromatogram of the triacylglycerol profile of an Iberian pig subcutaneous fat sample, where 1, PPP; 2, MOP; 3, PPS; 4, POP; 5, POPo þ PLP;
6, PLPo þ MLO; 7, PSS; 8, PSO; 9, POO; 10, PLO; 11, PLL þ PoLO; 12, SOS; 13, SOO; 14, OOO; 15, SOL; 16, OOL; 17, OLL. Reproduced from
Journal of Agricultural and Food Chemistry, 2012, 60, 1645–1651.
598 Fats: Classification and Analysis
mVolts
2
75
50
25 1
0
−9
5 10
Figure 2 Gas chromatogram of the polar fraction from 0 to 35 min corresponding to the subcutaneous fat of Iberian dry-cured ham. Labeled peaks are
identified as follows: 1, palmitic acid; 2, oleic acid; 3, 1-monoolein; 4, cholesterol; 5, 1,3-dimyristin (IS). Reproduced from Journal of Agricultural
and Food Chemistry, 2007, 55, 10953–10961.
the purification step, in which silica or diol SPE columns are
utilized.
Purification comprises several steps using different solvent
systems. The first is carried out passing a solution to eliminate
the major compounds (e.g., hexane/methylene chloride/ethyl
ether (89:10:1)) through the column, which is discarded. In
the second step, the diacylglycerol fraction is obtained by
passing a solution, generally chloroform/methanol (2:1), that
is evaporated to dryness in a rotary evaporator under reduced
pressure, and the residue is treated with 200 ml of the silylating
reagent and left at room temperature for a few minutes, obtain-
ing the corresponding trimethylsilyl derivatives of 1,2- and 1,3-
diacylglycerols. Monoacylglycerols are also extracted with the
diacylglycerol fraction, and they can be analyzed together by
GC using a proper temperature program and a proper column.
An example of this is shown in Figure 2.
The analysis of the silylated fraction is performed by GC-
FID using thermostable midpolarity GC columns (e.g., fused
silica capillary DB-17ht polyamide-coated column, 50%
phenyl methylpolysiloxane, 65% phenyl- and 35% dimethyl-
polysiloxane, and 65% methyl phenyl silicone). The peak of
1,2-diacylglycerols appears before their corresponding 1,3-
isomers.
With respect to monoacylglycerols, they are at trace levels in
fats, and their analytic determination is carried out with a
procedure similar to diacylglycerols, trimethylsilyl
derivatization, and GC-FID analysis but with nonpolar capil-
lary columns, or their combination as it has been mentioned in
the preceding text.
Phospholipids
Phospholipids are a class of lipids that have two fatty acyl
molecules esterified at the sn-1 and sn-2 positions of glycerol
and a head group linked by a phosphate residue at the sn-3
position. They have amphipathic property since the head
group forms a hydrophilic region that determines the type of
phospholipid, and the fatty acyl side chains are hydrophobic.
Diglycerides
5
15 20 25 30 Minutes
Two different categories are distinguished: glycerophospho-
lipids (phosphatidylethanolamine (PE), phosphatidylcholine
(PC), phosphatidylinositol (PI), phosphatidylserine (PS), and
cardiolipin (CL)), the derivatives of glycerol, and sphingopho-
spholipids (sphingomyelin: SPH), those that do not contain
glycerol.
Phospholipids have been determined traditionally by gravi-
metric methods and spectroscopic methods, like molecular or
atomic absorption spectroscopy. However, these are not usu-
ally used nowadays.
The thin-layer chromatography (TLC) technique using
adsorbent (silica) coated on glass strips is used for the separa-
tion of phospholipids as a separation technique previous to the
quantification of these by another procedure or even for their
direct quantification by densitometry or thincography. The type
of mobile phase used and whether the TLC is in one or two
dimensions are critical in the separation of the different groups.
This technique has some disadvantages, such as a limited capac-
ity to resolve the phosphatides in their different molecular
species, techniques of detection more or less complex with
significant errors in the quantification, and a relatively long
time period required for analysis.
Nowadays, the method used for the separation and determi-
nation of phospholipid classes is the HPLC. Silica columns are
the most used for such purposes since this can give excellent
results and is relatively inexpensive and robust. Nevertheless,
amino columns have been also used, but they need to be
periodically regenerated with ammonia solutions. So, they are
not widely used.
The most used detectors in the study of phospholipid clas-
ses are the UV and the ELSD. The response of UV detector is
dependent on the degree of unsaturation in the molecule, and
different wavelengths have been used, but the most frequently
used are 205 and 208 nm (absorption of the ester bond).
Besides, in these detectors, another problem is the absorption
of some mobile phases at these wavelengths. That is why there
are solvents that cannot be used with these detectors, like
chloroform and acetic acid. So, the quantification of
 Fats: Classification and Analysis 599

phospholipids using this detector is difficult. On the other

hand, ELSD gives a stable flat baseline and is very sensitive,
and its response is not dependent on the number of double
bonds in the molecule, showing the advantage of simplicity
and universality. To use this detector in quantitative analysis, 1
the response factors of the phospholipids must be calculated,
due to the minor components. 6
Referring to the mobile phases used, an isocratic eluant has
rarely been used. With a UV detector, an isocratic mode or a linear 2
gradient can be applied. Binary gradients can be employed using 4
ELSD, and the following have been used in several works: (a) 5
chloroform–methanol–ammonium hydroxide and (b) chloro-
3
form–methanol–water–ammonium hydroxide. More compli-
cated gradients of elution with three or four eluants have been
used, but to a lesser degree than the binary ones. A wide range of 7
different mobile phases have been used to separate the different
phospholipid classes, since unfortunately not all phases have
the same effectiveness for the different types of samples and the
composition of the mobile phase can become critical for each type 0 2.5 5 7.5 10 12.5 15 17.5 20 min
of matrix. Binary gradients that have been successfully used with
(a)
different types of matrix are (a) chloroform–methanol–ammonia
solution and (b) chloroform–methanol–triethylamine–water. An
example is shown in Figure 3. 2
The use of reversed-phase HPLC allows the separation of
the different molecular species of each phospholipid class.
Normally, the different phospholipid classes are separated
and collected by HPLC or TLC. One or two columns in series 5
can be used depending on the resolution desired and the
mobile phase used. Acetonitrile–methanol–water is the most 6.8 7 7.2 7.4 7.6 7.8 8 8.2 min
commonly used mobile phase in reversed-phase HPLC using
2
an RP18 column. 1
The last thing to mention about phospholipids is that they
can be purified previously to their chromatographic analysis
using SPE to isolate them, and the best results are obtained 6
3
when silica columns are employed, allowing a high recovery.
4
Waxes
Waxes are organic compounds that consist of long alkyl chains.
They are insoluble in water but soluble in organic nonpolar 0 2.5 5 7.5 10 12.5 15 17.5 20 min
solvents. The waxy material of the fats can include primary and (b)
secondary alcohols, diols, ketones, b-diketones, triacylglycer- Figure 3 HPLC chromatogram of phospholipid fractions (a) from the
ols, hydrocarbons, sterol esters, and aliphatic aldehydes. standard solution and (b) from the subcutaneous fat of Iberian pig. 1,
Silica gel column chromatography, TLC, SPE, and HPLC Cardiolipin; 2, phosphatidylethanolamine; 3, phosphatidylinositol; 4,
(using silica) have been used to separate them from other phosphatidylserine; 5, phosphatidylcholine; 6, sphingomyelin; 7,
lipid constituents and to isolate individual classes of waxes for lysophosphatidylcholine. Reproduced from Journal of Chromatography
more detailed analysis. On the other hand, high-temperature A, 2011, 1218, 3453–3458.
gas chromatography following trimethylsilylation has been
used in recent works, often in combination with mass spec- hydrated silica gel column after the addition of the internal
trometry, to achieve simultaneous identification and quantifi- standard (e.g., lauryl arachidate). The eluted fraction is recovered
cation of the various molecular species. and subsequently injected on-column in a GC capillary column.
Semipolar columns can be used for their analysis, although SE-54 or SE-52 semipolar columns can be used, although a better
a better resolution is obtained with phenyl methyl silicone resolution can be achieved with phenyl methyl silicone col-
columns, which can endure higher temperatures. umns, which can tolerate higher temperatures.
All the methods for the determination of waxes consist of a Waxes can be determined simultaneously with fatty
separation from other lipid constituents using silica gel column acid methyl esters and fatty acid ethyl esters by capillary
chromatography, SPE, or HPLC and subsequent quantification gas chromatography, which is a method widely applied in
using GC. In the official method, the waxes are determined laboratories.
by separating them according to the number of carbon atoms. A method based on through oven transfer
The fractionation step is carried out by chromatography on a adsorption–desorption (TOTAD) interface is another analytic
600 Fats: Classification and Analysis
alternative. The lipid extract, with an internal standard diluted
in n-heptane, is injected directly with no sample pretreatment
step other than filtration. The wax ester fraction is separated
from the triglycerides by normal-phase liquid chromatography,
and the TOTAD interface transfers it to the GC to be analyzed.
This method allows an automatic analysis of different wax
esters, being simpler and faster than other methods, although
its resolution is poorer.
A method in which samples are prepared by normal-phase
liquid chromatography has also been proposed for the analysis
of wax esters, but the precolumn is attached to the inlet of the
column in the GC  GC instrument by means of a press-fit
connector, the first dimension being performed with a PS-255
column (20 m  0.25 mm i.d.) and the second dimension with
a SOP-50 (50% phenyl polysiloxane) (1.5 m  0.15 mm i.d.).
Unsaponifiable Fraction
The main classes of compounds that constitute the unsaponifi-
able fraction of food are hydrocarbons, carotenes, tocopherols,
tocotrienols, linear fatty alcohols, triterpenic alcohols, methyl
sterols, and sterols. Other components that are part of the
unsaponifiable fraction are carotenoid pigments, triterpenic
dialcohols, diterpenic alcohols, and phytol, but they are not
always present in food matrices.
The unsaponifiable fraction is got doing a saponification
with ethanolic potassium hydroxide at high temperature. After
this, distilled water is added to the solution, and the mixture is
extracted several times with portions of a nonpolar solvent
such as hexane, heptanes, diethyl ether, chloroform, or their
mixtures thereof. The unsaponifiables will remain in the non-
polar phase, while the fatty acid salts will partition to the polar
phase. The nonpolar extracts are combined and washed several
times with portions of a mixture of ethanol–water (1:1), until
the wash reaches neutral pH. Finally, the hexane solution is
dried under a stream of nitrogen or by rotoevaporation and
redissolved in an appropriate amount of solvent for cleanup
by either TLC or SPE or directly for derivatization. The frac-
tionation of the different classes present in the extract can be
Linear Hydrocarbons
nRIU
300000
250000 Terpenic Hydrocarbons
200000
150000 Linear and Terpenic alcohols
100000
50000
–50000
–100000
0 5 10
Figure 4 HPLC chromatogram of unsaponifiable fraction from the subcutaneous fat of Iberian pig.
carried out by HPLC. In Figure 4, there is an example of the
fractionation using the HPLC technique. The same preparative
procedure is utilized for the analysis of all unsaponifiable com-
ponents. The trimethylsilyl derivatives (except for the hydrocar-
bons) of the purified fractions are analyzed using nonpolar
capillary gas chromatography columns. On the other hand, a
direct analysis of the silylated unsaponifiable components
obtained from different food products can be carried out
using a thermostable polar gas chromatography column (65%
phenyl–35% dimethylpolysiloxane).
Hydrocarbons
In the natural lipid systems of food matrices, hydrocarbons are
present in quite small amounts, and most of them have an odd
number of carbon atoms. Cyclic, polycyclic aromatic, and
ramified hydrocarbons are also present in small amounts.
A good separation of hydrocarbons is provided by silica TLC of
natural lipids using a mobile phase of n-hexane-diethyl ether (7:3,
v/v), eluting just after the solvent front. To improve the isolation
of the unsaponifiable fraction of lipids, TLC and liquid chroma-
tography are being replaced by HPLC methods, and HPLC-GC
off-line methods that allow isolation and quantification of the
hydrocarbon fraction have been reported. The column used for
such purposes is a silica column and the mobile phase is hexane/
ethyl acetate. SPE is also used, like in the case of polycyclic aro-
matic hydrocarbons, which are considered as contaminants.
After their purification, the hydrocarbons are analyzed by
GC-FID or GC-MS, the last technique allowing their simulta-
neous identification. For polycyclic aromatic hydrocarbon
analysis, low-bleed and low-polarity columns have been
used, like DB-XLB columns. Linear, branched, and terpenic
hydrocarbon analyses are performed using nonpolar columns
of (95%) dimethyl–(5%) diphenylpolysiloxane type. An exam-
ple of hydrocarbon analysis by GC-FID is shown in Figure 5.
Alcohols and Sterols
Linear alcohols are constituted by a homologue series of
primary fatty alcohols that generally contain 20–30 carbon
Sterols
15 20 25
min

I.S.
(a)
I.S.
5 C20H38
7
3
9 KAURENE
11
5´
3´ 7´
11´
1
0 10 20
(b)
Figure 5 Chromatograms of hydrocarbons obtained from the subcutaneous fat of Iberian pig by LC (a) and HPLC (b). Peaks: 1, 1-dodecene; 3,
1-tetradecene; 30 , tetradecane; 5, 1-hexadecene; 50 , hexadecane; 7, 1-octadecene; 70 , octadecane; 9, 1-eicosene; 11, 1-docosene; 110 , docosane; 13,
1-tetracosene; 130 , tetracosane; 15, 1-hexacosene; 150 , hexacosane; 17, 1-octacosene; 19, 1-triacontene; 21, 1-dotriacontene.
atoms. The triterpenic alcohols have a steroid structure and are
present at different levels in vegetal lipids. These are also
known as 4,40 -dimethylsterols.
Both linear and triterpenic alcohols are often badly sepa-
rated in silica TLC. To achieve a better TLC separation, a mul-
tiple development technique with a slightly different mobile
phase for the second development can be carried out. Then,
they are determined using the GC technique.
Sterols are steroid alcohols and they occur naturally in
plants (phytosterols), animals, and fungi. Cholesterol (chol-
est-5-en-3b-ol) is by far the most abundant sterol in animal
tissue. This can be esterified to long-chain fatty acids (choles-
terol esters). These cholesterol esters are much less polar than
free cholesterol. Cholesterol oxidizes slowly in tissues or foods
forming a range of different products with additional hydro-
peroxy, epoxy, hydroxy, or keto groups. These compounds are
called oxysterols, cholesterol oxides, or cholesterol oxidation
products (COPs). Enzymatic methods from commercially
available kits are used to determine the cholesterol content. It
is necessary to hydrolyze the cholesterol ester fraction to deter-
mine the total cholesterol content.
In the analysis of sterols, these are first isolated from lipid
extracts by TLC or column chromatography, following hydroly-
sis if necessary. Individual components can then be determined
by GC, often after conversion to trimethylsilyl ether derivatives
to give sharper peaks. The addition of an internal standard
allows obtaining accurate qualitative and quantitative results
by GC. The most common internal standards for sterol
quantification are 5a-cholestane, dihydrocholesterol (5a-
cholestan-3b-ol), epicoprostanol (5b-cholestan-3a-ol), and
Fats: Classification and Analysis 601
SQUALENE
13
15
17 19 21
13´ 15´
30 40 50 60
Minutes
betulin. The internal standard is typically added to the sample
prior to alkaline or acidic hydrolysis, undergoing the same
extraction conditions as the sterol fraction, or it can be added
after extraction but prior to derivatization.
Acidic hydrolysis is necessary to free the sterols when the
sample has a complex protein or carbohydrate matrix and/or
steryl glycosides. If this is needed, the internal standard is
added to the weighed sample, and then, it is hydrolyzed with
hydrochloric acid at 100  C. After the acidic hydrolysis, lipids
are extracted using a nonpolar solvent. After solvent removal,
saponification of the lipid fraction is carried out as described in
the preceding text.
Once saponification has been done, the unsaponifiable frac-
tion may contain other lipids besides sterols including hydro-
carbons, carotenoids, tocopherols, free fatty acids, and other
triterpenes. For the unsaponifiable fraction of a refined fat or
oil sample, it is usually acceptable to proceed with derivatization
and GC analysis without further sample cleanup, and no prob-
lems with interference by these compounds are presented. How-
ever, in other types of samples, the sterol fraction needs to be
purified. For such purpose, silica, C18, and aminopropyl SPE
cartridges have all been used.
Nowadays, sterols are usually analyzed as trimethylsilyl
ethers, since their volatility, peak shape, and response factors
improve. Direct injection of nonderivatized sterols results in
broader peaks and a lower FID response. The silylating reagents
used are N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and
N-methyltrimethylsilyltrifluoroacetamide (MSTFA), as well as
mixtures of pyridine, hexamethyldisilazane, and trimethyl-
chlorosilane (9:3:1, v/v/v). Derivatization reactions may proceed
602 Fats: Classification and Analysis
completely in several minutes (about 15 or 20 min) at room
temperature or heated to complete the reaction when the
hydroxyl group on the sterols is hindered in any way.
After derivatization, sterols can immediately be analyzed by
GC or after carefully evaporating the derivatization reagents
with nitrogen at low temperatures to prevent any sterol ethers
and redissolving them in an organic solvent, like chloroform, to
an appropriate concentration before injecting. Split mode
injection is usually used and the injector temperature usually
set from 250 to 300  C. Fused silica capillary columns with
polysiloxane phases of low to midpolarity are used for their
analysis. The most common column phase used for sterol
analysis is composed of (95%) dimethyl–(5%) diphenylpoly-
siloxane. A nonpolar phase of (100%) dimethylpolysiloxane
can be used, but a good separation of sterol peaks and their
respective stanol peaks is not achieved with this column phase.
Midpolarity columns (14% cyanopropyl-phenyl-methylpolysi-
loxane) have been suggested for a better resolution of
D5-saturated and D5-unsaturated sterols.
For the identification of individual components of sterol
fraction, mass spectrometry may be required.
HPLC can be employed for the analysis of sterols, using UV
and mass spectrometric detection for their quantification and
identification, respectively. An HPLC-/atmospheric pressure
chemical ionization-MS method has been developed for the
analysis of these compounds, in which no previous derivatiza-
tion is needed. Several stationary reverse phases (C8, C18, and
zirconia-based adsorbent) have been studied for this purpose,
and the best separation of sterols has been obtained with the
C8 phase. Increasing the column temperature accelerates their
elution markedly. Nevertheless, different mobile phases have
been used.
Rigorous attention in the analysis of the COPs should be
attempted because they tend to be present at rather low levels,
and there is a danger of further oxidation or side reactions
during the analytic process. Both hot and cold saponifications,
using either ethanolic or methanolic potassium hydroxide
(KOH), have been used in the analysis of COPs. Their enrich-
ment can be carried out using either silica or aminopropyl SPE.
Besides, the sample should be protected against light, oxygen,
and high-temperature exposures, since they promote lipid
oxidation. HPLC and GC techniques have been used for the
analysis of COPs, but GC has given better results than HPLC.
Samples are derivatized to trimethylsilyl ethers using BSTFA,
Tri-Sil reagent (Pierce Chemical Co., Rockford, IL), or the mix
of hexamethyldisilazane–trimethylchlorosilane–anhydrous
pyridine (3:1:9, v/v/v) prior to GC and GC-MS analyses.
Fused silica open tubular capillary columns coated with (5%)
phenyl methyl silicone and columns coated with (100%)
dimethylpolysiloxane, such as DB-1, have been used for their
analysis. Similar results are obtained using DB5-MS and DB1-
MS columns. Although GC has been the most used technique
for the analysis of COPs, some HPLC methods have been
reported. A method using reversed-phase LC/atmospheric pres-
sure chemical ionization mass spectrometry for the successful
determination of cholesterol and its oxidized compounds in
processed foods such as pork, beef, chicken, and eggs has been
described. Diode array ultraviolet detection and laser light scat-
tering detection (LLSD) have been compared, concluding that
the use of LLSD in the HPLC of cholesterol and its oxidized
products could provide a reliable tool for the determination of
these compounds, since the use of this allowed the detection
of cholestanetriol and a- and b-epoxides. Between the different
internal standards used for their quantification are 5a-coles-
tane, betulin, 19-hydroxycholesterol, and betamethasone-17,
19-dipropionate.
4-Methylsterol and 4,4-dimethylsterol fractions are ana-
lyzed in a similar way to other sterols.
Tocopherols and Tocotrienols
These compounds consist of a polar chromanol ring and a
hydrophobic 16-carbon side chain attached to the ring via
the C2 atom. The difference is that tocopherols contain satu-
rated phytyl side chains, while tocotrienols have isoprenyl side
chains with three double bonds. They display antioxidant
properties and are active as vitamins (vitamin E), making
them particularly important for human health. They are
amphipathic and lipid-soluble and are easily oxidized when
subjected to heat, light, and alkaline conditions. Tocotrienols
are present in small amounts in food lipids. However, palm oil,
grape-seed oil, and the annatto lipid fraction contain a rela-
tively high amount of these components.
The determination of the tocopherols and tocotrienols as
trimethylsilyl derivatives can be carried out on a short GC
column of medium polarity (OV-17), with both flame ioniza-
tion and mass spectrometric detection, within a short time of
analysis, achieving a very good separation of the two classes of
components. However, the methods that are usually recom-
mended involve HPLC with fluorescence detection.
These compounds can be extracted using solvent or alkaline
hydrolysis extractions. Supercritical fluid or pressurized liquid
extractions are alternative techniques to traditional solvent
extraction methods that may be used to extract tocopherols
and tocotrienols.
HPLC is the most widely used technique for the analysis of
these compounds since they are stable under HPLC conditions
and easy to dissolve in appropriate solvents, and there are
several detectors that can be combined with this technique to
detect them, fluorescence detection and UV (l ¼ 290–300 nm)
detection being the most commonly used. Aside from this,
both normal-phase chromatography and reversed-phase chro-
matography can be applied.
Normal-phase using silica columns is commonly applied
when all eight tocopherols and tocotrienols need to be sepa-
rated, achieving better selectivity when hexane is used with a
relatively strong polar modifier 1,4-dioxane in the mobile
phase than with a weak modifier such as tert-butyl methyl ether.
However, reversed-phase might also be utilized when only
some vitamins are of interest or when mixtures of fat-soluble
vitamins and free and esterified tocopherols and/or tocotrie-
nols have to be separated.
The external standard method is usually used to quantify
tocopherols and tocotrienols because it is difficult to find a
compound that would not interfere with their analysis.
See also: Fatty Acids: Determination and Requirements; Fatty Acids:
Fatty Acids; Triacylglycerols: Characterization and Determination;

Triacylglycerols: Structures and Properties; Vegetable Oils:
Composition and Analysis.
Further Reading
Abidi SL (2001) Chromatographic analysis of plant sterols in foods and vegetable oils.
Journal of Chromatography A 935: 173–201.
Hamilton RJ and Rossell JB (eds.) (1986) The analysis of oils and fats. London: Elsevier
Applied Science.
Harwood J and Aparicio R (2013) Handbook of olive oil: analysis and properties.
New York: Springer.
International Organization for Standardization, ISO 12228:1999 (1999) Animal and
vegetable fats and oils-determination of individual and total sterols content-gas
chromatographic method. Geneva, Switzerland.
Jurado JM, Jiménez-Lirola A, Narváez-Rivas M, Gallardo E, Pablos F, and León-
Camacho M (2013) Characterization and quantification of 4-methylsterols and 4,4-
dimethylsterols from Iberian pig subcutaneous fat by gas chromatography–mass
spectrometry and gas chromatography–flame ionization detector and their use to
authenticate the fattening systems. Talanta 106: 14–19.
Fats: Classification and Analysis 603
Kamal-Eldin A, Gorgen S, Pettersson J, and Lampi AM (2000) Normal-phase high-
performance liquid chromatography of tocopherols and tocotrienols – comparison
of different chromatographic columns. Journal of Chromatography A 881: 217–227.
Kuksis A (ed.) (1978) Handbook of lipid research: fatty acids and glycerides, vol. 1.
New York: Plenum Press.
Laakso P (2005) Analysis of sterols from various food matrices. European Journal of
Lipid Science and Technology 107: 402–410.
Lercker G and Rodriguez-Estrada MT (2000) Chromatographic analysis of
unsaponifiable compounds of olive oils and fat-containing foods. Journal of
Chromatography A 881: 105–129.
Narváez-Rivas M, Gallardo E, Rı́os JJ, and León-Camacho M (2011) A new high-
performance liquid chromatographic method with evaporative light scattering
detector for the analysis of phospholipids. Application to Iberian pig subcutaneous
fat. Journal of Chromatography A 1218: 3453–3458.
Nielsen MM and Hansen A (2008) Rapid high-performance liquid chromatography
determination of tocopherols and tocotrienols in cereals. Cereal Chemistry
85: 248–251.
Relevant Website
http://lipidlibrary.aocs.org/ – AOCS Lipid Library.
 Fats: Production and Uses of Animal Fats
SB Smith and DR Smith, Texas A&M University, College Station, TX, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The composition of lipids in fatty tissues of meat varies in
response to diet, the time at which the lipids are deposited,
and adipose tissue depot. The fatty acid composition of pork is
especially sensitive to dietary manipulation, whereas that of
beef and lamb is affected primarily by the age of the animal
and fat depot, although fatty acid composition of ruminant
tissues can be modified by dietary means. For example, pasture
or grass feeding causes fat depots to be high in saturated fatty
acids, whereas grain feeding increases the concentration of
monounsaturated fatty acids.
Between 45% and 50% of the entire live weight of livestock
species does not enter directly into the human food chain. The
by-products of animal slaughter and fabrication include mus-
cle trim and a large quantity of the carcass fat (especially
subcutaneous and internal fat depots) that are not used in
processed meats. In addition, many internal organs are
included in total by-products. Altogether, these by-products
contain approximately 20% extractable fat. With rendering,
some of this extractable fat can be recovered and converted
into more useful and profitable materials such as lard or tal-
low, allowing part of carcass fat to reenter the human food
chain. The quality, and especially the fatty acid composition of
the rendered materials, determines the fate of the finished
product.
Sources of Lipid in Meat
There are four sources of lipid in meat that are used for meat
processing or rendering: the muscle fibers; subcutaneous fat;
intermuscular (seam) fat between muscle groups; and intra-
muscular (interfascicular and marbling) fat. Very lean beef,
pork, or lamb, in which all subcutaneous and intermuscular
fat has been removed, contains approximately 1% extractable
lipid. At the other extreme, even lean trim from Japanese Black
(or wagyu) cattle can contain over 40% extractable lipid. This
extraordinary concentration of lipid can be attributed largely to
lipid within the muscle.
Fatty Acid Composition of Subcutaneous, Intramuscular,
and Muscle Lipids
Lipids from fat, lean trim, and organs destined for rendering
contain the saturated fatty acids palmitic acid (16:0) and stea-
ric acid (18:0) and their delta-9-desaturase products, the
monounsaturated fatty acids palmitoleic acid (16:1n 7) and
oleic acid (18:1n 9). Palmitic acid is produced by the com-
bined activities of acetyl-coenzyme A carboxylase and fatty acid
synthase, whereas stearic acid is produced by the addition of
two carbons to palmitic acid via a fatty acid elongase. Palmi-
toleic acid and oleic acid are the products of the delta 9
604 Encyclopedia of Food and Health
desaturation of palmitic acid and stearic acid, respectively,
catalyzed by the enzyme stearoyl-CoA desaturase (SCD). Pal-
mitic, palmitoleic, stearic, and oleic acids are always present in
all tissues of animals (Figures 1 and 2), but the relative con-
centration of each fatty acid is dictated primarily by the activity
of the enzyme SCD.
The polyunsaturated fatty acids typically consumed by live-
stock species consist primarily of linoleic acid (18:2n 6) and
a-linolenic acid (18:2n 3). In the rumen of cattle, sheep, and
goats, polyunsaturated fatty acids are extensively isomerized to
cis-9, trans-11 conjugated linoleic acid (CLA) and other CLA
isomers (Figure 3). However, the concentration of CLA iso-
mers in ruminant tissues does not exceed 2% of total fatty acids
because CLA is almost quantitatively hydrogenated to stearic
acid in the rumen, although small portions of trans-vaccenic
acid and other fatty acid trans-isomers escape the rumen and
are absorbed in the gastrointestinal tract. For this reason, rumi-
nant tissues are relatively high in stearic acid and naturally
occurring trans-fatty acids.
The contribution of both de novo fatty acid synthesis and
fatty acid desaturation to the fatty acid composition of fat
depots is especially apparent in bovine fat (Figure 1). Stearic
acid constitutes more than 80% of the fatty acids available for
absorption from the duodenum in beef cattle, yet the most
abundant fatty acids in bovine fat are palmitic acid (the prod-
uct of de novo synthesis) and oleic acid (from desaturation of
stearic acid). Typically, abdominal fat is more highly saturated
than subcutaneous or intramuscular fat. In beef cattle, intra-
muscular fat is also higher in stearic acid than is subcutaneous
fat (Figure 1). Unlike the situation for beef, intramuscular fat
from pork contains less stearic acid than does subcutaneous fat
(Figure 2).
As monogastrics, swine have adipose tissues with fatty acid
composition that more closely resembles that of dietary fatty
acids. Thus, diets enriched with oleic or linoleic acid will cause
the lipids associated with pork to become similarly enriched
with these fatty acids. Polyunsaturated fatty acids are especially
susceptible to oxidation during long-term storage or rendering.
This results in the formation of undesirable aldehydes and
ketones, causing the rendered fat to be less acceptable. Feeding
pigs diets enriched with polyunsaturated fatty acids also causes
the carcasses to be oily and may lead to production problems
such as soft pork bellies. Enrichment of carcass lean trim and
fat with oleic acid, which is a solid at typical freezer tempera-
tures, does not appear to accelerate lipid oxidation.
The majority of the fatty acids are stored as highly nonpolar
triacylglycerols (Figure 4). Triacylglycerols coalesce into the
large lipid vacuoles that are the central features of adipocytes.
The triacylglycerol structure consists of a glycerol (i.e., three-
carbon alcohol) backbone containing three fatty acids in ester
linkages. The glycerol primarily is derived from glycerol-3-
phosphate, which in turn is derived from the metabolism of
glucose or, in the liver, from the phosphorylation of free
http://dx.doi.org/10.1016/B978-0-12-384947-2.00273-7
 Fats: Production and Uses of Animal Fats 605

SUBCUTANEOUS
Desaturation of Fatty Acids
PERIRENAL
Whereas ruminal hydrogenation of unsaturated fatty acids
INTRAMUSCULAR
50 makes it difficult to enrich beef with unsaturated fatty acids,
desaturation of fatty acids available for digestion in the duo-
denum ensures that the plasma, muscle, and fat depots are not
WEIGHT PERCENTAGE

40 overly enriched with saturated fatty acids, especially stearic acid.

The activity of SCD has been demonstrated in the liver, muscle,
subcutaneous fat, and intestinal mucosa of pigs and cattle. The
30 greatest SCD activity is located within the subcutaneous fat.
However, substantial activity also is observed in intestinal
mucosal cells, where it may function to reduce the concentration
20 of stearic acid in chylomicrons and very-low-density lipoproteins
produced within mucosal cells. In this manner, SCD activity
within mucosal cells largely influences the composition of fatty
10 acids available for deposition in other tissues.

0 Fatty Acid Composition and Melting Points of Lipids

16:0 18:0 18:1 18:2
BOVINE ADIPOSE TISSUE FATTY ACIDS Longer-chain fatty acids have higher melting points than
shorter-chain fatty acids, and more saturated fatty acids have
Figure 1 Fatty acid composition of bovine adipose tissues. higher melting points than unsaturated fatty acids. Because of
Reprinted with permission from Rule, D. C., Smith, S. B. and Romans, the abundance of stearic and palmitic acids, animal fats are
J. R. (1995). Fatty acid composition of muscle and adipose tissue of
typically solids at room temperature. At 90–100  F, rendered
meat animals. In: Smith, S. B. and Smith, D. R. (eds.) Biology of fat in
pork fat will separate with a liquid upper layer and solid
meat animals: current advances, pp. 144–165. Savoy, IL: American
Society of Animal Science. material in the lower layer. The solid portion is known as
stearin (from the Greek for animal fat) and is composed pri-
marily of triacylglycerols enriched with stearic acid. The liquid
SUBCUTANEOUS
portion is composed of the glycerol backbone in the form of
PERIRENAL
INTRAMUSCULAR
esters with the less saturated fatty acids, primarily oleic acid,
60
INTERMUSCULAR and is known as lard oil or olein (from the Latin for oil).
Due to differences in fatty acid composition, the melting
50 points of fat from different species vary. Lamb fat typically has
WEIGHT PERCENTAGE

the highest melting point, followed by beef fat and then pork
fat. With a high polyunsaturated fat content, poultry fat has the
40 lowest melting point of the four species (80–110  F). Related
to the higher internal temperature, the internal fats have higher
saturated fatty acid content and correspondingly higher melt-
30
ing points. The fat in the outer layers of the animal body is less
saturated with lower melting points. This corresponds to the
20 lower temperature near the body surface and the need to
maintain a more liquid state. For example, beef kidney fat
has a melting point of 104–122  F, whereas the external sub-
10 cutaneous fat has a melting point of only 89–110  F. Beef
brisket subcutaneous fat has an unusually low melting point
0 (77  F), due to its high concentration of oleic acid. Pork leaf fat
16:0 18:0 18:1 18:2 has a melting point of 110–118  F; pork back fat has a melting
SWINE ADIPOSE TISSUE FATTY ACIDS point of 86–104  F. Tallow and lard melting points are stan-
dardized as titers, which are the minimum temperature at
Figure 2 Fatty acid composition of porcine adipose tissues.
which fat congeals. For edible tallow, the minimum titer is
Reprinted with permission from Rule, D. C., Smith, S. B. and
Romans, J. R. (1995). Fatty acid composition of muscle and adipose 105  F, whereas for edible lard, the minimum titer is 100  F.
tissue of meat animals. In: Smith, S. B. and Smith, D. R. (eds.) Biology Major differences in fatty acid composition are observed
of fat in meat animals: current advances, pp. 144–165. Savoy, IL: across fat depots of beef carcasses (Figure 5). Brisket fat con-
American Society of Animal Science. tains over 6% palmitoleic acid and less than 10% stearic acid,
whereas subcutaneous fat overlying the flank contains less than
glycerol by glycerol kinase. In adipose tissue and intestinal 3% palmitoleic acid and more than 15% stearic acid. The
mucosal cells, 2-monoacylglycerol (derived from partial highly consistent, negative relationship between palmitoleic
hydrolysis of triacylglycerols) provides a portion of the carbon acid and stearic acid demonstrated in several studies indicates
backbone for triacylglycerol synthesis. that the concentrations of these fatty acids are coordinately
606 Fats: Production and Uses of Animal Fats
Oleic acid
18:1c9
Linoleic acid
18:2c9, c12
Figure 3 Structures of linoleic acid, oleic acid, and the cis-9,trans-11 isomer of conjugated linoleic acid. Large filled circles represent carbon; large
shaded circles represent oxygen; small shaded circles represent hydrogen.
controlled by SCD. The concentration of stearic acid in adipose
tissue lipids is the primary determinant of lipid melting points
(Figure 6), and even lipids extracted from fat depots of the
same carcasses can exhibit a remarkable range of melting
points. These differences in lipid melting point can have
major practical importance.
Procedures for Rendering Fats
Early rendering involved the addition of water to the by-
products in an open kettle or the injection of steam into a
sealed autoclave. This type of process is referred to as ‘tanking’
and could be used for both edible and inedible products. The
fat produced by this method was relatively light in color, but
the increased presence of added water resulted in an elevation
in the free fatty acid content, reducing the quality and making
the product more susceptible to oxidation. Therefore, for eco-
nomic reasons, dry rendering is most commonly used today.
Conjugated
linoleic acid
18:2c9, t11
In the past, each slaughter facility had its own steam-
jacketed kettle and rendered its own fat. Although the kettle
was jacketed in steam, the by-products were cooked in their
own juices with no added water, and therefore, this was con-
sidered a dry rendering method. Currently, in the United
States, there has been a general shift to centralized rendering
operations that collect animal by-products and ship them to a
central rendering facility.
Rendering fat involves applying heat, extracting moisture,
and separating the fat. To accomplish this, by-products are first
ground into a consistent particle size and then cooked at
115–145  C for up to 90 min. With the heating process, micro-
organisms such as bacteria and viruses are inactivated (an
advantage over other waste product disposal methods). While
the by-products are cooked, the jacket steam pressure must be
controlled, the mixture agitated, and the temperature moni-
tored for the desired end point. Over the years, there have been
advances in the uses of the rendered products and in the
rendering methods. Today, more energy-efficient, continuous

Figure 4 Typical structure of a triacylglycerol: sn 1 fatty acid (on left),
oleic acid; sn 2 fatty acid, palmitic acid; sn 3 fatty acid, linoleic acid.
This triacylglycerol would be common in porcine adipose tissue. In
bovine and ovine adipose tissues, palmitic acid would occupy the sn 1
position and stearic or oleic acid would occupy the sn 2 position. Large
filled circles represent carbon; large shaded circles represent oxygen;
small shaded circles represent hydrogen.
processes are used to allow the reuse of process vapors to
preheat or dry the materials. Filtering and bleaching systems,
as well as refining equipment to remove free fatty acids, may be
part of the rendering process.
During rendering, the melted fat floats to the top of the unit by
virtue of its lesser density, whereas protein and bone solids settle
to the bottom of the rendering kettle. Traditionally, the rendered
fat then was skimmed from the top of the rendering kettle. This
has been replaced with a screw press that draws off the melted fat,
and the melted fat is stored and/or transported in tanks.
Chemistry of Rendered Fats
Rendered fats consist primarily of triacylglycerols (Figure 4).
The characteristics of the rendered fat are determined by the
composition of fatty acids attached to the glycerol backbone,
Fats: Production and Uses of Animal Fats 607
10
Palmitoleic acid (16:1n-7) g/100 g total fatty acids
8
0
0 5 10 15 20 25 30
Stearic acid (18:0), g/100 g total fatty acids
Figure 5 Palmitoleic acid (16:1n 7) plotted as a function of stearic
acid (18:0) in subcutaneous fat. Lipids were extracted from eight
subcutaneous fat depots, taken from 50 carcasses of unknown
background. Lipids from brisket fat contained the greatest concentration
of palmitoleic acid and least stearic acid, whereas lipids from flank fat
contained the least palmitoleic acid and the most stearic acid.
Reproduced with permission from Turk, S. N. and Smith, S. B. (2009).
Carcass fatty acid mapping. Meat Science 81: 658–663.
which in turn is determined by the source of the raw materials
used in rendering. The most abundant fatty acids of animal fat
triacylglycerols are palmitic, stearic, and oleic acids, typically
comprising 20–25%, 10–30%, and 30–55%, respectively, of
the total fatty acids in lean and fat trim. Significant quantities
of linoleic and a-linoleic acids are contained in triacylglycerols,
especially in rendered poultry fat. Lipids from ruminant tissues
also contain measurable amounts of odd-chained fatty acids,
branched-chain fatty acids, trans-fatty acids, and conjugated
fatty acids, all of which are products of ruminal fermentation.
Lard and tallow also contain free fatty acids (primarily oleic
acid), but these must be no more than 0.5% in edible lard and
0.75% in edible tallow.
When the rendered fat contains more saturated fatty acids,
the fat is referred to as hard fat. It is more solid at room
temperature and has a higher melting point due to its high
concentration of stearic acid. When the rendered fat contains
more unsaturated fatty acids, the fat is referred to as a soft fat,
which is not as solid at room temperature due to a greater
abundance of oleic acid.
Oxidative Rancidity
Rendered fat high in polyunsaturated fatty acids will react
chemically with oxygen, resulting in off-flavors and odors
608 Fats: Production and Uses of Animal Fats

50 fat. Tallow is primarily derived from rendered beef fat. Choice

Brisket white cooking grease is derived from pork fat. Yellow grease
Chuck (not to be confused with off-colored white cooking grease) is
Flank restaurant-quality grease or cooking oil and may be from
45 Loin
Plate blended sources. Rendered fat provides concentrated sources
Rib of energy for use in feed for poultry, aquaculture, and pets.
Round Other uses of rendered fat include soap, candles, and biodiesel.
Sirloin
40

Conclusions
Slip points, ⬚C

35
Fatty acid composition of animal fats varies with animal spe-
cies, diet, and fat depot. Stearic acid is the primary determinant
of lipid melting points, although high concentrations of lino-
30 leic acid can effectively reduce melting points. Rendered fats
have a great deal of functionality, depending on the source of
the fat and the separation techniques. Rendering provides an
outlet for animal by-products that would otherwise be unusa-
25
ble. Differential rendering of fat trims that vary in melting
points improves the functionality and usefulness of the ren-
dered fat.
20

See also: Adipose Tissue: White Adipose Tissue Structure and

15
4 6 8 10 12 14 16 18 20
18:0, g/100 g total fatty acids
Figure 6 Lipid melting point (slip point) plotted as a function of stearic
acid (18:0) in lipids from eight subcutaneous adipose tissue depots.
Lipids from brisket adipose tissue had melting points less than 17  C,
whereas lipids from the flank had melting points greater than 45  C.
Reproduced with permission from Turk, S. N. and Smith, S. B. (2009).
Carcass fatty acid mapping. Meat Science 81: 658–663.
known as rancidity. Oxidative rancidity is catalyzed by the
presence of heat, light, salt, and iron, as well as other elements.
Because rancidity is not chemically defined or quantifiable,
measurements of oxidation products are often used to indicate
quality of the product. One such test is the peroxide value test
that measures the milliequivalents (me) of peroxide per kilo-
gram fat. A low peroxide value (<10 me) indicates a nonrancid
fat. Lard and leaf lard (from pig abdominal fat) should have a
peroxide value no higher than 5 to be considered high quality.
Function; Beef; Fats: Classification and Analysis; Fatty Acids: Fatty
Acids; Fatty Acids: Metabolism; Triacylglycerols: Structures and
22 24 26
Properties.
Further Reading
Haberstroh C and Morris CE (eds.) (1991) Fat and cholesterol reduced foods,
technologies and strategies. Houston, TX: Gulf Publishing Company.
Meeker DL and Hamilton CR (2006) An overview of the rendering industry.
In: Meeker DL (ed.) Essential rendering: all about the animal by-products industry.
Arlington, VA: National Renderers Association.
North American Industrial Classification System. (2002). Animal fats rendering.
Available online at www.naics.com/censusfiles/ND311613.HTM.
Smith SB (2013) Functional development of stearoyl-CoA desaturase gene expression
in livestock species. In: Ntambi JM (ed.) Stearoyl-CoA desaturase genes in lipid
metabolism, pp. 141–159. Springer.
Smith SB and Smith DR (eds.) (1995) Biology of fat in meat animals: current advances.
Savoy, IL: American Society of Animal Science.
Smith SB and Smith DR (2014) Adipose tissue. In: Devine C and Dikeman M (eds.)
Encyclopedia of meat sciences, 2nd ed., pp. 225–238. Elsevier.
Turk SN and Smith SB (2009) Carcass fatty acid mapping. Meat Science 81: 658–663.

Fate of Rendered Fats Relevant Websites

The US rendering industry produces an estimated 0.9 billion kg http://fss.k-state.edu/FeaturedContent/CarcassDisposal/PDF%20Files/CH%204%20-
edible tallow, 1.5 billion kg inedible tallow, 0.6 billion kg %20Rendering.pdf – Carcass Disposal.
yellow grease, 0.1 billion kg lard, and 0.6 billion kg poultry http://www.sciencedirect.com/ – Science Direct.
 Fatty Acids: Determination and Requirements
M Narváez-Rivas and M León-Camacho, Instituto de la Grasa (C.S.I.C.), Seville, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Fatty acids, with a few rare exceptions, are the main compo-
nents of oils or fats. Generally, fatty acids are combined as
esters, and they are the main compounds of lipids. The most
common ester composed of fatty acids is the triacylglycerol,
which is a combination of glycerol and three fatty acids.
According to the last definition, acetic acid would be the
first of the fatty acids, but most scientists do not consider it that
way. They start the monocarboxylic aliphatic series of saturated
fatty acids with the propanoic or butanoic acid. That series
extends to an acid with 30 carbon atoms (melissic acid). Usu-
ally, fatty acids have an even number of carbon atoms, but
there are fatty acids that have an odd number of carbon atoms,
such as the common margaric acid, with 17 carbon atoms. In
general, the aliphatic chain is linear, but in some cases, fatty
acids have branched chains: isoacids and anteisoacids. The
unsaturated fatty acids are very important along the aliphatic
chain, and we can distinguish them based on characteristics of
the double bond: cis or trans (oleic acid or elaidic acid), posi-
tion, number (linoleic acid, arachidonic acid), and possible
conjugation. Most of the unsaturated fatty acids are cis and they
have no conjugation. The trans form appears more or less
frequently in the comestible oils and/or fats when they have
been subjected to high temperatures (the energy supplied to
the cis fatty acids allows isomerization). Because of this, the
degree of thermal change in oil and/or fat can be measured
using trans fatty acid content. Natural oils and/or fats with trans
fatty acids exist, and an example is the fat of ruminants’ milk.
Some of the natural fatty acids show different unsaturation
to the double bond: ethylenic bonds, a cycle of three (cyclo-
propane or cyclopropene) or five (cyclopentene) carbon atoms
(sterculic acid and Chaulmoogric acid). Between the fatty acids
with saturated cycles, the most common have structures of the
type: dihydrosterculic ([9-10] methylen, octadecanoic; 9D,
10L, cis). The ring of three carbon atoms makes a plane, and
the substituents can be put above or under it. In that case, the
first carbon atom of the cycle has four valences linked to four
different radicals: dH, d(CH2)dCOOH, dCH2R1, and
dCHR0 1(CH2)[(CH2)7CH3]. The next carbon atom bonds in
a similar manner: dH, d(CH2)7CH3, dCH2R2, and dCHR0 2.
The two hydrogens attached to these carbon atoms can be on
the same side of the plane (cis) or on opposites sides (trans).
Moreover, two different forms exist for the same isomer, one
being the mirror image of the other, depending on the position
of the radicals of each carbon atom.
Most of the fatty acids only have the acid role. Nevertheless,
in nature, fatty acids with other roles exist, because an organ-
ism has synthesized them through a specific biochemistry (e.g.,
ricinoleic acid, with an OH group; licanic acid, with a ceto
group). Due to the action of the oxygen, the mechanics of
oxidation and autoxidation produce oxidized fatty acids. The
oxygenated functional groups that are part of natural fatty acids
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00278-6
are hydroxy, ceto, oxo, and epoxy. Among the components of
the natural wax, we can find a-, or 2-, hydroxy acids and b-, or
3-, hydroxy acids, which are also part of bacteria lipids. Among
the monohydroxy natural acids, there are acids with different
chain lengths and saturations, such as the mentioned ricinoleic
acid. Among the polyhydroxy acids, there are fatty acids (most
of them saturated) with chain lengths between 14 and 24
carbon atoms (most of them from 16 to 18 carbon atoms),
having 2–5 OH groups. The most common positions for these
groups are the Ω carbon and the positions adjacent to the
unsaturations, when the fatty acid includes them.
Fatty Acid Analysis
Classical Methods
Before the emergence of gas chromatography (GC), the
methods for determining the fatty-acid composition and the
attributes of fat were basically volumetric methods based on
some physical–chemical attribute of the fatty acids that were
part of fats. Since the advent of GC, some of these analytical
methods are still in use. The determination of fatty acids is the
primary goal of fat analysis.
Saponification value. This value is defined by the quantity (in
milligrams) of potassium hydroxide needed to saponify 1 g
of fat. The value shows us the total amount of fat that is
contained in the sample, or the combined volume of free an
esterified fatty acids, and, in some ways, this value also
shows us the lengths of fatty acids in oils and fats.
Reichert–Meissl index and Polenske index. The first index is a
volumetric method that denotes the amount of sodium
hydroxide needed to neutralize the fatty acids included in
5 g of fat, which can be dissolved into and swept away by
water vapor. So, this index indicates the type and quantity
of volatile fatty acids (with short chains). The second index
refers to insoluble fatty acids in water.
Iodine value. Organic compounds with unsaturation show the
property of adding halogens, as well as other compounds,
in double bonds. This property is used for the quantitative
determination of unsaturation; determining the number of
double bonds identifies the type of unsaturated fatty acids
contained in an oil or fat. This parameter is determined by
using sodium thiosulfate to titrate the iodine excess that
remains after the addition to the double bond, with clear
starch serving as the indicator.
Chromatographic Methods
We have seen that some of these rates are connected to chain
length and the number of double bonds, in the fatty acids.
After the advent of chromatographic methods, such as GC,
the previous methods went out of favor, because GC can
609
610 Fatty Acids: Determination and Requirements
provide more information than the volumetric methods can.
GC can also be applied to the analysis of fatty acids and their
esters. In general, for fatty acids, direct GC is not commonly
used. Instead, a fatty-acid derivatization using methyl esters is
carried out, followed by their chromatographic analysis. It is
only used for very short-chain acids that are very difficult to be
esterified, such as formic acid, acetic acid, propionic acid,
butyric acid, and valerianic acid.
The knowledge of the composition, both in quantity and
quality, of the fatty acids in oil and fat has always been one of
the main issues, because of its importance in oil and fat
characterization and the determination of their possible adul-
teration. The methods for isolating and derivatizing these com-
pounds and the selection of the most appropriate techniques
for their determination have been discussed in many scientific
publications. The following section describes the GC analysis
of these compounds and how they should be prepared for such
analyses.
Most of the time, the fatty acids are not free; when they are,
they occur only in small amounts. They usually form esters,
most often with glycerol, to produce glycerides (mono-, di-,
and tri-acylglycerols) and phospholipids. They can also form
esters with aliphatic alcohols of linear structure (waxes) or
terpenic structure (terpene and sterol esters).
Derivatization of fatty acids
For fatty acid analysis by GC, these compounds have to be
analyzed as volatile esters (usually methyl). The usual way to
derivatize fatty acids is by forming their methyl esters, because
they are more volatile and less polar than free acids, and this
makes them easier to elute in chromatographic columns. The
reaction that takes place is as follows:
O solution in methanol and hexane and then heating by reflux
=
CH2OC-R1 CH2OH
O O
=
Catal.
= =
CHOC-R2 + 3CH3OH CHOH + 3R-C-O-CH3
O Q
CH2OH
CH2OC-R3
This interesterification reaction involves slow kinetics. The
presence of water slows down the reaction ratio and decreases
the output. The reaction has to be accelerated by means of
catalysis and/or by increasing the temperature to be efficient.
The most common methods for preparing the fatty-acid
methyl esters use a methanolic medium with alkaline catalysis,
with acid catalysis, with alkaline and acid catalysis, or with
diazomethane.
Methylation in a methanolic medium with alkaline catalysis
Methylation with sodium methylate is the most widely used
method. A solution of sodium methylate in methanol, pre-
pared via the direct reaction of metallic sodium with methyl
alcohol, is added to the oil sample. Reflux heating cleans and
homogenizes the solution, thus dissolving the methyl esters in
the reaction medium. Then, they are extracted by adding a
solvent, such as hexane or diethyl ether, and a watery solution
of sodium chloride at 10% to separate both phases without
their emulsioning. This method presents some drawbacks.
Even though the reaction takes place with high yield, there is
always the danger that the sodium salts of fatty acids (soaps)
might form in the alkaline medium. In addition, free fatty acids
do not methylate properly when this procedure is used. To
overcome this disadvantage, the fatty matter can be treated
with sodium methylate in a methanol solution inside a closed
tube and heated. Because of the increased pressure inside the
tube, the temperature rises above the boiling point of
methanol, up to 85–90  C. This increases the reaction yield.
The heating has to take place for a period of no less than 2 h,
however.
Another procedure that gives good results is methylation
with potassium hydroxide. It consists of treating a solution in
hexane of fat with a KOH 2N methanol solution, shaking it,
and letting it decant. It is rapid and has the advantage of not
needing heating, which requires eliminating the volatilization
losses of acids with chains shorter than C12. This method is
used for the methylation of milk fat, and it is widely used to
avoid the isomerization of unsaturated fatty acids (analysis of
trans-isomer fatty acid), but free acids do not derivatize well
when this technique is applied; therefore, its use is not advis-
able for high-acidity oils.
Methylation in a methanolic medium with acid catalysis
Transesterification with methanol can also be catalyzed in an
acidic medium. A common procedure consists of making oil
react with a hydrochloric acid solution in methanol in a pro-
portion of 2–3%. The acid has to be previously dried in a
closed tube at 100  C for about 1 h. Also with this procedure,
it has been observed that the reaction is not complete. The
esters are extracted with hexane before being injected.
A variation of the method involves adding a sulfuric acid
for 90 min. This method increases the output of the reaction,
thanks to the dehydrating action of the sulfuric acid, shorten-
ing the time employed in preparing the sample.
Lewis’s acidic catalysis is another procedure that efficiently
uses methylation with methanol that has been catalyzed with
boron trifluoride. The sample is reflux-heated with a metha-
nolic solution of sodium hydroxide until it is homogenized
(complete formation of soaps). Next, a methanolic solution of
boron trifluoride is added, and the heating continues for a few
moments. The esters are extracted by adding a saline solution
with heptane, and, thus, the saponification process is not
necessary for fatty acids.
Methylation in a methanolic medium with alkaline and acid
catalysis
As noted above, methylation in an alkaline medium, besides
being incomplete, has the disadvantage that oil or fat can
produce soaps (to saponify), and free acids do not methylate
well in the alkaline medium. On the other hand, glycerides do
not methylate properly in an acidic medium. Thus, a wide-
spread method to improve the yield of the methylation reac-
tion consists of combining both forms of catalysis.
Usually, the sample first undergoes alkaline methylation
with reflux heating. The medium is then neutralized and
subsequently acidified by means of the methanolic solution
of sulfuric acid, before being reheated by reflux. This process

guarantees the hydrolysis of the soaps that might have been
formed during the first step and the esterification of the free
acids.
Another procedure consists of first treating the sample with
a methanolic solution of potassium hydroxide, heated by
reflux. Then, dimethyl sulfate is added, and the solution is
shaken and heated again. The reaction is monitored using
bromocresol green as the indicator. When the reaction is com-
plete, the indicator changes from blue to yellow. Finally, a
methanolic solution of sulfuric acid is added to separate the
phases. The extract of methyl esters can be injected after puri-
fication with alumina.
Methylation with diazomethane
The apparently fast methylation using the diazomethane pro-
cedure has been used for free acids. The sample, after being
dissolved in methanol, is treated with an ethereal solution of
diazomethane, which has been prepared at the very moment of
the reaction and under a nitrogen stream. The process is fast,
but it requires the previous preparation of the diazomethane
from N-methyl-N-nitroso-p-toluene-sulfonamide and 2-(2-
ethoxyethoxy) ethanol in an alkaline medium. Diazomethane
is toxic and explosive; therefore, some safety measures must be
taken.
Formation of methyl esters without the previous extraction
of the fat
In some cases, the analyst might be in trouble when undertak-
ing the study of the composition of fatty acids, either because
the amount of oily material on hand is minimal or because of
extraction selectivity. Another factor to be considered is the
possibility that the alteration of the fat during the study itself
can induce the analysts to make serious mistakes in their
conclusions. Some techniques have recently been developed
in order to carry out the interesterification of some oily mate-
rials without the previous extraction of the fat. Even though
this method can also be useful for oil transmethylation, its
main advantage is that it can be directly applied to fresh tissues
containing a considerable amount of water, such as olives,
without the need for them to be previously dried, as in the
aforementioned methods. In this method, both methylation
with methanol and acid catalysis are carried out during a dis-
solving phase. The following reactive mixtures have been suc-
cessfully used:
1. Methanol:heptane:benzene:2,2-dimethoxypropane:sulfuric
acid (37:36:20:5:2, by volume)
2. Methanol:heptane:toluene:2,2-dimethoxypropane:sulfuric
acid (39:34:20:5:2, by volume)
3. Methanol:heptane:tetrahydrofurane:2,2-dimethoxypro-
pane:sulfuric acid (31:42:20:5:2, by volume)
The working temperature is 80  C, and the process takes place in
a closed tube. Both extraction and transmethylation are carried
out in a single phase because the working temperature is the
same for both. After the reaction takes place, the sample is cooled
at room temperature, and two different phases occur. The
organic (superior) phase containing the methyl esters is then
ready for chromatographic analysis. This method is extremely
Fatty Acids: Determination and Requirements 611
interesting and allows for a great deal of possibilities, such as
system automation when there are many samples to be analyzed.
GC analysis of the methyl esters of fatty acids
Once fatty acid methyl esters have been obtained by means of
any of the methods mentioned previously, GC analysis can
be undertaken with different aims. The first focus, however, is
the study of the different types of chromatographic columns and
the analysis of the results that can be obtained by using each of
them.
The material used to prepare packed columns is the main
factor determining the nature of the separations that can be
achieved. Liquid phases, such as SE-30, OV-1, JXR, or OF-1,
separate fatty acids according to their molecular weight; when
the amount in the stationary phase is low, however, fatty acids
can also be separated by their unsaturation. Phases such as
Apiezón-L separate by unsaturation for the same chain length,
with the previous elution of the unsaturated components.
The polyester liquid phases are quite appropriate because
they allow for the separation of esters of the same chain length,
from 0 to 6 double bonds, eluting the unsaturated components
after their corresponding saturated compound. These phases
can be classified into three groups:
1. High-polarity phases, such as polymers of ethylene glycol
succinate (EGS), diethylene glycol succinate (DEGS), and
copolymers of the former with a methylsilicone (EGSS-
X™), CP-Sil 84™, and CP-Sil 88™
2. Medium-polarity phases, such as polyethylene glycol adipate
(PEGA), polybutanediol succinate (BDS), and EGS copoly-
mers with a high proportion of methylsilicone (EGSS-Y™)
3. Low-polarity phases, such as polyneopenthylglycol succi-
nate (NPGS), EGS with a phenyl-silicone (EGSP-Z™), Car-
bowax 20M™, and Silar 5CP™
EGSS-X™, EGSS-Y™, and newer phases of equivalent polarity
are widely accepted as the most useful representatives of the
first and second groups, respectively, because of their rela-
tively high thermal stability, particularly in work with packed
columns. The phases from the group of highest polarity are
stable at temperatures above those possible with EGSS-X™,
and they produce excellent separations of polyunsaturated
fatty acids. Figure 1 shows the analysis using columns of
15% of EGSS-Y™ in a 100–120 mesh silanized and acid-
washed support.
The low-polarity phases are mainly utilized in wall-coated
open tubular (WCOT) and support-coated open tubular
(SCOT) capillary columns because the saturated and monoe-
noic compounds of the same chain length are separated rather
poorly when these phases are used in packed columns. At the
same time, the quality and nature of the separations are related
to the quantity applied to the stationary phase support. For
example, low polyester levels are advisable for eluting the
methylesters of long-chain polyunsaturated fatty acids at low
temperatures. The relative retention time of fatty acid esters
(generally 16:0 or 18:0) tends to shorten as the amount of
liquid phase in the support decreases. Unfortunately, variation
in the retention times for the esters of fatty acids is inevitable
when the column ages through use, as the stationary phase
polymerizes further from the column.
612 Fatty Acids: Determination and Requirements

The nature of the support also influences the quality of the
separations. Silanized and acid-washed materials are inert, and
the separations are controlled only by means of the liquid
phase. A minute particle size (160–120 mesh) is the best for
analytical columns.
Some losses of esters of polyunsaturated acids partly
respond to the use of very active supports or aged columns
and to the transesterification by the polyester liquid phase,
1 6 7
which is caused by the remnants of the catalyzer used in the
fabrication of the polyester.
The use of WCOT columns of fused silica has led to remark-
able improvements in separations with higher resolution,
higher analytical precision in quality and quantity, and a
higher sensitivity, as well as a shortening of analysis time and
a reduction in the preparation of analysis procedures. Polygly-
col phases based on Carbowax 20M™, FFAP™, Supelcowax-
10™, and SP-1000™ seem to have been the most utilized in
recent years. Figure 2 shows a chromatogram of olive oil fatty
acids carried out in a SP-2380 capillary column (60 m,
0.25 mm, i.d. 0.25 mm film thickness).
Stationary phases are the same as those used in packed
columns; the only difference is that, instead of impregnating
a support, they wet the interior wall of the capillary tube. Most
phases are now chemically bonded to the tube wall, which
decreases loss in the column and, in turn, increases duration
and temperature resistance and improves the resolution.

Trans-fatty-acid determination
Trans-fatty-acid isomers should be almost completely absent in
oils and fats, because they are formed during technological
treatments such as partial catalytic hydrogenation; the content
and distribution of the trans isomers depend on the hydroge-
5
nation parameters (i.e., temperature, hydrogen pressure, and
2
the kind of catalyst). Thus, the quantification of trans fatty
8 acids is of great relevance to the authentication of oils and fats.
910 12
34 11
Thin-layer chromatography with silver nitrate
Since its introduction in 1962, thin-layer chromatography
0 10 20 Min. (TLC) using silica gel coated with silver nitrate has been very
Figure 1 Chromatogram of fatty-acid methyl esters in packed column useful for lipid analysis, because fatty acids can be separated
EGSS-X. 1: Palmitic; 2: palmitoleic; 3: margaric; 4: margaroleic; 5: according to the number of unsaturations, the geometric con-
stearic; 6: oleic; 7: linoleic; 8: linolenic; 9: arachidic; 10: gadoleic; 11: figuration of the double bonds, and even the position of the
behenic; 12: lignoceric. double bond. Mobile phase systems using diethyl ether or

1 6 8 13

9
3

14
20
19
2 11 12
5
4
7 16 18
15
17
10 21

0 5 10 15 20 24 min
Figure 2 Chromatogram of fatty-acid methyl esters in capillary column SP-2380, 60 m. 1: palmitic; 2: trans palmitoleic; 3: cis palmitoleic; 4:
margaric; 5: margaroleic; 6: stearic; 7: elaidic; 8: oleic (o9); 9: oleic (o7); 10: linoleic (9t, 12t); 11: linoleic (9c, 12t); 12: linoleic (9t, 12c); 13:
linoleic (9c, 12c); 14: arachidic; 15: linolenic (9t, 12c, 15t); 16: linolenic (9c, 12c, 15t); 17: linolenic (9c, 12t, 15c); 18: linolenic (9t, 12c, 15c); 19:
linolenic (9c, 12c, 15c); 20: gadoleic; 21: behenic.

hexane are more useful for separating general kinds of methyl
esters of the fatty acids, although it is not possible to resolve
them if they have three or more double bonds.
In fact, separations with more than four double bonds are
not easy, and mixtures of hexane/ diethyl ether 90:10 (v/v)
divide compounds with two double bonds. The obtained
bandwidths are visualized under ultraviolet light, after
revealing them with 20 ,70 -diclorofluoresceine, and the com-
pounds with zero to two double bonds are eluted with diethyl
ether or chloroform. The chloroform/methanol 9:1 is neces-
sary to recover completely unsaturated compounds.
Monoenoic natural fatty acids, which contain trans double
bonds, can be studied through separation of cis compounds by
TLC with silver nitrate eluted with hexane/diethyl ether (9:1,
v/v) as a mobile phase and eluted together with saturated com-
pounds. With care, it is also possible to separate positional
isomers of the unsaturated fatty acids by TLC with silver nitrate.
With a high concentration of silver nitrate (in the order of 30%)
and developing with toluene at low temperatures ( 5 to 25  C),
the methyl esters 6-; 9-y 11-cis-octadecenoic are well-separated.
Infrared spectroscopy methods
Infrared spectroscopy was used to determine the amount of
trans isomers of fatty acids, replacing chromatographic absorp-
tion methods with silver nitrate, until the appearance of capil-
lary columns of great length (60–100 m) and very polar
stationary phase, which have allowed the determination of
these compounds. First, to carry out analysis using infrared
spectroscopy, fatty-acid methyl esters must be produced using
some of the mentioned procedures, before measuring absorp-
tion at wavelengths of 10.36 mm. Before any measurements are
carried out, it is necessary make a calibration with standard
solutions, however.
GC methods
The determination of trans fatty acids has been carried out by
infrared spectroscopy. However, the use of GC with great-length
capillary columns allows the determination of the percentage of
trans fatty acids, as well as the separation of individual fatty-acid
esters of a given chain-length that differ in degree of unsatura-
tion and in the positions of the double-bonds (e.g., two isomers
of 18:1 and of 18:3 are separated). In the analysis of the methyl
esters of polyunsaturated fatty acids in oil, it is important to note
that the shorter the distance between the last double bond and
the end of the molecule, the longer the retention time of the
isomer. A good correlation exists between the data supplied by
GC and infrared instruments.
To determine the composition of trans fatty acids by GC,
fatty-acid methyl esters should be prepared by means of any
previously mentioned procedure. However, it has been
observed that, for samples with low trans-isomer contents,
methylation methods using heating produce an increase in
those isomers. Therefore, for this determination, cold methyl-
ation with methanolic potassium hydroxide or diazomethane
is recommended. In addition, different injection temperatures
have been studied because there is always the possibility that
cis-fatty-acid isomerization might occur in the chromato-
graphic injector. The results of the analysis of a virgin olive
oil sample with low trans-isomer content (Table 1) are similar
for methods B and C, whereas method A yields results that are
Fatty Acids: Determination and Requirements 613
Table 1 Effect of injection temperature and methylation method on
the elaidic and linoleic (9c, 12t) acid content of a virgin olive oil
Methoda Methodb Methodc
T  C injector 200 300 200 300 200 300
C18:1 9t 0.026 0.042 0.017 0.030 0.016 0.024
C18:29c,12t 0.012 0.090 0.012 0.013 0.015 0.013
a
Methylation in a methanolic medium with alkaline and acid catalysis.
b
Methylation with sodium methylate.
c
Methylation with potassium hydroxide.
Source: León-Camacho, M. and Cert Ventulá, A. (1994). Recomendaciones para la
aplicación de algunos métodos analı́ticos incluidos en el reglamento cee 2568/91
relativo a las caracterı́sticas de los aceites de oliva y de orujo de oliva. Grasas y Aceites
45, 395–401.
slightly higher in elaidic acid (18:1t), which is in agreement
with the preference for isomerization in an acidic medium. On
the other hand, a remarkable increase in elaidic acid seems to
occur as the injection temperature increases.
After numerous injections, an analyst might also observe an
increase in the amount of elaidic acid that results from the
catalytic action of the residues accumulated in the injector. On
the contrary, the cleaner the injector, the lower the amount of
acid that will result. Consequently, cold methylation is used,
with a solution of potassium hydroxide in methanol at a
temperature no higher than 225  C.
Regarding GC analysis, the methods indicate that the
amount of injected sample must be such that a peak of arachi-
dic acid (C20:0) should represent over 20% of the full scale,
although it must not be higher than 50%. It has been proved
that overlapping between the peaks corresponding to both
elaidic and oleic acid and a quantitative distortion, either
through discrimination or saturation of the detector, can occur.
The column used by GC analysis should show a chromato-
graphic profile similar to that presented in Figure 2, because
the linolenic acid has a slightly inferior retention time than the
eicosenoic acid. The use of columns of lesser polarity produces
the appearance of peaks that interfere in the determination of
the trans isomers of linolenic acid. These peaks can resemble
ethyl or other esters. The position of the trans isomers, there-
fore, has to be precisely determined by means of the analysis of
a refined oil sample.
An HP-88 capillary column, coated with 88% cyanopropyl
aryl siloxane (100 m  0.2 mm i.d  0.2 mm film thickness), or
a Varian Chrompack CP-Sil 88 column, coated with cyanopro-
pyl polysiloxane (100 m  0.25 mm i.d.  0.2 mm film
thickness), is able to provide an accurate quantitation of
trans-fatty-acid isomers previously derivatized as methylesters.
A better peak resolution is obtained if the carrier gas is hydro-
gen instead of helium. Furthermore, no silver-ion pre-
fractionation of trans fatty acid (by TLC, SPE, or HPLC) is
required prior to GC analysis.
Free-fatty-acid determination
Acidity
Acidity was determined through a volumetric method based on
fatty-acid neutralization using a nonaqueous solution of
sodium or potassium hydroxide and an acid–base indicator
614 Fatty Acids: Determination and Requirements
(usually phenolphthalein) to determine the final point of the
titration.
Gas-chromatographic method
Free fatty acids, together with mono- and diacylglycerols, form
a part of the fat polar fraction, and their analysis can be carried
out using a methodology for suitable separation and quantifi-
cation. This fraction is isolated by solid-phase extraction using
diol columns and 1,3-dimyristin as the internal standard. A
solution of hexane:methylene chloride:ethyl ether (89:10:1
v/v) is first applied to the column to eliminate triacylglycerols.
Subsequently, a portion of chloroform:methanol (2:1 v/v) is
applied to the column and collected. This fraction contains free
fatty acids and mono- and diacylglycerols, and it is evaporated
to dryness in a rotary evaporator under reduced pressure. The
residue is silylated and left at room temperature for 15 min.
Then, the residue is injected into the GC system using a DB-
17HT column and a flame ion detector. In the chromatogram,
the free fatty acids appear in first place, followed by mono-
acylglycerols and subsequent diacylglycerols. An example of
this can be observed elsewhere in this encyclopedia.
See also: Fats: Classification and Analysis; Fatty Acids: Essential Fatty
Acids; Fatty Acids: Fatty Acids; Fatty Acids: Trans Fatty Acids;
Triacylglycerols: Characterization and Determination; Triacylglycerols:
Structures and Properties; Vegetable Oils: Composition and Analysis.
Further Reading
Christie WW (ed.) (2011) The AOCS lipid library. AOCS. Press Available from: http://
lipidlibrary.aocs.org/topics/ester_93/index.htm.
Grandgirard A, Sebedio JL, and Fleury J (1984) Geometrical isomerization of linolenic
acid during heat treatment of vegetable oils. Journal of the American Oil Chemists
Society 61: 1563–1568.
Hamilton RJ and Rossell JB (eds.) (1986) The analysis of oils and fats. London: Elsevier
Applied Science.
Harwood J and Aparicio R (2013) Handbook of olive oil: analysis and properties.
New York: Springer.
International Olive Council (IOC) (2001) Preparation of the fatty acids methyl esters
from olive oil and olive-pomace oil. Madrid: International Olive Council, COI/T.20/
Doc. No 24.
International Union of Pure and Applied Chemistry (IUPAC) (1987) Standard methods
for the analysis of oils, fats and derivatives. Oxford: Blackwell Scientific
Publications.
Kuksis A (ed.) (1978) Fatty acids and glycerides. In: Handbook of lipid research, vol. 1.
New York: Plenum Press.
León-Camacho M (1997) Isomerization of cis-trans fatty acids during the deodorization
of edible oils. PhD thesis, Spain: University of Seville.
León-Camacho M and Cert Ventulá A (1994) Recomendaciones para la aplicación de
algunos métodos analı́ticos incluidos en el reglamento cee 2568/91 relativo a las
caracterı́sticas de los aceites de oliva y de orujo de oliva. Grasas y Aceites
45: 395–401.
León-Camacho M, Ruiz-Méndez MV, Graciani-Constante M, and Graciani-Constante E
(2001) Kinetics of the cis-trans isomerizatión of linoleic acid in the deodorization
and/or physical refining of edible fats. European Journal of Lipid Science and
Technology 103: 85–92.
Martı́n MJ, Valdenebro MS, León-Camacho M, Gonzalez AG, and Pablos F (2001) Fatty
acids as discriminant parameters of coffee varieties. Talanta 54: 291–297.
Narvaez-Rivas M, Vicario IM, Graciani Constante E, and León-Camacho M (2007)
Changes in the concentrations of free fatty acid, monoacylglycerol and
diacylglycerol of the subcutaneous fat of Iberian ham during the dry-curing process.
Journal of Agricultural and Food Chemistry 55: 10953–10961.
Nimal Ratnayake WM, Hansen SL, and Kennedy MP (2006) Evaluation of the CP-Sil 88
and SP-2560 GC columns used in the recently approved AOCS official method Ce
1h-05: determination of cis-, trans-, saturated, monounsaturated, and
polyunsaturated fatty acids in vegetable or non-ruminant animal oils and fats by
capillary GLC method. Journal of the American Oil Chemists Society 83: 475–488.
Vicario IM, Griguol V, and León-Camacho M (2003) Multivariate characterization of
isomeric fatty acids in Spanish biscuits and bakery products. Journal of Agricultural
and Food Chemistry 51: 134–139.
Relevant Websites
http://lipidlibrary.aocs.org/ – AOCS Lipid Library.
 Fatty Acids: Essential Fatty Acids
B Lands, College Park, MD, USA
ã 2016 Elsevier Ltd. All rights reserved.
The Nature of Staple Foods
As human society slowly changed from hunter-gatherer life-
styles to more agricultural and urban lifestyles, food from
capture or culture remained the source of nutrient energy
needed for physical activity and of essential amino acids
(EAAs) and essential fatty acids (EFAs) needed for growth and
healthy tissue functions. Plants make all of these needed nutri-
ents, and they also contain the vitamins and minerals that
sustain healthy human life. Local community decisions about
soil and climate conditions have influenced the shift from
opportunistic capture to deliberate culture of a food, but the
logical basis by which people selected a regional staple is not
appreciably evident in any written history. Eventually, domes-
tication of plants and animals led to regional foods that were
available on a more reliable basis with less expenditure of
human energy. Having sufficient food energy (in carbohydrate
or fat) is one obvious feature of a staple food plus its stability
on storage over time. Awareness of EFA actions seems to have
played no role in creating the markets for staple foods that were
well established by the mid-twentieth century.
Vitamins and essential nutrients were not discovered until
the twentieth century after trial and error experiences had
already led to established staple foods with adequate protein
(EAA) to successfully support healthy human growth and
development. However, knowledge of EFA abundance and
action remains a topic of growing awareness and concern
among the biomedical community in the twenty-first century.
Current knowledge of metabolism and physiology of EFAs
indicates that some traditional ethnic lifestyles unknowingly
developed healthier daily supplies of EFA than others. Now,
careful use of recent information about EFA is likely to give
new insights and priorities for current staple foods.
Plants make the essential 18-carbon polyunsaturated fatty
acids (PUFAs) that are essential for growth, development, and
function of healthy animals and humans. Figure 1 shows how
two different desaturases (not found in animals) convert non-
essential omega-9 (n
9) oleic acid into the omega-6 (n
6)
and omega-3 (n
3) acids. The 18-carbon polyunsaturated
acids, predominantly linoleic acid (LA) and alpha-linolenic
acid, are made and stored in plant tissues. They are not made
in animals for which they are essential vitamin-like nutrients.
Small amounts of two other EFAs, stearidonic acid and
gamma-linolenic acid, are formed in a few plants by the action
of a D6 desaturase on the n
3 or n
6 precursor. A similar
desaturase enzyme occurs in animals.
Table 1 shows recent annual worldwide production of
major plant food staples along with the balance of their n
3
and n
6 EFAs. The Omega-3–6 Balance Scores are derived
from the difference in milligrams per kilocalorie (kcal) of
eleven different n
3 and n
6 nutrients in a food item. A
negative value means that there is more n
6 than n
3 nutri-
ent, whereas a positive value indicates more n
3 than n
6
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00279-8
nutrient. The grains listed at the top of Table 1 have high
contents of carbohydrates, which provide food energy. Grains
also have sufficient protein to provide needed EAAs. Impor-
tantly, the dry forms of grains are stable over long storage times
and can be transported fairly easily. The legumes in the center
of Table 1 have twice the amount of protein. The dried seeds
also are stable over long storage times and can be transported
fairly easily. The plant foods at the bottom of the table have a
higher water content and lower nutrient density that limits
their distribution away from local markets. In the absence of
a scientific rationale favoring any particular staple food, con-
venience, historical precedent, and financial marketing consid-
erations tend to drive the choice of staples among different
communities.
Ethnic Food Traditions Affect EFA Intakes
The low magnitudes of many Omega-3–6 Balance Scores in
Table 1 show that most major plant staples have relatively
balanced amounts of n
3 and n
6 EFAs. However, the very
large negative values (
36 and
59) for the processed oils
from these staples indicate a presence of much greater amounts
of n
6 than n
3 nutrients. These values show how important
nutritional differences can occur between a food and its pro-
cessed product. Later sections in this article note some unin-
tended consequences of processing staple foods into forms not
used earlier during the development of human societies.
The ability of plants to make their own carbohydrates,
amino acids, fatty acids, and vitamins from their surrounding
supply of nitrogen, carbon dioxide, minerals, and solar energy
allows each plant staple to have gene-determined predictably
consistent composition. The nutrient composition of most
plant-based foods has remained fairly constant throughout
the transition from prehominid times to the present. However,
some cultivars developed by selective breeding have special-
ized differences in composition and yield. Some high-oleic,
low-linoleic seed oils come from selected sunflower and saf-
flower strains, but they are not yet widely marketed. The major
change in staples as human populations increased was a
shift from the plant foods at the bottom of Table 1 toward
increased use of the staple commodities near the top. Plants
at the bottom of Table 1 were more common in tropical
Africa during early hominid times and may have been major
foods during early human evolution. Humans began to culture
grains as staples only about 10 000 years ago.
Soybeans and peanuts have much more fat than the other
plant staples, and they provide a profitable opportunity to
separate the oil from the protein to be stored and marketed
separately. Food oils also can be recovered as by-products from
other commercial activities (e.g., cottonseed oil or lard). Over
150 million tons of food oils were produced in 2012 (Table 2).
615
616 Fatty Acids: Essential Fatty Acids

Plants make 18-carbon polyunsaturated

essential fatty acids (PUFA)

Oleic acid (18:1n-9) OL

Plants insert the omega-6 bond

Linoleic acid (18:2n-6) LA

Plants insert the omega-3 bond

α-Linolenic acid (18:3n-3) ALA

Plants & animals insert the Δ6 bond

Stearidonic acid (18:4n-3) SDA γ- Linolenic acid (18:3n-6) GLA

Figure 1 Plants make 18-carbon polyunsaturated essential fatty acids (PUFAs).

Table 1 Production and composition of plant staples

Staple food name Annual production (million tons) Protein (%) Carbohydrate (%) Fat (%) Omega-3–6 Balance Scores

Corn, maize 823 Mt 9.4 76 4.7
3 oil,
59

Rice, white 690 Mt 7.1 79 0.7
0.3 oil,
36
Wheat 685 Mt 12.6 71 1.5
2 oil,
54
Sorghum 66 Mt 11.3 79 3.3
4
Millet 30 Mt 11 73 4.2
5
Oats 24 Mt 17 66 6.9
6
Soybeans 231 Mt 36 30 20
17 oil,
59
Peanuts 45 Mt 26 16 49
29 oil,
36
Beans 23 Mt 22 62 1.4
0.3
Peas 11 Mt 24 64 1.2
1
Cassava 233 Mt 1.4 38 0.3
0.1
Sweet potato 110 Mt 1.6 20 0.1
0.5
Yam 52 Mt 1.5 28 0.2
0.4
Plantain 34 Mt 1.3 32 0.4
0.1
Taro 7 Mt 0.5 26 0.1
3

Table 2 Production and composition of food oils and fats Importantly, the new staple food oils and fats continue to be
adopted without open public discussion and rigorous evalua-
Staple food Annual production (million Omega-3–6 Balance tion of the health impact of the relative contents of n
3
name tons) Scores
linolenate and n
6 linoleate EFAs.
Palm oil 53
10
Soybean oil 41
59
Rapeseed oil 24
12 Animal Metabolism Has Competing Actions of n
3
Sunflower oil 15
74 (
4, high oleic) and n
6 EFAs
Margarine 14
69 to
21
Cottonseed 5
59 When humans and animals eat plants, the EFAs are metabo-
Butter 5
1 lized by desaturase and elongase enzymes (see Figure 2) that
Coconut oil 3
2
convert the 18-carbon PUFAs into 20- and 22-carbon highly
unsaturated fatty acids (HUFAs). Most of the enzymes are
indiscriminate, allowing the n
3 and n
6 forms of EFA to
The introduction of hydrogenated vegetable oils at the start of compete similarly for reaction during each metabolic step. As
the twentieth century began providing cheap semisolid short- a result, the relative abundance of a competitor affects the
enings and margarines (Omega-3–6 Balance Scores ¼
21 to amount of conversion that occurs. The proportions of n
3

69) that partially replaced lard (
10) and butter (
1) for and n
6 in the HUFA accumulated in human and animal
household and commercial food preparation. Food marketing tissues predictably reflect the proportions of competing n
3
messages about food oils built new cultural lifestyles by the late and n
6 PUFA in the foods eaten. In contrast to what occurs in
twentieth century, and regional ethnic differences in food plants, the EFA composition in human and animal tissues is
habits gave way to global corporate marketing priorities. determined by both genetics and environment.
 Fatty Acids: Essential Fatty Acids 617

When animals accumulate 20- & 22-carbon

highly unsaturated fatty acids (HUFA)
OMEGA-3 ACIDS complete with OMEGA-6 ACIDS

α-Linolenic acid (18:3n-3) ALA Linolenic acid (18:2n-6) LA

Δ6 desaturation
Stearidonic acid (18:4n-3) SDA γ-Linolenic acid (18:3n-6) GLA
elongation

20- & 22-carbon highlyunsaturated fatty acids (HUFA)

(20:4n-3) Dihomo-γ -linolenate (20:3n-6) DGLA
Δ5 desaturation
Eicosapentaenoic acid (20:5n-3) EPA Arachidonic acid (20:4n-6) AA
elongation
Docosapentaenoic (20:5n-3) n-3DPA Adrenic Acid (22:4n-6)
Sprecher shunt

Docosahexaenoic (22:6n-3) DHA (22:5n-6) n-6DPA

Figure 2 When animals accumulate 20- and 22-carbon highly unsaturated fatty acids (HUFAs), the omega-3 acids compete with omega-6 acids.

Table 3 Production and composition of animal staples

Staple food name Annual production (million tons) Protein (%) Carbohydrate (%) Fat (%) Omega-3–6 Balance Scores

Meat, pig 109 22 0 4
4

Meat, chicken 93 20 0 3
8
Meat, cattle 63 20 0 9
3
Cheese 17 23 3 30 0
Fish, carp 25 18 0 6 þ14
Fish, freshwater 7.5 19 0 1 þ18
Clams 5.0 15 0 1 þ14
Oysters 4.7 6 3 2 þ50
Fish, tilapia 4.5 20 0 2 þ7
Shrimp 4.3 20 0 1 þ19
Salmon 3.2 20 0 6 þ73 (þ28, farmed)

The Sprecher shunt noted at the bottom of Figure 2
involves an elongase that acts faster with the n
3 than the
n
6 intermediate during a complex set of multiple metabolic
steps coordinated between subcellular regions, which gives 22-
carbon acids with a D4 double bond. This process leads to a
greater abundance of 22:6n
3 (DHA) than 22:5n
6
(n
6DPA).
An interesting human genetic haplotype that has faster D6
and D5 desaturation activities was apparently selected favor-
ably among humans in central and western Africa where plant
staples were like those at the bottom of Table 1. Faster conver-
sion of n
3 and n
6 PUFAs to tissue HUFAs makes African-
Americans more responsive than Caucasians or Asians to the
high proportions of n
6 nutrients in typical American diets.
Health-related consequences of these different nutrient
responses are noted later in this article.
By domesticating animals, agriculture provided a continual
supply of animal-based staple foods that do not require wide-
ranging efforts of hunting and gathering. Rather, the meat,
milk, and cheese acquired from livestock are eaten with short
storage times. However, one important (and often ignored)
nutritional aspect of these foods is that the EFA present in
domesticated animals is controlled by the EFA that the farmer
provides to the animal. As a result, different farming environ-
ments produce different balances of n
3 and n
6 EFAs in the
animal product. The food chain that provides EFA to humans
merits continuing careful attention and management.
Public attention to animal-based staple foods (Table 3) is
focussed primarily on protein content, even though plants do
provide appreciable protein. Animals store little carbohydrate,
and their energy reserves are mainly stored in fats that have
mostly nonessential palmitic, stearic, and oleic acids. Farmers
feeding grains to their domesticated animals are adding EFA
that accumulate in the animal fat. The negative Omega-3–6
Balance Scores for pig, chicken, and cattle reflect the impact of
feeding grains (scores near
3 to
6) in contrast to leaves,
grass, and foliage (scores near 0 to þ3). The milk, cheese, and
yogurt produced in high alpine meadows have more positive
scores than those from Japan (where there is limited pasture
acreage).
In the same manner, high positive scores for aquatic ani-
mals reflect the impact of the algal-based food web in the
aquatic environment. At present, wild and domestic fisheries
worldwide annually provide fish at a level of 91 million tons
(Mt) by capture and 67 Mt by culture plus aquatic plant
capture of 1 Mt and culture of 24 Mt. Fisheries remain the
618 Fatty Acids: Essential Fatty Acids
last large hunter-gatherer enterprise on Earth, and they are
increasingly being ‘managed’ by people hoping to sustain
some suitable level of capture for the growing human
population.
Eating captured seafoods provides humans with much
more n
3 than n
6 EFAs, and those n
3 EFAs are predomi-
nantly long-chain n
3 HUFAs rather than the 18-carbon
PUFAs found in domestic plants. However, modern large-
scale intensive culture of seafood can involve feeding grain
and plant foods that shift EFA proportions away from those
normally found in the less intensive natural algal-based food
web of oceans and lakes. These commercial, financial factors
strongly affect the scores for the cultured staples listed in
Table 3. As a result, scores in the table are conditional average
values subject to differing environmental variations. As wild-
caught seafoods continue to have increasing societal pressures
to diminish their capture, the issue of what balance of n
3 and
n
6 EFAs occurs in cultured animal staples will become an
increasingly serious topic to manage carefully.
Highly Unsaturated Fatty Acids Form Bioactive
Mediators
While linolenic acid is not a dietary essential for plants, it has
important physiological actions in plant survival. This occurs
by a lipoxygenase-catalyzed oxidative conversion of the acid to
the plant hormone, jasmonate, which optimizes plant growth,
development, and immunity. For animals and humans, the
linolenic acid and LA obtained in foods also undergo oxidative
conversion to many potent bioactive derivatives that optimize
growth, development, and immunity in animals.
One of the first recognized signs of severe EFA deficiency in
laboratory animals was an impaired epidermal water barrier
essential to effective growth and survival in low humidity. Two
lipoxygenases, ALOX12B and ALOXE3, participate in forming
ceramide and hepoxilin derivatives that strengthen the epider-
mal barrier. Fortunately, the amount of EFA in the diet needed
to prevent deficits in the barrier and in growth is <0.3% of
food energy. Because nearly all edible staples can provide this
amount, EFA deficiency is very rare among humans.
An associated biomarker of EFA deficiency is the occurrence
of more than 50% of tissue HUFA in the form of the n
9
HUFA, eicosatrienoic acid. This happens when the supply of
dietary 18-carbon EFA is too low to compete effectively with
the large amount of endogenous oleate for the elongation and
desaturation enzymes that form the n
3, n
6, and n
9
HUFAs. Again, most dietary staples provide enough EFA to
maintain the %n
9 in HUFA of tissues below 2% of HUFA.
In fact, the frequent occurrence of undetectable levels of n
9
HUFA in blood samples from Americans may indicate that
they are eating much more EFA than is needed for good health.
One of the most important features of EFA action in
humans is the oxidative conversion of HUFA into a large
group of potent hormonelike mediators called eicosanoids.
These induce signaling actions by many different specific
receptors that occur on nearly every cell and tissue of the
body. Such signaling is essential for coordinating tissue
responses, and excessive signaling can shift healthy physiology
toward pathophysiology. Potent hormonelike mediators
formed by the oxidative action of cyclooxygenases are prosta-
glandins (PGs) and thromboxanes (TXs). Mediators formed by
lipoxygenases include leukotrienes (LTs), hydroxyeicosatetr-
aenoic acids (HETEs), hydroxyoctadecadienoic acid (HODE),
lipoxins (LXs), resolvins (Rv’s), hepoxilins (Hx’s), maresins
(Ma’s), and protectins (PDs). Cytochrome P450 enzymes oxi-
dize HUFA to bioactive epoxides (EET, EEQ, and EDP). Also,
endocannabinoids are EFA derivatives with potent actions that
regulate neural signaling functions.
After the recognition of EFAs in 1929, biomedical research
on their actions progressed slowly until the introduction of
sensitive gas chromatographic methods by 1960 that gave
detailed insights to EFA occurrence and metabolism. Soon
thereafter, the 1964 discovery that n
3 and n
6 HUFAs can
form eicosanoids with potent physiological impacts led to an
explosive expansion of biomedical research as shown in
Figure 3. This interest was fueled by intensive pharmaceutical
company interest in developing new drugs to moderate exces-
sive actions of n
6 bioactive mediators formed by a set of
metabolic steps called ‘the arachidonic acid cascade.’ While
the pace of publication of papers on eicosanoids per se slowed
after 1990, interest in the physiological impacts of n
3 and
n
6 essential nutrients on a wide range of human health
conditions continues to expand.
The Balance of n
3 and n
6 HUFA Actions Affect
Health
The essential n
6 linoleic acid is a very potent and effective
nutrient at very low intake levels. However, in the absence of
n
3 nutrients, it seems to have a very narrow therapeutic
window that moves readily from healthy physiology to patho-
physiology. The window of safe efficacy is widened by dietary
n
3 nutrients. That makes the balance between n
3 and n
6
EFA nutrients a vital aspect of healthy human physiology. The
PubMed research reports for
EFA & eicosanoids
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
1930 1940 1950 1960 1970 1980 1990 2000 2010 2020
Figure 3 Research reports for EFA and eicosanoids. Squares
indicate annual reports retrieved by PubMed for the combined terms:
essential fatty acids or linoleic or linolenic or arachidonic or
eicosapentaenoic or docosahexaenoic. Diamonds indicate annual reports
retrieved for the term eicosanoids.

paradox of a therapeutic window involves both benefit and
harm from an agent.
Such a situation is apparent in dose-related actions of
aspirin-like NSAIDs. Increasing amounts diminish harmful
actions of n
6 eicosanoids, whereas doses above the level
that gives benefit can begin to impair needed eicosanoid-
mediated actions and increase the risk of adverse side effects.
While low-dose aspirin is widely used to decrease the risk of
n
6 thromboxane-mediated heart attacks, high doses of
NSAIDs have well-recognized adverse actions. In the case of
dietary n
6 linoleate, intakes from 0.05 to 0.50 en% increas-
ingly meet the need for essential tissue functions, whereas
intakes above those amounts increase the likelihood of n
6-
mediated overreactions with well-recognized adverse side
effects that we treat with medications. Such a transition from
benefit to harm is not evident for the dietary n
3 nutrients that
compete with and moderate the n
6 nutrients.
Figure 4 gives the context for the impact on health condi-
tions by n
3 and n
6 nutrients and their accumulated tissue
HUFA. The proportions of n
3 and n
6 nutrients determine
the proportions of n
3 and n
6 accumulated in tissue HUFA.
This makes the %n
6 in HUFA a useful biomarker for average
dietary EFA intakes. When the stored n
3 and n
6 HUFAs are
released in the tissues, they are converted by cyclooxygenases
to prostaglandins. Slower rates of the formation of n
3 than
n
6 prostaglandins and weaker actions of the n
3 than n
6
mediators at selective receptors cause greater proportions of
n
3 than n
6 HUFA to give less severe signaling (and patho-
physiology). These dynamics are evident in cardiovascular
disease (CVD) and heart attacks that are so prevalent in Amer-
icans. Excessive n
6 mediator signaling leads to greater inflam-
mation and oxidative stress in atherosclerotic vascular wall
plaques and in greater thromboxane-activated platelet-
FOOD
amino acids
carbohydrates
fatty acids
essential fatty acids
n-3 & n-6 in HUFA HUFA Biomarker
of phospholipids
n-3 & n-6 in HUFA release
Excessive n-6 signals
n-3 eicosanoids
n-6 eicosanoids
vessel wall
plaques
platelet
activation
ischemia
thrombosis
arrhythmia
Figure 4 Food-based imbalances in n-3 and n-6 HUFA proportions. The HUFAs accumulated in phospholipids are released by phospholipase and form
eicosanoids that amplify oxidative and inflammatory processes, which lead to many unwanted health conditions. Modified from Lands, B. (2014).
Historical perspectives on the impact of n-3 and n-6 nutrients on health. Progress in Lipid Research 55, 17–29. Internet: http://www.sciencedirect.com/
science/article/pii/S0163782714000253, with permission.
Fatty Acids: Essential Fatty Acids 619
mediated thrombosis. Such conditions are less frequent in
people with 50% n
6 in HUFA than with a typically American
value of 78% n
6 in HUFA. In this way, the %n
6 in HUFA is
a useful biomarker for health risk assessment (HRA).
When stored n
3 and n
6 HUFAs are released in the
tissues, they are also converted by lipoxygenases to leukotri-
enes. A 10- to 20-fold slower formation of n
3 than n
6
cysteinyl leukotrienes gives less intense signaling in broncho-
pulmonary conditions like asthma and chronic obstructive
pulmonary disorder. Also, the 50-fold weaker actions of the
n
3 leukotriene B5 (LTB5) compared to n
6 LTB4 at its selec-
tive receptor give much less intense amplification of inflam-
matory conditions in people with 50% n
6 in HUFA than
with 78% n
6 in HUFA. The potent amplification of
immune-mediated inflammatory conditions by n
6 LTB4
(rather than n
3 LTB5) causes many widespread and diverse
impacts on human health. These effects are mediated by
increasing the formation and action of inflammatory cytokines
and chemokines, which can further amplify many harmful
health conditions.
A growing number of biomedical research reports
(Figure 3) link imbalanced n
3 and n
6 EFA intakes and
excessive n
6 eicosanoid actions to unwanted health condi-
tions, including arthritis, asthma, atherosclerosis, bone loss,
cancer growth, heart attacks, depression, and suicide; length of
hospital stays; classroom disruptions; oppositional behavior;
unproductive workplace behavior; and annual healthcare
costs.
Three examples in Figure 5 show how the biomarker for the
balance of EFA intake, the %n
6 in HUFA, provides a useful
HRA measurement for predicting the risk of several unwanted
conditions. Coronary heart disease (CHD) mortality rates per
100 000 people are much higher for people with more than
Food-Based Imbalance in
n-3 & n-6 HUFA Proportions
arthritis
asthma
colon cancer
oxidant stress & length of hospital stay
inflammation & psychiatric disorders
cell proliferation &
workplace disruption
impaired nitirc
lost productivity
oxide action
health care costs
Sickness
& Death
620 Fatty Acids: Essential Fatty Acids
175
250
125
CHD Mortality
100
Quebec Cree
75
Quebec lnuit
50
25
Greenland
20 30
Figure 5 The risk of adverse conditions is higher for people with more than 50% n-6 in HUFA CHD mortality rates (per 100 000 people) for different
ethnic groups, indicated by circles, with closed circles representing the quintiles in the large US MRFIT study. Annual healthcare claims (in $US)
from diverse American groups are noted with triangles. Average hours per day of pain in a randomized, controlled clinical trial are noted with squares.
50% n
6 in HUFA than for those with less. People in the
lowest quintile of the large US MRFIT study had an estimated
HRA value near 62%, and they had nearly half the incidence of
CHD as those in the upper four quintiles. A similar relation-
ship was seen for CVD in two large clinical studies. Fragmen-
tary evidence of annual healthcare claim costs from diverse
American groups with HRA values ranging from 82% to 64%
shows lower annual expenses for people with lower HRA
values. In a recent randomized, controlled clinical trial that
carefully lowered the intake of competing omega-6 nutrients
while increasing the omega-3 nutrients, the HRA value went
from 77% to 61% n
6 in HUFA, and the average hours of pain
per day was lowered by 44% in 3 months.
Omega-6 Eicosanoids Amplify Transient Insults into
Chronic Injuries
The chronic inflammatory damage to the blood vessels during
atherogenesis is a well-recognized precursor of thrombotic
heart attacks. These conditions are known to be made worse
by the actions of n
6 eicosanoids. However, the contribution
of n
6 EFA to this situation has not been widely recognized in
public health messages that focus on predictive risk factors
related to food energy toxicity. High food energy density and
caloric imbalances were recognized long ago to accompany
higher risks of CVD, although explicit causal mechanisms
that need to be prevented remained uncertain. Decreasing
associated predictive factors without removing the major
causal factor seems unethical as it creates a sense of risk
removal while leaving the causal factor undiminished to con-
tinue causing harm and a need for treatment.
Many people regard obesity, the visible biomarker of an
imbalance in food energy intake and expenditure, to have a
causal role in the risk for CVD. Elevated blood cholesterol
levels, long recognized to be associated predictively with
CVD, are also regarded by many to be a cause of CVD. Massive
financial investments in biomedical studies have gathered
evidence to support or disprove both hypotheses. However, a
7000 12
USA
6000 10
Quebec Urban
5000
8
4000
6
3000
4
Janpan 2000
40 50 60 70 80 90
% n-6 in HUFA
clear causal mechanism by which these two biomarkers cause
death has not yet been identified and prevented.
Food energy per se poses little health problem if it enters the
body’s metabolism at a rate slow enough to be expended by
the normal biological actions of work, exercise, or building
body tissues. Unfortunately, modern lifestyles include much
time spent sleeping, eating, driving a car, riding public transit,
standing in line, using computer or Internet, reading, and
watching television, activities that tend to burn only
200 cal 3 h
1. As a result, a 1200 kcal meal (commonly con-
sumed when eating out) will provide an extra 1000 kcal during
the next 3 h that the liver will likely convert to triacylglycerols
and cholesterol (Figure 6). An important dimension of food is
energy density (kcal g
1) that causes an apparently small
amount of food to deliver many calories per mouthful and
easily give many more kcal per hour than is expended. Figure 6
shows some of the biomarkers and mediators for the resulting
food energy toxicity and how the food energy per hour inter-
acts with the food n
6 per n
3 nutrients.
The 1984 NIH Cholesterol Consensus Conference noted
that elevated blood cholesterol results from a food energy
imbalance. The conference urged that the first step in the
treatment of people who have blood cholesterol levels between
220 and 280 mg dl
1 should be diet therapy and caloric restric-
tion, because weight loss and moderate physical exercise can
lower the blood cholesterol biomarker. It also urged that the
use of drugs to lower blood cholesterol should be used only
after using the most rigorous diet modification appropriate for
the individual. The conference added that “even when use of
drugs seems appropriate, it is important to stress that maximal
diet therapy should be continued.”
Because high food energy density is associated with both
CVD and elevated blood cholesterol levels, blood cholesterol
has a logical predictive association with CVD. Even though the
association does not prove causality, the public perception is
that cholesterol causes CVD deaths. However, results from a
large, long-term multiethnic study indicate that cholesterol’s
predictive relationship may occur only to the degree that peo-
ple have more n
6 HUFA than n
3 HUFA and the resulting
 Fatty Acids: Essential Fatty Acids 621

Food Energy Toxicity: Mediators & Markers

work & CO2

FOOD ENERGY exercise
amino acids acetyl-CoA synthesis
carbohydrates
fatty acids fatty acyl-CoA &
triacylglycerols
HMG-CoA Biomarkers
VLDL & Triacylglycerolemia
squalene cholesterol Adipose/Obesity
mevalonate

NEFA & LDL lipoprotein

isoprenoids prenylated proteins
Insulin Resistance
Elevated glucose
Excessive n-6 signals oxidant stress &
inflammation & Amplified
vessel wall cell proliferation & Transient
plaques impaired nitric Postprandial
platelet oxide action Insults
activation
ischemia
Sickness
thrombosis & Death
arrhythmia
Figure 6 Food energy toxicity: Markers and mediators. Food energy that is not converted to work and CO2 forms cholesterol and triacylglycerols
that are released to plasma as very-low-density lipoproteins (VLDLs). Hydrolysis of VLDL produces nonesterified fatty acids that cause transient
postprandial insults that are amplified into fatal chronic disorders by excessive n-6 eicosanoid actions. Modified from Lands, B. (2014).
Historical perspectives on the impact of n-3 and n-6 nutrients on health. Progress in Lipid Research 55, 17–29. Internet: http://www.sciencedirect.com/
science/article/pii/S0163782714000253, with permission.

35

30
25-yr.CHD mortality (%)

25 No.Europe-80%
USA-75%
20 Serbia-65%
So.Europe-60%
15
Crete-50%
10 Japan-40%

0
100 150 200 250 300 350
Serum TC (mg/dl)
Figure 7 HUFA imbalance controls risk prediction by a food energy biomarker. Fatal heart attack risk may be predicted by blood cholesterol levels only
to the degree that n-6 exceeds n-3 HUFA. Modified from Lands, B. (2014). Historical perspectives on the impact of n-3 and n-6 nutrients on health.
Progress in Lipid Research 55, 17–29. Internet: http://www.sciencedirect.com/science/article/pii/S0163782714000253, with permission.

excess of n
6 eicosanoid mediators amplifies transient insult
of food energy toxicity into chronic inflammatory plaques with
their prothrombotic actions. Figure 7 shows that blood cho-
lesterol levels have no clear relationship to death rates in Japan,
where the n
3 and n
6 HUFAs are well balanced.
Cholesterol has a strong relationship with CVD for people
in northern Europe or the United States, where dietary habits
maintain the HUFA balance near 75–80% n
6 in HUFA. In
these people, every large meal with its transient postprandial
insult (Figure 6) has a higher probability of producing chronic
inflammatory conditions. This evidence can help the public
turn its attention away from decreasing the noncausal
predictive biomarker of cholesterol toward decreasing the
causal biomarker of the %n
6 in HUFA. This will likely lead
to a successful preventive nutrition intervention, which has
eluded the public for so long.
Using Knowledge of EFA Contents for Effective
Preventive Nutrition
The previously mentioned discussion of causal mechanisms by
which EFA balance affects health creates a high priority for
informing individuals of their current HUFA balance and the
622 Fatty Acids: Essential Fatty Acids
balance of n
3 and n
6 nutrients in the foods they typically
eat. With such information in hand, people can begin to
reverse the unintended consequences that come from includ-
ing certain staples in their diet. An important step in this
approach was to describe the quantitative way by which eleven
major dietary n
3 and n
6 EFAs affect the accumulated bal-
ance of HUFA in tissues. An empirical equation describing this
process was developed in 1992 and followed in 2011 by the
development of Omega-3–6 Balance Scores. The scores com-
press data on milligrams per kilocalorie for 11 different EFAs in
a food into a single value that indicates the likely impact of the
food on the HRA measure, %n
6 in HUFA. Positive values
indicate that the food will increase the %n
3 in HUFA, and
negative values indicate that the food will increase the %n
6 in
HUFA.
Hundreds of vegetables like potatoes, onions, and cabbage
have scores near 0, indicating a balance between n
3 and n
6
nutrients. However, potato salad (
21), potato chips (
33),
fried onion rings (
11), and coleslaw (
14 to
33) provide
much more n
6 than n
3 because of added food oils (
36
to
80) and mayonnaise (
32 to
68). While most staple
foods have balanced EFA, current processing and marketing
procedures have unknowingly altered the balance toward
harmful health consequences. Of the top 100 foods eaten by
Americans (average score ¼
6.2), eleven have high negative
scores between
17 and
50, and only five foods have posi-
tive scores (þ2 to þ5). No seafood (e.g., solid white tuna, þ46;
wild salmon, þ73; and herring, þ70) is on the list.
A hundred legumes (peas and beans) have scores near 0,
but soybeans (
17) and peanuts (
29) have very negative
scores plus a high energy density from their oil content
(Table 1). These two legumes and their oils may cause
Americans to have 76–80% n
6 in HUFA in contrast to a
value near 60% for people in Mediterranean coastal villages.
Some unintended consequences of such regional food habits
that affect HRA values are reflected in Figure 5. Annual health-
care costs may be much less for people with HRA values near
50% compared to those with values near 80%. Public health
experts estimate that more than half of American healthcare
costs are for preventable health conditions, and many of those
conditions are worsened by excessive n
6 eicosanoid actions.
Public recognition that dietary n
6 linoleate in the absence of
n
3 nutrients has a very narrow (to almost nonexistent) ther-
apeutic window may begin to build a broader awareness of
benefits from foods that help people NIX the 6 and EAT the 3.
See also: Cholesterol: Factors Determining Blood Cholesterol Levels;
Ethnic Foods; Fats: Production and Uses of Animal Fats; Fatty Acids:
Metabolism; Fish: Dietary Importance and Health Effects; Food–Herbal
Medicine Interface; Legumes in the Diet; Lipoproteins; Mediterranean
Diet; Nutritional Epidemiology; Phospholipids: Physiology; Risk
Assessment of Foods and Chemicals in Foods; Soy Beans: Dietary
Importance; Vegetable Oils: Dietary Importance.
Further Reading
Ameur A, Enroth S, Johansson A, et al. (2012) Genetic adaptation of fatty-acid
metabolism: a human-specific haplotype increasing the biosynthesis of long-chain
omega-3 and omega-6 fatty acids. American Journal of Human Genetics 90: 809–820.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376635/pdf/main.pdf.
Beydoun MA, Fanelli Kuczmarski MT, Beydoun HA, Hibbeln JR, Evans MK, and
Zonderman AB (2013) o-3 fatty acid intakes are inversely related to elevated
depressive symptoms among United States women. Journal of Nutrition 143(11):
1743–1752. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796345/pdf/
nut1431743.pdf.
Blasbalg TL, Hibbeln JR, Ramsden CE, Majchrzak SF, and Rawlings RR (2011) Changes
in consumption of omega-3 and omega-6 fatty acids in the United States during the
20th century. American Journal of Clinical Nutrition 93(5): 950–962. http://ajcn.
nutrition.org/content/93/5/950.full.pdfþhtml.
Hibbeln JR, Nieminen LR, Blasbalg TL, Riggs JA, and Lands WE (2006) Healthy intakes
of n-3 and n-6 fatty acids: estimations considering worldwide diversity. American
Journal of Clinical Nutrition 83(6 Suppl.): 1483S–1493S. http://ajcn.nutrition.org/
content/83/6/S1483.full.pdfþhtml.
Lands WEM (2005) Fish, omega-3 and human health, 2nd ed. Champaign, IL: American
Oil Chemists Society.
Lands B (2008) A critique of paradoxes in current advice on dietary lipids. Progress in
Lipid Research 47(2): 77–106.
Lands WEM (2009) Human life: caught in the food web. Chapter 14In: Arts MT,
Brett MT, and Kainz M (eds.) Lipids in aquatic ecosystems, pp. 327–354. Germany:
Springer.
Lands B (2014) Historical perspectives on the impact of n-3 and n-6 nutrients on health.
Progress in Lipid Research 55: 17–29.
Lands B and Lamoreaux E (2012) Describing essential fatty acid balance as 3–6
differences rather than 3/6 ratios. Nutrition & Metabolism 9: 46–54. http://www.
nutritionandmetabolism.com/content/pdf/1743-7075-9-46.pdf.
Ramsden CE, Faurot KR, Zamora D, et al. (2013) Targeted alteration of dietary n-3 and
n-6 fatty acids for the treatment of chronic headaches: a randomized trial. Pain
154(11): 2441–2451.
Relevant Websites
http://www.dhaomega3.org – DHA/EPA Omega-3 Institute website.
http://www.efaeducation.org/omega3-6Balanceapp.html – Omega 3-6 Balance Scores
App download site.
http://efaeducation.org – Essential fatty acid education website.
http://faostat.fao.org/site/567/desktopdefault.aspx?pageID¼567#ancor – FAO data on
world-wide food capture and culture.
http://www.fatsoflife.com – Fats of Life Newsletter.
http://ndb.nal.usda.gov/ndb/ – USDA Nutrient Database search portal.
 Fatty Acids: Fatty Acids
S Petrovic and A Arsic, Institute for Medical Research, University of Belgrade, Belgrade, Serbia
ã 2016 Elsevier Ltd. All rights reserved.
From Dietary Fats to FAs
Fatty acids (FAs) are the compounds of animal and vegetable
fats and oils, also found in cell membranes as a component of
phospholipids. They represent a class of long hydrocarbon
chain molecules (C4 to C28 atoms) with a terminal carboxylate
group. The common dietary source of FAs is dietary fats, which
are degraded to the final digestive products monoglycerides and
free FA. In the body, most FAs are stored as triacylglycerols
(TGs), the uncharged esters of glycerol and three FAs.
The term fats or lipids is a common name for large spectra
of chemical compounds, including TGs, phospholipids, and
cholesterol, originating from the organism or from food. The
main lipids in humans, such as TGs or less abundant glycer-
ophospholipids, glycoglycerolipids, and sphingolipids, are the
esters of FAs and different alcohols. TGs constitute 90% of
dietary lipid and provide 30–40% of daily energy intake in
the Western world. In addition, ordinary diet contains a
small amount of phospholipids and cholesterol esters (also
having FAs in their structure) and free cholesterol.
The digestion of TGs is dynamic and complex process sup-
ported by water-soluble digestive enzymes, bile acids, and
motion of the digestive tract, predominantly taking place in
the small intestine. Lingual lipase from saliva is the first
enzyme meeting fats in the mouth. Its enzymatic activity,
together with physical action of chewing, and emulsifying
activity of phospholipids represent the first step in the diges-
tion of TGs. As a result, the fats form the tiny droplets and
become more accessible to the digestive enzymes. Aided by
stomach’s churning and contractions, gastric lipase stars with
the breakdown of TGs into diglycerides and FAs, but almost
complete TGs digestion occurs in the small intestine. Due to
the action of the bile, lipid droplets in the intestine became
emulsified increasing the surface area over a thousandfold and
therefore being more exposed to lipase activity. Pancreatic
lipase is the most important fat digestive enzymes. It acts on
TGs at water–lipid interfaces of bile micelles and with the
assistance of colipase accesses the TGs in the inner lipid core
to hydrolyze the ester links in positions 1–3. The final products
of fat digestion are monoglycerides and free FAs, which are
absorbed into intestinal epithelial cells.
Triacylglycerols, Phospholipids, and Cholesterol
TGs are the uncharged esters of glycerol and three FAs. On one
hand, they are the predominant type of lipid ingested by
humans and consequently the primary source of free FAs; on
the other, they are synthesized in humans by two main biosyn-
thetic pathways, the sn-glycerol-3-phosphate pathway in liver
and adipose tissue and a monoacylglycerol pathway in the
intestines. FAs derived from triacylglycerols are absorbed into
enterocytes to be reesterified into TG. Packed with proteins and
phospholipids, TGs in the form of lipoprotein particles,
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00277-4
chylomicrons, enter the lymphatic system and the blood to be
transported from the small intestine to other tissues.
The phospholipids, the main component of bio-
membranes, are present in different foods but mostly in fats,
oils, meats, nuts, wheat germs, and egg yolks. Their structure is
similar to TGs, with a phosphorus molecule in the location of
one FA. Phospholipids from the diet are cleaved at position 2 by
pancreatic phospholipase A2, releasing the lysophospholipids
and the free FAs.
Ordinary diet also contains a small amount of cholesterol,
originating from animal sources (meat, egg yolks, diary fats, and
butter). Cholesteryl esters, in which cholesterol is esterified with
long-chain FAs (usually linoleic acid (LA)), are the main form
of cholesterol in the diet. The pancreatic enzyme cholesterol
esterase hydrolyzes cholesteryl esters to free cholesterol and FA.
Cholesterol by itself is a steroid compound that does not contain
FAs, but shows some physical and chemical characteristics of fat,
like fat origin and fat-like metabolism. Comparing to phospho-
lipids and TGs, it is poorly absorbed from the diet.
There are plenty of different FAs occurring in dietary fats, as
well as in plant and animal tissues. To be better described, they
are named and classified according to their composition,
structure, and properties.
FAs Nomenclature
The nomenclature of FAs relies on three main FA
characteristics:
- The length of the hydrocarbon chain
- The degree of unsaturation (double bond)
- The position of the double bonds in the chain
The systematic name for a single FA is the name of its parent
hydrocarbon with the final ‘e’ substituted with ‘oic.’
Parent hydrocarbon Fatty acid
Octadecane Octadecanoic C18
The number of double bonds is given with the prefix ‘en,’
‘dien,’ ‘trien,’ ‘tetraen,’ and so on, before ‘oic.’
C18 with: Name
No double bond Octadecanoic
One double bond Octadecanoic
Two double bonds Octadecadienoic
Three double bonds Octadecatrienoic
The position of double bond is derived from numbering of
FA carbon atoms that starts at the carboxyl terminus as a C1.
Carbon atoms 2 and 3 are often referred to as a and b, respec-
tively. In the same manner, the carbon farthest from carboxyl
terminus (C atom from methyl group) is called the o (Figure 1).
623
624 Fatty Acids: Fatty Acids

The symbol D with the superscript number (Dn) is used to

Short-chain fatty acids <8 carbons
represent the position of double bond, where n indicates
Medium-chain fatty acids 8–14 carbons
lower-numbered carbon of each pair. For example, the cis-D9
Long-chain fatty acids
16 carbons
means that there is a cis-double bond between carbon atoms 9
Very-long-chain fatty acids >22 carbons
and 10; trans-D2 means that there is a trans-double bond
between carbon atoms 2 and 3.
Altogether, the systematic name for a single FA (common According to the degree of desaturation, FAs are as follows:
name arachidonic acid (AA) and scientific name 5,8,11,
14-eicosatetraenoic acid) is derived like this: Saturated fatty acids (SFA) No C–C double bonds
Unsaturated fatty acids (USFA) At least one C–C double
Total carbons 20 bond
Double bonds 4 Monounsaturated fatty acids Only one C–C double
Double bonds positions D5,8,11,14 (MUFA) bond
Systematic name 20:4D5,8,11,14 Polyunsaturated fatty acids Two or more C–C double
(PUFA) bonds
Alternatively, FA could be named according to a double
bond distance from the o carbon atom, which is in that case All double bonds in naturally occurring FAs are in the cis-
numbered as 1. The known examples are o-3 and o-6 FAs. conformation, with two pieces of the carbon chain on the same
side of the molecule, both ‘up’ or both ‘down.’ Thus, due to
FA Composition and Structure cis-double bond (Figure 3), the carbon tails of unsaturated FAs
have the kinks.
FAs vary in the length of their carbon chains, mostly having an The trans-conformation mostly appears by the conversion
even number of carbons, typically between 12 and 24. Their of naturally occurring cis-bonds, when unsaturated plant oils
hydrocarbon chain may be saturated or it may contain one or are used for frying. In trans-bond, two pieces of the molecule
more double bonds separated by at least one methylene group are on opposite sides of the double bond, one ‘up’ and other
(Figure 2). ‘down,’ across from each other. The most common SFAs,
Based on the number of carbon atoms, FAs are as follows: MUFAs, and PUFAs are listed in Table 1.
There is also a group of FAs, branched-chain FAs, with acyl
branch(es) connected to the hydrocarbon chain (Figure 4).
3 1
CH2 Normally, their carbon chain is saturated with an odd number
H3C COO−
ω of carbon atoms, and the branch is a methyl group usually
β α
giving an even number of carbons in total. Branched-chain FAs
(CH2)n CH2 are common constituents of the bacterial lipids. They also
2
represent the 1–2% of total FAs in mammalian tissues, derived
Figure 1 The numbering of carbon atoms in fatty acid hydrocarbon mainly from bacteria from ruminant intestine, while they are
chain. very rarely found in the integral lipids of higher plants.

Typical long chain SFA:

O H H H H H H H H H H H H H H H H H
H O C C C C C C C C C C C C C C C C C C H
H H H H H H H H H H H H H H H H H
Stearic Acid

Typical MUFA:

O H H H H H H H H H H H H H H H
H O C C C C C C C C C C C C C C C C C C H
H H H H H H H H H H H H H H H H H
Oleic Acid- Monounsaturated fatty Acid

Typical PUFA:

H H H H H H H H H H H H H H O
H C C C C C C C C C C C C C C C C C C OH
H H H H H H H H H H H H H H H H
Linoleic acid
Figure 2 The structural formulas of typical long-chain saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid.
 Fatty Acids: Fatty Acids 625

FAs with the ring in the carbon chain structure are cyclic FAs
(Figure 5). They are derived from appropriate monounsatu-
rated FAs, such as oleic, linoleic, and linolenic acids. The ring
is commonly cyclopropene or cyclopentene. Cyclopropane FAs
(CPA-FAs) and cyclopropene FAs (CPE-FAs) have three-
member carbocyclic ring. They are found in bacteria and some
plants, especially in the seeds of plants belonging to families:
Bombacaceae, Malvaceae, Sterculiaceae, Leguminosae, and
Ranunculaceae.
Properties of FAs
The properties of FAs and lipids derived from them are mark-
edly dependent on their structure, especially on FAs’ chain
length and degree of desaturation. The difference occurs in
the temperature of melting point, fluidity at room temperature,
water solubility (or hydrophobicity), and susceptibility to oxi-
dation and reduction.
The temperature of melting point is influenced by FA struc-
ture in the way that it is lower for unsaturated FA compared
with saturated FA of corresponding size. For example, the
melting point of palmitic acid (C16) is 63  C, whereas that of
palmitoleic acid (C16, which contains one cis-double bond) is
0  C. It happens because the straight hydrocarbon chain of
O
saturated FA favors highly ordered molecules packing, increas-
ing the melting point of FA. On the other side, the cis-double
bond(s) introduces a kink in the shape of unsaturated FAs,
preventing FA molecules to be closely packed and therefore
leading to melting point decrease. In opposite, due to its dif-
ferent orientation, the trans-double bond favors again linear
molecular shape, which helps molecules to pack together in a
stable repeating array, resulting in the higher melting point of
trans-double bond isomers. For example, the trans-double
bond isomer of oleic acid (elaidic acid) has a melting point
of 45  C and cis-oleic acid 13  C. The fatty acyl chains interact
more strongly as the chain length extended with each newly
added –CH2-, increasing the melting points of long-chain FAs
compared with shorter-chain FA. In addition, the FA melting
points decrease with chain branching, being higher for FA with
odd-number carbon chains than for FA with even-number
carbon chains. The FA fluidity at room temperature is in strong
connection with their melting point. SFA and their derivatives
are usually in solid state at room temperature, while MUFA,
PUFA, and their derivatives are liquid.
COOH
Figure 4 The structure of typical branched-chain fatty acid.

OH
COOH
Oleic Acid
lacto bacillic acid
Figure 3 Oleic acid structure with one cis-double bond and a kink in
hydrocarbon chain. Figure 5 The structure of typical cyclopropane cyclic fatty acid.

Table 1 Examples of saturated, monounsaturated, and polyunsaturated fatty acids

Common name Chemical structure Scientific notation C: double bonds

Caprylic acid CH3(CH2)6COOH 8:0

Capric acid CH3(CH2)8COOH 10:0
Lauric acid CH3(CH2)10COOH 12:0
Myristic acid CH3(CH2)12COOH 14:0
Myristoleic acid CH3 ðCH2 Þ3 CH ¼ CHðCH2 Þ7 COOH 14:1n5
Palmitic acid CH3(CH2)14COOH 16:0
Palmitoleic acid CH3 ðCH2 Þ5 CH ¼ CHðCH2 Þ7 COOH 16:1n7
Stearic acid CH3(CH2)16COOH 18:0
Oleic acid CH3 ðCH2 Þ7 CH ¼ CHðCH2 Þ7 COOH 18:1n9
Vaccenic acid CH3 ðCH2 Þ5 CH ¼ CHðCH2 Þ9 COOH 18:1n7
Linoleic acid CH3 ðCH2 Þ4 CH ¼ CHCH2 CH ¼ CH 18:2n6
(CH2)7COOH
a-Linolenic acid CH3 CH2 CH ¼ CHCH2 CH ¼ CHCH2 CH ¼ CH 18:3n3
(CH2)7COOH
Arachidic acid CH3(CH2)18COOH 20:0
Arachidonic acid CH3 ðCH2 Þ4 CH ¼ CHCH2 CH ¼ CHCH2 CH ¼ CH 20:4n6
CH2 CH ¼ CHðCH2 Þ3 COOH
Eicosapentaenoic acid CH3 CH2 CH ¼ CHCH2 CH ¼ CHCH2 CH ¼ CHCH2 20:5n3
CH ¼ CHCH2 CH ¼ CHðCH2 Þ3 COOH
Behenic acid CH3(CH2)20COOH 22:0
Erucic acid CH3 ðCH2 Þ7 CH ¼ CHðCH2 Þ11 COOH 22:1n9
Docosahexaenoic acid CH3 CH2 CH ¼ CHCH2 CH ¼ CHCH2 CH ¼ CHCH2 22:6n3
CH ¼ CHCH2 CH ¼ CHCH2 CH ¼ CHðCH2 Þ2 COOH
Lignoceric acid CH3(CH2)22COOH 24:0
626 Fatty Acids: Fatty Acids
The water solubility of FA also depends on the length of
hydrocarbon chain. In general, FAs are poorly soluble in water,
with the increase of hydrophobicity parallel to chain extension.
Even more, the solubility of FA is additional hindered by their
tendency to form micelles and monolayers in aquatic environ-
ment. However, FA became more water-soluble when present in
the form of potassium or sodium salts, as well as in the medium
with increasing pH. At the same time, FAs will be well dissolved
in nonpolar solvents such as ethanol and chloroform.
In terms of the chemical susceptibility, SFAs are very stable,
whereas USFAs react more easily. Here states the rule: the more
double bonds, the greater reactivity.
Like other carboxylic acids, FAs can be esterified due to the
exchange of groups attached to the FA carboxyl. In biological
systems, this type of reactions converts FA to TGs and vice
versa. Ester exchange reactions, with TG as a starting point,
are also used in the manufacture of TGs enriched in medium-
chain FAs, methyl esters, glycerol, or biodiesel.
Despite hydrophobicity, in aquatic environment, at suit-
able temperatures and pressures, FA can undergo hydrolysis,
as used for the industrial production of glycerol and soaps.
However, FA hydrolysis usually needs to be catalyzed by acid,
base, or lipase. Lipases are enzymes that hydrolyze FAs from
lipid species (e.g., TGs and phospholipids) in vivo. They favor
the reaction of FAs with D9 double bond, when compared to
D4, D5, or D6 double bond. Being active in milder conditions
(less solvent and lower temperature and pressure), lipases are
used for large-scale reactions in the manufacturing of confec-
tionary fats and nutritional products (cocoa butter).
Both the double bonds and adjacent allylic carbons of FA
alkyl chain are susceptible to oxidation, with higher suscepti-
bility following higher degree of unsaturation. When exposed
to air (oxygen), unsaturated FAs undergo a process of auto-
oxidation, which is a free-radical chain reaction giving the
allylic hydroperoxides as a final product. The process becomes
much faster in the presence of light (photo-oxidation), natural
pigments (chlorophyll, hematoporphyrins, and riboflavin),
and dyes (erythrosine and methylene blue). Ex vivo, oxidation
leads to the deterioration of unsaturated oils and fats, reducing
their nutritional quality. Still, due to antioxidants in their
content (tocopherol), most vegetable oils can resist this pro-
cess. In vivo, enzyme-catalyzed oxidation is the initial step in
the production of eicosanoids and jasmonates.
The FA alkyl chain is susceptible to reduction at both
carbon–carbon double bond and the carboxyl group of FA.
From nutritional point of view, attention should be given to
catalytic partial hydrogenation. This is a process introducing H
atoms in the double bonds, accompanied by a variable degree
of cis- to trans-isomerization. Edible vegetable oils are trans-
formed by partial hydrogenation to the solid form of marga-
rines. The arising content of trans-FA in margarines is a subject
of concern because of their atherogenic properties.
There are some other reactions involving FAs but mainly
used for specific industrial handling and therefore not addi-
tional disused.
FA Metabolism
FAs in the body are organized in two forms: free or unesterified
and esterified. In the plasma, free FAs (5% of total FA) are
bound to albumin and to a lesser extent to globulins and
lipoproteins. Most of the FAs in circulation (95% of total FA)
are in the form of triglycerides, cholesterol esters, and phos-
pholipids. In biological systems, FAs usually contain an even
number of carbon atoms, mostly 14–24.
Synthesis of Saturated FA
Synthesis of FAs takes place in the cytosol of the liver, adipose
tissue, lung, and brain. Precursor of all carbon atoms is acetyl
CoA generated by oxidative decarboxylation of pyruvate, deg-
radation of amino acids, and b-oxidation of FA.
Prior to the FA synthesis, malonyl CoA is formed from acetyl
CoA by the action of acetyl CoA carboxylase, which has biotin as
prosthetic group. The reaction of carboxylation is carried out in
two stages. The first step is the formation of a carboxybiotin with
the presence of ATP and the second step involves CO2 transfer to
acetyl CoA and formatting of malonyl CoA:
HCO3 þ ATP þ Enzyme  Biotin
! Enzyme  Biotin  CO2 þ ADP þ Pi
Enzyme  Biotin  CO2 þ acetyl CoA
! malonil CoA þ Enzyme  Biotin
After synthesis of malonyl CoA, a series of four reactions in
repeating cycle follows: condensation, reduction, dehydration,
and reduction, which lead to the extension of the FA chain.
The FA synthesis starts with covalently binding of acetyl CoA
and malonyl CoA to the sulfhydryl group of an acyl carrier
protein (ACP) and formation of acetyl ACP and malonyl ACP.
Enzymes that catalyze these reactions are acetyl transacylase and
malonyl transacylase. Afterward, the reaction of condensation
follows, in which acetyl ACP and malonyl ACP form acetoacetyl-
ACP (b-ketoacyl ACP) under the action of acyl-malonyl-ACP
synthase (b-ketoacyl-synthase) and CO2 is released. In the third
step, acetoacetyl-ACP is reduced to D-b-hydroksybutyryl-ACP,
with NADPH as a reductant, by the action of b-ketoacyl-ACP
reductase. The following is the process of dehydration:
D-b-hydroksybutyryl-ACP to a, b-trans-butenoyl-ACP (crotonyl-
ACP) in the presence of 3-hydroxyacyl-ACP dehydratase. Finally,
crotonyl-ACP is reduced to butyryl-ACP by enoyl-ACP reductase
(Table 2). In the following reactions, butyryl-ACP reacts with
malonyl-ACP and the cycle is repeated.
The synthesis of FA is continued up to palmitoyl-ACP (a
total of 7 cycles) that is then cleaved to palmitate and ACP
under the influence of thioesterase:
Palmitoil  ACP þ H2 O ! palmitate þ ACP
The syntheses of SFA, of up to 16 carbons in length, are
catalyzed by the enzyme system that is called the fatty acid
synthase (FAS). Mammalian FAS is a homodimer, found in the
cytoplasm, consisted of the 250-kDa subunits containing three
domains each. Domain 1 includes enzymes acetyl transacylase,
malonyl transacylase, and acyl-malonyl-ACP, while domain 2
contains ACP, b-ketoacyl-ACP reductase, b-hydroxyacyl-ACP
dehydratase, and enoyl-ACP reductase. Domain 3 contains
thioesterase enzymatic activities. Unlike the eukaryotes where
FAS is a large polypeptide chain with seven different enzymatic
activities in two catalytic centers, in bacteria, FAS is a multien-
zyme complex consisting of six different enzymes plus a sepa-
rate ACP.

Table 2 Reaction in fatty acid synthesis
Reaction
Acetyl CoA þ ACP $ acetyl  ACP þ CoA
Malonyl CoA þ ACP $ malonyl  ACP þ CoA
Acetyl  ACP þ malonyl  ACP $ acetoacetyl  ACP þ ACP þ CO2
Acetoacetyl  ACP þ NADPH þ Hþ $ D-b-hydroxybutyryl  ACP þ NADPþ
D-b-Hydroxybutyryl  ACP $ crotonyl  ACP þ H2 O
Crotonyl  ACP þ NADPH þ Hþ ! butyryl  ACP þ NADPþ
The final product of FA synthesis by FAS complex, which
utilizes NADPH as reductant, is palmitate. In summary, stoi-
chiometry for synthesis of palmitate can be presented with the
following reaction:
8 acetyl CoA þ 14 NADPH þ 7 ATP
! palmitate þ 14 NADPþ þ 8 CoA þ 7 ADP þ 7 Pi
FA synthesis occurs in the cytosol using acetyl CoA, which is
generated in the mitochondria. Since the transport of acetyl
CoA through the mitochondrial membrane is not possible,
acetyl CoA is transferred to the cytosol with the use of citrate.
Citrate, formed in the mitochondrial matrix, in reaction of
acetyl CoA and oxaloacetate, passes across the inner mitochon-
drial membrane and enters the cytosol. In the cytosol, under
the influence of ATP citrate lyase, citrate is cleaved to acetyl
CoA and oxaloacetate.
Synthesis of Long-Chain FA
FAs in the cells, produced by FAS or taken up from the diet,
may be elongated into long-chain FAs containing 18 carbon
atoms or more. Synthesis of long-chain PUFA in mammalian
tissues is performed in the endoplasmic reticulum using 4
enzymes; three of these are on cytosolic side of the ER mem-
brane and the fourth is embedded in the membrane. The
formation of long-chain FAs starts the same as with FAS, with
the reaction of malonyl CoA and acyl CoA and formation of
b-ketoacyl CoA, in the presence of elongases. This is followed
by the reactions of reduction, dehydration, and repeated reduc-
tion to form the acyl chain that is extended with 2C atoms.
Three enzymes are involved: 3-ketoacyl CoA reductase, 3-
hydroxyacyl CoA dehydratase, and 2,3-trans-enoyl CoA reduc-
tase. In mammals, condensation reaction is catalyzed by
enzymes of elongation, known as elongases of very-long-
chain fatty acids (ELOVLs). There are seven ELOVLs including
elongases 1, 3, 6, and 7 involved in the elongation of SFA and
MUFA and elongases 2, 4, and 5 involved in the elongation of
PUFA. Microsomal cell vesicles containing the endoplasmic
reticulum fraction possess the highest enzyme activity for acyl
CoA elongation.
Unsaturated FAs are synthesized by introducing a double
bond at a specific position on the acyl chain of long-chain FAs,
using the enzyme acyl CoA desaturase. These desaturases are
present in the membrane of endoplasmic reticulum and use
fatty acyl CoA as substrates and cytochrome b5 as an electron
donor. The desaturases can be divided into two distinct fami-
lies referred to as stearoyl CoA desaturases (SCDs) or D9
desaturase and FA desaturases (FADS), which included D6
and D5 desaturases.
Fatty Acids: Fatty Acids 627
Enzyme
Acetyl transacylase
Malonyl transacylase
Acyl-malonyl-ACP synthase
b-Ketoacyl-ACP reductase
3-Hydroxyacyl-ACP dehydratase
Enoyl-ACP reductase
The conversion of saturated to monounsaturated FA is car-
ried by D9 desaturase. This enzyme introduces the first cis-
double bond at position 9, 10 from the carboxyl end of FAs.
The reaction is catalyzed by three enzymes: NADH-cytochrome
b5 reductase, cytochrome b5, and D9 desaturase:
NADH ! FAD ! cyt b5 ! desaturase
Stearoyl CoA þ NADH þ Hþ þ O2
! oleoyl CoA þ NADþ þ 2H2 O
The syntheses of PUFA are catalyzed by D6 desaturase and
D5 desaturase. The D6 desaturase can introduce a double bond
at position D6 of the essential FAs of the n6 (LA C18:2n6)
and n3 family (a-linolenic acid (ALA) C18:3n3) (Figure 6).
After the reactions of desaturation and elongation by D6 desa-
turase and elongase, D5 desaturase introduces another double
bond at position D5 of 20-carbon chain in 20:3n6 and
20:4n3 (Figure 6). The synthesis of docosahexaenoic (DHA)
(22:6n3) is carried out from the 22:5n3 through two
successive elongations, accompanied by D6 desaturation of
24:5n3 in 24:6n3, and followed b-oxidation in peroxi-
somes to DHA (Figure 6).
Opposite to plants, mammals lack D12 and D15 desa-
turases, which introduce double bonds at carbon atoms
beyond C9 in the FA chain, and cannot synthesize LA and
ALA de novo. Thus, these FAs are essential for mammals and
must be obtained from food.
Catabolism of FAs
FA Activation
FAs primarily enter a cell via FA protein transporters on the
cell surface. FA transporters include FA translocase, tissue-
specific FA transport proteins, and plasma membrane-bound
FA-binding protein. Depending on the tissue and its metabolic
demand, FAs in cell can be converted to either triacylglycerols
or membrane phospholipids or oxidized in the mitochondria
for energy production. Before this processes, FAs must be acti-
vated to acyl CoA. The reaction of activation, taking place on
the outer mitochondrial membrane, is catalyzed by acyl CoA
synthase (or FA thiokinase) to form an acyl adenylate. In the
second reaction, sulfhydryl group of CoA attacks the acyl aden-
ylate and forms acyl CoA and AMP:
R  COO þ ATP þ HS  CoA $ R  CO  AMP þ PPi
acyl adenilate
R  CO  AMP þ HS  CoA $ R  CO  CoA þ AMP
acyl CoA
628 Fatty Acids: Fatty Acids
Omega-6 fatty acids
Linoleic acid
(C18:2n−6; LA)
Δ-6 desaturase
γ-linolenic acid
(C18:3n−6; GLA)
ELOVL2
Dihomo-γ-linolenic acid
(C20:3n−6; DHGLA)
Δ-5 desaturase
Arachidonic acid
(C20:4n−6; AA)
ELOVL2 or ELOVL5
Adrenic acid
(C22:4n−6)
ELOVL2
Tetracosatetraenoic acid
(C24:4n−6)
Tetracosapentaenoic acid Δ-6 desaturase
(C24:5n−6)
β-oxidation
Docosapentaenoic acid
(C22:5n−6)
Figure 6 Biosynthesis of long-chain fatty acids.
Transport of FA into Mitochondria
The process of FA activation occurs at the outer mitochondrial
membrane, whereas the process of b-oxidation of FA takes
place in the mitochondrial matrix. Short- and medium-chain
acyl CoA are transported into the mitochondrial matrix
directly, but the transfer of long-chain acyl CoA through
inner mitochondrial membrane requires a special transport
mechanism. Carnitine and three proteins are included in the
FA transport into mitochondrial matrix. In the first reaction,
the acyl CoA reacts with carnitine and forms acyl carnitine.
Reaction is catalyzed by carnitine acyltransferase I (also called
carnitine palmitoyltransferase I, CTP-I) located in the outer
mitochondrial membrane. An acyl carnitine is transported
across the inner mitochondrial membrane by a translocase,
with simultaneous transfer of carnitine from the matrix to
cytosolic side. In the mitochondrial matrix, acyl carnitine is
converted back to acyl CoA in a reaction catalyzed by carnitine
acyltransferase II (CTP-II), releasing the carnitine. The acyl CoA
then undergoes the process of b-oxidation.
Oxidation of FA
FA b-oxidation is the process by which FAs are broken down to
produce energy. The b-oxidation of SFA is a process that
involves a cycle of four repeated reactions: oxidation by flavin
adenine dinucleotide (FAD), hydration, oxidation by NADþ,
and thiolysis by CoA, resulting in FAs shortened for two C
atoms. The first step is the oxidation of the acyl CoA to trans-
Omega-3 fatty acids
α-linolenic acid
(C18:3n−3; ALA)
Stearidonic acid
(C18:4n−3)
Eicosatetraenoic acid
(20:4n−3)
Eicosapentaenoic acid
(C20:5n−3; EPA)
Docosapentaenoic acid
(C22:5n−3; DPA)
Tetracosapentaenoic acid
(C24:5n−3)
Tetracosahexaenoic acid
(C24:6n−3)
Docosahexaenoic acid
(C22:6n−3; DHA)
D2-enoyl CoA catalyzed by enzyme acyl CoA dehydrogenase.
The second reaction is the hydration of the trans-double bond
in the presence of enoyl CoA hydratase and the formation of
the L-3-hydroxyacyl CoA. The third reaction leads to the con-
version of hydroxyl group at C3 into a keto group by L-3-
hydroxyacyl CoA dehydrogenase and generates NADH. The
final step is the cleavage of Ca-Cb-bond in the reaction of
thiolysis (Claisen ester cleavage), by using a thiol group of
the second molecule CoA, in the presence of b-ketothiolase
and the formation of acetyl CoA and acyl CoA shortened by
two C atoms (Table 3).
Acyl CoA then undergoes another process of b-oxidation. At
the final step of this cycle, an FA with an even number of
carbons yields two molecules of acetyl CoA. The complete
oxidation of palmitoyl CoA requires seven repeated cycles
and stoichiometry of oxidation is as follows:
Palmitoyl CoA þ 7FAD þ 7NADþ þ 7CoA þ 7H2 O
! 8 acetyl CoA þ 7FADH2 þ 7NADH þ 7Hþ
The FADH2 and NADH produced during the process of FA
b-oxidation are used in the electron transport chain to produce
ATP. Thus, the complete oxidation of a molecule of palmitate
yields 106 molecules of ATP.
Additional enzymes, isomerase and reductase are needed
for oxidation of USFA. The process of MUFA degradation
begins the same way as degradation of SFA. However, cis-D3-
enoyl CoA that is formed in the third round cannot be
 Fatty Acids: Fatty Acids 629

Table 3 Reaction in fatty acid oxidation

Reaction Enzymes
2
Acyl CoA þ E  FAD ! trans  D  enoyl CoA þ E  FADH2 Acyl CoA dehydrogenase
trans  D2  enoil CoA þ H2 O $ L-3-hydroxyacyl CoA Enoyl CoA hydratase
L-3-Hydroxyacyl CoA þ NADþ $ 3-ketoacyl CoA þ NADH þ Hþ L-3-Hydroxyacyl CoA dehydrogenase
3-Ketoacyl CoA þ HS  CoA $ acetyl CoA þ acyl CoA ðshortened by C2 Þ b-Ketothiolase

O O

C C
S CoA S CoA
linoleoyl-CoA
oleyl CoA
beta-oxidation (3 cycles) 3 Acetyl-CoA
beta-oxidation (3 cycles)
H
H O
H C S CoA
H O
cis-Δ3, cis-Δ6 acyl CoA
C S CoA Δ3, Δ2 enoyl CoA isomerase
cis-Δ3-enoyl CoA H
O
enoyl-CoA isomerase C S CoA
H trans-Δ2 cis-Δ6 acyl CoA
H
O
beta-oxidation (1 cycles) 1 Acetyl-CoA
C S CoA
H trans-Δ2-enoyl CoA O
C S CoA
trans-Δ2 cis Δ4 acyl CoA
beta-oxidation (5 cycles)
2,4-dienol-CoA reductase
O
6 acetyl-CoA
C S CoA
Figure 7 b-Oxidation of monounsaturated fatty acid. trans-Δ3 acyl CoA

enoyl-CoA isomerase
substrate for acyl CoA dehydrogenase. Therefore, isomerase
O
catalyzes the conversion of cis-double bond between C3 and
C S CoA
C4 into trans-D2 double bond and forms trans-D2enoyl CoA
trans-Δ2 acyl CoA
(Figure 7). This product is a regular substrate in FA oxidation
pathway and the process can be continued. beta-oxidation (4 cycles)

For oxidation of PUFA, a reductase is needed. After three

cycles of b-oxidation of linoyl CoA cis-D3, D6-enoyl CoA is
formed. The cis-D3 double bond is converted into a trans-D2
double bond by an isomerase. In another round, an acyl CoA
with a cis-D4 double bond is produced, and its dehydrogena-
tion yields a 2,4-dienoyl intermediate. This intermediate is not
a substrate for enoyl CoA isomerase, therefore it must first be
converted into trans-D3-enoyl CoA by the enzyme 2,4-dienoyl
CoA reductase, in the presence of NADPH. In the next step,
enoyl CoA isomerase converts trans- D3-enoyl CoA into the
trans-D2 form, a common intermediate in the b-oxidation
pathway. (Figure 8). In the FA, b-oxidation pathway produces
one acetyl CoA from each cycle of b-oxidation. This acetyl CoA
then enters the citric acid cycle. The citric acid cycle, also known as
the tricarboxylic acid (TCA) cycle or the Krebs cycle, is the final
common pathway for the oxidation of acetyl CoA derived from
carbohydrates, fats, and proteins into CO2 and H2O to generate a
form of usable energy. The NADH and FADH2 produced by both
b-oxidation and the TCA cycle are used by the electron transport
chain to produce ATP.
5 Acetyl CoA
Figure 8 b-Oxidation of polyunsaturated fatty acid.
The final products of b-oxidation of FA with odd-number C
atoms are the 3-carbon propionyl CoA and acetyl CoA. Pro-
pionyl CoA enters the citric acid cycle and is converted into
succinyl CoA.
Acetyl CoA, which is formed in the process of b-oxidation,
enters the citric acid cycle and forms citrate with oxaloacetate, if
it is available. In cases of increased lipolysis, as in unregulated
diabetes mellitus or in fasting, when oxaloacetate is used to
form glucose, the molecules of acetyl CoA form acetoacetate
and 3-D hydroxybutyrate, known as ketone bodies. Synthesis
of ketone bodies, acetoacetate, hydroxybutyrate, and acetone
occurs normally under all conditions, but their production
is dramatically increased during starvation. Ketone bodies
produced in the mitochondria of liver cells diffuse into the
blood that then transports them to peripheral tissues. They are
630 Fatty Acids: Fatty Acids
normal fuels of respiration and very important as sources of
energy. The acetoacetate is a major fuel source for the heart
muscle and kidney cortex and has the advantage over the
glucose, as energy source. Also, under conditions of starvation,
the brain may provide even 75% of the energy from the acet-
oacetate, by using b-ketoacyl CoA transferase, which converted
ketone bodies back to acetyl CoA.
The process of b-oxidation is also performed in the perox-
isomes that have a major role in the degradation of very-long-
chain FAs. In these vesicles, the end product of oxidation is
octanoyl CoA. Therefore, the products of b-oxidation are trans-
ferred from peroxisomes to mitochondria and this transport is
provided by carnitine. Acyl CoA oxidases in peroxisomes are
different from those in mitochondria because they transfer the
hydrogen to oxygen to produce H2O2 instead of producing
FADH2. The H2O2 is broken down to water by catalase. In
peroxisomes, enzymes for a-oxidation, which are necessary
for oxidation of some FAs with methyl branches, are also
present. Peroxisomes lack the Krebs cycle and cannot degrade
the acetyl CoA to produce CO2 and water.
In microsomes, very-long-chain FAs are oxidized to form
eicosanoids and epoxy and hydroxy FAs. Thus, prostaglandins,
thromboxanes, and leukotrienes known as eicosanoids are
formed from arachidonic and eicosapentaenoic acid by
enzymes cyclooxygenases and lipoxygenase. Eicosanoids act
as paracrine and autocrine hormones and each has specific
effect in the cells. They are involved in the regulation of local
inflammation, but they also modify the blood pressure, aggre-
gation of blood platelets, and cardiac function. Additionally,
eicosanoids inhibit the mobilization of FAs from adipose tis-
sue and are included in the functions of the central nervous
system and the contraction of smooth muscles.
Regulation of FA Metabolism
The availability of free FAs and the rate of utilization of acetyl
CoA are the crucial factors that control FA oxidation. The
release of FAs from adipose tissue is stimulated by glucagon
and epinephrine and inhibited by insulin. The metabolic pro-
cess that FA will undergo depends on cellular energy state.
If the energy charge of the cell is low, FA will be degraded by
b-oxidation reactions to yield acetyl CoA, FADH2, and NADH.
The process of b-oxidation is mainly allosteric-regulated, which
means that each of the b-oxidation enzymes is inhibited by the
specific fatty acyl CoA intermediate it produces. Malonyl CoA
is an essential metabolic intermediate in the regulation of FA
oxidation. A decrease in malonyl CoA level results in an
increased activity of CPT-I in mitochondria in the heart and
muscle, while an increase in malonyl CoA level inhibits CPT-I
activity. CTP-I catalyzes the commitment step in FA oxidation,
which links acyl CoA molecules to the hydroxyl group of
carnitine. In addition, activities of two other enzymes included
in b-oxidation pathway are allosteric-regulated; 3-hydroxyacyl
CoA dehydrogenase is inhibited by NADH and thiolase by
acetyl CoA.
The synthesis of FA is carried out if the energy charge is high
in the cells and when the level of FAs is low. Enzyme acetyl CoA
carboxylase plays an essential role in regulating FA synthesis
and degradation. This process is hormone-regulated. Insulin
activates carboxylase, while its activity is inhibited by glucagon
and epinephrine. In addition, levels of citrate, palmitoyl CoA,
and AMP within a cell also control acetyl CoA carboxylase. The
most important regulator of FA synthesis is the level of citrate
in the cytosol. Citrate stimulates acetyl CoA carboxylase to
form malonyl CoA and allows the synthesis of FAs. On the
other hand, palmitoyl CoA, as the end product of FA synthesis
by FAS, inhibits acetyl CoA carboxylase. AMP is also included
in the regulation of an acetyl CoA carboxylase activity. AMP
activates AMP-dependent protein kinase, which then performs
the phosphorylation of carboxylase and inhibits its enzymatic
activity.
There is adaptive control of FA metabolism regulation. The
rates of synthesis and degradation of the enzymes involved in
the FA synthesis change within a few days, depending on
dietary habits, such as fasting and low-fat or high-carbohydrate
intakes.
FAs, Human Nutrition, and Health
The general grouping of FAs as SFA, MUFA, and PUFA is
usually applied, when their effects on human health are dis-
cussed. This approach is based on the assumption that major
groups of FAs are associated with different health effects. How-
ever, despite the similarities of chemical structure, individual
FAs within the same group may have distinct biological prop-
erties and physiological activity.
Major SFAs in the diet are myristic acid (14:0), palmitic acid
(16:0), and stearic acid (18:0) that originate mainly from
animal fats. When predominant food sources of total fats are
milk and coconut oil, most SFAs range from C4 to C18. The
SFAs with shorter chain, such as butyric (C4:0), caprylic
(C8:0), capric (C10:0), and lauric (C12:0) acids, have positive
health effects through the prevention of cancer and antiviral
and antibacterial activity. On the other side, lauric, myristic,
and palmitic acids are known to have thrombogenic and ath-
erogenic effect. Long-chain SFAs are abundant in animal fats.
Their negative effect is related to increase in total and LDL
cholesterol leading to obesity and/or coronary disease. How-
ever, in the case of stearic acid, no atherogenic effect has been
shown. Moreover, recent results indicate the proapoptotic
effect of stearic acid in some cancer diseases.
Predominant MUFA in the diet is oleic acid (C18:1n9),
mostly supplied from olive oil. Oleic acid demonstrates large
spectra of biological activities being connected to many health
beneficial effects. On the cellular level, as a compound of
membrane phospholipids, oleic acid increases membrane flu-
idity and transport, stimulates enzymatic activity, and regulates
the activity of membrane receptors and signal transduction and
transcription of some genes. It is associated with the decrease
of plasma total cholesterol, LDL cholesterol, and triglyceride
concentrations, having positive effect in coronary artery dis-
ease. The reduced risk of cardiovascular and neurodegenerative
diseases as found in Mediterranean diet is also connected to
higher oleic acid content in this type of feeding. Even it is much
less present in ordinary diet, the palmitoleic acid (16:1n7) is
interesting because of its pro-lipogenic activity in the adipose
tissue. Due to this activity, palmitoleic acid could be beneficial
in the protection of other tissues and organs from fat accumu-
lation and consequent lipotoxicity. On the other side, its

antiapoptotic activity is undesirable in uncontrolled prolifera-
tion of cancer cells. In diets with common use of rapeseed and
mustard seed oil, the major MUFA is erucic acid (C22:1n9).
No negative health effects of erucic acid consumption have
been reported in humans.
Major PUFAs in the diet are LA, ALA, and long-chain PUFA
such as AA, eicosapentaenoic acid (EPA), docosapentaenoic
acid (DPA), and DHA. LA and ALA are essential FAs, which
must be supplied from dietary sources. LA, usually found in
sunflower and soybean oil, is significant as a precursor of PUFA
n6 family, while ALA, abundant in linseed oil, is precursor of
n3 PUFA. At the same time, long-chain PUFA can be provided
from the dietary sources. The main foods containing n6
PUFA AA are meat, poultry, and eggs, while fatty fish and fish
oils are the main sources of n3 PUFA, EPA, and DHA. The
n6 and n3 PUFAs have different roles in human’s physiol-
ogy and health. The AA is involved in the biosynthesis of pro-
inflammatory eicosanoids, having pro-inflammatory activity
in stress and injury. In opposite, eicosanoids derived from
n3 PUFA and EPA are anti-inflammatory, protecting organ-
ism from inflammatory diseases (lupus, arthritis, and asthma),
heart attack, and stroke. In addition, EPA acts as an inhibitor of
synthesis of pro-inflammatory cytokines. Due to the stimula-
tory effect on neuro- and synaptogenesis, DHA is crucial for
optimal neural function. Even more, n3 PUFAs by decreasing
lipid level and blood pressure, improving cardiac and endo-
thelial function, and manifesting antiplatelet and anti-
inflammatory effects have a crucial role in the control of car-
diovascular risk factors. In general, effects of PUFA are depen-
dent on n3 and n6 PUFA ratio, which is about 1:10 in
modern dietary habits compared with 1:4 as recommended
in terms of the health benefits. One or two fish meals weekly
could probably improve this situation.
The human diet contains a certain amount of trans-FA,
mostly 18:1 trans-isomers, derived from partially hydrogenated
or high-heat-treated vegetable oils, as well as produced by
bacteria from ruminant intestine. The increased intake of
trans-FA is associated with the HDL cholesterol decrease, an
increase of LDL cholesterol, and the higher risk of coronary
heart disease and atherosclerosis. The minor proportion of
‘unusual’ FA, as FA with branched-chain or non-methylene-
interrupted double bond structure, is also present in the diet.
In the case of increased concentration, the overall effects could
be potentially harmful for human health.
Fatty Acids: Fatty Acids 631
See also: Fats: Classification and Analysis; Fatty Acids: Essential Fatty
Acids; Fatty Acids: Metabolism; Fatty Acids: Trans Fatty Acids.
Further Reading
Berg JM, Tymoczko JL, and Stryer L (2002) Biochemistry, 5th ed. New York: W H
Freeman.
Food and Agriculture Organization of the United Nations (FAO) (2010) Fats and fatty
acids in human nutrition. Report of an expert consultation, Geneva 2008. FAO Food
and Nutrition Paper 91: 1–169.
Hanus LO, Goldshlag P, and Dembitsky VM (2008) Identification of cyclopropyl fatty
acids in walnut (Juglans regia L.) oil. Biomedical papers of the Medical Faculty of
the University Palacký, Olomouc, Czechoslovakia 152: 41–45.
Jakobsson A, Westerberg R, and Jacobsson A (2006) Fatty acid elongases in
mammals: their regulation and roles in metabolism. Progress in Lipid Research
45: 237–249.
Nakamura MT and Nara TY (2004) Structure, function, and dietary regulation of delta6,
delta5, and delta9 desaturases. Annual Review of Nutrition 24: 345–376.
Rizos EC, Ntzani EE, Bika E, Kostapanos MS, and Elisaf MS (2012) Association between
omega-3 fatty acid supplementation and risk of major cardiovascular disease
events: A systematic review and meta-analysis. Journal of the American Medical
Association 308: 1024–1033.
Roman O, Heyd B, Broyart B, Castillo R, and Maillard M (2013) Oxidative reactivity of
unsaturated fatty acids from sunflower, high oleic sunflower and rapeseed oils
subjected to heat treatment, under controlled conditions. LWT - Food Science and
Technology 52: 49–59.
Rustan AC and Drevon CA (2005) Fatty acids: structures and properties.
In: Encyclopedia of life sciences. Chichester, UK: Wiley.
Scrimgeour C and Scrimgeour C (2005) Chemistry of fatty acids. In: Shahidi F and
Shahidi F (eds.) Bailey’s industrial oil and fat products, 6th ed., pp. 1–43.
Chichester, UK: Wiley.
Wanders RJ, Vreken P, Ferdinandusse S, et al. (2001) Peroxisomal fatty acid alpha-
and beta-oxidation in humans: enzymology, peroxisomal metabolite
transporters and peroxisomal diseases. Biochemical Society Transactions
29: 250–267.
Relevant Websites
www.biochem.arizona.edu/Lec34-F08-Hando – Department of Chemistry and
Biochemistry at The University of Arizona.
www.els.net – Encyclopedia of Life Sciences.
www.library.med.utah.edu – The University of Utah.
www.lipidlibrary.aocs.org – American Oil Chemists’ Society.
www.lipidlibrary.aocs.org/animbio/fa-oxid/index.htm – American Oil Chemists’ Society.
www.ncbi.nlm.nih.gov/books/NBK22369 – National Center for Biotechnology
Information.
www.oregonstate.edu/dept/2kjan14lecturenotes.html – Oregon State University.
 Fatty Acids: Metabolism
PC Calder, University of Southampton, Southampton, UK
ã 2016 Elsevier Ltd. All rights reserved.
Fatty Acid Structure, Classification, and Nomenclature
A fatty acid is a hydrocarbon (acyl) chain with a carboxyl group
at one end and a methyl group at the other. A large number of
fatty acids exist in nature because the hydrocarbon chain may
be of varying lengths (i.e., containing a varying number of
carbon atoms linked together) and may have different degrees
of unsaturation (unsaturation refers to the presence of double
bonds between carbon atoms within the hydrocarbon chain).
The terminal carboxyl group is reactive and readily forms ester
links. Ester links between fatty acids and alcohol groups on
glycerol or cholesterol produce acylglycerols (e.g., mono-, di-,
and triacylglycerols are often called mono-, di-, and triglycer-
ides) and cholesteryl esters, respectively. Traditionally, the car-
bon of the terminal carboxyl group has been referred to as
carbon 1 with the carbon next to it (carbon 2) being referred
to as the a carbon and the methyl terminal carbon as the o
(sometimes called n) carbon. The most abundant fatty acids in
the human diet and in the human body have straight hydro-
carbon chains of an even number of carbon atoms, although
branched-chain, odd-numbered, and substituted fatty acids do
exist. Fatty acid chain lengths vary from 2 to 30, and based
upon chain length, fatty acids are classified as short chain (2–6
carbon atoms), medium chain (8–12 carbon atoms), and long
chain (14 or more carbon atoms); sometimes, fatty acids of 20
or more carbon atoms are referred to as very long chain. Fatty
acids are also classified according to the degree of unsaturation
(i.e., the number of double bonds in the hydrocarbon chain).
Fatty acids containing double bonds in the hydrocarbon chain
are called unsaturated fatty acids; a fatty acid containing two or
more double bonds is called a polyunsaturated fatty acid or
PUFA. Saturated fatty acids do not contain double bonds in the
hydrocarbon chain.
Fatty acids have systematic names that are based upon the
number of carbons in the hydrocarbon chain (Table 1). How-
ever, complications arise for the naming of unsaturated fatty
acids because there are multiple possibilities for the position of
double bonds within the hydrocarbon chain and because each
double bond may be in the cis or trans configuration. There-
fore, when naming an unsaturated fatty acid, it is important
that the exact positions of double bonds and their configura-
tions be specified. Traditionally, the position of double bonds
was denoted by identifying the carbon number (counting from
carbon 1 (the carboxyl carbon)) on which each double bond
occurs. Thus, octadecadienoic acid, an 18-carbon fatty acid
with cis double bonds between carbons 9 and 10 and carbons
12 and 13, is correctly denoted as cis-9, cis-12-octadecadienoic
acid or as cis, cis-9,12-octadecadienoic acid. More recently, an
alternative shorthand notation for fatty acids has come into
frequent use, particularly within the nutrition community. This
relies upon identifying the number of carbon atoms in the
hydrocarbon chain and the number of double bonds and
their position. Thus, octadecanoic acid is notated as 18:0,
632 Encyclopedia of Food and Health
indicating that it has a hydrocarbon chain of 18 carbons and
does not contain any double bonds. Unsaturated fatty acids are
named simply by identifying the number of double bonds and
the position of the first double bond counted from the methyl
terminus (with the methyl, or o, carbon counted as number 1)
of the hydrocarbon chain. The way the first double bond
is identified is as o
x, where x is the carbon number on
which the double bond occurs. Therefore, cis, cis-9,12-
octadecadienoic acid is also known as 18:2o
6. The o
x
nomenclature is sometimes referred to as n
x (e.g.,
18:2n
6). In addition to these nomenclatures, fatty acids are
often described by their common names (Table 1), which
sometimes reflect a common origin of the fatty acid, for exam-
ple, palmitic acid (16:0) from palm oil and oleic acid
(18:1n
9) from olive oil. In most PUFAs, each double bond
is separated by a methylene (–CH2
) group. However, this is
not always the case, and in some PUFAs, the double bonds are
conjugated (i.e., the two double bonds are separated by only
one single bond). Figure 1 shows the structure of several 18-
carbon fatty acids indicating the position of the double bonds
in the hydrocarbon chain and how this is reflected in their
naming.
Most commonly occurring unsaturated fatty acids contain
cis rather than trans double bonds. Trans double bonds do
occur in some intermediates in the fatty acid biosynthetic
pathway, in ruminant fats (e.g., cow’s milk), in plant lipids,
and in some seed oils, and they may also be introduced by
exposure of cis unsaturated fatty acids to high temperatures
such as when oils are used for deep fat frying. Cis, but not
trans, double bonds produce a kink in the molecule so that the
molecular shape of unsaturated fatty acids containing cis dou-
ble bonds is distinct from that of saturated (and trans unsatu-
rated) fatty acids. This has an effect on the physical properties
of the fatty acid. The melting point of a fatty acid is affected by
its chain length, its degree of unsaturation, and whether the
double bonds are cis or trans. Fatty acid melting point increases
with increasing chain length and decreases with increasing
degree of unsaturation. Thus, among the 18-carbon fatty
acids, stearic acid (18:0) has a higher melting point than
oleic acid (18:1n
9) and oleic acid has a higher melting
point than linoleic acid (18:2n
6). For a given chain length
and number of double bonds, a trans fatty acid has a higher
melting point than a cis fatty acid. Thus, elaidic acid (trans
18:1n
9) has a higher melting point than oleic acid. Therefore,
the fatty acid composition of a fat-rich substance will deter-
mine whether that substance is solid or liquid at room temper-
ature. Consequently, lard, beef tallow, and butter are solid at
room temperature because they contain a high proportion of
long-chain saturated fatty acids, while coconut oil and corn oil
are liquid at room temperature because they contain a high
proportion of medium-chain saturated fatty acids and PUFAs,
respectively. Melting point can be altered by the modification
of fatty acid composition. Hence, to produce margarine from
http://dx.doi.org/10.1016/B978-0-12-384947-2.00276-2
 Fatty Acids: Metabolism 633

Table 1 Nomenclature of fatty acids

Systematic name Trivial name Shorthand notation

Saturated
Ethanoic Acetic 2:0
Propanoic Propionic 3:0
Butanoic Butyric 4:0
Hexanoic Caproic 6:0
Octanoic Caprylic 8:0
Decanoic Capric 10:0
Dodecanoic Lauric 12:0
Tetradecanoic Myristic 14:0
Hexadecanoic Palmitic 16:0
Octadecanoic Stearic 18:0
Eicosanoic Arachidic 20:0
Docosanoic Behenic 22:0
Tetracosanoic Lignoceric 24:0
Monounsaturated
cis-9-Hexadecenoic Palmitoleic 16:1n
7
cis-11-Octadecenoic Vaccenic 18:1n
7
trans-11-Octadecenoic trans-Vaccenic t18:1n
7
cis-9-Octadecenoic Oleic 18:1n
9
trans-9-Octadecenoic Elaidic t18:1n
9
cis-11-Eicosenoic Gadoleic 20:1n
9
cis-13-Docosenoic Erucic 22:1n
9
cis-11-Docosenoic Cetoleic 22:1n
11
cis-15-Tetracosenoic Nervonic 24:1n
9
Polyunsaturated
cis-9, cis-12-Octadecadienoic Linoleic 18:2n
6
All-cis-6, 9, 12-Octadecatrienoic g-Linolenic 18:3n
6
All-cis-9, 12, 15-Octadecatrienoic a-Linolenic 18:3n
3
All-cis-6, 9, 12, 15-Octadecatetraenoic Stearidonic 18:4n
3
All-cis-11, 14, 17-Eicosatrienoic Mead 20:3n
9
All-cis-8, 11, 14-Eicosatrienoic Dihomo-g-linolenic 20:3n
6
All-cis-5, 8, 11, 14-Eicosatetraenoic Arachidonic 20:4n
6
All-cis-5, 8, 11, 14, 17-Eicosapentaenoic Timnodonica 20:5n
3
All-cis-7, 10, 13, 16-Docosatetraenoic Adrenic 22:4n
6
All-cis-4, 7, 10, 13, 16-Docosapentaenoic Osbond 22:5n
6
All-cis-7, 10, 13, 16, 19-Docosapentaenoic Clupanodonicb 22:5n
3
All-cis-4, 7, 10, 13, 16, 19-Docosahexaenoic Cervonicc 22:6n
3
a
Commonly called eicosapentaenoic acid.
b
Commonly called docosapentaenoic acid.
c
Commonly called docosahexaenoic acid.

vegetable oil, the process of hydrogenation is used to ‘harden’
the oil: vegetable oils are hardened by reacting them with hydro-
gen gas at about 60  C in the presence of a catalyst, which results
in the conversion of some of the double bonds to single bonds.
This process can also convert cis to trans double bonds.
Fatty Acid Biosynthesis
Biosynthesis of Saturated Fatty Acids
Saturated fatty acids can be synthesized from carbohydrates and
from proteins because the basic building block is acetyl-
coenzyme A (acetyl-CoA) produced from sugar and amino
acid metabolism. Acetyl-CoA is usually produced within mito-
chondria, while fatty acid biosynthesis occurs in the cytoplasm.
Thus, there is a transport system whereby mitochondrial acetyl-
CoA condenses with oxaloacetate to form citrate (catalyzed by
citrate synthase) – this is actually a reaction within the citric
acid (Krebs) cycle – and the citrate is transported across the
mitochondrial membrane to the cytoplasm. Here, citrate is
cleaved to produce oxaloacetate (which is converted to malate
and transported back into the mitochondria) and acetyl-CoA.
De novo synthesis of fatty acids is catalyzed by a multi-
enzyme complex called fatty acid synthase. The growing fatty
acid hydrocarbon chain is built up by successive addition of
two-carbon units. These are donated by malonate – a three-
carbon intermediate – linked to acyl carrier protein (ACP)
through the latter’s sulfhydryl group. Malonyl-ACP is formed
from malonyl-CoA that is, in turn, formed from acetyl-CoA.
Acetyl-CoA carboxylase catalyzes conversion of acetyl-CoA to
malonyl-CoA (Figure 2). This is the rate-limiting step of fatty
acid biosynthesis and is subject to hormonal regulation (e.g., it
is upregulated by insulin). The malonate group of malonyl-
CoA now condenses with ACP to produce malonyl-ACP. In the
634 Fatty Acids: Metabolism

H3C
Octadecanoic acid
COOH Stearic acid
18:0

H3C COOH cis 9-Octadecenoic acid

Oleic acid
18:1n-9

H3C
cis 9, cis 12-Octadecadienoic acid
COOH Linoleic acid
18:2n-6

H3C
cis 9, trans 11-Octadecadienoic acid
COOH Conjugated linoleic acid

cis 9, cis12, cis 15-Octadecatrienoic acid

H3C COOH α-Linolenic acid
18:3n-3

Mammals cannot insert double bonds in here

Plants can
Figure 1 Representation of the structure of five different 18-carbon fatty acids demonstrating the basis of different nomenclatures.

Acetyl-CoA carboxylase
COOHCH2CO-S-CoA CH3CO-S-CoA
Malonyl-CoA Acetyl-CoA
Malonyl-CoA: Acetyl-CoA:
ACP acyltransferase ACP acyltransferase

COOHCH2CO-S-ACP CH3CO-S-ACP
Malonyl-ACP Acetyl-ACP

3-Ketoacyl-ACP
CO2 synthase
NADPH + H+
NADP+ CH3COCH2CO-S-ACP
3-Ketoacyl-ACP
3-Ketoacyl-ACP
reductase
CH3CHOHCH2CO-S-ACP CH3CH2CH2CO-S-ACP
3-Hydroxyacyl-S-ACP Acyl-ACP

3-Hydroxyl-ACP Enoyl-ACP
dehydrase reductase
NADP+
CH3CH=CHCO-S-ACP
+
Enoyl-ACP NADPH + H
H2O

Figure 2 The pathway of de novo fatty acid biosynthesis catalyzed by the multienzyme fatty acid synthase complex. Malonyl-ACP (acyl carrier protein)
acts as the donor of a two-carbon unit to the growing acyl chain. The acceptor in the first step is acetyl-ACP, and in the subsequent steps, it is
the acyl-ACP produced during the previous round of reactions. The usual end product of the process is palmitoyl-ACP.

initial step of fatty acid biosynthesis, malonyl-ACP is con-
densed with acetyl-ACP to form the four-carbon acetoacetyl-
ACP with the loss of one carbon as CO2 (Figure 2). There
follows a sequence of hydrogenation (reduction), dehydration,
and hydrogenation (reduction) steps to produce the
four-carbon butyryl-ACP (Figure 2). The cycle is now repeated
with butyryl-ACP taking the role of acetyl-ACP from the first
cycle to ultimately produce a six-carbon acyl-ACP. The cycle is
repeated seven times to produce the 16-carbon palmitoyl-ACP,
which is hydrolyzed from the ACP to yield palmitic acid. The

reductive steps in the fatty acid biosynthesis pathway use
NADPH derived from the pentose phosphate pathway. The
overall reaction for palmitic acid biosynthesis is
Acetyl-CoA þ 7 malonyl-CoA þ 14NADPH þ 14Hþ
! palmitic acid þ 7CO2 þ 8CoA þ 14NADPþ þ 6H2 O
It is important to note that enzymes exist in some tissues
that act to release fatty acids of shorter chain length than
palmitic acid from the de novo biosynthetic pathway. For exam-
ple, in the mammary gland of some species, there are enzymes
that are responsible for the release of the medium-chain satu-
rated fatty acids such as caprylic acid (8:0) and capric acid
(10:0) that are characteristic of the milks of those species.
In eukaryotes, longer-chain saturated fatty acids (actually the
fatty acyl-CoA esters are formed) are produced from palmitic
acid (actually palmitoyl-CoA is the substrate) by elongation
reactions (the enzymes catalyzing these are termed elongases),
which occur on the endoplasmic reticulum or in mitochondrial
membranes. The mitochondrial system uses acetyl-CoA as the
two-carbon donor, and there is a requirement for NADH or
NADPH or both depending upon the tissue location. However,
the main location for fatty acid elongation is the endoplasmic
reticulum and this system uses malonyl-CoA as the two-carbon
donor and NADPH. Elongation converts palmitic acid to stearic
acid, and in neuronal tissue, large amounts of behenic (22:0)
and lignoceric (24:0) acids are produced through elongation
reactions. Thus, a large number of fatty acid substrates may be
elongated, and seven elongase enzymes, which differ in their
selectivity for fatty acids of different chain lengths and degrees of
unsaturation, have been identified. Elongases 1, 3, 6, and 7
preferentially catalyze the extension of saturated and monoun-
saturated fatty acids, while elongases 2, 4, and 5 act mainly on
PUFAs (see Further metabolism of linoleic and a-linolenic
acids). Elongation of palmitic acid to stearic acid is catalyzed
by elongase 6. Elongases 1, 3, and 7 catalyze the elongation of
stearic acid to lignoceric acid, while only elongases 1 and 3 can
further elongate lignoceric acid.
Biosynthesis of Monounsaturated Fatty Acids
The endoplasmic reticulum is a site for the introduction of
double bonds (‘desaturation’) into fatty acids. The pathway
used is almost universal, having been identified in bacteria,
yeasts, algae, higher plants, protozoa, and animals. Examples
are the conversion of stearic acid (18:0) to oleic acid (18:1n
9)
and of palmitic acid to palmitoleic acid (16:1n
7) by insertion
of a cis double bond between carbons 9 and 10; once again, it is
the fatty acyl-CoA that is the substrate for these reactions that use
NADPH and molecular oxygen. Because the double bond is
inserted between carbons 9 and 10 counting from the carboxyl
end of the hydrocarbon chain, the desaturase enzyme is known
as delta-9 desaturase (D9-desaturase), although sometimes, this
enzyme is referred to as stearoyl-CoA desaturase or SCD.
Biosynthesis of PUFAs
Biosynthesis of linoleic and a-linolenic acids
All eukaryotes and some bacteria can produce PUFAs. Plant
enzymes normally introduce a new double bond between an
Fatty Acids: Metabolism 635
existing double bond and the terminal methyl group of the
hydrocarbon chain, whereas animal enzymes normally intro-
duce a new double bond between an existing double bond and
the carboxyl group (see Figure 1). Insertion of a double bond
between carbons 12 and 13 (counted from the carboxyl car-
bon) of oleic acid yields linoleic acid (18:2n
6). The enzyme
that catalyzes this reaction is called D12-desaturase (Figure 3).
Linoleic acid can be further desaturated by insertion of a dou-
ble bond between carbons 15 and 16 (counted from the car-
boxyl carbon) by D15-desaturase to yield a-linolenic acid
(18:3n
3) (Figure 3).
Linoleic and a-linolenic acids are the simplest members of
the n
6 and n
3 families of fatty acids, respectively. As indi-
cated earlier, mammals lack the enzymes that introduce double
bonds at carbon atoms beyond carbon 9 in the acyl chain
(counting from the carboxyl carbon). Because these include
the D12- and D15-desaturases, this means that mammals can-
not synthesize linoleic or a-linolenic acids. Since these fatty
acids are required by mammalian cells, they are termed essen-
tial fatty acids, and there is a need for their consumption in
the diet.
Further metabolism of linoleic and a-linolenic acids
Although mammalian cells cannot synthesize linoleic or
a-linolenic acids, they can metabolize them by further desa-
turation and elongation; desaturation occurs at carbon atoms
below carbon number 9 (counting from the carboxyl carbon).
Linoleic acid can be converted to g-linolenic acid (18:3n
6) by
D6-desaturase, and then, g-linolenic acid can be elongated (by
elongase 5) to dihomo-g-linolenic acid (20:3n
6) (Figure 3).
Dihomo-g-linolenic acid can be further desaturated by
D5-desaturase to yield arachidonic acid (20:4n
6) (Figure 3).
Using the same series of enzymes as used to metabolize
n
6 PUFAs, a-linolenic acid is converted to timnodonic acid,
which is more commonly known as eicosapentaenoic acid
(20:5n
3) or EPA (Figure 3). In mammals, the pathway of
desaturation and elongation occurs mainly in the liver,
although it is also likely to occur in other cells and tissues
that have specific requirements for very-long-chain PUFAs.
The desaturation enzymes use NADPH and molecular oxygen
and are located on the endoplasmic reticulum. The genes
encoding △6- and △5-desaturase are known as fatty acid desa-
turase 2 and 1 (FADS2 and FADS1), respectively.
It is evident from the pathway shown in Figure 3 that there
is competition between the n
9, n
6, and n
3 fatty acid
families for metabolism. The D6-desaturase reaction is rate-
limiting in this pathway. The preferred substrate for
D6-desaturase is a-linolenic acid, followed by linoleic acid
followed by oleic acid. However, because linoleic acid is
much more prevalent in most human diets than a-linolenic
acid, metabolism of n
6 fatty acids is quantitatively the more
important. In the absence of intake of linoleic and a-linolenic
acids, metabolism of oleic acid is enhanced resulting in the
accumulation of mead acid (20:3n
9), which is normally
only found in tissues in trace amounts (Figure 3). Appearance
and accumulation of mead acid are taken to indicate dietary
essential fatty acid deficiency. The activities of D6- and
D5-desaturases are regulated by nutritional status, hormones,
and feedback inhibition by end products; for example, high-fat
diets and insulin downregulate the activities of these enzymes.
636 Fatty Acids: Metabolism

Stearic acid (18:0)

Δ9-desaturase
Δ12-desaturase Δ15-desaturase
Plants only Plants only
Oleic acid (18:1n-9) Linoleic acid (18:2n-6) α-Linolenic acid (18:3n-3)

Δ6-desaturase Δ6-desaturase Δ6-desaturase

18:2n-9 γ-Linolenic acid (18:3n-6) Stearidonic acid (18:4n-3)

Elongase Elongase Elongase

20:2n-9 Di-homo-γ-linolenic acid (20:3n-6) Eicosatetraenoic acid (20:4n-3)

Δ5-desaturase Δ5-desaturase Δ5-desaturase

Mead acid (20:3n-9) Arachidonic acid (20:4n-6) Eicosapentaenoic acid (20:5n-3)

Elongase Elongase

Adrenic acid (22:4n-6) Docosapentaenoic acid (22:5n-3)

Elongase Elongase

Tetracosatetraenoic acid (24:4n-6) Tetracosapentaenoic acid (24:5n-3)

Δ6-desaturase Δ6-desaturase

Tetracosapentaenoic acid (24:5n-6) Tetracosahexaenoic acid (24:6n-3)

β-oxidation β-oxidation

Osbond acid (22:5n-6) Docosahexaenoic acid (22:6n-3)

Figure 3 The pathways of biosynthesis of n
9, n
6, and n
3 unsaturated fatty acids.

Furthermore, polymorphisms in the FADS1 and FADS2 genes
have been associated with differences in the status of n
6 and
n
3 PUFAs, presumably due to the polymorphisms resulting
in different capacities of the pathway.
Further conversion of arachidonic acid to osbond acid
(22:5n
6) and of EPA, to cervonic acid, more commonly
known as docosahexaenoic acid (22:6n
3) or DHA, occurs
by a complex pathway. This involves chain elongation cataly-
zed by elongase 5, a second chain elongation catalyzed by
elongase 2 or 5, desaturation by D6-desaturase, and then
removal of two carbon atoms as acetyl-CoA by limited
b-oxidation in peroxisomes (Figure 3).
Fatty Acid Incorporation into Complex Lipids
Fatty Acids Are Esterified into Complex Lipids
Fatty acids do not usually exist in high concentrations in ‘free’
form; most often, they are esterified into acylglycerols like
mono-, di-, and triacylglycerols, into phospholipids or similar
complex lipids, or into cholesteryl esters. Fatty acids in foods
are mainly present in triacylglycerols, since this is how plants
and animals store fatty acids, although some dietary fatty acids
are present in phospholipids since these make up cell mem-
branes and many foods (e.g., meat) include intact cells.
Humans and other mammals store fatty acids esterified into
triacylglycerols in adipose tissue. Phospholipids are important
structural components of all cell membranes and phospho-
lipids contain fatty acids esterified at their sn
1 and sn
2
positions. Fatty acids circulate in the bloodstream mainly as
esterified lipids within lipoproteins. For example, dietary fatty
acids are carried in the bloodstream mainly as triacylglycerols
within chylomicrons, while very-low-density lipoproteins carry
triacylglycerols of hepatic origin (e.g., carrying fatty acids from
hepatic de novo biosynthesis that have been incorporated into
triacylglycerols). Low- and high-density lipoproteins are the
main cholesterol-carrying lipoproteins in the bloodstream,
and the majority of this cholesterol is present as fatty acid
cholesteryl esters. Within these lipoproteins, the hydrophobic
triacylglycerols and cholesteryl esters are within the core, which
is surrounded by a phospholipid monolayer, which serves to
solubilize the particle within the aqueous environment of the
bloodstream.
Incorporation of Fatty Acids into Triacylglycerols
The glycerol 3-phosphate pathway
Glycerol 3-phosphate is produced from glycerol in a reaction
catalyzed by glycerol kinase:
 Fatty Acids: Metabolism 637

Glycerol þ ADP ! glycerol 3-phosphate þ ADP The dihydroxyacetone phosphate pathway

There follows stepwise transfer of fatty acyl groups from
fatty acyl-CoA onto position 1 and then position 2 of glycerol
3-phosphate (Figure 4). The first acylation is catalyzed by acyl-
CoA/glycerol phosphate 1-O-acyltransferase, known as GPAT,
while the second acylation is catalyzed by acyl-CoA/1-acyl
glycerol phosphate 2-O-acyltransferase, known as LPAT
(because 1-acylglycerol 3-phosphate is also called lysopho-
sphatidic acid, hence lysophosphatidate acyltransferase).
GPAT has marked specificity for saturated fatty acyl-CoA,
whereas LPAT shows specificity toward mono- and dienoic
fatty acyl-CoA. Different isoforms of GPAT and LPAT are
found on the endoplasmic reticulum and outer mitochondrial
membrane, and this distribution differs between tissues.
The product of these two reactions is 1,2-diacylglycerol
3-phosphate (also known as phosphatidic acid). Phosphatidic
acid can also be formed by phosphorylation of 1,2-
diacylglycerol by diacylglycerol kinase using ATP as the phos-
phate donor. The next step of the glycerol 3-phosphate
pathway of triacylglycerol synthesis is the formation of 1,2-
diacylglycerol by the hydrolysis of phosphatidic acid by phos-
phatidate phosphohydrolase. The final step of this pathway is
fatty acylation of the now vacant third position of the diacyl-
glycerol catalyzed by diacylglycerol acyltransferase, known
as DAGAT. This enzyme has a broad specificity for fatty
acyl-CoAs.
Dihydroxyacetone phosphate is an intermediate in glycolysis.
The first reaction in the dihydroxyacetone phosphate pathway
for triacylglycerol synthesis is fatty acylation at position 1
catalyzed by dihydroxyacetone phosphate acyltransferase
(Figure 4). Although this enzyme is found in mitochondria,
peroxisomes appear to be a more important location. The next
step is the reduction of the keto group to form 1-acylglycerol 3-
phosphate, linking into the glycerol 3-phosphate pathway of
triacylglycerol synthesis. This reduction occurs on the cytosolic
side of the peroxisomal membrane and uses NADPH. Further
acylation requires transfer of the 1-acylglycerol 3-phosphate to
the endoplasmic reticulum.
The monoacylglycerol pathway
The main function of this pathway appears to be resynthesis of
triacylglycerols of dietary origin following their partial digestion
in the intestinal lumen. The 2-monoacylglycerol produced dur-
ing this hydrolysis is fairly resistant to further digestion and
appears to be absorbed intact. The pathway involves stepwise
transfer of fatty acyl groups from fatty acyl-CoA to positions 1
and 3 of the 2-monoacylglycerol (Figure 4). The first reaction is
catalyzed by monoacylglycerol acyltransferase, which appears to
have a preference for esterifying position. The second reaction is
catalyzed by diacylglycerol acyltransferase, which is specific for
1,2-diacylglycerols (i.e., for esterifying position 3).

H H O H O H
H C OH H C O C R1 H C O C R1 H C OH

HO C H 1 HO C H 3 O C H 2 O C H
H C O P H C O P H C O P H C O P
H H H H
Glycerol 3-Phosphate 1-acyl-G-3-P 1-acyl-DHAP Dihydroxyacetone
phosphate
4

H O
O H C O C R1
R2 C O C H
H C O P
H
1,2-diacyl-G-3-P

H H O H O
O H C OH O H C O C R1 O H C O C R1
R2 C O C H 6 R2 C O C H 7
R2 C O C H O
H C OH H C OH H C O C R3
H H H
2-monoacylglycerol 1,2-diacylglycerol Triacylglycerol
Figure 4 The pathways of triacylglycerol biosynthesis. Enzyme: 1, glycerol 3-phosphate acyltransferase (GPAT); 2, dihydroxyacetone phosphate
acyltransferase; 3, acyl-dihydroxyacetone phosphate reductase; 4, lysophosphatidate acyltransferase (LPAT); 5, phosphatidate phosphohydrolase; 6,
monoacylglycerol acyltransferase; 7, diacylglycerol acyltransferase. R1 and R2 represent fatty acyl chains; P represents phosphate.
638 Fatty Acids: Metabolism

Incorporation of Fatty Acids into Phospholipids Fatty Acid Release from Complex Lipids
Cell membrane phospholipids and related structures Fatty Acid Release from Triacylglycerols
All cells are bordered by a membrane composed of a lipid
Lipases are enzymes that hydrolyze fatty acids from acylglycerols;
bilayer that has both structural and functional roles. The most
lipases are a subclass of esterases and they are widespread in
common membrane lipids are based upon a glycerol back-
nature being found in microorganisms, plants, and animals.
bone with various moieties linked to the hydroxyl groups on
Because there are a large number of possible acylglycerol sub-
the glycerol; these are termed glycerolipids or glycerides. In
strates that have different tissue distributions, there are a
higher animals, phosphoglycerides (these are usually called
number of different lipases with differing specificities and loca-
‘phospholipids’) predominate, whereas in plants, glycosylgly-
tions. Triacylglycerol lipases typically split the fatty acids esterified
cerides are more important. In mammalian systems, the most
at positions 1 and 3 leaving the 2-monoacylglycerol intact. Hydro-
common phospholipids are the 1,2-diacylphosphoglycerides,
lysis of the latter will usually require a 2-monoacylglycerol
which have fatty acyl groups esterified at positions 1 and 2 of
lipase. Different triacylglycerol lipases have different fatty acid
the phosphoglyceride and usually another moiety (termed
specificities, meaning that different triacylglycerol substrates are
the ‘head group’) esterified to the phosphate group, which is
hydrolyzed at different rates and to different extents. In animals,
at position 3. These include phosphatidic acid, phosphatidyl-
triacylglycerol lipases can be classified as either extracellular or
choline (also called lecithin), phosphatidylethanolamine,
intracellular. Extracellular lipases are secreted from the cells in
phosphatidylserine, and phosphatidylinositol. Phospho-
which they are synthesized and they are involved in hydrolyzing
lipids with ether, rather than ester, linked fatty acids at posi-
triacylglycerols present in the extracellular environment enabling
tion 1 (and sometimes at position 2) also exist. These include
cellular uptake of the liberated fatty acids. Extracellular lipases
plasmalogens, which have an ether linkage at the sn
1 posi-
include pancreatic lipase, involved in hydrolyzing dietary triacyl-
tion and an ester linkage at the sn
2 position. In mammals,
glycerols in the intestinal lumen; lipoprotein lipase, involved in
the sn
1 position is typically derived from 16:0, 18:0, or
hydrolyzing lipoprotein triacylglycerols in the circulation; hepatic
18:1n
9 fatty alcohols, while the sn
2 position is most com-
lipase, involved in hydrolyzing lipoprotein triacylglycerols in the
monly occupied by a PUFA. The most common head groups
hepatic sinusoids (the liver’s equivalent of capillaries); and endo-
present in mammalian plasmalogens are ethanolamine
thelial lipase, which has both triacylglycerol lipase and phospho-
(called plasmanylethanolamines) or choline (called plasma-
lipase activities. The best characterized intracellular triacylglycerol
nylcholines). Platelet-activating factor has an sn
1 ether-
lipase is hormone-sensitive lipase found in adipocytes. Hormone-
linked acyl group, an sn
2 acetyl group, and choline as the
sensitive lipase acts on the surface of the triacylglycerol droplet
head group. Sphingolipids are membrane lipids based on
stored in the adipocyte to release stored fatty acids that can be
sphingosine rather than glycerol. These may include fatty
delivered to the circulation as ‘free’ fatty acids (or more correctly as
acyl groups or fatty acyl derivatives as side chains.
nonesterified fatty acids (NEFAs)) from where they are made
The fatty acid compositions of membrane lipids are usu-
available to tissues for b-oxidation (see succeeding text). As its
ally characteristic for the cell and membrane type, but may
name suggests, hormone-sensitive lipase is under tight regulation
change with the cell cycle, with age, in response to stimuli or
by various hormones, including adrenaline, noradrenaline,
to changes in the environment or the diet; these changes may
glucagon, and insulin. Insulin acts to inhibit its activity. In
have functional consequences. As many as 40 different fatty
contrast, adrenaline and glucagon increase the activity of lipopro-
acids can be incorporated into the sn
1 or sn
2 positions of
tein lipase. Therefore, through its concerted actions on the activi-
a phospholipid. Under normal conditions, the membrane
ties of lipoprotein lipase and on hormone-sensitive lipase, insulin
bilayer is in a ‘fluid’ state, meaning that membrane proteins
acts to make available fatty acids from circulating lipoproteins for
and lipids can migrate within the plane of the membrane.
storage in adipose tissue and to retain those fatty acids held there in
The fluidity of a membrane is strongly influenced by the fatty
stored triacylglycerols (Figure 6). Note too that through its actions
acid composition of its constituent lipids. In turn, membrane
on glucose transport into adipocytes, insulin makes available the
fluidity may affect cell function. This may be because fluidity,
precursor for the synthesis of the glycerol 3-phosphate that is
and hence protein movement, is required to establish
required for triacylglycerol synthesis. In contrast, an increase in
appropriate interactions between proteins or between pro-
adrenaline or noradrenaline concentration, for example, during
teins and lipids.
exercise, or in glucagon concentration, for example, during fasting,
enhances the activity of hormone-sensitive lipase, thus promoting
release of NEFAs from adipose tissue (Figure 6). The bulk of these
Phospholipid biosynthesis will be transported in the bloodstream noncovalently bound
Phospholipids are formed from the parent compound phos- to albumin and will be destined for energy generation via
phatidic acid, mentioned earlier as an intermediate in the b-oxidation in tissues like the skeletal muscle, heart, and liver
pathway of triacylglycerol biosynthesis. There is variation in (see succeeding text). Hormone-sensitive lipase also has choles-
the way in which the polar head groups are attached to the terol esterase activity. Adipose tissue also contains a lipase that is
phosphate of phosphatidic acid. Phosphatidic acid is the more active on monoacylglycerols than triacylglycerols.
precursor of phosphatidylglycerol, phosphatidylinositol,
and diphosphatidylglycerol, while the dephosphorylated
Fatty Acid Release from Phospholipids
derivative of phosphatidic acid 1,2-diacylglycerol is the pre-
cursor of phosphatidylcholine and phosphatidylethanol- Phospholipases are enzymes that selectively remove different
amine (Figure 5). components of the phospholipid molecule including the fatty
 Glycerol 3-phosphate
Acyl-S-CoA
Choline
1
(or Ethanolamine)
CoA-SH
ATP
1-Acyl glycerol 3-phosphate 11
(Lysophosphatidic acid)
ADP
Acyl-S-CoA
Phosphocholine
2
(or Phosphoethanolamine )
CoA-SH
CTP
1,2-Diacyl glycerol 3-phoshate
3
(Phosphatidic acid)
H 2O PPi
CTP
CDP-Choline
PPi 3 4 Pi (or CDP-Ethanolamine)

CDP-Diacylglycerol Diacylglycerol
Glycerol Inositol 12
3-phosphate CMP
5 8
CMP CMP
Phosphatidylcholine
Phosphatidylglycerol Phosphatidylinositol (or Phosphatidylethanolamine)
phosphate
ATP
H2O
6 9
ADP
Pi
Phosphatidylglycerol Phosphatidylinositol
4-phosphate
CDP-DAG ATP
7 10
CMP ADP
Diphosphatidylglycerol Phosphatidylinositol-
4,5-bis-phosphate
Figure 5 The pathways of phospholipid synthesis. Enzymes: 1, glycerol 3-phosphate acyltransferase; 2, lysophosphatidate acyltransferase; 3,
cytidylyltransferase; 4, phosphatidate phosphohydrolase; 5, phosphatidylglycerol phosphate synthase; 6, phosphohydrolase; 7, cardiolipin synthase; 8,
phosphatidylinositol synthase; 9, phosphatidylinositol 4-kinase; 10, phosphatidylinositol 4-phosphate 5-kinase; 11, choline (or ethanolamine)
kinase; 12, choline (or ethanolamine) phosphotransferase.

Insulin
Triacylglycerol
(carried in lipoproteins) Glycerol
+
Lipoprotein lipase

ADIPOCYTE Fatty acids

Fatty acids
Glycerol-3-phosphate Insulin
Hormone_
+ sensitive
lipase
+ Glucagon
+ Adrenaline
GLUT4
Triacylglycerol Noradrenaline
Glucose Glucose
+

Insulin
Figure 6 Scheme depicting triacylglycerol and fatty acid metabolism in adipocytes, the roles of lipoprotein lipase and hormone-sensitive lipase,
and the regulation by hormones including insulin. GLUT4, glucose transporter type 4.
640 Fatty Acids: Metabolism
Phosholipase B
H
Phospholipase A2
H C
R2 C O C H
O
H C
H O−
Phospholipase C
Figure 7 Sites of action of different phospholipase enzyme families toward a phospholipid substrate. R1 and R2 depict fatty acyl chains; X depicts the
phospholipid base.
acyl groups at positions 1 and 2 or the base (‘head group’) or
the entire phosphobase moiety. Phospholipases are classified
according to their site of action on the substrate phospholipid
(Figure 7). Type A phospholipases yield a monoacylphospho-
glyceride. Phospholipase A1 removes the fatty acid at the sn
1
position, while phospholipase A2 removes that at the sn
2
position. The phospholipase A2 enzymes are divided into
four groups based upon their properties. Phospholipase C
yields a 1,2-diacylglycerol and the phosphobase moiety,
while phospholipase D yields 1,2-diacylglycerol 3-phosphate
(phosphatidic acid) and the base. Type B phospholipases can
remove fatty acids from both sn
1 and sn
2 positions, but
these enzymes are quite rare. Phospholipases are important in
the digestion of dietary phospholipids, in cell membrane
remodeling, in generation of second messenger molecules,
and in provision of PUFAs for the production of bioactive
lipid mediators (see later section).
Fatty Acid b-Oxidation
b-Oxidation is the major metabolic pathway by which energy
is released from fatty acids. The rate of fatty acid b-oxidation is
controlled by the intracellular concentration of ‘free’ (i.e.,
unesterified) fatty acids, which in turn is determined by their
concentration in the blood, so that a rise in the concentration
of circulating NEFAs increases fatty acid oxidation in the tissues
that can use them (most aerobic tissues but not the brain).
NEFAs become important energy sources during starvation,
endurance exercise, and other situations where carbohydrate
supply is limiting.
Prior to b-oxidation, the fatty acid substrate is converted to
its CoA ester derivative:
Fatty acid þ ATP þ CoA ! acyl-CoA þ AMP þ PPi
This reaction is catalyzed by a cytosolic acyl-CoA synthase;
there are several such synthases with different specificities
according to fatty acid chain length.
Fatty acid b-oxidation occurs in the mitochondrial matrix,
and therefore, the fatty acid substrate (in the form of fatty acyl-
CoA) needs to be transported across the outer and inner
Phospholipase A1
O C R1
O
O
O P O X
Phospholipase D
mitochondrial membranes that are not permeable to fatty
acids or fatty acyl-CoAs with a hydrocarbon chain longer
than 12 carbons. Shorter-chain fatty acids can cross both the
outer and the inner mitochondrial membranes. Medium- and
long-chain acyl-CoAs require a special transport mechanism to
cross the mitochondrial membranes. Translocation of these is
a carnitine-dependent process involving the coordinate action
of isoforms of carnitine palmitoyltransferase (CPT) on
the mitochondrial outer and inner membranes (Figure 8).
CPT1 forms the fatty acylcarnitine at the outer mitochondrial
membrane, and this is then transported to the intramembrane
space via porin. The fatty acylcarnitine is transported through
the inner mitochondrial membrane via acylcarnitine translo-
cases in exchange for free carnitine. Once across the inner
mitochondrial membrane, the fatty acyl-CoA is reformed by
CPT2 (Figure 8). CPT1 is the rate-limiting step for mitochon-
drial b-oxidation. It is inhibited by malonyl-CoA, and thus,
conditions that promote malonyl-CoA synthesis (e.g., the fed
state) suppress fatty acid b-oxidation (and promote de novo
fatty acid biosynthesis). Conversely, conditions that promote
a decline in malonyl-CoA concentrations (e.g., fasting, starva-
tion, exercise, and insulin resistance) will act to promote fatty
acid b-oxidation (and suppress de novo fatty acid biosynthesis).
These actions at the level of CPT1 form part of integrated whole
body metabolic responses to changes in prevailing hormonal
conditions, for example, in fasting-to-fed and fed-to-fasting
transitions.
Fatty acid b-oxidation itself involves the progressive
removal of two-carbon units, as acetyl-CoA, from the carboxyl
end of the fatty acyl-CoA substrate in a series of four reactions
that act sequentially and repeatedly (Figure 9). Each round of
the cycle generates FADH2 and NADH in addition to acetyl-
CoA and an acyl chain that is two carbons shorter than
the original. The latter reenters the cycle. Thus, complete
b-oxidation of palmitoyl-CoA will generate eight acetyl-CoA,
seven FADH2, and seven NADH molecules:
Palmitoyl-CoA þ 7CoA þ 7FAD þ 7NADþ þ 7H2 O
! 8 acetyl-CoA þ 7FADH2 þ 7NADH þ 7Hþ
The acetyl-CoA produced by b-oxidation is normally oxidi-
zed in the citric acid (Krebs) cycle, while the FADH2 and
 Fatty Acids: Metabolism 641

OUTER INNER
MITOCHONDRIAL MITOCHONDRIAL
MEMBRANE MEMBRANE
Fatty acid
Acyl-S-CoA

ATP
Acyl-CoA
synthase CPT2
AMP+PPi

CoA-SH
Acyl-S-CoA Carnitine Carnitine

CPT1

Acyl- Trans-
CoA-SH locase Acyl-carnitine
carnitine

Figure 8 The carnitine palmitoyltransferase (CPT) system for transport of fatty acyl-CoA across the mitochondrial membranes into the matrix prior to
b-oxidation.

R-CH2-CH2-CH2-CO-S-CoA palmitic acid oxidized is 129 ATP. This is equivalent to approx-

imately 37 kJ or 9 kcal of energy per gram of fatty acid oxidi-
FAD
zed, over twice the energy yield for carbohydrate or amino acid
Acyl-CoA dehydrogenase
oxidation.
FADH2
Entry of acetyl-CoA into the citric acid (Krebs) cycle releases
R-CH2-CH=CH-CO-S-CoA CoA, and this is needed to maintain b-oxidation. This entry of
acetyl-CoA requires a supply of oxaloacetate for it to condense
H2O
to so producing citrate (the ‘first’ reaction of the cycle catalyzed
Enoyl-CoA hydratase
by citrate synthase). The supply of oxaloacetate comes from
pyruvate via the enzyme pyruvate carboxylase. Pyruvate car-
R-CH2-CHOH-CH2-CO-S-CoA boxylase is activated by acetyl-CoA, which indicates a lack of
oxaloacetate. In turn, the pyruvate is produced by glycolysis.
NAD+ Thus, some glucose metabolism is required to support contin-
3-Hydroxyacyl-CoA
dehydrogenase ued fatty acid b-oxidation. The conversion of pyruvate to oxa-
NADH + H+ loacetate is a type of anaplerotic reaction and, because of
R-CH2-CO-CH2-CO-S-CoA this requirement for some glucose metabolism to support
fatty acid b-oxidation, it is said that “fats burn in the flame of
CoA-SH carbohydrates.”
Thiolase The b-oxidation pathway outlined earlier operates for
straight-chain saturated fatty acids with an even number of
carbon atoms in the chain. Other specific enzymes are required
R-CH2-CO-S-CoA + CH3-CO-S-CoA for the oxidation of branched-chain, odd-numbered, or unsat-
urated fatty acids. b-Oxidation of odd-chain fatty acids yields
Figure 9 The pathway of b-oxidation of fatty acyl-CoA. Each cycle propionyl-CoA.
of four reactions produces acetyl-CoA and an acyl-CoA shortened
Although the process of b-oxidation was originally thought
by two-carbons that then reenters the cycle.
to occur exclusively in mitochondria, a second b-oxidation
system occurs in peroxisomes. Fatty acid transport into perox-
NADH pass their electrons to the mitochondrial electron trans- isomes involves the CPT system, which similar enzymes to
port chain. Hence, fatty acid b-oxidation generates a large mitochondrial CPT2 and regulation via malonyl-CoA. Com-
amount of ATP per mole of fatty acid and per mole of fatty pared with mitochondrial oxidation, peroxisomal b-oxidation
acid carbon oxidized: has a much broader substrate specificity and is especially active
toward very-long-chain fatty acids and also toward many fatty
Palmitoyl-CoA þ 23O2 þ 131Pi þ 131ADP
! CoA þ 131ATP þ 16CO2 þ 146H2 O acids with less common structural features, fatty acid
derivatives, and more complex lipids. Acyl-CoA oxidase is an
Because the activation of palmitate to palmitoyl-CoA con- important enzyme involved in catalyzing the first step of per-
sumes two ATP equivalents, the net gain per molecule of oxisomal b-oxidation. Peroxisomal b-oxidation is involved in
642 Fatty Acids: Metabolism

the biosynthesis of DHA from its precursor 24:6n
3 (Figure 3)
and in the conversion of DHA to EPA, a process sometimes
called retroconversion. Peroxisomal b-oxidation also seems
important in metabolism of other very-long-chain fatty acids
like erucic acid. Defects in acyl-CoA oxidase or in peroxisomal
b-oxidation cause clinical features such as mental retardation,
seizures, and leukodystrophy because of the inability to metab-
olize certain very-long-chain fatty acids that become elevated
in the circulation if not metabolized.
Ketone Body Synthesis
Ketone bodies are produced using acetyl-CoA derived from
fatty acid b-oxidation in the liver under specific metabolic
conditions. The two ketone bodies are acetoacetate and
b-hydroxybutyrate. Ketone body biosynthesis occurs in mito-
chondria and the pathway is shown in Figure 10. Two mole-
cules of acetyl-CoA are condensed to form acetoacetyl-CoA,
which is then conjugated to another molecule of acetyl-coA
to form 3-hydroxy-3-methylglutaryl-CoA (HMGCoA), which is
also an intermediate in de novo cholesterol biosynthesis.
HMGCoA is then cleaved to release one molecule of acetyl-
CoA and one molecule of acetoacetate. Some of the acetoace-
tate is reduced to b-hydroxybutyrate. Both acetoacetate and
b-hydroxybutyrate are readily soluble in the bloodstream and
are easily taken up by extrahepatic tissues, including the brain.
Some acetoacetate may be nonenzymatically decarboxylated
producing acetate, a volatile ketone that can appear in the
breath.
In target tissues, b-hydroxybutyrate is oxidized back to
acetoacetate, which is then converted to its CoA ester. This is
cleaved to yield two molecules of acetyl-CoA, which can enter
the citric acid (Krebs) cycle for complete oxidation. Thus,
hepatic ketone body synthesis allows the transfer of fatty acid
carbon to other tissues for oxidation and energy generation.
This is especially important for the brain, which cannot utilize
NEFAs as an energy source, but which can readily use ketone
bodies.
Hepatic ketogenesis is promoted in times of carbohydrate
shortage. As described earlier, glucose metabolism is required
to support continued entry of acetyl-CoA into the citric acid
(Krebs) cycle. However, during times of limited carbohydrate
availability, the liver diverts pyruvate and oxaloacetate into
gluconeogenesis (i.e., synthesis of glucose) and away from
the citric acid (Krebs) cycle; hence, anaplerosis is not sup-
ported. This creates a metabolic challenge to supporting the
recycling of CoA from acetyl-CoA back to fatty acids to enable
their b-oxidation. A solution to this is to divert acetyl-CoA into
another pathway that frees up the CoA to enable continuation
of b-oxidation. Ketogenesis, which converts acetyl-CoA to
acetoacetate and b-hydroxybutyrate, is this solution, and
furthermore, it produces metabolic substrates that are able to
be used by the brain when glucose is limiting.
Ketone bodies are synthesized during fasting (even after
overnight fasting), starvation, and prolonged exercise; in

LIVER CH3-CO-S-CoA + CH3-CO-S-CoA

CoA-SH Thiolase

CH3-CO-CH2-CO-S-CoA
CH3-CO-S-CoA
HMG-CoA synthase
CoA-SH
COOH-CH2-CH(CH3)(OH)-CH2-CO-S-CoA
Hydoxymethylglutyryl-CoA

HMG-CoA lyase
CH3-CO-S-CoA
HBDH
CH3-CO-CH2-COOH CH3-CH(OH)-CH2-COOH
Acetoacetic acid β-hydroxybutyric acid
BLOOD

EXTRAHEPATIC CH3-CO-CH2-COOH CH3-CH(OH)-CH2-COOH

TISSUES Acetoacetic acid HBDH β-hydroxybutyric acid
COOH-CH2-CH2-CO-S-CoA
3-Ketoacyl-CoA transferase
COOH-CH2-CH2-COOH
CH3-CO-CH2-CO-S-CoA

CoA-SH
Thiolase

CH3-CO-S-CoA + CH3-CO-S-CoA

Figure 10 The pathways of ketone body synthesis (ketogenesis) and oxidation. HMG, hydroxymethylglutaryl; HBDH, b-hydroxybutyrate
dehydrogenase.
 Fatty Acids: Metabolism 643

response to high-fat, low-carbohydrate diets (these are some-
times called ‘ketogenic diets’); and also by people with diabe-
tes. In the latter case, the marked elevation in blood and brain
ketone body concentrations can be pathological.
PUFAs as Substrates for Synthesis of Bioactive Lipid
Mediators
One of the key functional roles of PUFAs is as substrates for the
biosynthesis of bioactive mediators. These are oxidized deriv-
atives of PUFAs that are usually produced by enzyme-catalyzed
reactions, although some of them can be formed non-
enzymatically. Linoleic, dihomo-g-linolenic, arachidonic, eico-
sapentaenoic, osbond, clupanodonic, and docosahexaenoic
acids have all been shown to give rise to such mediators, and
it seems likely that all 18-, 20-, and 22-carbon PUFAs can act as
substrates for their formation.
Among the possible PUFA precursors, arachidonic acid
is usually the most prevalent in cell membranes, and
consequently, the most well described of these families of
PUFA-derived mediators are the eicosanoids formed from ara-
chidonic acid (eicosa is a prefix that denotes that a structure
contains 20 carbons). These include prostaglandins (PGs) and
thromboxanes (TXs), which together are termed prostanoids,
and leukotrienes (LTs), lipoxins (LXs), hydroperoxyeicosate-
traenoic acids (HPETEs), and hydroxyeicosatetraenoic acids
(HETEs). Prior to synthesis of eicosanoids, arachidonic acid is
released from the sn
2 position of membrane phospholipids
usually by the action of certain phospholipase A2 enzymes
(often cytosolic group IV phospholipase A2), although path-
ways involving phospholipase C and diacylglycerol lipase also
release arachidonic acid. The free arachidonic acid then acts as
a substrate for cyclooxygenase (COX; e.g., COX-1 and COX-2),
lipoxygenase (LOX; e.g., 5-LOX, 12-LOX, and 15-LOX), or
cytochrome P450 enzymes (Figure 11). COX enzymes lead to
PGs and TXs (2 series when formed from arachidonic acid),
LOX enzymes to LTs and LXs (4 series when formed from
arachidonic acid), and cytochrome P450 enzymes to HETEs
and other derivatives (Figure 11). Eicosanoids act through
binding to specific receptors, usually G protein-coupled recep-
tors, and thus, they act in a hormonelike manner working to
control cell and tissue responses. The range of activities of the
eicosanoids produced from arachidonic acid is extremely
broad and includes actions involved in controlling platelet
aggregation, blood clotting, inflammation, immune function,
smooth muscle contraction, renal function, neuron function,
bone turnover, and tumor cell proliferation. The physiology
and pathophysiology of arachidonic acid-derived eicosanoids
are widely explored.
One feature of increased dietary intake of the very-long-
chain n
3 PUFAs EPA and DHA is that they partly replace
arachidonic acid at the sn
2 position of cell membrane phos-
pholipids. The consequence of this is decreased availability of
arachidonic acid as a substrate for eicosanoid synthesis. EPA
and DHA may also inhibit arachidonic acid metabolism. These
effects of EPA and DHA are thought to be important in pre-
venting adverse pathological effects of eicosanoids produced
from arachidonic acid like thrombosis (excess blood clotting),
chronic inflammation, and tumor promotion and are thought
to underlie several of the health benefits of EPA and DHA and
foods that contain them like fatty fish. EPA is also a substrate
for the synthesis of PGs and TXs (3-series) and LTs (5-series)
but, in general, these are biologically weak compared to the
arachidonic acid-derived eicosanoids.
Although PGs, TXs, and LTs have been known for decades, a
relatively recent discovery has been of new families of bioactive
oxidized derivatives of EPA and DHA. These derivatives,

Arachidonic acid in various

membrane phospholipids

PGD2
Phospholipase A2
PGE2
COX CYT P450
PGI2 PGH2 PGG2 Arachidonic acid Various EETs, HETEs

TXA2 15-LOX 12-LOX 5-LOX

PGF2α
15-HPETE 12-HPETE 5-HPETE

15-HETE 12-HETE LTA4 5-HETE

Lipoxin A4 LTC4 LTB4

LTD4

LTE4

Figure 11 Overview of the pathways of conversion of arachidonic acid to eicosanoids. COX, cyclooxygenase; CYT P450, cytochrome P450; ETE,
eicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; PG,
prostaglandin; TX, thromboxane.
644 Fatty Acids: Metabolism
Hexose sugar
Lactate Pyruvate Succinate
Butyrate Acetyl-CoA Propionate
Acetate
Figure 12 Brief overview of the pathway of biosynthesis of short-chain
fatty acids from fermentation of simple sugars.
termed resolvins, protectins, and maresins, have been most
thoroughly explored in the context of inflammation, where
they have potent activity in resolution (‘turning off’) of on-
going inflammation. Hence, they have been termed specialized
proresolving mediators. E-series resolvins are produced from
EPA and D-series resolvins, protectins (sometimes called
neuroprotectins), and maresins from DHA. Synthesis of these
mediators involves the COX and LOX pathways often across
more than one cell type, with different epimers being produced
in the presence and absence of aspirin, and they act via specific
G protein-coupled receptors. The full range of the biological
activities of resolvins, protectins, and maresins has not yet been
explored.
Short-Chain Fatty Acid Metabolism
Short-chain fatty acids are volatile fatty acids produced mainly
by microbial fermentation. Their principal relevance to human
physiology is that they are produced in the proximal colon by
microbial fermentation of oligo- and polysaccharides that have
escaped digestion by mammalian enzymes further up the gas-
trointestinal tract (Figure 12). There are also some short-chain
fatty acids in ruminant milks. The major short-chain fatty acids
are acetic, propionic, and butyric. They are very soluble and are
absorbed easily from the gut lumen into the bloodstream. In
humans, acetate is usually the major short-chain fatty acid
present in the circulation. Increased dietary intake of substrates
for short-chain fatty acid producing microbes increases their
production resulting in higher concentrations in the gut con-
tents and in the bloodstream. Short-chain fatty acids can be
oxidized to produce energy, which is considered important for
cells of the gut lining, while they can also be used as substrates
for the synthesis of longer-chain fatty acids and ketone bodies.
See also: Adipose Tissue: White Adipose Tissue Structure and
Function; Energy Metabolism; Fatty Acids: Determination and
Requirements; Fatty Acids: Essential Fatty Acids; Fatty Acids: Trans
Fatty Acids; Fish Oils: Composition and Health Effects; Ketone Bodies;
Lipoproteins; Olive Oil: Its Role in the Diet; Phospholipids: Physiology;
Triacylglycerols: Structures and Properties; Vegetable Oils: Dietary
Importance.
Further Reading
Akram M (2013) A focused review of the role of ketone bodies in health and disease.
Journal of Medical Food 16: 965–967.
British Nutrition Foundation (1992) Unsaturated fatty acids: nutritional and
physiological significance. London: Chapman & Hall.
Buckley CD, Gilroy DW, Serhan CN, Stockinger B, and Tak PP (2013) The resolution of
inflammation. Nature Reviews Immunology 13: 59–66.
Burdge GC and Calder PC (2015) Introduction to fatty acids and lipids. World Review in
Nutrition and Dietetics 112: 1–16.
Calder PC (2015) Marine omega-3 fatty acids and inflammatory processes: effects,
mechanisms and clinical relevance. Biochimica et Biophysica Acta 1851: 469–484.
Coleman RA and Lee DP (2004) Enzymes of triacylglycerol synthesis and their
regulation. Progress in Lipid Research 43: 134–176.
Eaton S (2002) Control of mitochondrial beta-oxidation flux. Progress in Lipid Research
41: 197–239.
Guillou H, Zadravec D, Martin PG, and Jacobsson A (2010) The key roles of elongases
and desaturases in mammalian fatty acid metabolism: insights from transgenic
mice. Progress in Lipid Research 49: 186–199.
Gunstone FD, Harwood JL, and Padley FB (eds.) (1994) The lipid handbook, 2nd ed.
London: Chapman & Hall.
Gurr MI, Harwood JL, and Frayn KN (2002) Lipid biochemistry: an introduction, 5th ed.
Oxford: Blackwell Science.
Kuhna H, Banthiyaa S, and van Leyen K (2015) Mammalian lipoxygenases and their
biological relevance. Biochimica et Biophysica Acta 1851: 308–330.
Lafontan M and Langin D (2009) Lipolysis and lipid mobilization in human adipose
tissue. Progress in Lipid Research 48: 275–297.
Rådmark O, Werz O, Steinhilber D, and Samuelsson B (2015) 5-Lipoxygenase, a key
enzyme for leukotriene biosynthesis in health and disease. Biochimica et Biophysica
Acta 1851: 331–339.
Serhan CN (2014) Pro-resolving lipid mediators are leads for resolution physiology.
Nature 510: 92–101.
Sprecher H (2000) Metabolism of highly unsaturated n
3 and n
6 fatty acids.
Biochimica et Biophysica Acta 1486: 219–231.
Stubbs CD and Smith AD (1984) The modification of mammalian membrane
polyunsaturated fatty acid composition to membrane fluidity and function.
Biochimica et Biophysica Acta 779: 89–137.
Yamashita A, Hayashi Y, Nemoto-Sasaki Y, et al. (2014) Acyltransferases and
transacylases that determine the fatty acid composition of glycerolipids and the
metabolism of bioactive lipid mediators in mammalian cells and model organisms.
Progress in Lipid Research 53: 18–81.
Relevant Websites
http://www.aocs.org/index.cfm – American Oil Chemists’ Society (AOCS).
http://www.eurofedlipid.org/ – European Federation for the Science and Technology of
Lipids (Euro Fed Lipid).
http://www.goedomega3.com/ – Global Organization for EPA and DHA Omega 3s.
http://www.issfal.org/ – International Society for the Study of Fatty Acids and Lipids
(ISSFAL).
http://lipidlibrary.aocs.org/ – AOCS Lipid Library.
http://www.lipidmaps.org/ – Lipid Metabolites and Pathways Strategies (LIPIDMAPS).
 Fatty Acids: Trans Fatty Acids
AH Lichtenstein, Tufts University, Boston, MA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The relationship between dietary fat type and cardiovascular
disease risk was first identified soon after the turn of the
twentieth century. Since that time, the predominance of data
indicates that saturated fat is associated with the increased risk
of cardiovascular disease, whereas unsaturated fat (monoun-
saturated or polyunsaturated) is associated with the decreased
risk of cardiovascular disease. These conclusions come from
both observational studies and interventional studies and have
mechanistic underpinnings. However, within the category of
unsaturated fatty acids, there are two subtypes that have been
demonstrated to have unique effects with respect to cardiovas-
cular disease risk: fatty acids with the first double bond in the
omega-3 position of the acyl chain, associated with decreased
risk, and fatty acids containing one or more double bonds in
the trans configuration, associated with increased risk. The
focus of this article will be restricted to fatty acids containing
at least one trans double bond.
Terminology
The term ‘trans fat’ typically refers to edible fats that contain
trans-fatty acids. The term ‘trans-fatty acid’ refers to fatty acids
that have at least one double bond in the trans, as opposed to
the more comment cis, configuration, in their acyl chain
(Figure 1). Trans double bond containing fatty acids can be
either monounsaturated or polyunsaturated. Edible fats con-
tain a wide range of trans-fatty acid isomers, varying in the
location of the trans double bond(s) and the number of trans
double bonds in each acyl chain.
Food Sources of Trans-Fatty Acids
Ruminant Fat
Trans-fatty acids occur naturally in animal fat as a result of
anaerobic bacterial fermentation in the rumen. These trans-
fatty acids are absorbed from the gut and ultimately distributed
throughout the fat depots of the animal. Hence, animal fats,
such as meat and dairy fats, contribute to the trans-fatty acid
content of the diet. Of note, currently dietary guidelines to
reduce cardiovascular disease risk recommend limiting intake
of animal fat due to the relatively high concentration of satu-
rated fat. When these guidelines are adhered to, so too is trans-
fatty acid intake from this source limited.
Partially Hydrogenated Fat
Another major dietary source of trans-fatty acids is from
partially hydrogenated fat. The trans double bonds are formed
during the partial hydrogenation of liquid oil, typically vege-
table oil, which result from the breaking of cis double bonds
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00280-4
and their random reformation, some of which form trans
double bonds. In their unmodified state, vegetable oils are
made up of fatty acids with only cis double bonds. Vegetable
oils are partially hydrogenated to increase their viscosity by
changing them from a liquid state to a semiliquid or solid
state and to extend their shelf life by decreasing their suscepti-
bility to oxidation. Both of these properties are favorable to the
food industry.
Appearance of Partially Hydrogenated Fat in
the Food Supply
The initial impetus for developing the process of partial hydro-
genation of fat was to find an inexpensive substitute for butter.
The French chemist Paul Sabatier first developed the process of
partial hydrogenation around the turn of the twentieth cen-
tury. This process involved a nickel catalyst. Shortly thereafter,
the German chemist Wilhelm Normann developed a fat hydro-
genation process that involved hydrogen gas.
The use of partially hydrogenated fat accelerated from the
1960s to the 1980s as food producers responded to public
health recommendations to shift away from saturated fatty
acids, particularly animal fats and tropical oils, to unsaturated
fatty acids. At the time, partially hydrogenated fats appeared to
be a good substitute for fats high in saturated fatty acids because
their starting materials, vegetable oils, particularly vegetable oils
high in polyunsaturated fatty acids. In addition, relative to
vegetable oil in its natural state, partial hydrogenation resulted
in a product with more favorable stability and functional prop-
erties, similar to that of solid animal fat. The incorporation of
these fats into the food supply led to an increased trans-fatty acid
intake. During this period, there was little concern about the
effects of trans-fatty acids on lipoprotein profiles.
Estimating Trans-Fatty Acid/Partially Hydrogenated
Fat Intake
The trans-fatty acid content of foods is incompletely captured
in most nutrient databases, in part due to the rapidly changing
use of partially hydrogenated fat. Changes in use of partially
hydrogenated fat, primarily their decline, have been prompted
by evolving labeling regulations and legislative mandates.
Hence, there is likely a systematic misestimation of trans-fatty
acid intake when using food frequency questionnaires or food
records.
Plasma total trans-fatty acid, plasma phospholipid trans-
fatty acid, and erythrocyte membrane trans-fatty acid concen-
trations have been used as a biomarker of trans-fatty acid
intake, from both rudiment and partially hydrogenated fat
sources. The usefulness of this approach is limited to estimat-
ing relative intakes within or among populations. An
645
646 Fatty Acids: Trans Fatty Acids
Oleic Acid Elaidic Acid
Carbon
Hydrogen
Oxygen
Cis double
Trans double bond
bond
Figure 1 Structure of fatty acids containing a cis (left) and trans (right)
double bond.
additional option for estimating relative trans-fatty acid intake
from partially hydrogenated fat is to measure plasma dihydro-
phylloquinone concentrations. Dihydrophylloquinone is the
hydrogenated form of vitamin K and not naturally present in
vegetable oil. It is formed as an unintended consequence of
partial hydrogenation. However, as with biomarkers for trans-
fatty acid intake in plasma or erythrocyte membrane, the data
only provide an assessment of relative intakes within and
among populations.
More recently, an attempt to distinguish between the effects
of ruminant and partially hydrogenated fats using the plasma
proteome is being considered. At this time, it has met with
limited success. In the development phase are attempts to iden-
tify potential protein biomarker panels for specific trans-fatty
acids. This approach is based on changes in the regulation of
proteins elicited by the addition of elaidic acid to HepG2 cells.
Current Sources and Trends in Dietary Trans-Fatty
Acid Intake
Sources
The major source of trans-fatty acids in the food supply has
come from foods made with partially hydrogenated fat, pri-
marily commercially prepared fried and baked products. Over
the past decade, the trans-fatty acid intake in the United States
has declined. This trend has been prompted by a number of
factors, including legislative changes at the federal and local
levels and grassroots pressure to remove partially hydrogenated
fat from the food supply. In the mid-2000s, the Food and Drug
Administration mandated that by 2006, the trans fat content of
packaged food must be listed in the Nutrient Facts label. This
ruling appears to have prompted a re-formulation of some
products. Between 2005 and 2010, the US Department of
Agriculture documented a dramatic decline in the trans fat
content of newly introduced foods and a rise in the use of ‘no
trans fat’ claims on food packages. By 2011, it was estimated
that about 66% of the products surveyed had lower trans-fatty
acid content than previously. Of those, the vast majority, 82%,
had <0.5 g per serving of trans-fatty acids. Overall, the mean
trans-fatty acid content of the products surveyed decreased by
49% between 2007 and 2011. Of note, at the time mandatory
trans fat labeling was enacted, the Food and Drug Administra-
tion ruled that products containing <0.5 g trans fat per serving
could be labeled as 0 g trans fat or trans fat-free.
Around that same time that the FDA mandated trans fat
labeling on packaged foods, some US localities restricted the
use of partially hydrogenated fats by restaurant chains. As a
result, in one such city (New York City), between 2006 and
2008, the proportion of restaurants using partially hydroge-
nated fat declined from 51% to 2%. More recently, in late
2013, the Food and Drug Administration published a prelimi-
nary determination that partially hydrogenated fat, which is
currently included on the Food and Drug Administration’s
‘generally recognized as safe’ list, will be removed. A final ruling
is expected to become available in 2015. If put into effect, this
ruling will likely precipitate additional product reformulation in
a variety of food categories and result in a further decline in the
trans-fatty acid content of commercially prepared foods.
Trends
Although it is difficult to assess the potential public health
impact of changes in the trans-fatty acid content of the food
supply, fragmentary data suggest that the legislative mandates in
Scandinavia and the United States have contributed to a secular
decline in trans fat intake in those places. For example, it has
been estimated that between the years 2000 and 2009, plasma
trans-fatty acid concentrations in non-Hispanic, white adults
living in the United States declined by 50%. In the Framingham
Offspring Study cohort, between 1999 and 2006, erythrocyte
trans-fatty acid content declined by 23%. Similar downward
trends have been seen in Canada, the United Kingdom, Costa
Rica, and Western Europe, but trans-fatty acid intake remains
high in Eastern Europe and appears to have increased in Greece,
North Africa, the Middle East, and South Asia.
Trans-Fatty Acids and Lipoprotein Profiles
and Metabolism
Lipoprotein Profiles
Early work in the 1950s through 1970s evaluating the effect of
trans-fatty acids on plasma cholesterol concentrations suggested
a neutral effect. When differences were observed, they were
attributed, in part, to decreases in the level of polyunsaturated
fatty acids and increases in saturated fatty acids. In the early
1990s, reports started to appear suggesting that, similar to satu-
rated fatty acids, trans-fatty acids increased plasma low-density
lipoprotein (LDL) cholesterol concentrations and, in contrast to
saturated fatty acids, trans-fatty acids did not increase high-
density lipoprotein (HDL) cholesterol concentrations. Taken
together, these changes resulted in a less favorable LDL choles-
terol/HDL cholesterol ratio with respect to cardiovascular disease
risk. Subsequent work confirmed these findings and, with few
exceptions, demonstrated a dose–response relationship between
dietary trans-fatty acids and LDL cholesterol concentrations.
A trend toward increased triglyceride concentrations resulting
from trans-fatty acid intake has been reported in some studies.

Other Cardiovascular Disease Risk Factors
Small, dense LDL particles have been associated with a higher
risk of cardiovascular disease than large, buoyant LDL particles.
Dietary partially hydrogenated fat, compared to saturated or
polyunsaturated fat, has been reported to shift LDL particle
distribution from the large, buoyant particles toward small,
dense LDL particles. Changes in fasting insulin and glucose
concentrations resulting from dietary partially hydrogenated
fat have been reported to be small and, hence, would not
be predicted to have a large impact on glucose homeostasis.
No significant effect of partially hydrogenated fat intake on
C-reactive protein concentrations, lipoprotein (a), blood pres-
sure, visceral adiposity, metabolic syndrome, inflammatory
factors, and susceptibility of LDL particles to oxidation has
been reported. When effects have been observed, they tend to
be small and occur at relatively high intakes of trans-fatty acids,
and the data are not as consistent as the effects on LDL and
HDL cholesterol concentrations.
Trans-Fatty Acid Metabolism
Changes in trans-fatty acid intake are associated with propor-
tional changes in tissue levels. For example, plasma and eryth-
rocyte trans-fatty acid content declines when intake declines,
making these useful measures for assessing trends in trans-fatty
acid intake. These data also suggest that humans have adequate
capacity to metabolize trans-fatty acid isomers.
Early work that focussed on identifying the mechanism by
which trans-fatty acids elevate LDL cholesterol concentrations
led to the conclusion that higher rates of de novo cholesterol
synthesis were not a contributing factor, leading to the specula-
tion that an impairment in the catabolic pathway was involved.
This speculation was confirmed with the use of stable isotope-
labeled leucine to characterize lipoprotein kinetics in response
to partially hydrogenated fat intake. Relative to butter (saturated
fatty acids) and soybean oil (polyunsaturated fatty acids),
partially hydrogenated fat increased LDL concentrations by
decreasing the catabolic rate, resulting in a larger pool size. Little
effect of partially hydrogenated fat on LDL production rate was
observed. In contrast, partially hydrogenated fat lowered HDL
concentrations by increasing the catabolic rate, resulting in a
smaller pool size. Again, little effect on HDL production rate was
observed. Additional work on the effect of trans-fatty acids on
HDL metabolism indicated that differences in cholesteryl ester
transfer protein activity, phospholipid transfer protein activity,
or the fractional esterification rate of cholesterol in HDL did not
account for the differences in observed concentrations.
In some cases, dietary partially hydrogenated fat has been
reported to increase plasma triglyceride concentrations. In an
attempt to determine the mechanism by which trans-fatty acids
alter plasma lipoprotein profiles, the effect of partially hydro-
genated fat on triglyceride uptake by adipose tissue was esti-
mated by measuring plasma acylation-stimulating protein
(ASP) concentrations. The data indicated that a diet high in
partially hydrogenated fat resulted in lower plasma ASP con-
centrations compared with a diet high in polyunsaturated fat.
Differences in remnant-like particle concentrations, measured
in both the fasting and nonfasting states, did not account for
differences in triglyceride concentrations.
Fatty Acids: Trans Fatty Acids 647
Trans-Fatty Acid and Cardiovascular Risk
The negative effect of partially hydrogenated vegetable oil and
the resulting trans-fatty acids on plasma lipoprotein profiles
bears out consistently in multiple research studies conducted
throughout the 1990s. A meta-analysis of prospective cohort
studies and retrospective case-controlled studies resulted in an
estimated 2% increase in energy intake from trans-fatty acids is
associated with a 23% increase in the relative risk of coronary
heart disease. Similar estimates on the basis of multiple trials
have been made for the substitution of trans-fatty acids for
saturated, monounsaturated, and polyunsaturated fatty acids,
all of which appear to have a more favorable effect on LDL
cholesterol and HDL cholesterol concentrations, and the total
cholesterol/HDL cholesterol ratio.
The relationship of dietary trans-fatty acids and cardiovascu-
lar disease risk has been well documented and reviewed. Recent
work supports these earlier findings. Trans 18:2 isomer intake
has been associated with less favorable indexes of heart rate
variability in adults from two cohorts: the Cardiovascular
Heart Study, involving older subjects (mean age 72 years), and
a cohort of younger Portuguese subjects (mean age: 19 years).
Trans 18:1 isomers were associated with more favorable heart
rate variability in the older, but not the younger, cohort. Simi-
larly, a study of middle-aged and older Chinese volunteers
concluded that higher erythrocyte 18:2 trans-fatty acid concen-
trations were associated with dyslipidemia, whereas higher 18:1
trans-fatty acid concentrations were associated with lower risk of
type 2 diabetes. In the Nurses’ Health Study, trans-fatty acid
intake, estimated from food frequency questionnaires, was not
associated with sudden cardiac death. However, when the data
were assessed on the basis of women with and without coronary
heart disease, trans-fatty acid intake was positively associated
with sudden cardiac death in those women who had clinical
atherosclerosis. Data from the Reasons for Geographical and
Racial Differences in Stroke (REGARDS) cohort, a US-based
prospective cohort study of white and black women and men,
indicate that trans-fatty acid intake, as assessed using food fre-
quency questionnaires, was associated with higher all-cause
mortality. Using plasma phospholipid profiles from two
cohorts, the Cardiovascular Health Study and the Estrogen
Replacement and Atherosclerosis Study, trans-fatty acids were
associated with increased cardiovascular disease risk.
Ruminant Versus Partially Hydrogenated Fat
and Cardiovascular Disease Risk
Some observational data have suggested that dietary trans-fatty
acids derived from partially hydrogenated fat are more closely
associated with cardiovascular disease risk that dietary trans-fatty
acids derived from ruminant fat. However, these data have not
been consistent.
The difference between ruminant and partially hydroge-
nated fat sources of trans-fatty acids is reflected in the relative
distribution of trans-fatty acids isomers. Conjugated linolenic
acid and vaccenic acid predominate in ruminant fat. The
results of clinical studies comparing these two sources of
trans-fatty acids have been mixed. Some have reported no or
only small differences between ruminant and partially
648 Fatty Acids: Trans Fatty Acids
hydrogenated fat sources of trans-fatty acids on lipoprotein
profiles, whereas others have reported that partially hydroge-
nated fat results in less favorable lipoprotein profiles than
ruminant fat. Interpreting these data can be challenging,
because in many studies, the level of ruminant fat trans-fatty
acid isomers achieved in the test diets could not be achieved
with current forms of ruminant fat in the food supply.
Conclusions
The data supporting a negative effect of dietary trans-fatty acids
on cardiovascular disease risk are consistent. The primary die-
tary sources of trans-fatty acids include partially hydrogenated
fat and ruminant fat. Due to legislative changes and public
pressure, intake from the former source has declined over the
past decade. Adherence to current recommendations to restrict
the intake of saturated fat from meat and dairy products would
result in reduction in trans-fatty acid intake from the latter
source. These factors have contributed to a secular decrease in
the United States and some other countries worldwide. The
adverse effect of trans-fatty acids on plasma lipoprotein profiles
is consistent; the effect of trans-fatty acids on other cardiovas-
cular risk factors is less clear. Observational data consistently
demonstrate an adverse effect of trans-fatty acids and partially
hydrogenated fat on cardiovascular disease risk. Yet to be
resolved is the issue of whether trans-fatty acid isomers predo-
minating in ruminant and partially hydrogenated fats have
differential effects in terms of health outcomes. This uncer-
tainty should not detract from a clear and consistent public
health message to restrict the intake of trans-fatty acids. Recent
trends in the food supply toward less use of partially hydroge-
nated fat have accelerated this change.
See also: Fatty Acids: Fatty Acids; Fatty Acids: Metabolism; Soybean
Oil; Vegetable Oils: Composition and Analysis; Vegetable Oils: Dietary
Importance.
Further Reading
Angell SY, Cobb LK, Curis CJ, Konty KJ, and Silver LD (2012) Change in trans fatty
acid content of fast food purchases associated with New York City’s restaurant
regulation. Annals of Internal Medicine 157: 81–86.
Aronis KN, Joseph RJ, Blackburn GL, and Mantzoros C (2011) Trans-fatty acids, insulin
resistance/diabetes, and cardiovascular disease risk: should policy decisions be
based on observational cohort studies, or should we be waiting for results from
randomized placebo-controlled trials? Metabolism, Clinical and Experimental
60: 901–905.
Aronis KN, Khan SM, and Mantzoros CS (2012) Effects of trans fatty acids on glucose
homeostasis: a meta-analysis of randomized, placebo-controlled clinical trials.
American Journal of Clinical Nutrition 96: 1093–1099.
Brouwer IA, Wanders AJ, and Katan BM (2010) Effect of animal and industrial trans fatty
acids on HDL and LDL cholesterol levels in humans – a quantitative review. PLoS
One 5: e9434.
Chiuve SE, Rimm EB, Manson JE, et al. (2009) Intake of total trans, trans-18:1, and
trans-18:2 fatty acids and risk of sudden cardiac death in women. American Heart
Journal 158: 761–767.
Gebauer SK, Chardigny JM, Jakobsen MU, Lamarche B, Lock AL, Proctor SD, and
Baer DJ (2011) Effects of ruminant trans fatty acids on cardiovascular disease and
cancer: a comprehensive review of epidemiological, clinical, and mechanistic
studies. Advances in Nutrition 2: 332–354.
Kiage JN, Merrill PD, Robinson CJ, et al. (2013) Intake of trans fat and all-cause
mortality in the Reasons for Geographical and Racial Differences in Stroke
(REGARDS) cohort. American Journal of Clinical Nutrition 97: 1121–1128.
Krogager TP, Nielsen LV, Bak S, et al. (2012) Identification of a potential biomarker
panel for the intake of the common dietary trans fat elaidic acid (trans9-C18:1).
Journal of Proteomics 75: 2685–2696.
Laake I, Pedersen JI, Selmer R, et al. (2012) A prospective study of intake of trans-fatty
acids from ruminant fat, partially hydrogenated vegetable oils, and marine oils and
mortality from CVD. British Journal of Nutrition 108: 743–754.
Lichtenstein AH, Ausman LA, Jalbert SM, and Schaefer EJ (1999) Comparison of
different forms of hydrogenated fats on serum lipid levels in moderately
hypercholesterolemic female and male subjects. New England Journal of Medicine
340: 1933–1940.
Mozaffarian D, Katan MB, Ascherio A, Stampfer MJ, and Willett WC (2006) Trans fatty
acids and cardiovascular disease. New England Journal of Medicine
354: 1601–1613.
Rahkovsky, I., Martinez, S. and Kuchler, F. (2012). New food choices free of trans fats
better align U.S. diets with health recommendations. Economic Information Bulletin
Number 95, USDA.
Remig V, Franklin B, Margolis S, Kostas G, Nece T, and Street JC (2010) Trans fats in
America: a review of their use, consumption, health implications, and regulation.
Journal of the American Dietetic Association 110: 585–592.
Soares-Miranda L, Stein PK, Imamura F, et al. (2012) Trans-fatty acid consumption and
heart rate variability in 2 separate cohorts of older and younger adults. Circulation:
Arrhythmia and Electrophysiology 5: 728–738.
 Fermented Foods: Composition and Health effects
D Ansorena and I Astiasarán, University of Navarra, Pamplona, Navarra, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Fermentation is a biotechnological process used by man since, at
least, the Neolithic period (around 10 000 years BC). The fermen-
tation process is carried out by microorganisms (filamentous
fungi, yeast, bacteria, or a combination of them) in anaerobic
conditions and consists of the conversion of fermentable carbo-
hydrates into end-metabolites such as organic acids, alcohols,
and carbon dioxide. Louis Pasteur defined the fermentation
process as ‘la vie sans l’air,’ which means ‘life without air.’
Although the main aim for its application was initially food
preservation and an increase in the shelf life, other effects such
as the improvement of safety, nutritional value, and organo-
leptic quality of the foods are simultaneously achieved.
The discovery of microorganisms in the nineteenth century
enabled the improvement of the production of fermented
foods using specific starter cultures that can be added in a
large number to accelerate and steer the fermentation process
and to obtain a broad variety of fermented foods with different
organoleptic and nutritional properties. Spontaneous fermen-
tation is still used in some cases, such as in traditional foods and
foods in which the microorganisms responsible for the fermen-
tation are not well known. The microorganisms that can be
used as starter cultures because of their proved safety, techno-
logical role, and/or beneficial use are gathered in lists elabo-
rated by different institutions such as the International Dairy
Federation (IDF), the European Food and Feed Cultures
Association (EFFCA), the European Food Safety Authority
(EFSA), and the Food and Drug Administration (FDA). Recently,
an updated inventory of microorganisms used in food fermen-
tations covering a wide range of food matrices (dairy, meat, fish,
vegetables, legumes, cereals, beverages, and vinegar) was
presented. The inventory of microbial food cultures (live bacte-
ria, yeasts, or molds used in food production) contains 195
bacterial species and 69 species of yeasts and molds. There are
two main fermentation processes: alcoholic fermentation, which
results in the production of ethanol and is carried out predom-
inantly by yeasts, and lactic acid fermentation, which gives rise to
a great variety of products, occurs mainly in dairy products and
cereals, and is carried out by lactic acid bacteria (LAB). Also,
acetic acid and alkali fermentation processes take place in
alcoholic products and in fish and seeds, respectively.
In the last years, special attention has been paid to the
nutritional and health effects of fermented foods derived
from microbial metabolic activities. This article is mainly
focussed on these aspects. A brief revision of the composition
of the different groups of fermented foods and the currently
known main health aspects related to the biocompounds
resulting from the fermentation process is reported.
Fermented Foods: Composition and Nutritional Value
Approximately one-third of human food intake corresponds to
fermented foodstuffs. The major groups of fermented foods
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00285-3
worldwide are dairy products, beverages (beer, wines, cider,
coffee, and tea), and cereal-based products (bread, pancakes,
and donuts) and several fermented foods derived from meat,
fish, fruits, and vegetables.
The nutritional and organoleptic properties that character-
ize each type of these products depend on the composition of
the food matrix, the type of microorganism responsible for the
fermentation, and the processing conditions.
Dairy Products
The group of dairy products encompasses some of the most
typical fermented foods, especially in Europe, North America,
and the Middle East and also in Africa and Southeast Asia.
The most common subcategories are cheeses and acid-
fermented milks.
There are more than 1000 different types of cheeses
elaborated with milk from different animal species (cow,
sheep, goat, camel, and buffalo) and different maturation
degrees, giving rise to a wide variety of textures and flavors.
From a nutritional point of view, cheeses are in general high-
density products with high energetic value and significant fat,
protein, vitamin B, and calcium contents. Also, if they are
subjected to large ripening processes, high amounts of
biogenic amines (BAs) might be present. These compounds,
resulting from microbial decarboxylation of amino acids, may
generate toxic effects such as headache and hypertensive crisis
and also act as inhibitors of histamine-detoxifying enzymes (as
monoamine oxidase). Selection of appropriate starter cultures
(decarboxylase-negative activity) has been suggested as a good
strategy to try to reduce BA formation during fermentation
processes.
Acid-fermented milk (e.g., yogurts and kefir) have different
nutritional profiles depending on the ingredients used: type of
milk (full fat vs. skimmed milk), added sugars or flavors, etc.
The LAB responsible for their fermentation give rise to a degree
of acidity that inhibits the growth of other types of microor-
ganisms. In addition, they can also produce several bacterio-
static compounds and compounds responsible for the
characteristic organoleptic properties of these types of
products. One of the main changes that occur during the
fermentation of milk is the conversion of lactose (milk sugar)
into the more digestible lactate. Effectively, there are a growing
number of individuals that are intolerant to lactose, which
benefit from the fermentation of milk. There are other benefi-
cial effects related to the intake of LAB (Lactobacillus and
Streptococcus) and Bifidobacterium that are also used as starter
cultures in fermented milk. Some of these microorganisms are
considered as probiotics and can be positively related to an
improvement of the intestinal tract health, an enhancement of
the immune system, a decrease in the prevalence of allergy in
susceptible individuals, and even a risk reduction of certain
cancers. As for cheeses, fermented milk are also rich in calcium
and B vitamins.
649
650 Fermented Foods: Composition and Health effects
Fermented Beverages
Different beverages result from the fermentation process of
vegetable products. Wines, beers, and cider have a rich
composition that is dependent on the fermentation process
(predominantly carried out by yeasts) and also on the raw
materials (grapes or fruit juice and barley or other cereals)
that serve as substrates. Ethanol is a common end product in
beverage fermentation and has been classified as carcinogenic
to humans by the International Agency for Research on Cancer
(IARC). However, there are several epidemiological studies
suggesting that moderate consumption of alcohol reduces
overall mortality, mainly from coronary diseases. In general,
the positive effects of alcoholic beverages are due to the pres-
ence of multiple polyphenols, bioactive compounds present in
the vegetables or even their metabolites as a consequence of the
fermentation process. Wine, being the richest in very active
polyphenols such as resveratrol, is associated, when consumed
in moderate amounts, with a decreased incidence of cardiovas-
cular disease, hypertension, diabetes, and certain types of can-
cer. These positive effects could also be due to the presence of
other phytochemicals, such as melatonin and phytosterols.
Additionally, moderate consumption of beer has been associ-
ated with these effects to a lesser degree, probably because of its
lower amount of polyphenols. Nevertheless, beer is rich in
nutrients such as carbohydrates, amino acids, minerals, and
vitamins. In the case of distillate drinks (spirits), they hardly
have other interesting compounds apart from ethanol and the
aromatic compounds responsible for their organoleptic prop-
erties. However, it is worthy to note that favorable changes in
several cardiovascular biomarkers (higher levels of high-
density lipoprotein cholesterol and adiponectin and lower
levels of fibrinogen) provide indirect pathophysiological
support for a protective effect of moderate alcohol use on
coronary heart disease, independently of its source of supply.
Tea and coffee constitute another interesting group of fer-
mented beverages characterized by their supply of stimulating
compounds. It should be pointed out however that, although
microorganisms (basically fungi) are involved in their proces-
sing, the main changes are due to endogenous polyphenol
oxidase activity and other biochemical reactions that take
place during fermentation. Although the intake of caffeine,
theophylline, and theobromine has been associated with life-
styles related to risk of cancers, those beverages have shown
some beneficial effects. Tea is the second most popular bever-
age in the world after water. Around 75% of tea consumed
worldwide is black tea (obtained from the fermentation of
green tea). There are many studies demonstrating protective
effects of tea over chronic diseases as cardiovascular patholo-
gies, and also, there are some studies, although not conclusive,
demonstrating a chemopreventive effect. These effects are
related to the high amounts of polyphenolic compounds,
especially catechins. Effectively, during the fermentation pro-
cess to obtain black tea, catechins are converted in a great
extent to polymeric complexes known as theaflavins and
thearubigins, which seem to be also effective in the prevention
of cardiovascular disease. Coffee is very rich in several biolog-
ical compounds as caffeine, diterpenes, chlorogenic acids, and
melanoidins, which may affect human health. The health ben-
efits of coffee consumption regarding the cardiovascular
system and metabolism mostly depend on its antioxidant
compounds; meanwhile, diterpenes and caffeine may produce
harmful effects by raising lipid fraction and affecting endothe-
lial function, respectively. The variety of coffee species, roasting
degree, and brewing method determine the final composition
and the derived effects.
Fermented Meat and Fish Products
Meat fermentation processes in combination with drying and
smoking probably developed empirically over 5000 years ago
and have given rise to a broad variety of products: dry, semidry,
mold-ripened, and undried products all around the world. The
fermentation process is carried out basically by LAB and
coagulase-negative staphylococci. Fermented meat products
have the nutritional advantages related to the intake of meat,
which is a significant amount and quality of proteins depend-
ing on the type and percentage of raw meat used in the formu-
lation, iron (50–60% in the heme form), zinc, and vitamin B.
On the other hand, they have some arguably negative aspects
related to a generally high amount of fat (with a high saturated
fat proportion), cholesterol, and sodium. Moreover, the pres-
ence of nitrites/nitrates used as nitrificants (curing process) in
their formulations has to be taken into account. As the result of
the fermentation process (due to proteolytic and lipolytic activ-
ities of microorganisms in concertation with the action of meat
enzymes), a wide variety of compounds with significant impact
on the sensory properties are formed: amino compounds,
aldehydes, alcohols, acids, alkanes, alkenes, ketones, and
even BAs. Some of these compounds have been pointed out
as functional compounds, in particular some of the peptides.
Furthermore, there are studies that have pointed out that
fermented sausages could be considered as probiotic foods
although much more studies are needed to establish the actual
health benefits. Moreover, different studies trying to improve
the composition of fermented sausages in order to get healthier
products are being carried out.
Fermented fish products are especially consumed in Asia
and Oceania countries. These products include very popular
sauces and pastes, which are used as condiments and ingredi-
ents in a great variety of processed foods. They are rich in
proteins and amino acids but sometimes also in BAs, which
have to be controlled. Also, they have in general high amounts
of salt (around 20–25%, except for a traditional product
sikhae, which contains 6–8%).
Cereal-Based Fermented Products
Cereals are a good microbial substrate, so the fermentation
process takes place in a natural way involving mixed cultures
of yeast, bacteria (in general LAB), and fungi (Aspergillus, Fusar-
ium, Penicillium, and Trichothecium, among the most frequent).
Fermentation of cereals gives rise to very interesting changes in
composition, namely, a decrease in the level of carbohydrates
and nondigestible oligosaccharides, a synthesis of some
amino acids, an increase in the availability of the vitamin B
group, and a degradation of phytate (a quelant of iron, zinc,
and calcium). As a consequence of the fermentation of wheat
and rye flour by LAB (sourdough process), a reduction in the
starch digestibility is noticed, leading to a lower glycemic

index. This effect is mainly attributed to the formation of organic
acids (lactic acid), to the greater release of peptides and amino
acids, or to the increment in the amount of free phenolic com-
pounds, among other factors. All these changes improve the
nutritional properties and even can lead to the development of
functional products. Effectively, the products formed as a conse-
quence of the fermentation process and the presence of some
compounds with health beneficial effects, as it is the case of
b-glucan (especially present in oat), make fermented cereals a
group to be taken in consideration in the context of functional
foods. Moreover, cereals have also been evaluated as good sub-
strates for the growth of probiotic strains, giving rise to some new
probiotic foods (e.g., Yosa and Dogik) with supposed beneficial
effects, although these aspects are still a research challenge.
Furthermore, the fact that cereal-based fermented products are
major contributors to energy intake in developing countries has
to be considered.
Fermented Fruits and Vegetables
Fresh vegetables and fruits are recommended in a healthy diet
because they are fundamental sources of vitamins, minerals,
dietary fiber, and biocompounds as phytosterols (hypocholes-
terolemic effects) and polyphenols (antioxidant properties).
However, there are some traditional and emerging fermented
products resulting from lactic acid fermentation that represent
a suitable way for increasing the consumption of vegetables
and fruits. For instance, kimchi is a traditional fermented
vegetable product of Korea, which is consumed in winter
when the dietary intake of fresh vegetables might be more
difficult. Other examples include sauerkraut, cocoa, fermented
olives, pickles, and fermented radish.
Bioactive Compounds Derived from the Fermentation
Processes
Fermentation leads, among other changes, to the release and/
or synthesis of different bioactive compounds that are respon-
sible for some of the health benefits attributed to this type of
foods.
Table 1 gathers some of the most common bioactive com-
pounds resulting from fermentation processes and the foods in
which they are present.
Bioactive Peptides
Fermentation is one of the mechanisms by which bioactive
peptides can be present in foods. Effectively, a number of
papers describe the release of these compounds during fermen-
tation of a wide variety of foods, ranging from milk, legumes,
meat, soybean, to sourdough. These compounds, formed by
2–20 amino acid sequences, have been related to several phys-
iological functions able to influence health.
Antihypertensive properties have been described for IPP
(isoleucine–proline–proline) and VVP (valine–proline–proline)
tripeptides released during the fermentation of milk by different
proteolytic strains of Lactobacillus helveticus (CP790, LBK-16H,
and ASC474), Streptococcus thermophilus, and Saccharomyces cere-
visiae. The formation of these and other peptides can be affected
Fermented Foods: Composition and Health effects 651
by fermentation time and temperature and by pH and calcium
concentration in medium. The main mechanism involved in
their biological activity deals with the inhibition of ACE
(angiotensin-converting enzyme). It has been postulated, for
instance, that after a 2-week consumption of 150 ml day 1 of
fermented milk, hypertensive patients reduced systolic blood
pressure approximately by 7 mmHg. Other studies do not clearly
support this finding. Nevertheless, according to the EFSA,
more studies are needed to conclude about the cause–effect
hypotensive activity. In addition, not only the production but
also the stability of bioactive peptides has to be taken into
account during fermentation.
Antioxidant, antimicrobial, and immunomodulatory prop-
erties and anti-inflammatory potential have also been
described for peptides formed during the fermentation of
milk and other dairy products such as kefir or cheese.
Soybean is also an important source of bioactive peptides.
Thus, the proteases and peptidases of Bacillus, Aspergillus, and
Rhizopus strains have been shown to hydrolyze soy proteins
into active hypotensive peptides. In addition, soy-fermented
products such as nattō and tempeh have yielded antioxidant
peptides. Less explored sources of bioactive peptides are lentils
or porcine skeletal muscle proteins, whose fermentations by
Lactobacillus plantarum or Lactobacillus sakei and Lactobacillus
curvatus, respectively, were able to release also ACE inhibitory
peptides during in vitro studies.
Exopolysaccharides
Exopolysaccharides (EPSs) are homopolymers or heteropoly-
mers with a wide diversity of structures, capable of modifying
the sensory properties of foods. Some strains of LAB species
(Lactobacillus acidophilus, Lactobacillus delbrueckii subsp.
bulgaricus, Lactococcus lactis, Lactobacillus plantarum, Lactobacil-
lus casei, etc.) are EPS producers, and in fact, one of these
bacteria, Leuconostoc mesenteroides, excretes dextran, authorized
as a novel food ingredient to be used in bakery products
(Decision 2001/122/EC). Besides its technological property
as thickener in bakery and dairy products, prebiotic potential-
ities have been associated with this polymer, as gut microbes
metabolize it to produce propionic acid, which is able to
reduce cholesterol and triglyceride levels. In addition, EPSs
have generally been suggested as blood cholesterol reducers,
antioxidants, immunomodulators, and antitumor and antiul-
cer agents. One of the most studied strains for the antitumor
activity of its EPS has been Lactobacillus plantarum 70 810,
whereas the EPS from Lactobacillus helveticus MB2-1 with
whey as matrix was associated with strong scavenging activity
in in vitro tests. Kefir grains are also able to release EPSs from
soy milk. However, there is a lack of clinical evidence demon-
strating these putative prebiotic characteristics and beneficial
effects after oral administration of EPS in functional foods.
Arabinoxylans
During the fermentation of cereal-derived processed food such
as bread or beer, the enzymatic hydrolysis of arabinoxylans,
yielding arabinoxylan oligosaccharides, can occur. These com-
pounds have been associated with prebiotic and other health
effects (improved gut transit, reduced serum triglyceride and
652 Fermented Foods: Composition and Health effects

Table 1 Nonexhaustive list of examples of compounds produced during fermentation of foods

Compound Health effect Substrate Microorganisms References

Peptides Hypotensive Lentils Lactobacillus plantarum CECT 748 T Torino et al.

(2013)
Peptides Hypotensive Soybean meal Bacillus subtilis natto Wang et al.
(2013)
Peptides Hypotensive Fermented milk Lactobacillus helveticus LBK-16H Seppo et al.
(2003)
Peptides Hypotensive Fermented milk Lactobacillus helveticus 1188 Elfahri et al.
(2014)
Exopolysaccharide Antitumor Chinese pao cai Lactobacillus plantarum 70 810 Wang et al.
(fermented (2014a,b)
cabbage)
Exopolysaccharide Scavenging activity Whey Lactobacillus helveticus MB2-1 Li et al. (2014)
Phenolic compounds Antioxidant activity Buckwheat, Lactobacillus rhamnosus and yeast Dordevic et al.
wheat germ, Saccharomyces cerevisiae (2010)
barley, and
rye
Phenolic compounds Antioxidant activity Red cabbages Lactobacillus plantarum ATCC 8014 Hunaefi et al.
(2013)
Folates, free phenolic Varied Germinated rye Saccharomyces cerevisiae Katina et al.
acids, total (2007)
phenolic
compounds,
lignans, and
alkylresorcinols
Isoflavone aglycone Improvement of glucose metabolism, Soy milk Lactobacillus rhamnosus C6 Hati et al.
antioxidant activity, alleviation of (2015)
hormonal disorders in postmenopausal
women, etc.
GABA Hypotensive Lentils Lactobacillus plantarum Torino et al.
(2013)
GABA Hypotensive Cheeses Lactobacillus brevis CECT 8183, CECT 8181, Diana et al.
and CECT 8182 and Lactococcus lactis (2014)
subsp. lactis CECT 8184
CLA Varied Pickle Lactobacillus plantarum Liu et al.
(2011)
CLA Varied Milk Lactococcus lactis (best among 155 LAB Ashish et al.
cultures) (2012)
Ubiquinone Antioxidative Jeotgal Bacillus subtilis, Leuconostoc mesenteroides, Pyo and Oh
Pediococcus halophilus, Torulopsis sp., (2011)
Sarcina sp., Saccharomyces sp.
Folate Vitamin Oat bran Saccharomyces cerevisiae and Candida milleri Korhola et al.
(2014)
Vitamin B2 Vitamin Pasta and bread Lactobacillus plantarum Capozzi et al.
(2011)
Probiotic mixture Modification of activity of the brain regions Fermented milk Bifidobacterium animalis subsp. lactis, Tillisch et al.
that control the central processing of product with Streptococcus thermophilus, Lactobacillus (2013)
emotion and sensation probiotic delbrueckii subsp. bulgaricus, and
(FMPP) Lactococcus lactis subsp. lactis
Probiotics Gut health Fermented Mainly Lactobacillus and Bifidobacterium Smug et al.
foods strains (2014)

cholesterol levels, and immunomodulating and antioxidative
effects), although they have not been extensively studied yet.
Polyphenols and Flavonoids
Health beneficial effects are attributed to polyphenols and fla-
vonoids present in vegetable products. These compounds are
predominantly found as glycosides in nature. However, their
bioavailability in the organism increases when they are not
glycosylated. Some food-fermenting LAB can remove glycoside
residues by means of enzymatic activity (a-rhamnosidase,
b-galactosidase, and b-glucosidase), resulting in increased
bioavailable bioactive compounds. Some examples of LAB
causing the hydrolysis of healthy phenolic compounds are the
following: Lactobacillus plantarum, Lactobacillus pentosus,
Lactobacillus brevis, and Pediococcus pentosaceus. Strains of these

LAB species have been shown to hydrolyze oleuropein, the
most prevalent polyphenol present in olives, to which some
of the beneficial effects of unrefined olive oil are attributed. It
has been described as an antioxidant and with antitumor and
anti-inflammatory properties.
Soy fermentation induces a relevant increase in free isofla-
vones (daidzein, glycitein, and genistein) from their conju-
gated forms (daidzin, glycitin, and genistin). b-Glucosidase
activity during soy fermentation has been described for
selected strains of Lactobacillus rhamnosus, Lactobacillus acidoph-
ilus, Lactobacillus casei and Enterococcus faecium. As soy isofla-
vones are structurally similar to estrogen, similar health effects
to these compounds have been reported. They have been pos-
tulated to play a role in the prevention of cardiovascular
disease, metabolic syndrome, and type 2 diabetes; in relieving
postmenopausal symptoms such as osteoporosis; or in the
decrease in risk of breast and prostate cancers. However, in
addition to evidences from in vitro biochemical tests and also
from epidemiological studies, mostly done in populations
consuming high amounts of soya, long-term interventional
studies are needed to conclude on the health effects of these
compounds. In this sense, the EFSA has considered that, based
on the currently available studies, the following claimed ben-
eficial health effects lack a clear enough cause and effect rela-
tionship: maintenance of normal blood LDL cholesterol
concentrations, protection of DNA, proteins and lipids from
oxidative damage, maintenance of bone mineral density,
and reduction of vasomotor symptoms associated with
menopause.
In the case of cereals, it has been shown that fermentation
by Lactobacillus rhamnosus and Saccharomyces cerevisiae can
increase antioxidant activity and total phenolic content of
buckwheat, wheat germ, barley, and rye as compared with
their unfermented counterparts. Thus, tailored fermentation
to improve the antioxidant capacity of resulting foods can be
accomplished.
g-Aminobutyric Acid
g-Aminobutyric acid (GABA) is a nonprotein amino acid that
has several well-known physiological functions including anti-
oxidant, antihypertension, and antidepression effects.
Although most of the clinical applications of GABA are
theoretical, based on data extrapolated from drug interven-
tions, there are some experiments in humans and mostly in
animal studies, in which fermented foods have shown these
physiological effects. A significant decrease in blood pressure
was noticed after the administration of fermented milk rich in
GABA to mild hypertensive patients (using Lactobacillus casei
strain Shirota and Lactococcus lactis YIT 2027) and also in rats
(with Lactobacillus paracasei subsp. paracasei NTU 101 and
Lactobacillus plantarum NTU 102). Administration of products
fermented with the fungus Monascus to rats demonstrated their
antidepressant effect.
LAB are known food-associated GABA producers, based on
their glutamic acid decarboxylase activity, which is the enzyme
that catalyzes the decarboxylation of glutamate to GABA. Lac-
tobacillus brevis gives particularly high GABA production yields
compared with other LAB. A wide range of GABA-enriched
fermented food products have been described, including
Fermented Foods: Composition and Health effects 653
cheeses and other dairy products (e.g., koumiss and diverse
fermented milk), soy sauces, kimchi, sourdough, black rasp-
berry juice, red seaweed beverage, and fermented fish and
meat. Also, model fermentation systems aiming at an increase
in GABA and other bioactive compounds have been success-
fully assayed using lentils and kidney bean extracts as matrices.
Conjugated Linoleic Acid
Conjugated linoleic acid (CLA) (C18:2, with c9, t11 and t10,
and c12 being the most common isomers) is naturally present
in milk. In addition, several Lactobacillus, Propionibacterium,
Bifidobacterium, and Enterococcus strains extensively synthesize
CLA during the fermentation of milk, whereby the final
concentration is influenced by the processing conditions.
Often, CLA is considered as a healthy compound, to which
several potent physiological properties were attributed,
including anticarcinogenic, antiobese, antidiabetic,
immunomodulator, and antihypertensive effects. However,
other studies point out as well an increase in oxidative markers
after CLA supplementation, and controversial results on
glucose metabolism or liver function problems have been
obtained. Thus, safety concerns regarding the use of CLA in
humans persist and need further investigation, with better
experimental designs that will clarify the mechanisms of CLA
activities. In this sense, the EFSA has considered that, with the
studies currently available, the previously mentioned claimed
beneficial health effects for these molecules have no clear-
enough cause–effect relationship.
Vitamins and Cofactors
The (over)production of vitamins by LAB provides a very
attractive approach to improve the nutritional composition
of fermented foods. Fermentation can indeed modulate the
vitamin content of foods in diverse ways, via either increases
or decreases. For instance, yeast fermentation has been shown
to increase the folate content in the production process of
bakery goods based on wheat, oat, and rye. In the particular
case of rye, the level of folate was increased up to sevenfold and
that of free phenolic acids up to tenfold after germination and
fermentation by Saccharomyces cerevisiae. Not only yeasts but
also LAB (Lactobacillus plantarum riboflavin-overproducing
strains) have been successfully used for the preparation of
bread and pasta with enhanced levels of vitamin B2. In milk,
folate levels can significantly increase after fermentation with
different LAB, as shown for milk fermented with Lactobacillus
acidophilus ATCC 4356 and Streptococcus thermophilus MC. Also,
higher amounts of ubiquinone have been found in Korean and
Japanese fermented foods than in unfermented foods, due to
their diverse microbiota during the process of fermentation
(LAB, yeast, and fungi). Foods such as kimchi and jeotgal (a
fermented fish) may show high values of this compound. The
deficiency of CoQ10 has been implicated in several clinical
disorders, including chronic heart failure and hypertension.
Polyols
Other beneficial compounds that might result from fermen-
tation processes are polyols. Modulation of the redox balance
654 Fermented Foods: Composition and Health effects
during sugar metabolism by LAB can result in the synthesis of
sorbitol, mannitol, and xylitol, which play an important role
in foods as reduced caloric sweeteners and anticariogenic
agents.
Fermented Foods as Probiotics
Probiotics are ‘live microorganisms that, when administered in
adequate amounts, confer a health benefit on the host.’ Several
Lactobacillus and Bifidobacterium species have been investigated
and even commercialized, and certain strains of Enterococcus,
Propionibacterium, and Bacillus species and also from the yeast
Saccharomyces boulardii have been considered as microorganisms
with probiotic properties. Some of the health benefits tradition-
ally associated with probiotic consumption include the
improvement of intestinal tract health (contributing to the con-
trol of irritable bowel syndrome and inflammatory bowel
disease, the suppression of pathogens, and the control of trave-
lers’ diarrhea or antibiotic-caused diarrhea), enhancement of the
immune system, synthesis and enhancement of the bioavailabil-
ity of nutrients, reduction of symptoms of lactose intolerance,
reduction of serum cholesterol levels, and reduction of the risk
of certain cancers. Several specific criteria should – arguably – be
fulfilled by a microorganism to be considered a probiotic: it
should remain viable during the shelf life of the product, with-
stand transit through the GI tract, and stabilize the intestinal
microbiota.
One of the difficulties dealing with confirming the health
benefits associated with probiotics is the fact that their biolog-
ical effects are strain-specific, which makes the data compari-
son among studies difficult. In addition to this, the lack of
evidence for cause–effect relationship has caused the EFSA to
reject probiotic health claims. In many of the opinions given
by the EFSA on probiotics, it was argued that the outcomes
measured did not equate a beneficial physiological effect. Nev-
ertheless, some EU member states have national nutrition
guidelines or recommendations that include either probiotics
or fermented milk with live bacteria.
Due to the fact that some fermenting microorganisms have
probiotic properties, they have been used as starter cultures in
attempts to formulate healthier foods. A consensus document
elaborated in 2014 makes a reflection about whether the
probiotic framework should include traditional fermented
foods containing live microbes or not. According to the latter
opinion, it is not always possible to clearly distinguish the
health contribution of the pure cultures from that caused by
the consumption of fermented foods. Consequently, it has
been concluded that conventional fermented products are
best described as ‘containing live and active cultures’ but
should not be called probiotic. Nevertheless, fermented foods
containing live cultures might also qualify as a ‘probiotic’ if
they meet the criteria for that category.
Conclusion
Fermented foods include a wide variety of foodstuffs, whose
composition strongly determines their nutritional value.
Additionally, several metabolites resulting from fermentation
processes are currently being researched as potential functional
compounds that contribute to the health properties of this type
of foods. Among others, compounds such as bioactive pep-
tides, EPSs, arabinoxylans, polyphenols and flavonoids, and
GABA can be highlighted.
See also: Acidophilus Milk; Alcohol: Metabolism and Health Effects;
Alcohol: Properties and Determination; Beer: Fermentation; Beer:
History and Types; Beer: Raw Materials and Wort Production; Beverage:
Health Effects; Bifidobacteria in Foods: Health Effects; Brandy and
Cognac: Consumption, Sensory and Health Effects; Bread:
Breadmaking Processes; Bread: Types of Bread; Cheese: Chemistry and
Microbiology; Cheese: Composition and Health Effects; Cheese:
Processing and Sensory Properties; Cheese: Types of Cheese –
Medium; Cheese: Types of Cheeses – Hard; Cheese: Types of Cheeses
– Soft; Cured Foods: Health Effects; Dairy Products: Dietary and
Medical Importance; Fermented Foods: Fermented Meat Products;
Fermented Foods: Fermented Milks; Fermented Foods: Fermented
Vegetables and Other Products; Fermented Foods: Origins and
Applications; Fermented Foods: Use of Starter Cultures; Tea: Health
Effects; Wines: Wine and Health; Yogurt: Dietary Importance; Yogurt:
The Product and its Manufacture.
Further Reading
Beena Divya J, Kulangara Varsha K, Madhavan Nampoothiri K, Ismail B, and Pandey A
(2012) Probiotic fermented foods for health benefits. Engineering in Life Sciences
12(4): 377–390.
Beermann C and Hartung J (2013) Physiological properties of milk ingredients released
by fermentation. Food and Function 4: 185–199.
Blandino A, Al-Aseeri ME, Pandiella SS, Cantero D, and Webb C (2003) Cereal-
based fermented foods and beverages. Food Research International 36(6):
527–543.
Borresen EC, Henderson AJ, Kumar A, Weir TL, and Ryan EP (2012) Fermented
foods: patented approaches and formulations for nutritional supplementation
and health promotion. Recent Patents on Food, Nutrition and Agriculture 4(2):
134–140.
Bourdichon F, Casaregola S, Farrokh C, et al. (2012) Food fermentations:
microorganisms with technological beneficial use. International Journal of Food
Microbiology 154(3): 87–97.
Chilton SN, Burton JP, and Reid G (2015) Inclusion of fermented foods in food guides
around the world. Nutrients 7(1): 390–404.
FAO-WHO (2006). Probiotics in food. Health and nutritional properties and guidelines
for 339 evaluation. Paper 85.
Gupta S and Abu-Ghannam N (2012) Probiotic fermentation of plant based products:
possibilities and opportunities. Critical Reviews in Food Science and Nutrition
52(1–3): 183–199.
Hill C, Guarner F, Reid G, et al. (2014) Expert consensus document. The international
scientific association for probiotics and prebiotics consensus statement on the
scope and appropriate use of the term probiotic. Nature Reviews. Gastroenterology
and Hepatology 11(8): 506–514.
Leroy F and De Vuyst L (2014) Fermented food in the context of a healthy diet: how to
produce novel functional foods? Current Opinion in Clinical Nutrition and Metabolic
Care 17(6): 574–581.
Pihlanto A and Korhonen H (2014) Bioactive peptides from fermented foods and health
promotion. In: Holzapfe W (ed.) Advances in fermented foods and beverages:
improving quality, technologies and health benefits, pp. 9–74. South Korea:
Woodhead Publishing.
Shiby VK and Mishra HN (2013) Fermented milks and milk products as
functional foods-a review. Critical Reviews in Food Science and Nutrition 53(5):
482–496.

van Hylckama Vlieg JE, Veiga P, Zhang C, Derrien M, and Zhao L (2011) Impact
of microbial transformation of food on health — from fermented foods to
fermentation in the gastro-intestinal tract. Current Opinion in Biotechnology 22(2):
211–219.
Waters DM, Mauch A, Coffey A, Arendt EK, and Zannini E (2015) Lactic acid bacteria
as a cell factory for the delivery of functional biomolecules and ingredients in
Fermented Foods: Composition and Health effects 655
cereal-based beverages: a review. Critical Reviews in Food Science and Nutrition
55(4): 503–520.
Yang, H. J., Park, S., Pak, V., Chung, K. R. and Kwon, D. Y. (2011). Fermented soybean
products and their bioactive compounds. In: El-Shemy, H. (ed.) Soybean and health.
Available from http://www.intechopen.com/books/soybean-and-health/fermented-
soybean-products-and-their-bioactive-compounds.
 Fermented Foods: Fermented Meat Products
F Leroy and L De Vuyst, Vrije Universiteit Brussel, Brussels, Belgium
ã 2016 Elsevier Ltd. All rights reserved.
Introduction: From Necessity to Delicacy
Fermented meat products have been known as highly appreci-
ated foods throughout the ages. They are found in many
regions worldwide as a part of traditional diets, where they
are perceived as attractive gastronomic entities contributing to
cultural and geographic distinctiveness (Table 1). In Europe,
fermented meats can be traced back to, at least, classical antiq-
uity where they served to preserve meat after slaughter. The
production of dry-fermented sausages, consisting of seasoned
and salted minced meat that is stuffed into intestinal casings,
has been documented in historical sources as a Roman craft.
The technique was, however, largely based on the art of dry
ham production developed by the Celts and Gauls, consisting
of the salting and drying of hind legs of wild boars and pigs.
Over the years, fermented meats came to exist in an over-
whelming variety, characterized by large differences in ingredi-
ents, shape and caliber, and processing conditions. Several
types of meat have been applied, including pork, beef, horse,
donkey, deer, poultry, and ostrich. Well-known examples of
dry-fermented sausages include different types of salami (e.g.,
salame Brianza), chorizo (e.g., chorizo de Pamplona), and saucis-
son sec (e.g., rosette). In addition, some fermented meats are
semidry or even moist, such as German mettwurst, which have
distinctive texture properties but are generally displaying a
decreased stability. Besides Europe and its expansion to the
New World, Asia also has a remarkable tradition of meat
fermentations, although this has been less documented and
explored. Yet, all local fermented meat specialties have origins
in more or less remote pasts, with unique stories to tell, but
eventually trace back to the same ancestral need for meat
preservation. In fact, meat fermentation originally took off as
an empirical preservation method of a primary commodity, in
a similar manner as the preservation of milk led to the produc-
tion of cheese and fermented milks of all sorts.
Due to its highly proteinaceous, energy-dense, and overall
nutritious character, meat originating from hunted or domes-
ticated animals has been a strategic foodstuff for most of
human history and continues to be so. Nonetheless, before
the introduction of the cold chain, the perishable nature of
meat made it difficult to achieve prolonged storage in times of
scarcity. The stabilization of meat through salting and fermen-
tation therefore led to large logistic benefits. The end products
became stable to such a degree that they could even be used as
trading goods and provisions for travelers and soldiers, widely
circulating throughout the European continent and facilitating
expeditions and military campaigns. Clearly, preservation is no
longer the most important raison d’être, at least in the Western
world. Still, the convenience factor and unique sensory prop-
erties of fermented sausages continue to provide them with a
steady share of the meat market. Over the centuries, the basic
ingredients have remained the same, that is, minced meat, fat,
curing salt, and spices. These ingredients are stuffed into
656 Encyclopedia of Food and Health
casings, from either intestinal or synthetic origin, and subse-
quently subjected to a microbial fermentation process, in most
cases followed by a drying stage for further stabilization and
maturation of flavor complexity (Figure 1).
Meat Fermentation as a Spontaneous Process
As for all raw and salted meat products, the endogenous activ-
ity of meat enzymes, in particular proteases and lipases, con-
tributes to the aging and ripening of the product being
subjected to drying. In contrast to dry-salted hams, the original
production of dry-fermented sausages involved a desirable and
spontaneous acidification of the meat matrix that serves as an
extra preservation hurdle in addition to the primary salting and
drying, mostly because a comminuted mixture of meat trim-
mings and fat particles is less stable than whole muscle. Fer-
mentative acidification to pH values that are usually situated
between 4.8 and 5.2 inhibits the growth of undesirable micro-
organisms that are present on the raw meat and denatures the
salt-solubilized protein fractions to a characteristic sliceable
and gel-like texture.
Under appropriate temperature conditions (e.g., between
18 and 28
C), the combination of curing salt and anaerobic
conditions that are prevailing in the sausage casings strongly
select for specific and desirable microorganisms. As a result, the
initial microbial species diversity that is present on the raw
materials is narrowed down within a couple of days to a few
persistent microbial groups. Largely dominating bacteria are
certain mesophilic species of Lactobacillus, in particular
Lactobacillus sakei and, albeit to a lesser extent, Lactobacillus
curvatus or Lactobacillus plantarum. Other lactic acid bacteria
that may occasionally be present but do not or only marginally
affect the production process of most traditional, spontane-
ously fermented sausages include enterococci, lactococci, ped-
iococci, and leuconostocs. In addition to the dominance of
meat-associated lactobacilli, a considerable subdominant pop-
ulation consists of catalase-positive cocci, mostly coagulase-
negative staphylococci and sometimes kocuriae. Depending
on the applied raw materials and processing conditions, differ-
ent species of catalase-positive cocci may be encountered dur-
ing spontaneous meat fermentations. In most cases,
Staphylococcus equorum, S. saprophyticus, and/or S. xylosus are
prevailing. Yet, minor levels of, for instance, S. carnosus,
S. sciuri, S. simulans. S. succinus, and S. warneri are not uncom-
mon. Whereas lactic acid bacteria chiefly cause acidification
through lactic acid production, catalase-positive cocci prevent
oxidative deterioration, generate stable nitrosomyoglobin pig-
ments via nitrate reductase activity, and contribute to an attrac-
tive aroma via amino acid and fatty acid conversions. These
precursor amino acids and fatty acids are liberated via protease,
peptidase, and lipase activities by muscle enzymes, although
minor contributions by bacteria have also been described.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00283-X

Table 1 Illustrative but nonexhaustive list of some different types of well-known fermented sausages and their particularities
Type Country of origin
Mediterranean dry- Italy (e.g., salami), France (e.g.,
fermented sausages rosette), Spain (e.g., fuet), etc.
Chorizo Spain
North European dry- Germany, Norway, Belgium
fermented sausages (saucisson d’Ardennes), etc.
Teewurst, mettwurst Germany
Hungarian salami Hungary
Lap Cheong China, Vietnam, etc.
Technological ingredients Traditional fermentation (smoking) maturation
and additives (optional) ingredients
Carbohydrates
Raw meat
Nitrate and/or nitrite
Starter cultures
Fat
Sodium ascorbate
Bulking agents
Curing salt
Colorants
Phosphates
Sodium glutamate Seasonings
Figure 1 Common ingredients used in the meat fermentation process and targeted quality characteristics of the end products.
Flavor maturation of fermented sausages thus correlates
with the amounts of branched-chain aldehydes and alcohols
from the conversion of branched-chain amino acids and
branched-chain and straight-chain methyl ketones derived
from microbial b-oxidation of fatty acids.
Besides bacteria, yeasts and molds can be present in some
sausage types. Yeasts, such as Debaryomyces hansenii, are prob-
ably of a rather minor importance in flavor development but
could accentuate certain aromas. In contrast, molds are crucial
elements in the elaboration of south European fermented sau-
sages where they develop as a desirable outside layer on the
product surface after fermentation, during which they generate
additional aroma and contribute to color stabilization through
catalase activity, oxygen consumption, and shelter against
light. The mycobiota of fermented sausage is diverse, but Pen-
icillium is usually the predominant genus (e.g., Penicillium
nalgiovense). In traditional northern European fermented sau-
sages, molds are mostly undesirable. This is due to the rather
humid climate conditions in these regions, which are unsuita-
ble for proper, controlled, and safe mold development. As a
consequence, such products are frequently smoked after fer-
mentation, as an empirical manner to add additional preser-
vative action and to prevent the unbalanced growth of
detrimental molds. Meanwhile, the smoked flavor became a
desirable sensory attribute for northern European consumers.
In addition, northern European varieties of fermented sausages
are generally more acidified to obtain enhanced microbial
Fermented Foods: Fermented Meat Products 657
Particularities
Large variety of recipes and types, often heavily seasoned with pepper, garlic, and
herbs; mildly fermented, extensively dried and matured, and mostly molded
Contains paprika; variable drying periods and moist contents but often semidry
and smoked; high-end variants contain Iberico pork
Large variety of recipes and types; usually rather acid and often heavily smoked
without molding, although salami-type sausages are also produced
Moist and spreadable pork sausages that are only moderately or not dried; usually
smoked and no molding; highly perishable
Salami-like but with an unusual combination of intense smoking and molding
Different types of dried and usually smoked pork or poultry sausages; sometimes
containing rice and seasoned with soy sauce, rose water, or sake
Lactobacillus sakei Visual appeal
Catalase-positive cocci Colour
(Yeasts)
Flavour
(Moulds)
Texture
Microbial Safety
selection
Healthiness
Nutritional value
Artisan hue
Stability
inhibition, since they are usually more moist and, hence,
more vulnerable to hazards than their drier southern
counterparts.
Technological Optimizations
Over the years and particularly since the mid-twentieth cen-
tury, several technological adaptations have been introduced,
as to adjust the traditional meat fermentation process to the
requirements of an ever industrializing and globalizing world.
This led to the emergence of large factories in the 1960s,
producing fermented meat products of consistent quality and
safety on an industrial scale. Adaptations range from the sim-
ple addition of ascorbate as an antioxidant and of sugar and
manganese as bacterial stimulants, over the introduction of
climate chambers and microbial starter cultures for improved
control, to a multitude of cost reduction attempts via the use of
artificial casing and additives of all sorts. Also, technological
adaptations have been brought in to reduce the potential gen-
eration of toxic compounds, for instance, by dipping sausages
in a sorbate solution to avoid undesirable mold growth and by
optimizing potentially hazardous smoking procedures.
One of the key breakthroughs has been the gradual intro-
duction of controlled-climate chambers in the 1950s, which
enable a rigid control of temperature and relative humidity.
Accordingly, the unpredictability of fluctuations in weather
658 Fermented Foods: Fermented Meat Products
conditions can be ruled out, and a more precise tuning to
optimal process requirements can be obtained. This is particu-
larly important not only for a correct drying process but also to
steer fermentation and molding.
More or less in the same period, another major advance-
ment has been achieved through the development and addi-
tion of starter cultures. In the 1940s, Jensen and Paddock
explored the use of Lactobacillus cultures onto raw meat,
which was followed by inoculations with Pediococcus cerevisiae
in the United States and Micrococcus M53 in Europe. Such
starter cultures permit to mostly overcome the problem of
failed fermentations, which may be occurring in traditional
processes where producers rely on a spontaneous development
of the indigenous meat microbiota. Meat starter cultures now-
adays usually consist of a predefined microbial consortium of
lactic acid bacteria for acidification purposes, mostly
Lactobacillus sakei and/or Pediococcus species, accompanied by
selected catalase-positive cocci, mostly S. xylosus, S. carnosus,
and/or Kocuria varians. These combined cultures are usually
lyophilized and added as a powder that has been resuspended
in water to the meat batter prior to stuffing and fermentation.
In addition, a surface mold culture may be applied to control
the molding process during maturation. As a result of starter
culture addition, the production process is accelerated, and
end products are more uniform. In North America, further
speeding up has been achieved by increasing the fermentation
temperature from the traditional range below 28
C toward
temperatures above 35
C. As a result of this enhanced fermen-
tation temperature, pediococci take over from the mesophilic
lactobacilli. Even faster processes have been obtained with
chemical rather than microbial acidification, mostly by using
glucono delta-lactone or encapsulated lactic acid as acidulants.
Yet, the ever-increasing attempts to speed up fermentation
and maturation and to minimize ingredient and processing
costs have been counterbalanced by severe losses in product
quality. Flavor, for instance, is the result of a complex and
delicate interplay between endogenous meat enzymes, micro-
bial action, and physicochemical effects. The dynamics of this
interplay depend strongly on the process conditions, in partic-
ular the allowed time frame for biochemical conversions and
the rate of acidification. On the one hand, cathepsin D, which
is a major meat proteinase acting on the myosin and actin
fractions, is activated by acidity (with optimal activity at pH
4.5). On the other hand, low pH values strongly inhibit the
metabolic performance of meat microorganisms, including
their production of peptidyl peptidases and aminopeptidases
and their enzymatic conversions of amino acids and fatty acids.
With respect to the postfermentation processing, too short
maturation periods may impede the proper flavor-generating
chemical changes that lead to fatty acid oxidation products and
Strecker degradation of amino acids.
In conclusion, excessive acceleration of the production pro-
cess, on the level of both fermentation and maturation, is
known to compromise flavor development to a large extent,
despite the obvious advantages related to standardization and
cost reduction. As a technological response to partially conceal
cost-induced quality losses on the level of texture, color, and
flavor, several additives have been introduced in the meat
fermentation process. Commonly applied examples include
the use of bulking agents (e.g., milk powder), phosphates,
sodium glutamate, added flavors (e.g., liquid smoke flavo-
rings), colorants (e.g., cochineal), and sorbates.
From a safety point of view, controlled use of nitrite and/or
nitrate salts as additives to fermented meats has become a
common practice, as these curing compounds contribute to
the inhibition of meat-associated pathogens. Moreover, they
are involved in the generation of a typical cured flavor, inhibit
lipid oxidation, and lead to stable color formation via the
nitrosylation of myoglobin. Yet, because of their involvement
in carcinogenic nitrosamine formation, maximum levels have
been introduced by regulation. In addition, their chemical
conversions can be tuned technologically, for instance, by
adding the reductant ascorbic acid, which is to be added in
levels exceeding 550 ppm in the United States.
Understanding the Metabolic Functioning of Meat
Microorganisms
Improving the meat fermentation process to yield even more
attractive end products is a matter not only of muscle
biochemistry but also of a better insight into the microbial
community dynamics and metabolism. Although the overall
roles of the fermented meat microbiota are well understood,
the more subtle aspects of their metabolism need further explo-
ration. For instance, recent whole-genome analyses of fermen-
ted meat bacteria have identified specialized metabolic
repertoires that may explain why certain community members
are so strikingly dominant. The specialized adaptation by
Lactobacillus sakei to the fermented meat environment has
been linked to specific metabolic properties via genome anal-
ysis, metabolite kinetics, modeling, and gene expression stud-
ies. Consequently, the use of arginine and nucleosides by
Lactobacillus sakei as alternative, nonglucose compounds as
energy sources that naturally occur in meat has been suggested
as relevant for bacterial dominance. This property has also
been shown for coagulase-negative staphylococci, possibly
explaining differences on the inter- and intraspecies levels.
Variability in competitiveness between coagulase-negative
staphylococci is particularly intriguing, since this forms the
basis for the eventual selection of a specific bacterial diversity
associated with a certain product. As a result, repercussions on
the overall bacterial metabolic activity and hence the final
sensory quality are to be expected. It has, for instance, been
shown that differences in sensitivity to pH lead to different
growth or survival kinetics, when comparing S. carnosus to the
more acid-sensitive S. xylosus. Also, the replacement of a smok-
ing treatment by molding may cause a shift from
S. saprophyticus to S. equorum, possibly because of pH increases.
As different staphylococcal species display variations in both
the absolute and specific production of volatile compounds,
this can affect sausage aroma measurably. Enhanced techno-
logical control on the staphylococcal community could thus
represent an opportunity for tailored flavor modulation.
Interactions between microorganisms, the enzymes of the
muscle tissue, the applied ingredients, and the process tech-
nology determine the quality, safety, and stability of the end
products. New metabolic insights will thus contribute to pro-
cess optimization and the further development of the so-called
functional starter cultures, which should lead to improved

sausage fermentation with respect to flavor, safety, processing,
technology, or health, as compared with traditional starter
cultures that are merely aiming at standardization and proces-
sing speed. Such novel insights are to be obtained from the use
of state-of-the-art methodologies, for instance, omics
approaches, which are increasingly being used in the domain
of food science and technology.
Toward a New Generation of Fermented Meat
Products?
Although the meat fermentation process has been dramatically
optimized through technological innovations, several chal-
lenges are still to be undertaken. Preferably, these strategies
need to make use of ‘natural’ options, avoiding the use of
commonly shunned chemical additives. Some of the existing
concerns have to do with the safety and health implications
upon consumption of the end products.
First, several incidences with foodborne pathogens have
been reported despite the fact that acidification, salting, and
drying are important microbiological hurdles. Problems are
sometimes encountered due to the presence and ingestion of
Salmonella and Escherichia coli or enterotoxins produced by
Staphylococcus aureus. Listeria monocytogenes, although not
linked to foodborne illness by fermented meat consumption
till now, is also of concern. Several countries, such as the
United States, have adopted a zero-tolerance policy with
respect to the presence of this bacterium in ready-to-eat
foods. This is problematic because Listeria monocytogenes has
been shown to survive several meat fermentation processes and
can sometimes be recovered from nonheated European end
products. In general, the presence of foodborne pathogens may
be due to high loads in the raw materials or failing production
processes. Novel strategies that could eliminate potential resid-
ual levels of undesirable bacteria are being sought for. The use
of lactic acid bacteria starter cultures that produce natural
antimicrobials, in particular bacteriocins, has been frequently
explored. Although this mode of action offers considerable
potential against Listeria monocytogenes, bacteriocins by lactic
acid bacteria are generally inefficient toward gram-negative
pathogens. Meat-compatible microorganisms with targeted
antimicrobial actions, in particular toward Salmonella and
E. coli, would thus offer considerable food safety advantages.
Other microbial concerns are related to the use of the fermen-
tative microbiota itself, since certain lactobacilli, staphylococci,
and enterococci present during fermentation may be produc-
ing biogenic amines. Likewise, surface molds may in principle
be toxicogenic. This is particularly of concern during
meat spontaneous fermentation processes, since commercial
starter cultures have normally been screened to demonstrate
the absence of biogenic amine, mycotoxin, and antibiotic
formation.
A second major concern is related to the potential implica-
tions of fermented meat consumption on human health.
Although meat products are characterized by a high intrinsic
nutritional value, mostly due to their high contents of quality
protein, iron, zinc, and vitamins, some unease has been
expressed with respect to their fat and sodium contents.
Although the mostly pork-based fat fraction is rather
Fermented Foods: Fermented Meat Products 659
unsaturated and not necessarily problematic, high-fat products
are energy-dense and do not meet the current market demand
for low-fat foods. More importantly, fermented meat products
have been charged for their potential association with the risk
of colon cancer. The World Cancer Research Fund specifically
advises to avoid all processed meats as part of its ten recom-
mendations to combat cancer. The mechanisms for this asso-
ciation are not fully understood, but a part of the effect could
be due to the use of mostly red muscle and the presence of
nitrate and/or nitrite in the curing salt. Several attempts have
already been made to partially counter all aforementioned
concerns, although this frequently implies loss of sensory
excellence. Reduction of sodium results in a loss of flavor
intensity and texture development, whereas the replacement
of potassium and/or magnesium may sometimes lead to
metallic or bitter taste defects. Likewise, fat reduction leads to
poor mouth feel and aroma losses, whereas fat replacement by,
for instance, vegetable oils often results in texture setbacks.
Also, the increased production of poultry-based fermented
meat products or products based on meat replacers, as an
attempt to counter the consumption of red muscle, is leading
to end products that display weaker hedonistic profiles. Up till
now, attempts to lower the added nitrite levels have been
limited to the indirect addition of nitrate via vegetal fractions
such as celery or spinach extracts, which is then further con-
verted to nitrite by the meat staphylococci. Although this may
be useful for labeling purposes, this approach is mainly aiming
at concealment and does not contribute to the health debate.
In an endeavor to counter the negatively perceived health
status of fermented meats, several trials have been made to
include health-promoting compounds in the original formu-
lations. Examples include the use of probiotics, prebiotics, and
other fibers, lycopene, antioxidants, olive oil, and calcium and
attempts to increase the content of conjugated linoleic and
linolenic acids. Although commercial examples exist, techno-
logical and legislative bottlenecks often remain, and questions
arise on the conceptual and nutritional relevance of such
products.
Paradoxically, part of the innovation of fermented meat
products is based on the reintroduction of artisan features.
Such features are increasingly used in marketing strategies for
contemporary fermented meat products, in a process that can
be categorized as ‘reemergence of tradition.’ The use of labels
that claim artisanship and tradition is largely employed to give
a feel of trustworthiness in a globalizing food market in crisis.
Conclusions
Despite the fact that fermented meats are rooted in an age-old
tradition, several innovations and technological adaptations
have been introduced over time. The advent of modern food
technology has greatly improved processing speed and effi-
ciency, although in many cases, a too pronounced focus on
cost reduction has resulted in inferior sensory quality. As a
result, several opportunities can be identified to improve the
quality of fermented meats, especially when produced on a
cost-effective industrial level. Further research, based on state-
of-the-art methodologies and taking into account the com-
bined disciplines of microbiology, meat science, and
660 Fermented Foods: Fermented Meat Products
processing technology, is needed if any breakthroughs are to be
achieved. In addition, the nutritional aspects of fermented
meat consumption will likely become more important in the
future. Finally, sociological approaches and consumer studies
may help to better understand the complex relationship
between apparently conflicting demands for traditional char-
acteristics and innovative elements in fermented meats.
See also: Bacteriocins; Beef; Biogenic Amines: Toxicology and Health
Effect; Cancer: Diet in Cancer Prevention; Carcinogenic: Carcinogenic
Substances in Food; Cholesterol: Factors Determining Blood
Cholesterol Levels; Cured Foods: Health Effects; Drying: Physical and
Structural Changes; Drying: Principles and Types; Fat Replacer;
Fermented Foods: Composition and Health Effects; Fermented Foods:
Origins and Applications; Fermented Foods: Use of Starter Cultures;
Foodborne Pathogens; Functional Foods; Ham: Dry-cured Ham; Horse
Meat; Hypertension and Diet; Iron: Physiology of Iron; Lactic Acid
Bacteria; Listeria: Properties and Occurrences; Low-fat Foods: Types
and Manufacture; Low-salt Foods: Types and Manufacture; Meat:
Conversion of Muscle into Meat; Meat: Eating Quality and Preservation;
Meat: Role in the Diet; Meat: Structure; Mycotoxins: Occurrence and
Determination; Nitrites and Nitrates; Nutrition and Health Claims for
Food: Regulatory Controls, Consumer Perception, and Nutrition
Labeling; Oxidation of Food Components; pH: Principles and
Measurement; Pork Meat Quality, Production and Processing on;
Poultry: Processing; Prebiotics; Preservation of Foods; Preservatives:
Classifications and Analysis; Preservatives: Food Use; Probiotics;
Protein: Food Sources; Salmonella: Properties and Occurrence;
Skeletal Muscle; Smoked Foods: Principles and Production;
Staphylococcus: Occurrence and Properties; Traditional Foods.
Further Reading
Campbell-Platt G and Cook P (eds.) (1995) Fermented meats. Glasgow, UK: Blackie
Academic and Professional.
Chaillou S, Daty M, Baraige F, Dudez AM, Anglade P, Jones R, et al. (2009) Intraspecies
genomic diversity and natural population structure of the meat-borne lactic acid
bacterium Lactobacillus sakei. Applied and Environmental Microbiology
75: 970–980.
Cocolin L, Dolci P, and Rantsiou K (2011) Biodiversity and dynamics of meat
fermentations: the contribution of molecular methods for a better comprehension of
a complex ecosystem. Meat Science 89: 296–302.
Demeyer D, Honikel K, and De Smet S (2008) The World Cancer Research Fund report
2007: a challenge for the meat processing industry. Meat Science 80: 953–959.
Fadda S, López C, and Vignolo G (2010) Role of lactic acid bacteria during meat
conditioning and fermentation: peptides generated as sensorial and hygienic
biomarkers. Meat Science 86: 66–79.
Janssens M, Myter N, De Vuyst L, and Leroy F (2012) Species diversity and metabolic
impact of the microbiota are low in spontaneously acidified Belgian sausages with
an added starter culture of Staphylococcus carnosus. Food Microbiology
29: 167–177.
Janssens M, Myter N, De Vuyst L, and Leroy F (2013) Community dynamics of
coagulase-negative staphylococci during spontaneous artisan-type meat
fermentations differ between smoking and moulding treatments. International
Journal of Food Microbiology 166: 168–175.
Leroy F, Verluyten J, and De Vuyst L (2006) Functional meat starter cultures for improved
sausage fermentation. International Journal of Food Microbiology 106: 270–285.
Leroy F, Geyzen A, Janssens M, De Vuyst L, and Scholliers P (2013) Meat fermentation
at the crossroads of innovation and tradition: a historical outlook. Trends in Food
Science and Technology 31: 130–137.
Ravyts F, Steen L, Goemaere O, Paelinck H, De Vuyst L, and Leroy F (2010) The
application of staphylococci with flavour-generating potential is affected by
acidification in fermented dry sausages. Food Microbiology 27: 945–954.
Rimaux T, Vrancken G, Vuylsteke B, De Vuyst L, and Leroy F (2011) The pentose moiety
of adenosine and inosine is an important energy source for the fermented-meat
starter culture Lactobacillus sakei CTC 494. Applied and Environmental
Microbiology 77: 6539–6550.
Talon R and Leroy S (2011) Diversity and safety hazards of bacteria involved in meat
fermentations. Meat Science 89: 303–309.
Tjener K, Stahnke LH, Andersen L, and Martinussen J (2004) The pH-unrelated
influence of salt, temperature and manganese on aroma formation by
Staphylococcus xylosus and Staphylococcus carnosus in a fermented meat model
system. International Journal of Food Microbiology 97: 31–42.
Toldrá F (2006) The role of muscle enzymes in dry-cured meat products with different
drying conditions. Trends in Food Science and Technology 17: 164–168.
Toldrá F (ed.) (2007) Handbook of fermented meat and poultry. Ames, IA:
Wiley-Blackwell.
Relevant Websites
http://www.fao.org/docrep/010/ai407e/ai407e11.htm – Food and Agriculture
Organization.
http://www.meatscience.org/page.aspx?id¼403 – American Meat Science Association.
http://www.salumi-italiani.it/en/ – Istituto Valorizzazione Salumi Italiani.
 Fermented Foods: Fermented Milks
CD Khedkar, College of Dairy Technology, Pusad, India
SD Kalyankar, Government College of Dairy Technology, Udgir, India
SS Deosarkar, College of Dairy Technology, Pusad, India
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
A chronological appraisal of the origin of fermented milks
(FMs) (e.g., Bulgarian milk, Yogurt, Kefir, Koumiss, Laben,
Dahi, Skyr, and sour milk) shows that they date back to early
civilizations around 10 000 BC. As the way of life of humans
changed, it led to the domestication of the cow, sheep, goat,
buffalo, and camel. A wide range of indigenous FM products
are traditionally made in rural areas worldwide. Most of these
products rely primarily on spontaneous fermentation due to
the presence of indigenous microflora (mainly lactic acid
bacteria, LAB) in the milk. In the Asiatic Steppes, containers
made from animal hides containing microorganisms from the
previous production batch are used during the manufacture of
traditional Koumiss, Bulgarian milk, etc. This reliance on con-
tainers or utensils to provide an initial inoculum of starter
organisms is widespread.
During the past 90 years, considerable attention has been
directed sporadically on benefits derived from the consump-
tion of milk products containing LAB. The role of FM in human
nutrition is well documented. Man knew the virtues of fermen-
ted foods even during the early days of civilization. In earlier
days, these foods were produced by natural fermentations with
the main objective of preserving milk.
In many areas of the world, the fermentation process is
initiated by the adventitious indigenous microorganisms or
through ‘backslopping’ with mixed or unknown cultures.
Often, this type of fermentation may be slow or unpredictable.
Nevertheless, it affords a vital means of preserving highly perish-
able foods, especially where refrigeration is lacking. In Europe,
Asia, and Africa, sour milk was known as being more stable and
advantageous than fresh milk. It preserved the high-quality nutri-
ents present in milk in a relatively more stable form.
From prehistoric times, man learned to use milk as food.
Though the origin of the art of preserving milk by lactic acid
fermentation has been lost in antiquity, the biochemical and
microbiological knowledge of fermentation is of compara-
tively recent date. In Indian subcontinent, the conversion of
milk into Dahi in every household by souring with the leftover
of previous day’s sour milk has been a common practice ever
since the Aryans inhabited the land. In this way, the life and
utility of milk nutrients were extended.
Today, the practice of preserving milk by fermentation has
become a routine household technology, and FMs are almost
compulsory in this subcontinent. In developed countries, fer-
mentation is initiated with pure starter cultures, which have
predictable performance potentials. Recent scientific and tech-
nological advances in starter culture management and process
control have yielded a variety of products. These products are
with superior chemical, physical, nutritional, and sanitary
qualities.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00286-5
A number of FMs, cereal-based products, and beverages are
prepared through the action of LAB. Nearly every civilization
has consumed FMs. Over the past two decades, the per capita
consumption of FMs has increased more than 45-fold due to
their image as nutritious and healthful foods. Likewise, the per
capita production and consumption of cheese have recorded
notable growth in the United States, Europe, and Indian sub-
continent. Thus, FMs have been and still are of extreme impor-
tance in the nutrition of people throughout the world. The FMs
like Acidophilus milk, Bifidus milk, Yogurt, Kefir, Koumiss,
Leben, and Skyr are also used as dietary adjuncts. The FMs
represent an important component of functional foods and
value-added probiotic products.
Nowadays, intense research efforts are under way to
develop dairy products into which probiotic organisms are
incorporated to make them more valuable. This article pro-
vides an overview of the different types of FMs and the starter
cultures used in their preparation and health benefits, which
can be derived by the consumers through their regular intake.
Raw Materials
The milk of several species, including cow, buffalo, goat, sheep,
mare, camel, and reindeer has been used for the preparation of
FMs. Each type of FM has its own characteristic texture, body,
flavor, and composition, depending on the type of milk used,
the type of starter cultures (LAB, with or without aroma pro-
ducers, and yeasts), and the procedures followed in its prepa-
ration. The FMs popularly used in various countries may be
classified broadly into four categories: (1) moderately sour
types with a pleasant aroma associated with diacetyl, for exam-
ple, cultured milk; (2) sour and very sour types owing to high
acid production, for example, yogurt and Dahi; (3) ethanol in
addition to lactic acid, for example, Koumiss and Kefir; and (4)
probiotic FMs, for example, acidophilus milks especially pre-
pared with human strains of L. acidophilus.
Definition
FM is a milk product obtained by the fermentation of milk,
where milk may have been manufactured from products
obtained from milk with or without compositional modifica-
tion as limited by the provisions in law, by the action of
suitable microorganisms resulting in reduction of pH with or
without coagulation (isoelectric precipitation). These starter
microorganisms shall be viable, active, and abundant in the
product to the date of minimum durability. If the product is
heat-treated after fermentation, the requirement for viable
microorganisms does not apply.
661
662 Fermented Foods: Fermented Milks
Types of FM
Concentrated FM
Concentrated FM is an FM of which the protein has been
increased prior to or after fermentation to minimum 5.6%.
Concentrated FMs include traditional products such as
Stragisto (strained yogurt), Leben, Ymer, and Ylette.
Flavored FMs
Flavored FMs are composite milk products, which contain a
maximum of 50% (m m 1) of nondairy ingredients (such as
nutritive and nonnutritive sweeteners, fruits, and vegetables as
well as juices, purees, pulps, preparations and preserves
derived therefrom, cereals, honey, chocolate, nuts, coffee,
spices, and other harmless natural flavoring foods) and/or
flavors. The nondairy ingredients can be mixed in prior to or
after fermentation.
Drinks Based on FM
Drinks based on FM are composite milk products, obtained by
mixing FM with potable water with or without the addition of
other ingredients such as whey, other nondairy ingredients,
and flavorings. Drinks based on FM contain a minimum of
40% (m m 1) FM. Other microorganisms than those consti-
tuting the specific starter cultures may be added.
Starter Cultures Used in the Preparation of FM
Although the evolution of the process was strictly intuitive, the
production of FM products became an established pattern of
preservation; since the early 1900s, defined microorganisms
have been used to prepare these products on a large industrial
scale. The microorganisms that are employed in FMs (includ-
ing probiotic products, alcoholic/lactic beverages, cultured
cream, and products containing molds) and the cheese indus-
try are used singly, or in pairs or multiples, or in a mixture, thus
giving the industry the opportunity to manufacture different
products. Certain FMs are characterized by specific starter
culture(s) used for fermentation; Table 1 depicts the starter
cultures used in preparation of FM.
Nutritional Significance of FMs
Lactobacilli use the nutrients in milk for their own metabolism
and growth and multiply from 1 to 10 million cells per millil-
iter. The microorganisms are present in the FM not only as
viable cells but also as autolyzed cells, which give rise to
primary and secondary metabolites and enzymes, which they
have produced during fermentation and storage.
The nutritive value (NV) of an FM depends on the avail-
ability and digestibility of nutritive constituents and the
changes in these constituents due to microbial growth and
fermentation processes (Table 2).
Similarly, the NV of a fermented food also depends on the
nutrient content and bioavailability of the nutrients. Impact of
fermentation of milk on its constituents is depicted in Table 3.
Generally, the energy value of FM is similar to that of the milk
from which it was prepared, yet it is claimed that the FM is
more nutritious (the composition of milk and yogurt is given
in Table 4). Fermentation enhances availability of some of the
nutrients for absorption, as shown below:
Effect of Fermentation on Milk Constituents
Milk Proteins
The proteins in milk are of excellent biological quality, and
both caseins and whey proteins are well endowed with essen-
tial amino acids (EAA). The fact that the protein content of FM,
like yogurt, is often elevated by concentration or addition of
skim milk solids means that it is an even more attractive source
of protein than liquid milk. It is important that the proteins in
FMs are totally digestible due to the fact that the starter organ-
isms cause some degree of initial proteolysis. The extent of this
breakdown depends on the strains of bacteria being employed,
but, in general, at least some release of amino acids and pep-
tides can be expected during incubation and storage.
FM products contain significantly higher levels of free
amino acids than milk, due to the heat treatment and proteo-
lytic activity of the starter organisms. The native milk proteins,
which form a hard curd in the stomach, are converted into a
soft curd containing finely dispersed casein particles resulting
from the action of starter organisms in the FM. The FM proteins
are useful for children, old people, and those with stomach
ulcers, because the soft curd in FM is easier to digest than the
milk (Table 3).
It has been observed that the rats fed with diets containing
FM products had higher feed consumption, greater weight
gains, and a more efficient utilization of their feed. Some
medical practitioners advocate the use of Dahi and buttermilk
by children not only for treatment of common intestinal ail-
ments but also for their general nutrition. In tropical countries
like India, where milk is exposed to heavy bacterial contami-
nation, it is better to convert it into FM in order to preserve
its NV.
Milk Fats
Although much of the FM sold in industrialized countries is
produced from skim milk, traditional materials have always
contained some 3–4% milk fat. It has been proposed that the
digestibility of fat is improved via the action of the bacteria
present during the fermentation process, but whether this
effect really exists is highly debatable. The overall energy con-
tent of FM reflects both the fat content of the milk from which
it was made and whether or not there has been the addition of
ingredients such as cream and sugar. Humans have a double
requirement for lipids. It acts as storage fat, which is composed
of saturated fatty acids, and serves as a source of energy or
protection for vital organs.
In addition to this, it also plays an important role by acting
as structural fat, which, with proteins, forms many of the
essential membranes in animal cells, particularly in areas like
brain and nerve cells. These are essential to many physiological
processes. Some essential nutrients are fat-soluble and are
 Fermented Foods: Fermented Milks 663

Table 1 Starter cultures used for various fermented milks

Sr. Fermented
no. milks Starter cultures used

1. Yogurt Symbiotic cultures of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus
2. Acidophilus L. acidophilus
milk
3. Kefir Starter culture prepared from kefir grains, L. kefir, species of the genera Leuconostoc, Lactococcus, and Acetobacter
growing in a strong specific relationship. Kefir grains constitute both lactose-fermenting yeasts (Kluyveromyces
marxianus) and nonlactose-fermenting yeasts (Saccharomyces unisporus, Sac. cerevisiae, and Sac. exiguus)
4. Koumiss L. delbrueckii subsp. bulgaricus and Klu. marxianus. Other microorganisms than those constituting the specific starter
culture(s) specified above may be added
5. Bulgarian L. delbrueckii subsp. bulgaricus
buttermilk
6. Dahi Thermophilic lactic cultures
Str. salivarius subsp. thermophilus
L. delbrueckii subsp. bulgaricus
Mesophilic lactic cultures
Lactococcus lactis subsp. lactis
Lac. lactis subsp. cremoris
Citrate fermentators
Leuconostoc mesenteroides subsp. citrovorum, Leu. dextranicum
7. Shrikhand Str. salivarius subsp. thermophilus
L. delbrueckii subsp. bulgaricus
8. Lassi Str. salivarius subsp. thermophilus,
L. delbrueckii subsp. bulgaricus
Lac. lactis subsp. lactis
Lac. lactis subsp. cremoris
Leu. mesenteroides subsp. citrovorum (any two LAB and one citrate-fermenting culture organism)
9. Cultured Lac. lactis subsp. lactis or Lac. lactis subsp. cremoris and Str. cremoris subsp. diacetylactis
buttermilk
10. Leben Lac. lactis subsp. lactis
Str. salivarius subsp. thermophilus
L. delbrueckii subsp. bulgaricus
Lactose-fermenting yeasts
11. Cheese(s) Lac. lactis subsp. lactis, Lac. lactis subsp. cremoris, Lac. lactis subsp. diacetylactis
Str. salivarius subsp. thermophilus
L. delbrueckii subsp. bulgaricus
Propionibacterium freudenreichii subsp. shermanii
Penicillium roqueforti, Pen. camemberti, etc.
12. Yakult L. casei strain Shirota
13. Filmjolk Lac. lactis subsp. lactis or Lac. lactis subsp. cremoris and Leu. mesenteroides subsp. citrovorum or Leu.
mesenteroides subsp. dextranicum
14. Viili Lac. lactis subsp. cremoris
Lac. lactis biovar. diacetylactis
Leu. mesenteroides subsp. cremoris and Geotrichum candidum
15. Skyr Str. salivarius subsp. thermophilus
L. delbrueckii subsp. bulgaricus

Table 2 Fermentation induced changes in milk constituents

Constituents Changes

Milk proteins Coagulated into a smooth curd with casein particles finely dispersed. Partially peptonized (0.1–0.7%) and utilized for microbial
(3–4.5%) growth, resulting in the buildup of microbial cell proteins, increase in nonprotein nitrogen, and release of peptides and
amino acids
Lactose (4.5–5%) Partially utilized (1–2%) by starter bacteria producing mainly lactic acid (0.6–2%), volatile acids, flavor compounds, and CO2
may also be formed by heterofermentative types. Alcohol and CO2 may be produced by lactose-fermenting yeasts
Milk fat (3.5–7%) Fermentation leads to partial digestion of lipids
Mineral matter No direct appreciable change
(0.7–0.8%)
Vitamins No change in fat-soluble vitamins A, D, E, and K. Slight decrease in B-complex vitamins depending on the strains used

Source: Khedkar, C. D., Khedkar, G. D., Chavan, N. V. and Kalyankar, S. D. (2003). Fermented milks: Dietary importance. In: Encyclopaedia of Food Science and Nutrition (2nd ed.).
London: Elsevier.
664 Fermented Foods: Fermented Milks

Table 3 Composition of milk and yogurt (per 100 g)

Whole Whole milk plain Low-fat plain Very low-fat/low-calorie plain Greek Plain fromage
Nutrients milk yogurt yogurt yogurt yogurt frais

Energy (kcal) 66 79 56 41 115 113

Protein (g) 3.1 5.7 5.1 4.3 6.4 6.8
Fat (g) 3.9 3.0 0.8 0.2 9.1 7.1
Calcium (mg) 115 200 190 130 150 89
Zinc (mg) 0.4 0.7 0.6 0.4 0.5 0.3
Phosphorus 92 170 160 110 130 110
(mg)
Magnesium 11 19 19 13 12 08
(mg)

Source: Khedkar, C. D., Khedkar, G. D., Chavan, N. V. and Kalyankar, S. D. (2003). Fermented milks: Dietary importance. In: Encyclopaedia of Food Science and Nutrition (2nd ed.).
London: Elsevier.

the case, as skim milk powder or nonfat milk solids are some-
Table 4 Effect of fermentation on vitamin content (mg/100 g)
times added during the manufacture of some of these products.
Whole Sour Cottage During fermentation, 20–50% of the lactose in milk is
Vitamins milk Buttermilk cream Yogurt cheese hydrolyzed to its component monosaccharides, glucose and
galactose, by the starter culture organisms. The production of
Thiamin 30 34 35 44 21 ß-galactosidase increases during the fermentation of yogurt,
Riboflavin 170 154 149 214 165 reaching a maximum after four hours of incubation. This
Niacin 100 58 67 114 128
enzyme may be released from the microbial culture during
Pantothenic 300 275 360 591 215
acid
digestion, indicating that it may be present in the intestine of
Vitamin B6 40 34 16 49 68 persons consuming FM. Lactose-intolerant subjects had a
Folacin 6 Traces 11 11 12 much lower rise in blood glucose and thus a much lower
Vitamin B12 0.40 0.22 0.30 0.56 0.63 lactose utilization efficiency. A lower lactose content would
presumably help tolerance of the product to those with a
Source: Khedkar, C. D., Khedkar, G. D., Chavan, N. V. and Kalyankar, S. D. (2003). reduced ability to digest lactose. But clearly the explanation is
Fermented milks: Dietary importance. In: Encyclopaedia of Food Science and Nutrition not this simple because FM with fairly high lactose content is
(2nd ed.). London: Elsevier.
also better tolerated by such individuals than the equivalent
amount of lactose in milk. Lactic acid is produced as a by-
product of the fermentation process. It acts as a preservative. It
found primarily in foods that contain fat. These nutrients are influences the physical properties of casein curd by inducing a
essential fatty acids and fat-soluble vitamins. The LAB possess finer suspension, which promotes digestibility of casein. It also
improves utilization of calcium and other minerals and
lipase activity evidenced by the lipids in the cultured products
being partially digested. Their assays for lipase activity, how- inhibits the growth of potentially harmful bacteria.
ever, were performed using culture organisms with tributyrin
emulsions as substrate, and they did not prove that the bacte-
Milk Vitamins
rial lipases acted on lipids in the cultured products. A signifi-
cant increase in free fatty acids over the starting milk has been Milk is a rich source of vitamins, particularly A, D, and some
observed in FM. The lactic acid fermentation and enzymatic B-complex vitamins. Breed, diet, climate, geographic location,
action on amino acids also produce small quantities of volatile age and stage of lactation, and other factors can influence the
fatty acids (Table 3). vitamin content of the milk. This, in turn, will affect the vita-
min content of the FM. There is conflicting evidence about
whether there is an increase, decrease, or no change in level
Milk Carbohydrates
of vitamins during the fermentation of milk. The various
The milk carbohydrate, lactose, causes intestinal problems in a manufacturing treatments may affect the content of labile
number of persons who are lactose-intolerant. These persons vitamins while there is metabolism of vitamins by the LAB
are deficient in the intestinal enzyme, ß-galactosidase, and thus during the log phase of their growth and subsequent synthesis
must restrict their intake of milk and dairy products. Milk does by the same bacteria. International Dairy Federation gave an
not contain this enzyme. Lactose intolerance is normally indication of the changes due to heat treatment, fermentation,
defined as the occurrence of gastrointestinal symptoms after and storage.
administration of single test dose, about 50 g lactose in aque- The vitamin content of FM varies with the type of milk used
ous solution. In a large proportion of world’s population, (particularly the fat content of the milk, which influences the
ß-galactosidase activity is low or absent. This enzyme is present amount of vitamin A and other fat-soluble vitamins present),
in sufficient quantities in lactic FMs. Consequently, the lactose the strain of bacteria, and the fermentation conditions. Vitamin
level in FM can be lower than milk, although this is not always C is heat- and light-labile, but it is more stable in the acid

conditions of FM than normal milk. Levels of some B-complex
vitamins are reduced due to the requirement of some of the
LAB. Losses of up to 90% of vitamin B12 have been reported
with specific bacterial strains, but losses are not always so large
and can be reduced considerably by the use of ‘supplementary
culture’ capable of synthesizing significant amounts of vitamin
B12. On the other hand, several cultures are able to synthesize
the B vitamin, folic acid, increasing the level of this vitamin
present in the final product, for example, Prop. freudenreichii
subsp. shermanii, which is used as a starter culture in some of the
cheeses, is known to synthesize vitamin B12.
In the current scenario, when there are concerns about the
folate intake of women during the early stages of pregnancy,
this aspect may prove to have nutritional advantage. Increase
in vitamin B and P (riboflavenoid) group vitamins has been
observed in Kefir due to the activity of yeasts and acetic acid
bacteria. Fermentation has been reported to increase folic acid
in buttermilk, sour cream, Yogurt, Bifidus milk, Koumiss, Dahi,
lassi, laben, skyr, and kefir. Effect of fermentation on the
vitamin content in some of the FMs is given in Table 4.
Milk Minerals
Most experiments performed recently did not confirm the
improved absorption of calcium because of the more acidic
pH in the intestine following consumption of FM. When bio-
availability of essential minerals and trace elements was inves-
tigated in rat experiments using diets based on milk, yogurt,
and pasteurized yogurt and a commercial diet, it was observed
that bioavailability (as measured by intestinal absorption, uri-
nary excretion, and bone content) of calcium, phosphorus,
magnesium, and zinc from all diets was superior than the
commercial diet. In experiments with albino rats, availability
of calcium and phosphorus from FM was increased by about
7% and 11%, respectively, compared to milk. The results are
explained by the fact that colloidal calcium complexes and
lactic acid, both present in FM, enhance calcium absorption.
Bioactive peptides derived from the tryptic hydrolysates of
casein known as caseinophosphopeptides (CPP) possess
physicochemical properties that enable chelation of various
bi- and trivalent minerals to be carried out, thereby enhancing
mineral solubility in the lower small intestine. It has been
suggested that moderate and exchangeable binding of calcium
to CPP is responsible for the high absorbability of calcium
from milk. Results of experiments conducted at Melbourne
Dental School, the University of Melbourne, have proved that
there is a significant reduction in the tooth decay in laboratory
animals that are fed with CPP (Table 3).
Formation of Single-Cell Proteins in FMs
In recent years, the nutritional importance of microbial single-
cell proteins (SCPs) has received considerable attention. When
FM is consumed, a large number of bacterial cells (about
107–109 CFU g 1) and the products released from these cells
enter the digestive tract. Most of the living cells are inactivated
in the stomach by the action of hydrochloric acid and pepsin.
It has been estimated that about 60% of the SCP nitrogen
content (excluding cell wall material and nucleic acids) can
Fermented Foods: Fermented Milks 665
be utilized by the body. The EAA content of SCP in FM ranges
from 2.0 to 6.5 mg/100 g of product. The SCPs of some bacte-
ria were found to be rich in methionine, lysine, and cystine.
Studies on amino acid patterns of acid hydrolysates of SCP
have revealed the presence of all the known amino acids. The
SCPs of mixed cultures contained higher concentrations of
many amino acids than those found in cells of individual
cultures.
There were also considerable species variations in the pat-
tern of amino acids in the SCP. When the cells were subjected
to proteolytic enzymes simulating to some extent the condi-
tions during passage in the gastrointestinal tract (GIT), signif-
icant differences were found between species with regard to the
extent of hydrolysis and concentration of EAA released. Cells of
L. acidophilus and mixed cultures of Str. salivarius subsp. thermo-
philus and L. delbrueckii subsp. bulgaricus released the highest
concentrations of EAA. The biological activity of many EAA in
FM protein is much higher than that of corresponding amino
acids in milk. These results indicate that the SCPs of starter
bacteria consumed along with FM are likely to be hydrolyzed
partially or fully in the digestive tract, releasing EAA, and some
of the starter cultures are most useful than others in this
respect.
Biological Value of FMs
The cumulative effect of the available EAA from milk proteins
and SCP is reflected in the protein quality (biological value) of
the total protein content isolated from FM prepared with dif-
ferent cultures. The protein quality of Dahi was assessed by
microbiological assay procedure using Str. zymogenes as the test
organism. It was found to be higher (3–30%) than that of milk
used for preparing dahi. Samples prepared by using mixed
cultures of Lac. lactis subsp. lactis, Lac. lactis subsp. cremoris,
Str. salivarius subsp. thermophilus, and L. delbrueckii subsp. bul-
garicus gave higher values than those containing other cultures.
Incorporation of the culture of Prop. freudenreichii subsp. sher-
manii along with the starter cultures caused a further improve-
ment in protein quality. It has also been found that there is an
increased secretion of digestive enzymes by salivary glands
when stimulated by the curd particles and that the FM proteins
are twice as digestible as milk proteins.
Biopeptides in FMs
Several of the peptides derived from milk proteins represent
‘extranutritional’ substances, which display significant physio-
logical influence. There are several modes of release of these
peptides from precursor proteins or synthesis thereof from
simpler molecules. The starter and nonstarter bacteria are com-
monly used in the manufacture of fermented dairy products.
They take advantage of their proteolytic system, which contains
at least 16 peptidases. During the microbial fermentations
involved in the manufacture of FM, milk proteins undergo
controlled proteolysis under the combined influence of native
microflora (comprising of some spore producers) and the
starter bacteria.
666 Fermented Foods: Fermented Milks
Additionally, proteinases produced by the starter organisms
could complement the complex enzyme system of starter
microorganisms, leading to the accumulation of beneficial
peptides. The angiotensin-I-converting enzymes inhibiting
peptides derived from casein are termed as casokinins, whereas
those derived from whey proteins are called as lactokinins.
Cheeses and processed cheese products, therefore, offer the
potential for developing specialized functional food products
displaying prophylactic properties, besides their natural nutri-
tional virtues. This represents a hitherto unexplored area of
food research, which has tremendous future potential, in
both academic and commercial perspectives.
The folklores regarding the nutritional, healthful, and ther-
apeutic aspects of FM have been fully substantiated through
recent biochemical, nutritional, and physiological research.
With the growing awareness of beneficial attributes of FM
products, there has been a tremendous growth of market for
probiotic FM products in the European countries, Japan, and
developing countries. Prospectus for the genetic modification
of starter organisms to enhance the NV and probiotic attributes
has opened challenging vistas for the new product develop-
ment and diversification. The FM, as a potential carrier for
natural source of biological peptides, is a fascinating area for
technological development.
FMs in the Health-Aware Era
When milk is fermented to obtain any FM, it has billions of
live microbial cells, some of which can sustain the drastic
conditions in the stomach and impact the GIT microflora.
The human GIT microbiota, has been investigated since the
beginning of microbiological studies, when Antonie van Leeu-
wenhoek, the father of microbiology, investigated the micro-
organisms in his own stools. The human GIT microbiota
comprises trillions of microbes distributed in various niches
throughout the intestinal tract and is one of the most complex
microbiological ecosystem on Earth. The host and its micro-
biota have coevolved together and considering the staggering
numbers and diversity.
There have been major scientific advances especially in
human intestinal microbiology in the recent past. Early studies
were limited to description of the culturable microbes, which,
as we now realize, made up only a minority of the GIT
microbes. Due to the development of molecular biological
techniques over the last two decades, microbes can now be
detected and studied to a large extent, without the need for
culturing.
The vast extent of human GIT microbiota is only being
revealed by virtue of the advancement of modern molecular
biological tools. It is stated that 90% of the DNA of GIT is
contributed by this microbiota. The number of microorgan-
isms within the intestine greatly exceeds human cells, resulting
in one of the most diverse and dynamic microbial ecosystems.
Associations among the microbes and between the microbiota
and the host have a profound influence on all concerned. The
GIT offers various alcoves with nutrients, those ingested and
spawned by the host, and a relatively nonhostile environment
to the microorganisms. The microbiota plays essential roles in
a wide variety of therapeutic, nutritional, developmental, and
immunologic processes and therefore notably contributes to
the well-being of the human beings. During the last two
decades, specific bacterial isolates capable of surviving and
establishing in the GIT known as probiotics have been exten-
sively used to modulate the microbiota of GIT to benefit the
consumers. Today, there is swaying evidence for probiotics in
prevention and/or treatment of a number of GIT-related ail-
ments, especially for reducing attack of diarrhea, providing
relief for lactose-intolerant human beings and also atopic dis-
eases. In order to rationally use synbiotics or other functional
foods as therapeutic agents, in-depth knowledge of the struc-
ture, dynamics, and function of the bacterial populations of
the GIT microbiota is crucial.
Studying the microbial ecology of the intestinal content
involves the determination of the abundance and diversity of
the microorganisms present, their activity within this niche,
and their interactions with each other and their host. The GIT
microbiota have been extensively investigated by culture-based
methods, but our knowledge about the culturable fraction
of this community is limited. It is due to the challenges of
obtaining pure cultures of GIT inhabitants, which are hindered
by the largely anaerobic nature of this community and the
paucity of suitable enrichment strategies to simulate intestinal
conditions.
The advent of molecular techniques based on 16s ribo-
somal (rRNA) gene analysis is now allowing a more complete
assessment of this complex microbial ecosystem by unraveling
the extent of diversity, abundance, and population dynamics
of this community. The techniques like metagenomics have
extended our view of those microorganisms that have proved
difficult to culture and that play an important role in gut
physiology. This huge intestinal microbial reservoir is esti-
mated to contain above 1500 bacterial species and as much
as 1014 cells. Besides studying the diversity, it is essential to
identify these microbes based upon their ecophysiological
traits, that is, those that are functionally active versus those
that are effectively redundant and play little or no role at a
particular time or at a given site of the intestinal tract. Thus, in
the present era of health consciousness, the FMs have opened a
new vista for multifarious development of these products as
dietary adjuncts.
Future Developments in FM Industry
Mechanization and Automated Controls
In the era of aggressive marketing, a strong trade name and a
good company image are of utmost importance in order to
succeed as a food producer in the increasingly competitive
global food market of the FMs. One of the essential aspects
when building and protecting a brand name is to produce and
deliver a product that is consistent in quality. Consumers
expect their favorite product to have the same taste and texture
every time it is consumed, regardless of when or where it was
purchased and under what circumstances it was manufactured.
In just a few decades, the fermented dairy product industry
has developed from small production units operated by a
number of skilled dairy personnel, performing most of the
operations manually, to large-scale production lines where
predetermined product quality and production efficiency are

equally essential for success. Mechanization and the introduc-
tion of automation and production management systems have
major roles in these developments. The main advantage of
having an automated FM production line is that each proce-
dure in the operation is repeated in exactly the same controlled
manner every time, which ensures high precision of repeatabil-
ity and safety.
At present, the rapid developments in computer-assisted
information technology and electronics facilitate much more
than just the process control to be built into an automated
production system, for example, production management sys-
tems with work execution, data collection and data analysis,
and integration to local or global business systems. The bene-
fits from such developments in the FM industry are not only
improved quality, safety, and control but also increased possi-
bilities to map, visualize, analyze, develop, and optimize the
product and the process as well as the production management
and the production facilities.
Food Safety Aspects
The LAB are ubiquitous in fermented and nonfermented foods
and are common components of the human commensal
microflora. This long history of human exposure and con-
sumption has led to the reasonable conclusion that they are
generally safe. Attention has also been focussed on their pos-
sible role as probiotic bacteria, promoting beneficial health
effects. There have, however, been a number of reports of
human infections caused by LAB and these are reviewed. In
most cases, the source of the infection was the commensal LAB
flora rather than ingested bacteria, and the patient had some
underlying disease or predisposing condition.
Even opportunistic pathogens, the LAB, with the notable
exception of the enterococci, are much less successful than a
number of other members of the commensal microflora. The
use of new strains for probiotic use is likely to require more
detailed evidence for their safety, particularly if the strains have
been genetically modified or have been derived from animals.
Procedures that have been proposed for assessing the safety of
new strains are described. Consumers around the world are
raising their voices on this issue, and local health authorities
are introducing regulations that require all food producers to
ensure that food is handled appropriately and safely. All these
demands from a global consolidating food industry drive
the development of tools that are accurate, competent, and
effective that can assist the human brain and body when it
comes to managing a food processing enterprise in most
advantageous way.
Future Prospectus
Anyone walking around a supermarket will notice the promi-
nence given to dairy products like cheeses, and FMs appear to
be prominent dietary components. The sheer variety and
Fermented Foods: Fermented Milks 667
consistent quality of such products soon become evident as
well and attribute to the technical and scientific expertise of the
manufacturers. How markets will develop in the future
remains to be seen, for the consumers’ tastes are not always
predictable. In order to meet such changes in demand, what is
important is that fundamental knowledge about the fermenta-
tion of milk continues to expand, for only then can product
diversity and quality achieve the standards that consumers
deserve.
See also: Acidophilus Milk; Cheese: Chemistry and Microbiology;
Fermented Foods: Composition and Health Effects; Fermented Foods:
Origins and Applications; Fermented Foods: Use of Starter Cultures;
Functional Foods; Lactic Acid Bacteria; Malnutrition: Prevention and
Management; Probiotics; Protein: Digestion, Absorption and
Metabolism; Protein Quality and Amino Acids in Maternal and Child
Nutrition and Health; Single Cell Proteins; Vitamins: Overview; Yogurt:
Dietary Importance; Yogurt: The Product and its Manufacture.
Further Reading
Backhed F, Ley RE, Sonnenburg JL, Peterson DA, and Gordon JI (2005) Host-bacterial
mutualism in the human intestine. Science 5717: 1915–1920.
Falk PG, Hooper LV, Midtvedt T, and Gordon JI (1998) Creating and maintaining the
gastrointestinal ecosystem: what we know and need to know from gnotobiology.
Microbiology and Molecular Biology Reviews 62: 1157–1170.
International Dairy Federation (1983) Cultured foods in human nutrition, Doc. No.
159: pp. 18–28. Brussels: International Dairy Federation.
Khedkar CD and Ouwehand AC (2006) Gastrointestinal microbiology. New York: Taylor
& Francis, pp. 315–327.
Khedkar CD, Khedkar GD, Chavan NV, and Kalyankar SD (2002) Fermented milks:
dietary importance. In: Encyclopaedia of food science and nutrition, 2nd ed.
London: Elsevier.
Madureira AR, Tavares T, Gomes AMP, Pintado MI, and Malcata FX (2010) Invited
review: physiological properties of bioactive peptides obtained from whey proteins.
Journal of Dairy Science 93(2): 437–453.
Ouwehand AC and Vaughan EE (2006) Gastrointestinal microbiology. New York: Taylor
& Francis, pp. 51–60.
Ouwehand AC, Salminen S, and Isolauri E (2002) Probiotics: an overview of beneficial
effects. Antonie Van Leeuwenhoek 82: 279–289.
Rawls JF, Samuel BS, and Gordon JI (2004) Gnotobiotics zebrafish reveal evolutionarily
conserved responses to the gut microbiota. Proceedings of the National Academy of
Sciences of the United States of America 101: 4596–4601.
Shahani KM and Chandan RC (1979) Nutritional and healthful aspects of cultured and
culture-containing dairy foods. Journal of Dairy Science 62: 1685–1694.
Tamime AY (2006) Fermented milks. Oxford: Blackwell Science Publishing Company.
Yadav JS, Grover S, and Batish VK (1993) A comprehensive dairy microbiology. New
Delhi: Metropolitan.
Yamauchi K (1992) Biologically functional proteins of milk and peptides derived from
milk proteins. IDF Bulletin 272: 51–58.
Relevant Websites
http://aem.asm.org/content/66/9/3898.short.
http://www.pubfacts.com/detail/22265282/Technological-and-probiotic-role-of-
adjunct-cultures-of-non-starter-lactobacilli-in-soft-cheeses.
http://www.sciencedirect.com/science/article/pii/S0958694607000696.
 Fermented Foods: Fermented Vegetables and Other Products
R Di Cagno, P Filannino, and M Gobbetti, University of Bari Aldo Moro, Bari, Italy
ã 2016 Elsevier Ltd. All rights reserved.
Lactic Acid Bacteria as Cell Factories for the
Production of Health-Promoting Compounds
Food fermentations are typically driven by microbial consortia,
which may lead to significant changes in the health-promoting
properties of fermented foods. Besides the potential direct role
of food-borne microorganisms on the gut microbiota ecosys-
tem, their indirect role in the modification of the bioavailabil-
ity of certain food elements may also have a biogenic effect.
The latter depends on the specific metabolic traits of the micro-
organisms, whereby several biochemical mechanisms allow
them to fulfill the role of efficient cell factories for the synthesis
and release of health-promoting compounds. As a result, fer-
mentation may lead to a marked increase of the concentration
of vitamins or amino acids, a higher bioavailability of phyto-
chemicals and minerals, and an improvement of the nutri-
tional qualities of foods by increasing digestibility and
removing antinutrients (e.g., oxalate, protease and a-amylase
inhibitors, lectins, condensed tannins, and phytic acid). The
proteolytic system of lactic acid bacteria may also contribute to
the liberation of bioactive peptides. Based on the previously
mentioned considerations, lactic acid fermentation represents
a simple and valuable biotechnology to exploit the health-
promoting properties of fruits and vegetables.
Fruits and Vegetables as Carriers for Probiotic
Microorganisms
Raw, minimally treated, and, especially, fermented vegetables
are the vehicles of high-value nutrients and new lineages of
commensal bacteria that interact with the gastrointestinal res-
ident microbiota. Few and, in some cases, controversial in vivo
studies have shown the direct causal relationship between
microbes conveyed by fermented vegetable foods and gut
microbiota. The consumption of fermented kimchi, for
instance, delivers  8 log cfu g 1 of autochthonous lactic acid
bacteria and can lead, after administration of about 60 g day 1
for 2 weeks, to a positive effect on the number of beneficial
human intestinal microbiota (Lactobacillus sp. and Leuconostoc
sp.). In vivo intervention studies based on short-term consump-
tion of diets, consisting entirely of plant foods, have shown
that food-borne bacteria associated to diet can transiently col-
onize the gut. Although there is still debate on the link between
fermented vegetables and other fermented foods on the one
hand and the gut microbiota on the other hand, it is conceiv-
able that ingestion of fermented foods is a source of beneficial
microbes that remain viable during transit and at least interact
with the gastrointestinal microbiota. More than other food
ecosystems, raw fruits and some vegetables possess intrinsic
chemical and physical parameters that, for some traits, mimic
those of the human gastrointestinal tract, such as the very acid
environment, buffering capacity, presence of antinutritional
668 Encyclopedia of Food and Health
factors (e.g., tannins and phenols), and high concentration
of indigestible compounds (fiber, inulin, and fructo-
oligosaccharides). Indigestible molecules protect probiotic
microorganisms from the acidic environment of the stomach
and are a source of nutrients for the microbiota, which
positively influences bacterial survival. Plant tissues have a
microstructure composed of intercellular spaces, stomas, lenti-
cels, capillaries, irregularities naturally occurring on the surface
tissue, and tissue lesions. These sites favor microbial internal-
ization and offer protection. Thus, raw fruits and vegetables
may harbor functional strains able to survive gastric and
intestinal fluids and capable of adhering to the gut epithelium
and hydrolyzing dietary constituents, which cannot be metab-
olized by the host.
Microbiota of Raw Vegetables and Fruits
Vegetables and fruits, providing a variety of nutrients, are
attractive hosts for microbes that colonize surfaces (epiphytes)
and tissues (endophytes). Type, availability, and concentration
of substrate; buffering capacity; competing microorganisms;
and natural plant antagonisms provide a unique environment
harboring a rather constant microbiota. However, farming and
storage conditions may influence the composition and abun-
dances of microbial communities found on raw vegetables and
fruits. The microbial population of these raw matrices
fluctuates between 5 and 7 log cfu g 1, which may include
spoilage (e.g., Erwinia carotovora), pathogenic (e.g., Listeria
monocytogenes), and beneficial (i.e., lactic acid bacteria)
microbes. As also shown by deep sequencing approaches
(high-throughput pyrosequencing analysis of the 16S rRNA
gene), the microbiota of fruits is dominated by yeasts and
fungi, and that of vegetables mainly consists of bacteria, espe-
cially aerobes (e.g., pseudomonads, enterobacteria, and coryn-
eforms). Lactic acid bacteria are only a relatively small part
(2–4 log cfu g 1) of the autochthonous microbiota of raw veg-
etables and fruits. Hetero- and homofermentative species
belonging to species of Leuconostoc, Lactobacillus, Weissella,
Enterococcus, and Pediococcus have been variously identified,
depending on the species of vegetables. Nevertheless, Weissella
cibaria/W. confusa and, especially, Lactobacillus plantarum are
the most frequently found species. Lactobacillus plantarum is
considered to be a ubiquitous and metabolic versatile bacte-
rium, which is largely found in fruits and vegetables. Recently,
fructophilic lactic acid bacteria (FLAB) were described as bac-
teria that prefer fructose to glucose as growth substrate. They
are found in fructose-rich niches such as fruits and fermented
foods made from fruits and flowers. Only two Lactobacillus spp.
have been classified as fructophilic until now, that is, Lactobacil-
lus plantarum and Lactobacillus kunkeei. The latter has originally
been isolated from grape juice. All other FLAB were grouped into
the genus Fructobacillus, which contains Fructobacillus durionis,
http://dx.doi.org/10.1016/B978-0-12-384947-2.00284-1
 Fermented Foods: Fermented Vegetables and Other Products
F. ficulneus, F. fructosus, F. pseudoficulneus, and F. tropaeoli. All
these species are phylogenetically related to the genera Leuconos-
toc, Oenococcus, and Weissella. F. pseudoficulneus was isolated
from figs and banana. FLAB species are separated into two
groups: obligately and facultatively fructophilic. Obligately
FLAB grow well on fructose, whereas external electron acceptors
such as fructose, pyruvate, and oxygen are needed for glucose
metabolism. Molecular typing of lactic acid bacteria isolated
from vegetables and fruits is useful to group them into clusters
that reflect their distribution in the raw matrix and, presump-
tively, their way of entry. Besides, intraspecific differentiation is
the important preliminary step to select starter cultures.
Fermentation of Vegetable Products
When favorable conditions of anaerobiosis, water activity, salt
concentration, and temperature occur, raw vegetables and
fruits may be subjected to spontaneous lactic acid fermenta-
tion. Alcoholic fermentation may take place concomitantly.
Usually, Gram-negative bacteria are inhibited starting from
the early stage of fermentation. Spontaneous fermentation
may be optimized through back-slopping, commonly used
for sauerkraut production (i.e., inoculation of the raw material
with a small quantity of a previously performed successful
fermentation). Hence, back-slopping results in the dominance
of the best-adapted strains and represents a way of using a
selected microbial consortium to shorten the fermentation
and to reduce the risk of fermentation failure. The same effect
is achieved through the use of starter cultures, especially when
these are selected within the autochthonous microbiota. In
either cases (spontaneous and with starters), the composition
of the microbiota completely changes, showing high cell den-
sities of lactic acid bacteria towards the end of the fermentation
Table 1 Examples of vegetables and fruits successfully subjected to fermentation by selected autochthonous lactic acid bacteria
Vegetables and fruits Autochthonous lactic acid bacteria
Almagro eggplant Lactobacillus brevis, Lactobacillus
plantarum
Leek Leuconostoc mesenteroides and
Lactobacillus plantarum
Carrots, French Lactobacillus plantarum, Leuconostoc
beans, or marrows mesenteroides, and Pediococcus
pentosaceus
Red and yellow Lactobacillus plantarum, Lactobacillus
peppers curvatus, and Weissella confusa
Pineapple Lactobacillus rossiae and Lactobacillus
plantarum
Sweet cherry puree Pediococcus pentosaceus and Lactobacillus
added of stem plantarum
infusion
Tomato juice Lactobacillus plantarum
Kimchi Lactobacillus sakei
669
(9 log cfu g 1). Under these conditions, humans are solely
exposed to beneficial bacteria without the need for drastic
processing.
Nowadays, the use of starter cultures is increasing in vege-
table fermentation technology. Yet, contrary to other fermen-
ted foods (e.g., dairy, meat, and baked goods), only few
cultures are used for fruit and vegetable fermentations, with
Lactobacillus plantarum as the most frequently used one. It has
been shown that the use of selected autochthonous lactic acid
bacteria as starters, when contrasted to allochthonous/com-
mercial cultures or to spontaneous fermentation, guaranteed
the prolonged shelf life of fermented vegetables and fruits and
maintained agreeable nutritional, rheological, and sensory
properties. These effects may be the consequence of the mod-
ified profile of organic acids, the synthesis of lactic and acetic
acids, and the metabolism of free amino acids. Recently, a
number of vegetables and fruits were successfully subjected to
fermentation by selected autochthonous lactic acid bacteria
(Table 1). Adaptation to the intrinsic features of the raw matri-
ces guarantees high survival ( 8–9 log cfu g 1) during fermen-
tation and storage (c.40 days), whereas allochthonous strains
show longer latency phases of growth and acidification with
respect to selected autochthonous strains. Autochthonous cul-
tures may also synthesize bacteriocins to improve competitive-
ness and contribute to food safety.
Main Fermented Vegetable Products
Many fruits and vegetables are processed through lactic acid
fermentation for the production of a wide range of traditional
fermented products in various worldwide regions, mainly in
the Mediterranean area. Sauerkraut, kimchi, and fermented
cucumbers are the most studied lactic acid-fermented
Effects
Synthesis of most preferred end products with regard to sensory properties
High microbiological quality, extensive consumption of carbohydrates, and
synthesis of various end products
Rapid decrease of pH, marked consumption of fermentable carbohydrates,
and inhibition of Enterobacteriaceae and yeasts
Rapid decrease of pH, marked consumption of fermentable carbohydrates,
inhibition of Enterobacteriaceae and yeasts, high firmness, high color
indexes
Inhibition of yeasts, maintaining agreeable sensory and nutritional features
Evident consumption of organic acids (e.g., malic acid) and free amino acids,
especially throughout storage and preservation of phenolic compounds
Synthesis of exopolysaccharides and high viscosity; preservation of elevated
values of ascorbate, glutathione, and total antioxidant activity during
storage; synthesis of volatile compounds having a positive log odor value
(e.g., 3-methyl-3-butan-1-ol, 2,3-butanedione, and 3-hydroxy-2-butanone)
Mild acidifying properties, synthesis of volatile compounds (diacetyl, acetoin,
acetaldehyde, secondary alcohols, esters, and lactones) affecting the
sensory properties
670 Fermented Foods: Fermented Vegetables and Other Products
vegetables mainly due to their commercial importance. The
manufacturing protocols for some traditional fermented vege-
tables are described in the following.
Fermented Olives
Table olives are one of the main pickled products with industrial
importance. The trade standard applying to table olives of
the International Olive Oil Council defines table olives as
“the sound fruit of varieties of the cultivated olive trees
(Olea europaea L.) that are chosen for their production of olives
whose volume, shape, flesh-to-stone ratio, fine flesh, taste, firm-
ness and ease of detachment from the stone make them partic-
ularly suitable for processing; treated to remove their bitterness
and preserved by natural fermentation; or by heat treatment,
with or without the addition of preservatives; packed with or
without covering liquid.” Table olive processing has been a very
old tradition in all Mediterranean countries and, from the begin-
ning of this century, has become popular all over the world. The
high nutritional value of table olives is well known, since they
contain several nutritional components (e.g., vitamins E and A
and polyphenols), which largely depend on the olive variety, the
maturation stage of the olive fruit, the cultivating conditions, and
the processing method. Because of the bitterness caused by the
phenolic glycoside oleuropein, raw olives are not suitable for
consumption without any processing step. The main commer-
cial preparations, namely, (a) treated green olives (Spanish
style), (b) naturally black olives (Greek style), and (c) ripe olives
processed by alkaline oxidation (Californian style), are among
the first three processing methods defined by the standard.
Spanish-style olive manufacture combines a lye treatment with
a NaOH solution (1–3.5% w/v) and a fermentation step. It has
been generally established that lactic acid bacteria are responsi-
ble for the fermentation of treated olives. However, lactic acid
bacteria and yeasts compete for the fermentation of natural
olives. The predominant bacterial genus is Lactobacillus. Other
genera of lactic acid bacteria have also been isolated to a lesser
extent (e.g., Weissella, Pediococcus, Enterococcus, and Leuconostoc).
Yeasts play a minor role, though they may contribute to the
flavor and aroma of table olives. According to the Greek-style
process, the fermentation occurs in a brine solution with high
NaCl content (6–15% w/v), which promotes the growth of
yeasts (mainly Candida spp., Debaryomyces spp., Pichia spp., and
Rhodotorula spp.). The predominant lactic acid bacteria genera
are the same as the ones involved in Spanish-style processing.
Californian-style processing is based mostly on a lye treatment to
remove the bitterness and may include only a weak fermentation
step.
Microbial starter selection, bacteriocin production, avail-
ability of nutrients, probiotication, and adjustment to deter-
minants affecting the fermentation process (e.g., pH,
temperature, NaCl, and polyphenol content) are some of the
main current challenges for the table olive industry.
Sauerkraut
Sauerkrauts, widely consumed in many European countries and
in the United States, are manufactured by spontaneous lactic
fermentation of white cabbage (Brassica oleracea L. var. capitata).
After removal of the core and outer leaves, fresh cabbage is
shredded and mixed with 2–3% (w/w) salt before allowing for
natural fermentation. Cabbages are quickly surrounded by
brine and covered with plastic sheeting draped over the tank, to
ensure air exclusion. Sauerkraut production typically relies on a
sequential microbial process that involves hetero- and homo-
fermentative lactic acid bacteria, such as Leuconostoc spp. and
Weissella spp. in the early phase and Lactobacillus spp., Lactococcus
lactis, and Pediococcus spp. in the subsequent phases. Lactobacillus
plantarum prevails during late fermentation, leading to further
acidification and a final pH of 3.5–3.8. Sauerkraut brine is an
important by-product of the cabbage fermentation industry and
may be used as a substrate for the production of carotenoids by
Rhodotorula rubra. Sauerkraut fermentation lasts several weeks,
depending on the fermentation conditions, sensory attributes,
the autochthonous microbiota of cabbages, and the related sugar
contents. Secondary fermentations, such as those carried out by
yeasts, are undesirable.
Kimchi
Kimchi is a Korean traditional fermented vegetable made from
Chinese cabbage (beachu), radish, green onion, red pepper pow-
der, garlic, ginger, and fermented seafood (jeotgal), which is
traditionally made at home and served as a side dish at meals.
Kimchi is a generic term indicating a group of traditional lactic
acid-fermented vegetables in Korea. Kimchi fermentation tradi-
tionally represents an efficient method of preserving the fresh and
crispy texture of vegetables during winter, when fresh vegetables
are not available. Actually, kimchi is a globally popular food, and
its market size is gradually increasing. Thanks to the physiological
effects of its ingredients and its microbial metabolites, kimchi is
widely perceived as a health-promoting food. Vegetables are
soaked with water, cut, and placed in the salt solution (5–7%)
for 12 h, until the salt content in the tissues reaches the value of
 2.0–4.0% of the total weight. Further, vegetables are rinsed
several times with freshwater and drained. The mixed ingredients
are then placed in fermentation vessels, covered with macerated
cabbage leaves, and pressed by stones to ensure anaerobic condi-
tions. Kimchi fermentation is a temperature-dependent process. It
ripens in 1 week at 15  C but takes only 3 days at 25  C. Low
temperature (2–6  C) is nevertheless preferred in kimchi fermen-
tation to prevent the production of strong acidity and over-
ripening, as well as to extend the period of optimum taste. The
time for the fermentation of kimchi may thus vary from few
days to several months, depending on the temperature and
concentration of NaCl. Kimchi fermentation is carried out by
various autochthonous microorganisms. The microbiota is largely
affected by the fermentation conditions (e.g., anaerobiosis, ingre-
dients, salt concentrations, and temperature). Generally,
Leuconostoc mesenteroides and Lactobacillus plantarum have been
identified as the predominant species, but several studies suggest
that lactic acid bacteria contributing to kimchi fermentation also
include Leuconostoc citreum, Leuconostoc gasicomitatum, Lactobacil-
lus brevis, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis,
Pediococcus pentosaceus, W. confusa, and W. koreensis.
Pickled Cucumbers
The manufacture of pickled cucumbers requires that fully ripe
cucumbers are washed and drained and, in some cases, sliced.
 Fermented Foods: Fermented Vegetables and Other Products
Further, regular-shaped cucumbers are dipped into brine
(5–7% of NaCl). Industrial fermentations are carried out by
the spontaneous growth of lactic acid bacteria. The fermenta-
tion lasts 2–3 weeks, depending on the temperature
(20–27  C), which allows to reach a final pH of  3.1–3.5.
Because of the high concentration of NaCl, the growth of
Leuconostoc mesenteroides and other less salt-tolerant species is
inhibited. Only homofermentative lactic acid bacteria, such as
Pediococcus pentosaceus and Lactobacillus plantarum, are respon-
sible for fermentation. Strictly heterofermentative species are
undesirable. The synthesis of CO2 leads to texture defects and
bloater damage, with serious economic losses. Since CO2
results also from the cucumber respiration or the malolactic
fermentation by Lactobacillus plantarum, fermentation vessels
are sometimes purged with inert nitrogen gas. Salt, consump-
tion of fermentable carbohydrates, and low values of pH pre-
vent undesirable secondary fermentations by yeasts and other
lactic acid bacteria (e.g., Lactobacillus buchneri), which often
cause cucumber spoilage. Blanching of cucumbers is also
used to decrease the concentration of NaCl (4% or less), par-
tially inhibit the autochthonous microbiota, and improve the
texture, through the inactivation of endogenous pectin esterase
and glycosidases. Partial or complete replacement of NaCl by
CaCl2 has been suggested as an efficient method to prevent
softening.
Household-Level Fermentations and Traditional
Products
Besides the semi-industrial and globally popular products, a
large variety of fermented vegetables are manufactured around
the world at household level as fundamental components of
the daily diet, representing a culture heritage. Perishable and
seasonal raw plant substrates are fermented into edible prod-
ucts using indigenous methods of biopreservation. Lactic acid
fermentation is the simplest way to preserve fruits and
vegetables and helps in solving with the issue of food security.
A brief description of some traditional fermented vegetable
products from different regions of the world is reported later
in the text.
Africa
Fermented foods have a long history in Africa. Substrates used
in fermented plant food production include vegetables, fruits,
seeds, cereals, roots, and tubers. Some fermented products,
usually obtained from carbohydrate-rich raw matrices, serve
as main course meals or as beverages, while others, resulting
from the fermentation of protein-rich seeds, are used as food
condiments.
Fermented seeds are an important cheap source of dietary
protein. Examples of traditional fermented seeds used as fla-
voring agents include iru (fermented Parkia biglobosa seeds),
ogiri (fermented Citrullus vulgaris seeds), dawadawa (fermen-
ted soybean (Glycine max) seeds), and okpehe (fermented Pro-
sopis africana seeds). Often, Bacillus species are predominant
during these fermentations.
Pickled lemons are widely consumed in North Africa. Fer-
mentation helps in dealing with the issue of seasonality. Cured
671
lemon, called msayer or msir, is consumed in Morocco, while
lamoun makbouss are pickled lemons consumed in Libya and
Egypt. According to Moroccan style, the salted lemons are
stored in sealed glass container, kept in a cool dry place for
4–6 weeks, to allow the leakage of the lemon juice and the
dissolving of salt. The resulting strongly salty and acidic brine
promotes the growth of lactic acid bacteria and yeast (e.g.,
Candida parapsilosis and unclassified Saccharomycetales).
Cassava (Manihot esculenta Crantz) is one of the most
important root crops in many African countries and ensures
the energetic food security for many millions of people. Sweet
varieties of cassava may be consumed directly, whereas bitter
varieties are traditionally processed into a wide range of tradi-
tional products: gari (partially gelatinized granular cassava
meal), fufu (pounded boiled cassava), lafun (fine powdery
cassava), kokonte (dried cassava flour), kivunde (retted cas-
sava), and cingwada (retted cassava). Lactic acid fermentation
may significantly prolong the short shelf life (only 5 days after
harvesting) of raw cassava and may have a role in cyanogen
reduction in bitter cassava. Lactobacillus plantarum typically
dominates the fermentation. Some Lactobacillus plantarum
strains isolated from fermented cassava are able to hydrolyze
resistant starch, thereby increasing the energy density.
Asia
Plant-based foods represent the core of the daily diet in Asian
countries, which are the first in the world for per capita con-
sumption of fruits and vegetables. Food fermentation in Asia is
strongly dependent on the availability of seasonal vegetables.
Ethnic fermented foods are linked to the Asian culture and
tradition, and hundreds of different forms of fermented vege-
tables are prepared by the different ethnic groups. A wide range
of substrates is subjected to fermentation, which includes veg-
etables, fruits, legumes, rice, and bamboo shoots. Processing is
still carried out at household-scale through spontaneous fer-
mentations (e.g., alcoholic, lactic, propionic, and acetic acid
fermentations).
Gundruk is a fermented leafy vegetable commonly pre-
pared by the Nepalis of the Himalayan region prepared during
the months of December to February, when the weather is less
humid and there is a wide supply of vegetables. For gundruk
preparation, fresh leaves of local cabbage, mustard, and cauli-
flower are wilted for 1–2 days. The dried leaves, after a mild
crushing procedure, are soaked briefly in hot water and hand-
pressed in an earthen pot to remove surplus water. Then, they
are kept in a warm and dry place for 15–22 days. Generally, a
spontaneous fermentation is started by Lactobacillus brevis and
Pediococcus pentosaceus and dominated by Lactobacillus plan-
tarum in the late stage. After a desirable fermentation, gundruk
is removed and sun-dried. Other examples of Asian fermented
leafy vegetables include hum-choy, produced in the south of
China from a local vegetable called gay-choy; anishi, goyang,
and ziang-sang, made in northeast India from local leafy veg-
etables; sunki, prepared from red turnip leaves in Japan; and
pak-gard-dong, produced in Thailand from mustard.
Khalpi is a fermented pickle consumed in the Himalayan
region. For its preparation, mature and ripened cucumbers are
cut and sun-dried for 2 days. Then, they are fermented into
sealed bamboo vessels for 4–7 days at room temperature.
672 Fermented Foods: Fermented Vegetables and Other Products
Khalpi is eaten as a pickle after mixing with mustard oil,
powdered chilies, and salt. The microbiota of khalpi is charac-
terized mainly by Leuconostoc fallax, Lactobacillus brevis, and
Pediococcus pentosaceus in the early stage and Lactobacillus plan-
tarum in the last stage of fermentation.
Sinki is prepared from radish taproots, and it is very popu-
lar in Nepali communities. The protocol for its manufacturing
is similar to that of gundruk. The fermentation may last up to
30–40 days and is commonly started by Lactobacillus fermentum
and Lactobacillus brevis and later replaced by Lactobacillus
plantarum.
Pickled fruits, such as lime, lemon, and mango, are very
popular in Asia. Burong mangga is a green mango pickle pro-
duced in the Philippines. For its manufacturing, peeled and
sliced fruits are fermented in brine for 3 days. Lactic acid
bacteria involved during burong mangga fermentation include
mainly Leuconostoc mesenteroides, Lactobacillus brevis, Pediococcus
cerevisiae, and Lactobacillus plantarum.
Tempoyak is a traditional Malaysian fermented condiment
made from the pulp of the durian fruit (Durio zibethinus).
Fermentation is carried out by lactic acid bacteria, such as
Lactobacillus brevis, Lactobacillus mali, Lactobacillus fermentum,
Lactobacillus durianis, and Leuconostoc mesenteroides.
Kombucha is a refreshing beverage obtained by the fermen-
tation of sugared tea by a symbiotic mixture of bacteria (acetic
acid and lactic acid bacteria) and yeast. Native of China, kom-
bucha is currently common around the world. The many ben-
efits of kombucha for health were reported, such as anticancer
activity, detoxifying effects, and antibiotic activity. Its proper-
ties are mainly attributed to the acidic composition, to the
capacity of glucuronic acid to bind to toxin molecules, and to
the content of B-complex vitamins and phenols.
South America
Water kefir, also known as tibicos or sugar kefir, is a probiotic
beverage native of Mexico and currently common worldwide.
Water kefir is obtained through fermentation of sweetened
water with a symbiotic culture of lactic acid bacteria, bifido-
bacteria, and yeast. Figs and lemons are traditionally added for
additional flavor and nutrients. Microorganisms are intro-
duced through water kefir grains (polysaccharide matrix in
which microorganisms are imbibed), handed down from
household to household. Water kefir grains are thought to
have originated from fermented sap on the pads of the Opuntia
cactus.
Eastern Europe and Middle East
Researchers and food manufacturers have become increasingly
interested in Turkish fermented nonalcoholic beverages due to
their value as probiotic source. Shalgam, for instance, is a lactic
acid-fermented beverage that is very popular in Turkey. Its
manufacturing requires that bulgur flour, sourdough, and salt
are mixed with water and left to ferment by lactic acid bacteria
and yeasts for 3–5 days at room temperature. Then, vegetables
(e.g., carrots) and water are added to the fermented product of
the first stage. Fermentation is carried out in wooden barrels for
3–10 days, mainly by Lactobacillus plantarum, Lactobacillus para-
casei subsp. paracasei, Lactobacillus fermentum, and Lactobacillus
brevis, although yeasts such as Saccharomyces cerevisiae may con-
tribute to flavor development. Hardaliye is a Turkish grape-based
nonalcoholic traditional beverage. Pressed grapes and cherry
leaves are mixed with pressed mustard and fermented in barrels
at room temperature for 5–10 days. After incubation, the mixture
is filtered and kept in a cool place. The predominant lactic acid
bacteria species are Lactobacillus paracasei subsp. paracasei and
Lactobacillus casei subsp. pseudoplantarum.
Innovative Plant-Based Fermented Products
During the last decade, innovation in food biotechnology has
led to an increase in the number of new products with func-
tional claims for an estimated growth rate of 28% per year. This
is paralleled by a rise in the amount of scientific papers dealing
with this topic. The global market of functional foods is a
growing field of the food industry, driven by modern
consumer’s demand for foods that may improve well-being
and reduce the risk of disease. The ongoing trend of vegetari-
anism, the increasing prevalence of lactose intolerance, and the
cholesterol effects of dairy products drive the research towards
nondairy functional foods. The suitability of several vegetables
and fruits as raw materials for the manufacturing of functional
products has opened new perspectives. There is a strong inter-
est in the development of plant-based functional foods, as
juices, smoothies, and yogurt-mimicking beverages. Lactic
acid fermentation represents an innovative and attractive
approach to improve the nutritional, functional, and hedonis-
tic features of plant material, although several applications
often originate from or are inspired by ethnic food (e.g.,
Asian). In order to develop novel fermented functional foods,
implementation strategies relate to the exploitation of micro-
bial functionalities, the testing of novel formulations with
ingredients that either are of natural origin or originate from
industrial by-products as functional ingredients, and the forti-
fication with probiotics and prebiotics.
Fruit and Vegetable Juices
One of the most attractive opportunities is the development of
lactic acid-fermented fruit and vegetable juices, also named
‘lacto-juices.’ Since the latter have flavor profiles that are pleas-
ing to all age groups and are perceived as healthy and refresh-
ing beverages, they promise a considerable market value. Fruit
and vegetable juices are ideal substrates for functional beverage
making, due to their content of essential nutrients. Further-
more, formulation with different substrates may provide dif-
ferent combinations of health-promoting components (e.g.,
antioxidants, dietary fiber, minerals, and vitamins). The tech-
nological options for the production of fermented vegetable
juices mainly include (i) spontaneous fermentation by autoch-
thonous microbiota, (ii) fermentation by selected starter cul-
tures added to raw vegetables, and (iii) fermentation of mildly
heat-treated vegetables by starter cultures. Selection of lactic
acid bacteria that are able to adapt to the inherent features of
each raw material (e.g., availability of nutrients and physic-
ochemical features) may assure fermented vegetable juices with
optimal characteristics. Fruits and vegetables tested in novel
lacto-juices include watermelon, sapodilla, carrot, beetroot,
 Fermented Foods: Fermented Vegetables and Other Products
pepper, parsley, lettuce, lemon, cabbage, spinach, tomato,
pomegranate, blackcurrant, orange, grapes, and sweet pota-
toes. Pomegranate juices fermented with selected strains of
Lactobacillus plantarum have, in general, improved health-
promoting and sensory properties. Besides higher phenolic
concentration and antioxidant activity, a positive immunomo-
dulation activity distinguishes fermented pomegranate juices
from unfermented ones. Fermentation by autochthonous lac-
tic acid bacteria also positively affects the health-promoting
and sensory properties of tomato juice, leading to improved
viscosity, color, and, especially, ascorbic acid, glutathione, and
total antioxidant contents. A fermented juice based on vegeta-
bles, seeds, and sprouts of germinated lentils and cowpeas was
proposed as potential functional beverage, due the content of
ACE-inhibiting substances, bile acid-binding activities, and
hemagglutinating activities. Fermented carrot juice, in addition
to being microbiologically stable, has good sensory properties
and a potentially high nutritional value, due to the improved
iron solubility and overall mineral availability. The preserva-
tion of pigments and the synthesis of health-promoting sub-
stances, such as aglycones and b-carotene, have been shown in
lactic acid-fermented beetroots and sweet potatoes. Red beets
were also evaluated as a potential substrate for the production
of probiotic beet juice by four species of lactic acid bacteria
(Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus del-
brueckii, and Lactobacillus plantarum). Although the probiotica-
tion of several fruit and vegetable juices with lactic acid bacteria
and bifidobacteria seems promising, some cultures may nega-
tively affect the sensory properties of the juices or show sensi-
tivity towards the low pH of the raw matrices and
postfermented products, fermentation metabolites, processing
temperature, and gastrointestinal tract conditions. The addi-
tion of yeast autolysate (e.g., spent brewer’s yeast) into the
juices may increase the number of lactic acid bacteria during
fermentation, reduce the time of fermentation, and enrich the
juices with amino acids, vitamins, minerals, and antioxidants.
Thus, a proper selection of the fruit matrices and probiotic
strains and, if required, the addition of other ingredients are
decisive steps to produce a palatable beverage that may be
consumed regularly, providing functional benefits.
Fruit and Vegetable Smoothies
As an alternative to the consumption of fresh fruits and vege-
tables, the food industry offers the manufacture of smoothies,
which consist of mixtures of fruits and vegetables processed to
pulp or puree. Recently, a novel protocol for the manufacture
of smoothies was set up, based on lactic acid fermentation of
white grape juice and Aloe vera extract mixed with fruit and
vegetable purees. Lactic acid fermentation by autochthonous
selected starters positively affects the content of antioxidant
compounds and enhances the sensory attributes of smoothies.
An alternative technological option for processing sweet
cherries involves the lactic acid fermentation of cherry puree
supplemented with cherry stem infusion, as to exploit the
nutritional, antioxidant, and sensory features of the raw mate-
rials. Likewise, broccoli puree fermented by Lactobacillus spp.
for the production of functional foods is enriched in phenolic
derivatives with increased bioavailability and biological
activity.
673
Minimally Processed Fruits and Vegetables
Minimally processed fruits and vegetables are products
obtained by trimming and/or peeling and/or cutting and
packaging and are characterized by convenience and ability
to maintain their freshness. Fermented pineapple slices by
autochthonous lactic acid bacteria, also without any heat treat-
ment, is an example of a shelf-stable minimally processed fruit
with elevated numbers of viable lactic acid bacteria and agree-
able nutritional and sensory properties. Lactic acid fermenta-
tion may promote the storage of peppers at room temperature
for extended shelf life. A protocol that includes blanching at
85  C for 2 min, fermentation at 35  C for 15 h in brine (1%,
w/v) with selected autochthonous starters, and heat treatment
at 85  C for 15 min allows the storage at room temperature for
30 days with better texture and sensory properties compared to
the ‘unstarted’ peppers. Moreover, the survival of Lactobacillus
plantarum and Lactobacillus fermentum strains on pineapple
pieces has been demonstrated throughout storage. A protective
effect against relevant food-borne pathogens was also success-
fully reported. Artichokes may act as a valuable vector, as they
are capable of supporting viable bacteria for at least 90 days. The
anchorage of strains to artichokes also improves the survival of
probiotic microorganisms during gastrointestinal digestion.
Fruit- and Vegetable-Based Yogurt-Like Products
The so-called vegetable milks might be used as raw materials to
develop yogurt-mimicking products. They are mainly obtained
from soy, cereals, and nuts. Other minor raw materials may be
hemp, sunflower, lupin seeds, and tubers (e.g., tiger nuts).
Although the production of yogurt-mimicking products may
differ according to the raw material and the microbial starters,
the main steps are (i) milk conditioning to the optimal growth
temperature of the starters, (ii) inoculation and incubation pro-
cedures, and (iii) cooling to 4  C. Protein coagulation and serum
separation during storage may cause physical stability problems.
The addition of hydrocolloids, such as xanthan gum, modified
starches, pectin, and cellulose derivatives, promotes physical
stability. In situ production of oligosaccharides and exopolysac-
charides by lactic acid bacteria positively affects the texture, since
these compounds act as emulsifiers or stabilizers and increase
viscosity and mouthfeel. Prebiotic compounds (e.g., starch and
fibers) are sometimes added to promote the survival of probiotic
starters, due to their nutritional contribution and protective
action, since they act as protective barriers within the human
gastrointestinal tract. The most popular vegetable milk derives
from soy. Soy-based beverages have a huge potential as func-
tional foods, since they may have a role in the prevention of
some chronic degenerative diseases, are poor in cholesterol and
saturated fats, and contain high levels of quality protein and
amino acids, dietary fiber, magnesium, phosphorus, vitamin K,
riboflavin, thiamine, folic acid, isoflavones, and other flavo-
noids. The low concentration of soluble carbohydrates of soy
milk may limit the growth and acidification of lactic acid
bacteria and make the addition of glucose and fructose indis-
pensible. Probiotic bacteria have been successfully incorporated
in soy milks, with a positive impact on the intestinal ecosystem.
Nut-derived milks also represent suitable raw substrates to
develop healthy yogurt-mimicking beverages, since they are
674 Fermented Foods: Fermented Vegetables and Other Products
rich in mono- and polyunsaturated fatty acids, proteins, dietary
fibers, phytosterols, phenolics, vitamins, and minerals. Probio-
tication of hazelnut milk through fermentation with Lactobacillus
rhamnosus and Streptococcus thermophilus has been successful,
ensuring high probiotic survival throughout cold storage
(108 cfu ml 1) and an in vitro simulation of the digestion
process (105 cfu ml 1). Minor raw materials, such as tuber
milk, may also be exploited for the production of yogurt-
mimicking products. Fermented tiger nut milk with good nutri-
tional, chemical, and sensory properties was produced through
pasteurization at 90  C for 15 min and fermentation at 45  C for
18 h using mixed cultures of Lactobacillus plantarum, Lactococcus
lactis, and Lactococcus thermophilus.
Conclusion
Fermented fruits and vegetables are associated with several
social and cultural aspects of different human communities.
Due to their high nutritional value, fermented vegetable foods
and beverages are estimated to constitute about one-third of
the foods daily consumed worldwide. Studies have shown that
fruits and vegetables may serve as a suitable carrier for probio-
tics. In addition, fermented fruits and vegetables contain a
large range of prebiotic compounds that can stimulate the
growth of beneficial bacteria. Therefore, exploitation of the
axis ‘fermented foods – human health’ based on plant fermen-
tations is a promising and conceivable strategy for the future.
Basic understanding of the relationship between food, benefi-
cial microorganisms, and human health can be important to
improve food quality and to prevent disease.
See also: Fermented Foods: Composition and Health Effects;
Fermented Foods: Use of Starter Cultures; Fruit Juices; Functional
Foods; Lactic Acid Bacteria; Probiotics.
Further Reading
Corbo MR, Bevilacqua A, Petruzzi L, Casanova FP, and Sinigaglia M (2014) Functional
beverages: the emerging side of functional foods. Comprehensive Reviews in Food
Science and Food Safety 13: 1192–1206.
Das AJ and Deka SC (2012) Fermented foods and beverages of the North-East India.
International Food Research Journal 19: 377–392.
Di Cagno R, Cardinali G, Minervini G, Antonielli L, Rizzello CG, Ricciuti P, and
Gobbetti M (2010) Taxonomic structure of the yeasts and lactic acid bacteria
microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous
starters for minimally processing. Food Microbiology 27: 381–389.
Di Cagno R, Coda R, De Angelis M, and Gobbetti M (2013) Exploitation of vegetables
and fruits through lactic acid fermentation. Food Microbiology 33: 1–10.
Di Cagno R, Surico RF, Paradiso A, et al. (2009) Effect of autochthonous lactic acid
bacteria starters on health-promoting and sensory properties of tomato juices.
International Journal of Food Microbiology 128: 473–483.
Do Espı́rito Santo AP, Perego P, Converti A, and Oliveira MN (2011) Influence of food
matrices on prebiotic viability: a review focusing on the fruity bases. Trends in Food
Science and Technology 22: 377–385.
Granato D, Branco GF, Nazzaro F, Cruz AG, and Faria JA (2010) Functional foods and
nondairy probiotic food development: trends, concepts, and products.
Comprehensive Reviews in Food Science and Food Safety 9: 292–302.
Kabaka B and Dobson ADW (2011) An introduction to the traditional fermented
foods and beverages of Turkey. Critical Reviews in Food Science Nutrition
51: 248–260.
Lee CH (1997) Lactic acid fermented foods and their benefits in Asia. Food Control
8: 259–269.
Leff JW and Fierer N (2013) Bacterial communities associated with the surfaces of fresh
fruits and vegetables. PLOS One 8: e59310.
Swain MR, Anandharaj M, Ray RC, and Rani RP (2014) Fermented fruits and vegetables
of Asia: a potential source of probiotics. Biotechnology Research International
2014: 2090–3138.
Tamang JP, Tamang B, Schillinger U, Franz CM, Gores M, and Holzapfel WH (2005)
Identification of predominant lactic acid bacteria isolated from traditionally
fermented vegetable products of the Eastern Himalayas. International Journal of
Food Microbiology 105: 347–356.
Vitali B, Minervini G, Rizzello CG, et al. (2012) Novel probiotic candidates for humans
isolated from raw fruits and vegetables. Food Microbiology 31: 116–125.
Vorholt JA (2012) Microbial life in the phyllosphere. Nature Reviews Microbiology
10: 828–840.
Relevant Websites
http://www.fao.org/docrep/ – Fermented fruits and vegetables.
http://www.fao.org/documents/ – Traditional fermented food and beverages for
improved livelihoods.
http://www.fda.gov/Food/GuidanceRegulation/
GuidanceDocumentsRegulatoryInformation/ – Draft Guidance for Industry: Acidified
Foods.
http://www.fda.gov/Food/GuidanceRegulation/
GuidanceDocumentsRegulatoryInformation/ – Guidance for Commercial Processors
of Acidified & Low-Acid Canned Foods.
http://nchfp.uga.edu/index.html – Pickle.
 Fermented Foods: Origins and Applications
A Bevilacqua, M Sinigaglia, and MR Corbo, University of Foggia, Foggia, Italy
ã 2016 Elsevier Ltd. All rights reserved.
What Is Fermentation? Food Fermentation Versus
Biochemical Fermentation
Fermentation is one of the oldest methods of food preservation
and preparation; however, the word has many meanings. It is
derived from the Latin word fermentum, which is the past parti-
ciple of fervere (to boil). A first microbiological definition relied
upon the expression ‘la vie sans air’ by Louis Pasteur (life in the
absence of air, in particular oxygen). In biochemistry, fermenta-
tion is thus a microbial metabolism that produces energy from
carbohydrates in the absence of oxygen. Alternatively, fermenta-
tion can be defined as an incomplete oxidation of sugars in the
absence of oxygen that uses an internal organic compound as an
electron acceptor. In 1957, Prescott and Dunn defined it “a
process in which chemical changes are brought about in an
organic substrate, whether carbohydrate or protein or fat or
some other types of organic materials through the action of
biochemical catalysts known as enzymes, elaborated by specific
types of living organisms.” Based on the latter broad definition,
fermentation describes any biological process whether oxygen is
present or not, that is, the production of wine, vinegar, yogurt,
beer, table olives, and many other foods, as well as of different
organic fine chemicals (e.g., antibiotics, amino acids, citric acid,
and monosodium glutamate). The main trait of a fermentation is
the conversion of complex organic substrates into simpler com-
pounds with the production of chemical energy as adenosine
triphosphate.
At least three kinds of fermentation can be distinguished:
(i) solid-state (e.g., tempeh), (ii) liquid-state (or submerged)
(e.g., fermentation of corn steep liquor), and (iii) semisolid-
state, where the product is submerged (e.g., shoyu or soy sauce).
Moreover, fermentation can be spontaneous and uncontrolled
(raw materials get fermented by microbiota that are indigenous
or that originate from the equipments and environment) or
controlled and inoculated (the fermenting microbiota mostly
originates from the inoculation of starter cultures). Controlled
fermentation is based on either a monoculture (using a single
and pure strain) or multiculture (using biculture or
multiculture strains of microorganisms or mixed inocula).
The definition of spontaneous or controlled fermentation is
closely related to the definition of starter cultures; a starter
culture consists of microorganisms that are inoculated directly
into food materials to overwhelm the existing microbiota and
bring about desired changes in the finished product. These
changes may include novel functionality, enhanced preserva-
tion, reduced food safety risks, improved nutritional or health
value, enhanced sensory qualities, and increased economic
value. Table 1 lists the most important microorganisms of
starter cultures.
Fermentation is at the basis of the production of fermented
foods, which can be defined in different manners:
1. Foods that are fermented until at least one of the constitu-
ents has been subjected to the action of microorganisms for
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00281-6
a period, so that the final products have often undergone
significant changes in physicochemical composition and
sensory scores, due to microbial and enzymatic changes
2. Foods that have been subjected to the action of microor-
ganisms or enzymes, so that desirable biochemical changes
cause significant modification
3. Palatable and wholesome foods prepared from raw or
heated materials by microbial fermentation
There are at least 5000 major and minor or ethnic fermented
foods; they generally bear the 3A or A3 connotation that pin-
points acidic, alkaline, or alcoholic traits. Thus, the most
important sensory and physicochemical properties of fermen-
ted foods are as follows:
1. Acidic taste (low pH and lactic acid fermentation) (e.g.,
yogurt, fermented milks, gundruk, kimchi, and table olives)
2. Alkaline nature (high pH) (e.g., kinewa, dawadawa, and
pidan)
3. Alcoholic taste (moderate-to-high concentration of etha-
nol) (e.g., wine, beer, sake, and pulque)
There are different classifications of fermented foods, relying
upon different criteria. Hereby, we propose a simple classifica-
tion (Table 2) based on three questions:
1. Is the fermented food an alcoholic product/beverage?
2. What is the raw material?
3. Which kind of starter culture was applied?
Historical and Cultural Aspects
Our ancestors already consumed food products that had been
subjected to food fermentation; they did not know the role of
microorganisms, but they recognized the preservative and
nutritional qualities of fermented foods, as well as their palat-
ability. We do not know when humans began to use fermen-
tation intentionally, but archaeological data suggest an
intentional fermentation of honey, fruits, and rice about
10 000 years ago. Moreover, we have some evidence of bread
and cheese making, as well as of grape fermentation, around
6000–7000 BC.
One of the first descriptions of a fermented food is the
Hymn to Ninkasi by Sumerians; it dates back c.1900 years BC
and describes the production of beer. Fermented foods were
widespread in all the ancient populations and there is an
important document for the production of fermented foods
by ancient Indian: the sacred book Rig Veda reported on the
production of a fermented beverage to gain immortality. For
centuries, fermentation has been an empirical process until
Louis Pasteur described the role of microorganisms in 1857.
Table 3 proposes a short historical overview of fermentation
from the Neolithic era to the twentieth century.
675
676 Fermented Foods: Origins and Applications

Table 1 Starter cultures

Bacteria Lactic acid bacteria (Lactobacillus spp., Lactococcus spp., Streptococcus spp., Leuconostoc spp., Pediococcus spp., Leuconostoc spp.,
Oenococcus oeni), Propionibacterium spp., Bifidobacterium spp., acetic acid bacteria (Acetobacter spp., Gluconobacter spp.), Bacillus
spp., Brevibacterium linens, catalase-positive cocci (coagulase-negative Staphylococcus spp., Kocuria spp.)
Yeasts Saccharomyces cerevisiae, Sacc. pastorianus, Zygosaccharomyces spp., Candida spp., Pichia spp.
Molds Penicillium camemberti, P. roqueforti, P. chrysogenum, P. nalgiovense, Aspergillus oryzae, Rhizopus microsporus subsp. oligosporus

Table 2 Classification of fermented foods

Nonalcoholic fermented foods Alcoholic fermented foods

Fermented vegetables Nondistilled mild alcoholic beverages produced by amylolytic startersa

Fermented soybean and legumes Nondistilled and filtered alcoholic beverages produced by amylolytic starters
Fermented cereals Distilled alcoholic beverages produced by amylolytic starters
Fermented milks Alcoholic beverages produced using human saliva as amylolytic starter
Fermented fish Alcoholic beverages produced by mono-fermentation
Fermented meat Alcoholic beverages produced from honey
Fermented root/tuber products Alcoholic beverages produced from plants
Miscellaneous products Alcoholic beverages produced from malting or germination
Alcoholic beverages produced from fruits without distillation
Distilled alcoholic beverages without amylolytic starters
a
Ethnic preparations containing filamentous fungi able to hydrolyze starch.

Table 3 Historical overview of fermentation

When What

7000 BC Cheese- and breadmaking practiced

6000 BC Winemaking
3500 BC Breadmaking in Egypt
2000–3000 BC Beer fermentation in Egypt and Near East
1900 BC The Hymn to Ninkasi by Sumerians describes the production of beer
1500 BC The sacred book of Hindus (Rig Veda) describes Soma, a fermented beverage for immortality, although in Indian subcontinent,
fermentation has been used since 6000 BC
1000–2000 BC Preparation of fermented meat by Babylonians
300 BC Preparation of fermented vegetables in China
54 BC Julius Caesar reports cheddar cheese being produced in Britain
48 BC Sausage making introduced into Rome by Caesar
277 Emperor Probus introduces wine in Alsatia
617 Kumyss, an ethanol-containing fermented mare’s milk product, is introduced in Asia
1070 Roquefort cheese discovered
1383 A brewery, later named as Löwenbräu in 1552, opens in Bavaria
1637 The Gekkeikan Sake Co. begins producing sake in Kyoto, Japan; today, it is the world’s largest producer of sake
1697 Introduction of white grape in Southern California
1698 Dom Pierre Pérignon (a French monk) develops a method to retain carbon dioxide in wine (production of Champagne)
1857 Pasteur realizes that fermentation is a microbial process
1864 Production of Heineken beer in Amsterdam by using a customized yeast
1873 Lister isolates Lactococcus lactis from milk
1881 Publication of some papers on the preparation of koji and the production of sake
1907 Publication of book Prolongation of Life by Metchnikoff describing therapeutic benefits of fermented milks
1900–30 Use of defined cultures for fermentation
1950 Identification of bacteriophages in New Zealand
1996 Saccharomyces cerevisiae genome sequenced
2001 Lactococcus lactis genome sequenced

Not surprisingly, the ancient roots of fermented foods have
led to the incorporation of the concept in cultural and religious
frameworks. For instance, fermented foods are mentioned in
many parts of the Holy Bible, thus suggesting that they must be
already known by those civilizations that lived during that
time. In the Bible, we found that Noah planted a vineyard
immediately after the floodwaters had receded; later, he was
described as drunk and naked (Genesis 9, 20). Later in Genesis
(18, 8), three angels visited Abraham who offered them some
refreshments, including curds. The most important mention of

fermented foods in the Bible was in the Passover story: Hebrew
slaves were thrust out of Egypt and could not tarry; therefore,
dough could not rise. Fermented foods also played a major role
in the New Testament. The first miracle of Jesus was at a
wedding in Cana (John 2, 1–11), where he turned water into
wine. Another important miracle was the feeding of 5000
men with five loaves of bread and two fishes (John 6, 5–15).
Finally, the sacrament of Holy Communion is represented by
bread and wine.
Effects of Fermentation on Food Properties
Biosafety and Preservation
As mentioned earlier, food fermentation originally emerged as
a way to preserve perishable foods when few other preserving
methods existed (e.g., salting). However, even today, preserva-
tion is still an important feature of fermented foods. Often, this
effect is related to the microbial end products of fermentation
(lactic acid, acetic acid, ethanol, etc.), the resulting decrease of
pH up to the threshold value of 4.5 in the case of acidic
fermentations, the production of specific antimicrobial com-
pounds (such as bacteriocins and antibiotics), and competi-
tion for nutrients. Fermentation is thus responsible for the
inhibition not only of spoiling microorganisms, such as
Pseudomonas spp. but also of foodborne pathogens. The latter
include Staphylococcus aureus, Clostridium botulinum, Salmonella
spp., Listeria monocytogenes, Escherichia coli, Yersinia enterocoli-
tica, Campylobacter jejuni, Bacillus cereus, Mycobacterium
tuberculosis, M. bovis, Brucella abortus, and Br. melitensis.
Safety of fermented foods is also linked to the ability of
fermenting microorganisms to remove or decrease the content
of harmful compounds from the raw materials. Examples
include the hydrolysis of limarin (a cyanogenic glucoside
found in the leaves and roots of plants such as cassava) by
Lactobacillus spp. and the degradation of mycotoxins (e.g.,
aflatoxins and ochratoxin A) by lactic acid bacteria and Saccha-
romyces cerevisiae.
Flavor Generation
Aroma compounds produced throughout fermentation com-
prise different molecules, synthesized through the degradation
of proteins, lipids, and carbohydrates by the concerted actions
of microorganisms and the endogenous enzymes of the raw
materials. The most important groups include acids, alcohols,
aldehydes, esters, lactones, amines, pyrazines, and sulfur
compounds.
Acids
This group comprises fatty acids from the hydrolysis of lipids,
short-chain fatty acids (SCFA) produced from the degradation
of amino acids or carbohydrates, and organic acids (lactic,
acetic, and succinic acid), which are the major or minor prod-
ucts of some pathways of carbohydrates.
Long-chain fatty acids (C > 12) do not contribute to aroma
because they have a high threshold in the perception value,
while aroma is strongly affected by SCFA and organic acids.
Besides their effects on aroma, acids also contribute to taste
and overall flavor complexity.
Fermented Foods: Origins and Applications 677
Alcohols
The contribution of primary and secondary alcohols is very
important to aroma development; they can be produced
throughout different pathways: (i) carbohydrate metabolism
(ethanol is the end product of the primary metabolism in
many yeasts and a minor end product in some hetero-
fermentative lactobacilli), (ii) amino acid metabolism
(amino acids undergo an oxidative deamination and produce
a-keto acids, which are the intermediate compounds to pro-
duce aldehydes and alcohols), (iii) reduction of methyl
ketones from fatty acid metabolism, and (iv) degradation of
long-chain fatty acids (linoleic and linolenic acids) by lipox-
ygenase and hydroperoxide lyase enzymes.
Aldehydes
Aldehydes are usually produced through two different routes:
(i) amino acid deamination or transamination, giving rise to
a-keto acids, which are then decarboxylated into aldehydes;
and (ii) Strecker degradation of amino acids. Aldehydes are
generally intermediate products, as they are converted into
alcohols or acids; however, their contribution to aroma is
significant.
The most important aldehydes recovered from fermented
foods are ethanal, 2-methyl-propanal, 2-methyl-butanal, and
benzaldehyde.
Esters
Esters are produced through the esterification between SCFA
and alcohols (generally ethanol). They play a significant role in
fermented beverages, as they have a very low perception thresh-
old in water (some ppb). The most important notes are melon,
pear, banana, apricot, pineapple, floral, rose, honey, and wine.
Lactones
Lactones are cyclic esters produced by the condensation of an
alcohol and acid function of the same molecule. They play a
significant role in the aroma development of some fermented
fruit, dairy, and meat products. They are generally described as
fruity notes (peach, pear, apricot, and coconut) or yielding a
milky and creamy flavor; the most important compounds
are d- and g-octalactone, d- and g-decalactone, and d- and
g-dodecalactone.
Amines and nitrogen compounds
Amines come from the decarboxylation of amino acids; these
pathways could be a great threat as they can produce biogenic
amines, whose toxic effects are well known. An important
pathway is the synthesis of L-glutamine from L-glutamic amino
acid, reported in yogurt and miso.
Pyrazines
Pyrazines are heterocyclic compounds containing nitrogen;
they are responsible for different flavors (nutty, roasty, and
toasty), depending on the alkyl group. The two most important
pyrazines in fermented foods are 2,5-dimethylpyrazine (abun-
dant in Camembert cheese) and tetramethylpyrazine (abun-
dant in Chinese black vinegar); threonine is the precursor
of 2,5-dimethylpyrazine, whereas tetramethylpyrazine is
produced from acetoin and ammonium.
678 Fermented Foods: Origins and Applications

Sulfur compounds surface-ripened cheeses, and blue-veined cheeses. Some kinds

Sulfur compounds are end or intermediate products of the
routes of degradation and synthesis of some amino acids,
mainly methionine. The flavor of these compounds has been
described as similar to garlic, cabbage, cooked cabbage, cauli-
flower, and cooked cauliflower; moreover, they have a very low
perception threshold.
Color
Optical perception is one of the main sensory traits of impor-
tance for food quality, as many consumers decide to buy a
product for its appearance. It is related to three different cate-
gories: color, cesia, and spatial properties. Color is linked to the
optical power spectral properties of the stimulus detected by
observers, while cesia includes transparency, translucence,
gloss, luster, haze, lightness, opacity, and matt and depends
on the properties of reflecting, transmitting, or diffusing light.
Finally, spatial properties rely upon the angle of observation
and the optical properties of surface.
The color of a fermented food is strongly affected by its
composition, storage, and physical parameters (e.g., pH and
Aw). Microorganisms can also play an important role. The latter
is, for instance, clear in the case of dry-cured fermented sausages;
their bright red color is due to nitrosylmyoglobin (MbFeIINO)
formation, in which an axial ligand nitric oxide (NO) is coordi-
nated to the central FeII in the myoglobin heme group. In such
products, color development depends on the reduction of
nitrate into nitrite and subsequently into NO. Meat-associated
coagulase-negative staphylococci are able to reduce nitrate con-
tained in the curing salt into nitrite, using a membrane-bound
nitrate reductase. This nitrite is then further reduced to yield NO,
which takes place under acidic conditions or can be performed
by some coagulase-negative staphylococci. Thus, nitrate reduc-
tion is an important trait for the selection of strains of Staphylo-
coccus xylosus and Staphylococcus carnosus intended as starter
cultures for dry-cured fermented sausage production.
Aside from fermented meats, starter cultures or indigenous
microbiota play a significant role in the color development of
different types of fermented foods. For example, microbial
metabolism is important for the color of mozzarella cheese,
of mozzarella cheese are used for baking (pizza); thus, the
presence of residual galactose (a monocarbonyl) can modify
the color of melted cheese due to its participation to Maillard
reactions. The selection of galactose-positive lactobacilli could
thus minimize mozzarella browning. As another example,
Brevibacterium linens is associated with smear surface-ripened
cheeses; it produces yellow-to deep-orange colonies, due to the
synthesis of carotenoids. Some other important microorgan-
isms for cheese color are the following: Brevibacterium aurantia-
cum, Corynebacterium casei, Microbacterium gubbeense,
Staphylococcus saprophyticus, Debaryomyces hansenii, Candida
spp., Penicillium roqueforti, and P. camemberti.
Rheology
Fermentation can significantly affect the rheological properties
of foods through some main routes, including the production
of exopolysaccharides (EPS) and the gelation of casein and
other proteins. EPS are extracellular polysaccharides of micro-
bial origin; they might be both bound to the surface of micro-
bial cells and secreted in the extracellular environment in the
form of suspension or mucilage. They exhibit thickening,
thixotropic, and pseudoplastic properties in yogurt and in
some other fermented (dairy) products (e.g., desserts, and
dressings). Another important effect of fermentation on rheol-
ogy relies upon the gelation of casein and whey proteins; this
change is associated with the isoelectric point, but the changes
in the electric charges may affect the overall structure of pro-
teins and consequently their functionality.
Fermented Foods and Health
Food fermentation is often associated with the production of
some biologically active compounds with a potential impact
on health, usually referred as nutraceuticals. They can be
synthesized through different pathways, for example, oxida-
tion, reduction, hydrolysis, condensation, and isomerization.
Table 4 reports some examples of biologically active
compounds produced throughout fermentation.

Table 4 Nonexhaustive list of biologically active compounds and their producer microorganisms in fermented foods

Compound Producer microorganisms

EPS: homopolysaccharides (a-D-glucans, b-D-glucans,
fructans, polygalactan) and heteropolysaccharides
Angiotensin-1-converting enzyme (ACE)-inhibiting peptides
Bacteriocins (e.g., reuterin, nisin, sakacin, and plantaricin)
Peptides with cholesterol-lowering activity
Conjugated linoleic acid (CLA)
Vitamins (group B)
Phenolic antioxidants
Antibiotics
Leuconostoc mesenteroides subsp. mesenteroides, Ln. mesenteroides subsp.
dextranicum, Streptococcus mutans, Str. sobrinus, Str. thermophilus, Pediococcus
spp., Lactobacillus kefiranofaciens, Lb. delbrueckii subsp. bulgaricus, Lb. casei, Lb.
acidophilus, Lb. helveticus, Bifidobacterium spp.
Lactobacillus. helveticus, Lb. rhamnosus GG, Lb. delbrueckii subsp. bulgaricus,
Lactococcus lactis subsp. diacetylactis, Str. thermophilus, Saccharomyces
cerevisiae
Lactobacillus reuteri, Lb. acidophilus, Lb. brevis, Lb. delbrueckii subsp. bulgaricus, Lb.
helveticus, Pediococcus spp., Lb. sakei, Lb. plantarum, Lactococcus lactis subsp.
lactis
Bifidobacterium spp.
Lb. acidophilus, Bifidobacterium spp.
Lactobacillus spp., Bacillus spp., Saccharomyces cerevisiae
Acetobacter spp., species of Saccharomyces, Schizosaccharomyces, Kloeckera,
Zygosaccharomyces, Brettanomyces, Pichia
Streptomyces spp.

An interesting topic is the effect of fermentation on health.
Some authors found that health disorders were characterized
by chronic and low-grade oxidative stress; moreover, they sug-
gested the existence of an ‘intestinal inflammatory
microbiome,’ significantly contributing to altered mood via
intestinal permeability, systemic LPS burden, and even direct-
to-brain microbe communication. This kind of inflammatory
microbiome is enhanced by some Western habits, for example,
high-fat and high-sugar or low-nutrient-value foods. 70–80
years ago preliminary studies in rodents and humans pin-
pointed the consequences of inflammatory microbiome on
health; nowadays, it is possible to find some answers toward
the potential of fermented foods. It is well known that fermen-
tation can magnify protein quality and the bioavailability of
vitamin B, zinc, and magnesium; these compounds exert a
significant effect on mental health as they act on mood regu-
lation. Moreover, there is a strong evidence on the fact that
some lactic acid bacteria, mainly some strains of Lb. plantarum,
provide a strong protection toward oxidation. Fermentation of
herbal extracts by Lb. plantarum was shown to preserve phenols
and vitamin C, thus enhancing the free scavenging effect of
fermented foods; a similar phenomenon was also found in
fermented soy milk by lactobacilli and/or bifidobacteria.
A frontier goal is the production of bioactive compounds;
some of these compounds act on immune system, influence
glycemic index, possess anti-inflammatory activities, and to
some extent can show beneficial mental activities. One of the
most important compound is GABA (gamma-aminobutyric
acid); the use of synthetic GABA has a limited benefit due to
adsorption issues, while the consumption of fermented foods
containing GABA produced in situ by Lb. hilgardii contributes
to the reduction of blood pressure and of anxiety. In addition,
GABA-rich red yeast rice has been reported as a strong
antidepressant.
Fermentation could act on health toward its effect on fla-
vonoids; some authors suggested the ability of some starter
cultures to produce a biotransformation of phenols and this
phenomenon is the leading cause of a beneficial shift in growth
stimulation of ‘good bacteria,’ mainly bifidobacteria.
A particular promising aspect is provided by the possible
influence of fermented foods on mental health, related to the
emerging and promising trend of nutritional psychiatry. The
effects of fermented foods on mental health could be due to
three leading factors: (i) the presence of specific nutrients, (ii)
the ingestion of beneficial microorganisms, and (iii) the mod-
ulation of the intestinal microbiota. Ingestion of beneficial
microorganisms may have the potential to affect and improve
the body responses to some stressful conditions via a whole set
of direct and indirect actions, including the following:
1. Direct protection of intestinal barrier
2. Influence on local and systemic antioxidant status and
reduction in lipid peroxidation
3. Production of neurochemical compounds, such as GABA
4. Indirect influence on neurotransmitter or neuropeptide
production
5. Prevention of stress-induced changes in the intestinal
microbiota
6. Activation of neural pathways between the gut and brain
7. Reduced production of cytokine
8. Modulation of neurotrophic chemicals
Fermented Foods: Origins and Applications 679
9. Limitation of carbohydrate malabsorption
10. Improvement of nutritional status
11. Inhibition of gastric and intestinal pathogens
12. Limitation of overgrowth of bacterial growth in the small
intestine
13. Analgesic properties
Moreover, some placebo-controlled researches suggest that
beneficial microbes can influence mood and fatigue, decrease
anxiety and perception of stress, and improve mental outlook;
these effects could be linked to their ability to increase periph-
eral tryptophan levels and alter dopamine and serotonin
turnover in the frontal cortex and in the limbic system. Besides
the mere ingestion of beneficial microorganisms as such, fer-
mented foods can also display the ability to modulate the
qualitative composition of the intestinal microbiota. In 1986,
one of the first studies focusing on the effect of traditional
dietary patterns examined the differences in the fecal micro-
biota of rural Japanese versus Canadian urbanites. The
researchers found higher counts of Lactobacillus spp. and
Bifidobacterium spp. in the Japanese group that maintained a
traditional diet rich in fiber, fermented foods, fish, and vege-
tables. Several studies have confirmed these preliminary data
and have reported a high diversity of fecal microbiota in people
consuming fermented foods, whereby the improvement of
intestinal balance has been linked directly or indirectly to
brain functionality.
A final remark on the most important result on the effect of
fermentation on mental health: 3 years ago, some authors were
able to show a direct correlation between the administration of a
strain of Lb. pentosus var. plantarum (isolated from kimchi, a
traditional fermented cabbage) and hippocampal production
of brain-derived neurotrophic factor in mice with a protective
effect toward scopolamine-induced memory deficit. This result is
a challenge for the future, as it shows that the effects of fermented
foods on mental health are not hypothesis, but possibilities.
Concluding Remarks
Fermentation is one of the oldest method of food preservation;
moreover, the consumption of fermented foods affects both
well-being and cultural traits of life, as it is a part of religion,
society, and tradition all over the world. Apart from the effect
of fermentation on safety and quality of foods, a new trend is
the focus on the link between fermentation and mental health;
the expected outcomes of this frontier goal could be a
great threat and a challenge for future. We are only scratching
the surface in our understanding of the relationship between
potentially beneficial microbes and brain health; going inside
this phenomenon could lead to some interesting findings and
possibilities.
In conclusion, fermented foods are a significant part of our
life and culture, and we could presume that discovery,
optimization, and focus on fermentation were and will be
some leading factors of mankind evolution.
See also: Bacteriocins; Beer: History and Types; Bifidobacteria in
Foods: Health Effects; Bioactive Peptides in Foods; Biogenic Amines;
Cheese: Chemistry and Microbiology; Ethnic Foods; Fermented Foods:
680 Fermented Foods: Origins and Applications
Composition and Health Effects; Fermented Foods: Use of Starter
Cultures; Functional Foods; Lactic Acid Bacteria; Preservation of
Foods; Probiotics; Traditional Foods; Yeasts.
Further Reading
Bourdichon F, Casaregola S, Farrokh C, et al. (2012) Microorganisms with technological
beneficial use. International Journal of Food Microbiology 154: 87–97.
El-Mansi EMT, Bryce CFA, Dahhou B, Sanchez S, Demain AL, and Allman AR (2012)
Fermentation microbiology and biotechnology, 3rd ed. Boca Raton, FL: CRC Press/
Taylor & Francis Group.
Farnworth ER (2008) Handbook of fermented functional foods, 2nd ed. Boca Raton, FL:
CRC Press/Taylor & Francis Group.
Hutkins RW (2006) Microbiology and technology of fermented foods. Oxford: Blackwell
Publishing.
Jung IH, Jung MA, Kim EJ, Han MJ, and Kim DH (2012) Lactobacillus pentosus var.
plantarum C29 protects scopolamine-induced memory deficit in mice. Journal of
Applied Microbiology 113: 1498–1506.
Metha BM, Kamal-Eldin A, and Iwanski RZ (2012) Fermentation. Effects on food
properties. Boca Raton, FL: CRC Press/Taylor & Francis Group.
Montville TJ and Matthews KR (2008) Food microbiology. An introduction. New York:
ASM Press.
Parkar SG, Trower TM, and Stevenson DE (2013) Fecal microbial metabolism of
polyphenols and its effects on human gut microbiota. Anaerobe 23: 12–19.
Selhub EM, Logan AC, and Bested AC (2014) Fermented foods, microbiota and mental
health: ancient practice meets nutritional psychiatry. Journal of Physiological
Anthropology 33: 2.
Tamang JP and Kailasapathy K (2010) Fermented foods and beverages of the world.
Boca Raton, FL: CRC Press/Taylor & Francis Group.
Relevant Websites
http://www.culturesforhealth.com/lacto-fermentation-method-food-preservation.
http://www.fao.org/docrep/x0560e/x0560e07.htm.
http://www.nourishingdays.com/2009/07/the-benefits-of-fermented-food-lacto-
fermented-vegetables/.
http://www.npr.org/2012/06/13/154914381/fermentation-when-food-goes-bad-but-
stays-good.
 Fermented Foods: Use of Starter Cultures
PM Malo, Westat Research Corporation, Los Angeles, CA, USA
EA Urquhart, National Exposure Research Laboratory, Office of Research and Development, US EPA, Durham, NC, USA
ã 2016 Elsevier Ltd. All rights reserved.
Background
Fermentation has been used to extend the shelf life of foods for
over 6000 years. Fermented foods have played an important
role in the diets of almost every society throughout the world
and are known to offer a variety of benefits. While initially
utilized for food preservation, fermentation has numerous
benefits that include the improvement of sensory characteris-
tics, acceptability, nutritional value, and safety of foods while
also providing diversification to the diet. The number of
fermented food products is nearly limitless.
Fermentation is the anaerobic metabolism of carbohydrates
by microorganisms. The production of many frequently con-
sumed foods and beverage such as bread, cheese, yogurt, sauer-
kraut, beer, and wine involves fermentation. Numerous lesser
known fermented foods are used in cultural foods throughout
the world. The popularity of fermented foods is increasing due
to their health benefits and technological advancements that
allow for their production on an industrial scale.
A starter culture is a preparation of microbiological cultures
that performs or initiates fermentation. Starters usually consist
of a cultivation medium, such as grains, seeds, or nutrient
liquids that have been well colonized by the microorganisms
used for the fermentation. Initially, starter cultures needed to
be prepared just prior to use; today, they may be frozen and
lyophilized or dried and prepared on an industrial scale.
The choice of starter culture is dependent upon the substrate
or raw material being fermented. A starter culture may consist of
bacteria, molds, yeasts, or a combination thereof. Lactic acid
bacteria are of critical importance in fermentation. Desirable
properties of starter cultures include rapid acidification, predict-
able fermentation processes, desirable sensory characteristics
(including taste, texture, aroma, and consistency), and the reduc-
tion of harmful microbiota. The first isolation and cultivation of
starter cultures occurred in 1890 for cheese and sour milk in
Denmark and Germany. Modern technological advancements
have led to the development of functional starter cultures. A
functional starter culture provides at least one functional charac-
teristic to improve the food safety, nutrition, or quality of the food
product.
Purpose of Fermentation
Fermentation serves five primary purposes:
Preservation: Historically, fermentation has predominantly
been used as a method of food preservation. Fermentation
allows the preservation of substantial amounts of food
through lactic acid, alcohol, acetic acid, and alkaline
fermentations.
Diet enrichment: Fermentation enriches the diet through the
development of a diversity of flavors and textures in food
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00282-8
substrates. Examples of this include carbon dioxide produc-
tion by fermentation in leaven bread and the formation of
eyes in cheese or foam in beer and buttermilk.
Biological enrichment: Fermentation enables biological enrich-
ment of food substrates with protein, essential amino acids,
and vitamins.
Detoxification: Fermentation can reduce toxic or undesired
food components in foods, such as cyanide, phytic acid,
and oxalic acid.
Efficiency: Unlike other means of food preservation, fermenta-
tion decreases cooking times and cuts back on fuel
requirements.
Types of Fermentation
There are three primary types of fermentation most used in
traditional and industrial fermentation practices around the
world: lactic acid fermentation, alcohol fermentation, and
alkaline fermentation.
Lactic Acid Fermentation
Lactic acid fermentation, commonly referred to as lacto-
fermentation, is one of the most common and easiest methods
of home preservation. Lactic acid fermentation was a method
used to preserve dairy products, vegetables, and meat for
extended periods of time before the advent of refrigeration
and modern canning practices and today is also utilized in
industrial fermentation. Lactic acid bacteria such as Lactobacil-
lus spp., lactococci, Streptococcus thermophilus, and leuconostocs
are examples of lactic acid bacteria that have the ability to
convert sugars into lactic acid. Lactic acid inhibits the growth
of subsequent and potentially harmful bacteria of other
species. It also creates favorable conditions for yeast activity, a
property that is utilized in the production of wine and beer.
The chemical reaction of lactic acid fermentation can be
written as
C6 H12 O6 ! 2C3 H6 O3 þ 2CO2 þ 2ATP
Glucose ! lactic acid þ carbon dioxide þ energy
Alcohol Fermentation
Alcohol fermentation, also referred to as ethanol fermentation,
is the anaerobic process in which yeasts convert simple sugars
such as glucose, fructose, and sucrose into cellular energy
where ethanol and carbon dioxide are produced as metabolic
end products. While yeasts typically function under aerobic
conditions, they are also capable of anaerobic respiration, in
which sugars are metabolized in the absence of oxygen.
The net amount of alcoholic fermentation by yeast can be
written as
681
682 Fermented Foods: Use of Starter Cultures
C6 H12 O6 þ zymase ! 2C2 H5 OH þ 2CO2
Glucose þ zymase ! ethanol þ carbon dioxide
The first step in alcoholic fermentation involves the process
in which the enzyme invertase breaks the glycosidic linkage
between glucose and fructose molecules. Each glucose mole-
cule is then broken down into two pyruvate molecules via the
chemical process known as glycolysis. Glycolysis causes the
reduction of two NADþ molecules to NADH. Lastly, two ADP
molecules are converted to two adenosine triphosphate (ATP)
and two water molecules via substrate-level phosphorylation.
The last step uses the enzymes pyruvate decarboxylase and
alcoholic dehydrogenase. Zymase is the enzyme complex that
catalyzes ethanol fermentation.
Yeast fermentation
The most commonly used microorganism for food production
domestically is Saccharomyces cerevisiae, also known as ‘baker’s
yeast.’ This species was selected due to its ability to produce a
large volume of carbon dioxide and a pleasant flavor and to be
shelf-stable during storage. When mixed with bread dough, the
resulting product is sugar-ferment. Pure cultures of S. cerevisiae
are isolated and maintained in a laboratory and then inocu-
lated into a medium that often contains molasses and corn
steep liquor. (Corn steep liquor is a by-product of wet-milling
corn. It is a viscous substrate that contains carbon, nitrogen,
and mineral salts.) The inoculated medium is then incubated
and aerated, and yeast cells are removed by centrifugation and
then washed and mixed with starch or cornmeal. They can be
pressed into yeast cakes or freeze-dried to extend their viability.
Yeasts are common epiphytic residents on the surfaces of
fruits, vegetables, and cereals. It is the fermentation activity of
these yeasts that will lead to their eventual spoilage. Storage
and drying decrease the amount of yeast present. Dry flours can
also harbor yeasts, though less than whole grains (cereals) that
have not had their hull removed.
Alkaline Fermentation
Alkaline fermentation occurs when the protein in a food
is broken down into amino acids and peptides. During
alkaline fermentation, ammonia is released and pH increases.
Alkaline fermented food products are primarily found in Asia
and Africa. Examples include natto, Thai thua-nao (and
kinema–Thai thua-nao), kinema (made from cooked
soybeans), dawadawa (made from African locust beans),
ugba (made from African oil beans) kawal (made from fresh
legale leaves), and pidan (made from fresh poultry eggs).
Spontaneous cultures are most frequently used to make these
foods; however, pure cultures can also be used.
Combination Fermentation
Combination fermentation results from the presence of multi-
ple fermentative organisms. Mold-ripened cheeses and salami,
cocoa, vinegar, certain fermented milks, and sourdough bread
are examples of products that use multiple types of fermenta-
tion. Specifically, vinegar is produced using both yeast and
acetic acid bacteria. Sorghum beer in Africa is made through
lactic acid and alcoholic fermentation. Cocoa is made using
lactic acid bacteria, acetic acid bacteria, and yeast.
Traditional Versus Commercial Fermentation
The earliest record of fermentation dates as far back as 6000
BC, long before microorganisms were discovered. Nearly every
civilization since has included at least one form of fermented
food in its culinary heritage. In some cases, fermentation is a
critical component of food safety beyond preservation. Links
between food fermentation and human health have been
found as early as ancient Rome and China. As cultures and
times have changed over the centuries, so have the ways in
which we process and preserve foods through fermentation.
Traditional Fermentation and Starter Cultures
Fermentation can occur either through traditional methods or
via industrial production. Early methods of fermentation
occurred in small batches and involved spontaneous fermen-
tation from microorganisms present on raw goods (i.e., vege-
tables, fruits, milk, meat, and grains) under the proper
conditions. The outcome and taste of the fermented product
in traditional fermentation is dependent on the quality and
range of microbiota contained in the natural starter culture. For
other fermentation processes, inoculation is necessary and a
small sample of a previously fermented product is saved and
used as inoculate for new raw material. Referred to as back-
slopping, this method is still used today in the making of some
artisanal cheeses, kombucha, sourdough, and sauerkraut.
Commercial Fermentation and Starter Cultures
While methods of traditional fermentation are still being used
in various cultures today, the majority of fermented goods are
now produced through large-scale industrial techniques. With
the discovery of microorganisms, recent insight into their
genetics, metabolism, and interaction with raw material has
allowed for the ability to improve products and the process of
fermentation by using specifically designed and isolated starter
cultures. Bread, alcohol, and vinegar were among the first to be
‘produced’ from starter cultures in the nineteenth century, later
leading to the industrialization of the process. Dairy and meat
fermentation followed a century later. Today, manufacturers of
fermented foods can either use highly concentrated ready-to-
use starter cultures or propagate the starter culture in factory
production. The decision to use one starter culture method
over another depends on various factors including economic
value, the number of products produced, the degree of auto-
mated fermentation, and the level of microbiology expertise.
Variations in commercial starter cultures include direct vat
inoculates, frozen or freeze-dried cultures, or starter cultures
formulated with stabilizer and carrier genes. Lactic acid bacte-
ria constitute the majority of the volume and value of modern
starter cultures. A commercial culture starter with direct inoc-
ulation results in the highest level of safety and yield flexibility.
 Fermented Foods: Use of Starter Cultures 683

Fermented Foods and Starter Cultures
Due to the variation of raw materials, starter cultures, and
fermentation conditions, there are a wide array of fermented
food products available. Fermented products are often catego-
rized by the food category of the raw material. See Table 1.
lactic acid is acceptable to consume, lactic acid starter cultures
may contain milk.) With bean ferments such as soy products,
enzymes convert proteins into amino acids, which are more
digestible and readily assimilated by humans. Lastly, with
meats and fish products, bacterial and fungal enzymes help
tenderize foods.

Removal of Antinutritional Factors

Health Benefits
Fermented foods, as a group, are known to be highly
nutritious, and a vast number of health benefits associated
with consuming fermented foods are well documented.
Predigestion
As bacteria and yeast feed on the food in their immediate
environments, they release enzymes that break down mole-
cules into smaller ones, releasing energy for cellular metabolic
use in the form of ATP along the way. Thus, as the larger
polysaccharides and protein molecules that hold cells together
are degraded, large food particles are ultimately digested into
usable pieces. In this way, microorganisms partially digest and
physically break down food before it is consumed. Different
types of foods are metabolized and broken down in different
ways. With grains, gluten is cleaved to nonallergenic fragments.
With dairy, predigestion occurs as lactic acid bacteria convert
lactose into lactic acid. (For those with a milk allergy, while
Fermentation is known to remove a number of harmful and
toxic compounds from foods by several means of detoxifica-
tion. Most notably, fermentation increases the nutritive quality
of plant foods by metabolically reducing certain types of anti-
nutrients found in foods such as cyanide, phytic acid, and
oxalic acid. For example, the fermentation process that pro-
duces kawal, a Sudanese meat substitute, removes harmful
toxins from cassava leaves, ensuring them safe for human
consumption. Phytates, or phytic acids, are the phosphorus-
bound organic acids that protect grains, beans, nuts, and other
seeds from premature germination and are well-known anti-
nutrients that can cause mineral deficiencies and digestion
issues over time. Common health effects of a phytate-rich
diet include tooth decay, lack of appetite, digestive problems,
and nutrient deficiencies. For regular consumption of phytate-
containing foods in the absence of negative health effects, it is
necessary to remove the phytates and other antinutrients
through processing techniques such as sprouting, soaking,
and fermenting.

Table 1 Nonexhaustive list of types of fermented foods, including the final product, raw material, and type of starter culture

Food category Product Raw material Starter culture

Bean Tempeh Soybean Molds (Rhizopus oryzae or Rhizopus oligosporus)

Miso Soybean Koji (Aspergillus oryzae)
Natto Soybean Bacillus subtilis var. natto
Soy sauce Soybean Mold (Aspergillus), lactic acid bacteria
Grains/cereals Bread Various cereals Yeast, lactic acid bacteria
Injera Teff Lactic acid bacteria and yeast
Beer Various cereals Yeast (Saccharomyces)
Sorghum beer Sorghum Yeast (Saccharomyces cerevisiae and K. marxianus), lactic acid
bacteria
Sake Rice Lactic acid bacteria, yeasts (and occasionally mold)
Vegetables Kimchi Cabbage Lactic acid bacteria
Sauerkraut Cabbage Lactic acid bacteria
Pickled Cucumbers, olives, Lactic acid bacteria
vegetables and more
Fruits that underwent alcoholic Wine Grape juice Yeast, lactic acid bacteria
fermentation Hard cider Apple juice Yeast
Vinegar Grapes Acetic acid bacteria (Acetobacter)
Meat Fermented Sausage Lactic acid bacteria, catalase-positive cocci (and sometimes molds)
sausage
Dairy Yogurt Milk Lactic acid bacteria
Kefir Milk Lactic acid bacteria and yeast
Cheese Milk Lactic acid bacteria (and sometimes propionic acid bacteria,
brevibacteria, and molds)
Pima Milk Lactic acid bacteria
Tea Kombucha Tea Lactic acid bacteria and yeast
Honey Mead (honey Honey Yeast
wine)
Root Tanzanian Cassava Lactic acid bacteria
kivunde
684 Fermented Foods: Use of Starter Cultures
Probiotics and Gastrointestinal Immune Function
Probiotics refer to microorganisms that when ingested in ade-
quate amounts offer health benefits to the host and host phys-
iology. Lactic acid bacteria and bifidobacteria are thought to
promote healthy gut bacteria; this is why lacto-fermented
foods (fermented dairy foods such as yogurt and dietary sup-
plements) containing these bacteria are considered to have
probiotic characteristics. Research suggests that ingestion of
probiotic-containing ‘health’ and functional foods can alter
and improve the human gastrointestinal microflora and host
immunity. In the absence of mechanistic data, exactly how
particular bacterial species contributes to immune system func-
tion is not completely understood; however, improvements
may include nonspecific defenses against infection, regulation
of the balance of T-helper type 1 and T-helper type 2 cells, and
increased activity of white blood cells. Some probiotics have
been shown to produce antimicrobial compounds, affect
immune response or prevent autoimmunity, and/or alleviate
lactose intolerance in humans.
Enhancement of Micronutrients and Functional Compounds
During the process of predigestion, vitamins are produced and
excreted by the yeast and/or bacteria. In particular, fermenta-
tion can increase the amount of several vitamins (e.g., niacin,
thiamine, and biotin) in some plant and animal products. In
addition to increasing vitamins and their absorption availabil-
ity in humans, fermentation produces a unique array of micro-
nutrients or functional compounds not found in raw foods.
One example of this involves the phytochemical glucosinolate,
which is broken down into anticarcinogenic compounds as a
result of cabbage fermentation.
While the literature on the health benefits of fermented
foods continues to grow, there is still much to be understood.
For example, the understanding of how ingestion of live cul-
tures affects, mediates, and regulates physiological processes in
the human body is still elementary.
Risks of Fermented Food Consumption
Although the benefits of fermented food consumption have
been well documented, biogenic amine content, the possibility
of antibiotic resistance in fermented meat products, and the
uptake of ethanol and high sodium content are among the
risks associated with the overconsumption of fermented foods.
Biogenic Amines
While lactic acid bacteria produced during food fermentation
are typically considered to be nontoxic and nonpathogenic to
humans, some species of lactic acid bacteria have been found
to convert amino acids into potentially harmful amine-
containing compounds referred to as biogenic amines. Over-
consumption of foods containing a high level of amines is
known to have toxicological effects. High levels of biogenic
amines are found in a wide array of foods including fermented
food products (fish, meat, dairy products, and vegetables) and
fermented beverages (wine, beer, and cider). Of the many
biogenic amines, histamine and tyramine are thought to be
the most important amines occurring in fermented foods.
Minimizing or avoiding the presence of amine producing
organisms can be achieved through the use of fresh, hygienic
raw food materials and, if applicable, addition of microbial
controls. Furthermore, precautions should be taken against
organisms intended for use in starter cultures confirming non-
production of biogenic amines and the ability to withstand
production and storage conditions.
Antibiotic Resistance
The emergence and rise in antibiotic-resistant bacterial strains
in food products, specifically in meat products, is an ever-
increasing public health concern. Starter cultures help drive
fermentation in meat products by decreasing fermentation
time and improving the overall quality and safety of the prod-
ucts. However, bacteria used as starter cultures, including lactic
acid bacteria, are known to pose safety risks by way of trans-
mission of antimicrobial resistance genes. Fermented meat
meant for consumption is typically not heat-treated and thus
can serve as a direct route for antimicrobial-resistant bacterial
transmission from food to human gut microbiota. Studies have
found antibiotic resistance in lactic acid bacteria from meats
and meat products, with particular mention of strains involved
in sausage fermentation such as L. plantarum, L. curvatus, and
L. sakei.
Other Risks
As foods ferment, or decompose, certain waste by-products are
produced by the bacteria that break down our food. Of the
various by-products, alcohol is one by-product that can lead to
ethanol uptake due to overconsumption of fermented foods.
Fermented foods may also contain excess sodium and not be
appropriate for individuals on a low-sodium diet.
Future Possibilities
Biotechnology is advancing the understanding of fermentation
and starter cultures rapidly. Pangenomics, or the analysis of the
genetic sequences of members of the same species, is now
possible with the reduction in cost and time of DNA sequenc-
ing. Pangenomics compares complete genome sequences of
members within the same species. This opens vast new possi-
bilities to understand and improve industrial starter cultures,
which can help with strain screening, strain improvement,
safety, and process improvements. For example, thousands of
tons of commercial starter cultures are used every year to
produce cheese, yogurt, and other dairy products. Further
developments and understanding fermentation and starter cul-
ture technology will deepen our awareness of this fascinating
field.
See also: Fermented Foods: Composition and Health Effects;
Fermented Foods: Fermented Meat Products; Fermented Foods:
Fermented Milks; Fermented Foods: Fermented Vegetables and Other
Products; Fermented Foods: Origins and Applications.

Further Reading
Bachmann H, Pronk JT, Kleerebezem M, and Teusink B (2015) Evolutionary
engineering to enhance starter culture performance in food fermentations. Current
Opinion in Biotechnology 32: 1–7.
Capozzi V, Russo P, Dueñas MT, López P, and Spano G (2012) Lactic acid bacteria
producing B-group vitamins: a great potential for functional cereals products.
Applied Microbiology and Biotechnology 96(6): 1383–1394.
Cogan TM, Beresford TP, Steele J, Broadbent J, Shah NP, and Ustunol Z (2007) Advances
in starter cultures and cultured foods. Journal of Dairy Science 90(9): 4005–4021.
Ganzle MG (2009) From gene to function: metabolic traits of starter cultures for improved
quality of cereal foods. International Journal of Food Microbiology 134: 29–36.
Garrigues C, Johansen E, and Crittenden R (2013) Pangenomics – an avenue to
improved industrial starter cultures and probiotics. Current Opinion in
Biotechnology 24(2): 187–191.
Gullo M and Giudici P (2009) Acetic acid bacteria: taxonomy from early descriptions to
molecular techniques. In: Vinegars of the world, pp. 41–60. Italy: Springer-Verlag/
S.r.l. Milan.
Fermented Foods: Use of Starter Cultures 685
Hansen EB (2002) Commercial bacterial starter cultures for fermented foods of the
future. International Journal of Food Microbiology 78: 119–131.
Holzapfel WH (2002) Appropriate starter culture technologies for small-scale
fermentation in developing countries. International Journal of Food Microbiology
75(3): 197–212.
Hotz C and Gibson RS (2007) Traditional food-processing and preparation practices to
enhance the bioavailability of micronutrients in plant-based diets. Journal of
Nutrition 137(4): 1097–1100.
Katz S (2012) The art of fermentation: an in-depth exploration of essential concepts and
processes from around the world. White River, VT: Chelsea Green Publishing.
Leroy F and De Vuyst L (2004) Lactic acid bacteria as functional starter cultures for
the food fermentation industry. Trends in Food Science and Technology 15(2):
67–78.
National Center for Home Food Preservation (2014). Fermenting. Accessed December
20, 2014. http://nchfp.uga.edu/how/can6a_ferment.html.
Ng EW, Yeung M, and Tong PS (2011) Effects of yogurt starter cultures on the survival
of Lactobacillus acidophilus. International Journal of Food Microbiology
145: 169–175.
 Fish Oils: Composition and Health Effects
C Jacobsen, Technical University of Denmark, Lyngby, Denmark
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Fats and lipids constitute an important part of the daily diet,
and they have a number of essential biological functions.
Lipids have an important structural function in cell mem-
branes, they act as energy storage in the cell, they work as
signaling molecules, and some of them even act as a kind of
hormones. Lipids and fats constitute a broad range of mole-
cules among which triacylglycerols (TAGs) and phospholipids
are the most important in our diet. TAGs are composed of a
glycerol backbone on which three fatty acids are esterified.
Phospholipids can be divided into three classes: glyceropho-
spholipids, ether glycerolipids, and sphingophospholipids.
Glycerophospholipids represent the most widespread
phospholipid class, and they differ in their polar head groups
esterified to the sn-3 position of the glycerol backbone. Fatty
acids occupy the two other positions (sn-1 and sn-2) on the
glycerol backbone. Fatty acids can be classified into saturated,
monounsaturated, and polyunsaturated fatty acids (PUFAs),
depending on the number of double bonds in the fatty acid.
PUFA can be classified into omega-3 and omega-6 fatty acids,
which can be further subdivided into various subclasses.
Marine lipids are a major source of the two very long-chain
omega-3 fatty acids eicosapentaenoic acid (EPA; C20:5 n-3)
and docosahexaenoic acid (DHA; C22:6 n-3), while plant oils
such as flaxseed oil are a major source of alpha-linolenic acid
(ALA; C18:3 n-3), which has a shorter carbon chain and fewer
double bonds. The molecular structure of EPA and DHA is
shown in Figure 1. EPA and DHA are not naturally found in
plant oils. These two fatty acids have a wide range of well-
established health benefits including lowering of triglyceride
levels in the blood and reducing the risk of arrhythmia. ALA
can be converted to EPA and DHA in the human body, but for
people on a normal Western diet with a medium to high intake
of omega-6 fatty acids, this only happens to a low extent. The
conversion of ALA to EPA and DHA is particularly low in men.
This article will provide an overview of the major sources of
EPA and DHA and will also discuss the health benefits of
marine oils in the different stages of life.
Sources and Production
Sources of Fish Oils
Marine oils refer to fish body oils, fish liver oils, crustacean oils,
marine mammal oils, and cephalopod oils. Fish body oils are
the predominant product in this category representing almost
all of the production with small amounts of marine mammal
and squid oils. The fish body oils are sourced from pollack,
salmon, tuna, hoki, sand eel, mixed white fish, catfish, dogfish,
sprat, horse mackerel, sardines/pilchards, and mackerel,
among others. The fatty acid compositions of some of the
important fish body oils are shown in Table 1. These fish oils
686 Encyclopedia of Food and Health
are usually extracted from fatty fish in connection with the
production of fish meal. The fish that are processed to produce
crude fish oil (and fish meal) can usually be categorized as
follows:
(1) Offal and waste from the edible fisheries, for example,
cutting from filleting industry. The cuttings or trimmings
from the edible fisheries constitute
50% of the fish bio-
mass intended for food use. These by-products are com-
posed of 15% heads, 14% frames, 4% skin, and 17%
viscera (including the livers), and they represent a poten-
tial of about 42–44 million tons of waste. Only a portion
of that potential resource is currently utilized.
(2) Fish of a quality that is not high enough to make the fish
suitable for human consumption.
(3) Fish types that are not considered acceptable or aestheti-
cally pleasing for human consumption. The latter are
caught especially for reduction to fish meal and fish oil.
This category represents 15–20% of the landings or
approximately 16–21 million tons of fish. This raw mate-
rial, which is landed for direct production of marine oil
and fish meal, is 100% utilized.
In the EU, the so-called hygiene legislation requires that fish
oils intended for human consumption must be produced from
fish that are fit for human consumption, meaning that some of
the raw materials that have previously been used for fish oil
production for human consumption can no longer be used.
The predominant source of fish liver oils is cod liver. Other
sources are hake, halibut, and shark. Fish liver oils constitute
< 3% of the total production of fish oils.
The other species used for the production of marine oils
include krill, squid, and to a lesser extent marine mammals.
The main producers of marine oils are Japan, Scandinavia,
Chile, Peru, and Japan (Table 2). The most important coun-
tries in the ‘Others’ category include Russia, Vietnam, and
China.
The total annual world production of fish oil during the last
10 years has been
1–1.25 million tons. The annual produc-
tion in Chile and Peru and thereby also the total world pro-
duction are greatly affected by the El Niño events, which can
reduce production significantly.
Most of the fish oil is going into salmonid production in
Norway, Chile, Canada, and various European countries, but
an increasing proportion is being used for dietary supplements
and functional foods as will be discussed later. Due to the
growth in both aquaculture and direct human consumption
of fish oil, it is expected that the total demand for fish oil may
soon exceed the production. However, as already indicated
earlier, a large proportion of the cuttings and trimmings from
the fish industry are currently being wasted and could be
utilized for fish oil production. Moreover,
25–30 million
tons of fish is discarded annually and it represents another
potential source of fish oils.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00295-6
 Fish Oils: Composition and Health Effects 687

COOH
EPA (C20:5 (n-3))
H3C

COOH DHA (C22:6 (n-3))

H3C

Figure 1 Molecular structure of EPA and DHA.

Table 1 Sources of fish oil and their fatty acid compositions

Fish species

Sand Sardine/ Horse

Capelin Herring Norway pout Mackerel eel Menhaden pilchard mackerel Anchovy Sprat

Main sources Barents N. Atlantic N. Sea, N. Atlantic N. Sea US East Off S. S. Africa Off S. and N. Sea
Sea N. Sea, N. Atlantic, Pacific Coast, Africa, Pacific W.
N. Norwegian Barents Ocean, Gulf of Chile, coast of Africa,
Atlantic Sea, Sea N. Sea Mexico Peru, South Chile,
Pacific Japan, America Peru,
Ocean Atlantic and
coasts Mexico
of (Pacific
Canada coast)
and the
United
States
Fatty acids
14:0 7 7 6 8 7 9 8 8 9 –
16:0 10 16 13 14 15 20 18 18 19 16
16:1 10 6 5 7 8 12 10 8 9 7
18:1 14 13 14 13 9 11 13 11 13 16
20:1 17 13 11 12 15 1 4 5 5 10
22:1 14 20 12 15 16 0.2 3 8 2 14
20:5 8 5 8 7 9 14 18 13 17 6
22:6 6 6 13 8 9 8 9 10 9 9
Total 86 86 82 84 88 75 83 81 83 78
(principals)

Source: Allen, D. A. (1995). Fish oil compositions. In: Hamilton, R. J. and Rice, R. D. (eds.) Fish oil. Technology, nutrition and marketing. Wycombe: PJ Barnes & Associates,
pp. 95–108.

market for several years. Currently, DSM uses Schizochytrium

Other Sources of EPA and DHA
Anchovy and sardine oils are the major sources of EPA and
DHA products. These two species are currently responsible for
80% of the omega-3 products on the market. Fish liver oils,
salmon, tuna, algae, or single-cell oils, krill, and yeast account
for the remaining 20%. However, the production of EPA
and DHA from alternative sources such as microorganisms
and plants is expected to grow and is therefore further dis-
cussed in the succeeding text.
Several microorganisms are capable of producing EPA and
DHA including lower fungi, bacteria, and marine microalgae.
Marine microalgae are able to accumulate high amounts of
omega-3 PUFAs and they are therefore regarded as a very
promising source. Algal oil contains higher levels of DHA, for
example, up to 52% compared to fish oil.
Microalgae are cultivated either in photoautotrophic or in
heterotrophic production systems. EPA and DHA produced in
heterotrophic production systems have been available on the
sp. and Crypthecodinium cohnii for the production of DHA.
They have obtained novel food approval from the European
Commission for using the Schizochytrium sp. oil in products
such as dairy products, spreads, dressings, breakfast cereals,
and food supplements. The C. cohnii DHA oil is used in several
infant formula products. The advantage of the heterotrophic
production system is that it can take place in closed fermenta-
tion systems. In contrast, photoautotrophic algae have to be
produced in open systems as they require the presence of light.
If the production is carried out in closed photobioreactors, the
scale-up of the production is to some extent limited by the
ability to effectively introduce light. However, recent develop-
ment in photobioreactor technologies has reduced this
problem, and commercial EPA and DHA products produced
in photobioreactors or a combination of photobioreactors and
raceway ponds are available.
Genetically modified organism (GMO) plants that can pro-
duce EPA and DHA are an alternative source that is being
688 Fish Oils: Composition and Health Effects

Table 2 Global fish oil production in major countries, 10-year volume. The remaining 30.0% is consumed as dietary supple-
average ments and functional foods.
The most natural way of consuming EPA and DHA is of
1000 metric tons
course via fish consumption, and therefore, a few comments
EU 25a 48.32 4.76% on the contribution of fish consumption to EPA and DHA
Scandinavia 207.58 20.44% intake are given in the succeeding text. A single lean fish
Peru 274.25 27.00% meal, such as one serving of cod, could provide about
Chile 161.74 15.93% 0.2–0.3 g of EPA and DHA. On the other hand, a single oily
The United States 76.68 7.55% fish meal, such as one serving of salmon or mackerel, could
Japan 112.49 11.08% provide 1.5–3.0 g of these fatty acids. However, in most West-
Morocco 30.31 2.98% ern countries, the fish intake is low and the intake of lean fish is
Canada 5.15 0.51%
more common than of oily fish. For example, the median
Mexico 18.65 1.84%
intake of fish was only 16 g fish per day in Denmark during
Panama 8.81 0.87%
Ecuador 5.78 0.57% the period from 2003 to 2008. Among the Danes,
25% eat
S. Africa 5.55 0.55% < 5 g fish per day. Consequently, intakes of EPA and DHA
Others 60.27 5.93% among adults in several Western countries may be as low as
Total 1015.58 0.1–0.2 g day1. The International Society for the Study of
Fatty Acids and Lipids suggests a minimum intake of
a
Denmark and Sweden included in Scandinavia and not EU27. 500 mg day1. In Australia and New Zealand, the recom-
Source: Bimbo, A. (2013). Sources of omega-3 PUFA. In: Jacobsen, C., Horn, A. F.,
mended intake is 430–610 mg day1. Recently, the Food and
Sørensen, A.-D. M., Nielsen, N. S. (eds.) Food enrichment with omega-3 fatty acids.
Agriculture Organization (FAO) of the United Nations and the
Cambridge, England: Woodhead Publishing Ltd. pp. 27–107.
European Food Safety Authority (EFSA) recommended a min-
imum intake of 250 mg EPA þ DHA per day for adult males
intensively investigated these years. A major challenge is to and for nonpregnant/nonlactating adult females. For pregnant
obtain satisfactory yields. Commercial products are expected or lactating females, the minimum intake was recommended
to occur on the market within the next 5 years. The biotech to be 300 mg EPA þ DHA per day of which at least 200 mg
company Monsanto has developed a GMO soybean plant that should be DHA. They also made recommendations for DHA
can produce stearidonic acid (SDA) (C18:4 n-3). The advan- intake for infants aged up to 2 years. For children, the recom-
tage of this fatty acid is that it has one double bond more than mendations for EPA þ DHA in mg per day were 100–150 for
ALA. This means that SDA is more efficiently converted in the those aged 2–4 years, 150–200 for those aged 4–6 years, and
human body to EPA than current plant sources (ALA) because 200–250 for those aged 6–10 years.
it bypasses a step in the conversion process. The SDA oil is the
first step towards EPA and DHA from GMO plants.
Absorption and Metabolism

Patterns of Consumption
Among the different omega-3 fatty acids (EPA, DHA, and ALA),
marine oils were the most used form in 2007. It thus consti-
tuted ca.86% of the ingredients market. A consumer study
conducted in 2008 showed that 67.0% of the existing users of
omega-3 products identified marine oils as the most preferred
source of omega-3.
The marine oils were mainly consumed as dietary supple-
ments, that is, gelatin capsules and to a lower extent liquid
supplements. The total consumption of EPA- and DHA-rich
oils was
100 000 metric tons in 2010 and increased to
123 800 tons in 2013. It has been predicted that total global
human consumption will increase to 241 000 tons by 2020.
Among the marine oils, fish oils are consumed as triglycer-
ide oils (80% of total sale), low concentrates (2.3% of total
sale), middle concentrates (5.2% of total sale), and high
concentrates (2.6% of total sale).
As previously mentioned, commercial algal DHA oils are
now on the market. However, in 2007, algal DHA oils only
accounted for 1.3% of the volume share in omega-3 fatty acid
market. However, with more producers entering the market
and thereby increasing availability, the consumption of algal
oil is expected to increase. Algal oils are primarily used in infant
formulas as this application sector accounts for
70% of the
Absorption and metabolism of omega-3 fatty acids that have
been orally consumed follow the same routes as other dietary
fatty acids. In brief, they are hydrolyzed from the TAGs or
phospholipids, absorbed into enterocytes, and reesterified
back into TAGs that are assembled into chylomicrons (lipo-
proteins). The chylomicrons are released into the lymphatic
circulation and then enter the bloodstream. Omega-3 fatty
acids from chylomicron TAGs will primarily be stored in
adipose tissue, but a minor part of the omega-3 fatty acids
will be cleared by the liver. Here, they may be oxidized, metab-
olized to other omega-3 fatty acids, or resecreted into the
bloodstream.
Several studies have investigated whether EPA and DHA
from fish are more available than from fish oil supplements
in the form of TAGs or ethyl esters. In a recent study, the
bioavailability of EPA þ DHA (3.1–3.6 g day1) as reesterified
triacylglycerides, ethyl esters, or free fatty acids was compared
with that of natural fish oils (triacylglycerides) in a human
study. Omega-3 fatty acids were taken as capsules. EPA and
DHA contents were lowest in capsules with natural fish oils,
but this was adjusted for by the number of capsules taken per
day. The bioavailability was highest (124%) in the reesterified
triacylglycerides compared with natural fish oil, whereas the
bioavailability from ethyl esters was inferior (73%). Free fatty
acid bioavailability (91%) did not differ significantly from
 Fish Oils: Composition and Health Effects 689

natural fish oils. The availability of EPA and DHA from TAG
has also been compared with that from phospholipids.
Although several studies seem to suggest that EPA and DHA
may be more bioavailable when ingested from phospholipids,
more studies are needed to finally confirm this conclusion.
In the human body, different cells and tissues have different
fatty acid compositions. The EPA and particularly the DHA
contents differ significantly among these pools. EPA content
is usually lower than the DHA content. Tissues such as parts of
the brain and eye have a particularly high level of DHA. This is
due to the fact that DHA plays an important role in maintain-
ing a normal function of these tissues. Importantly, supple-
mentation with omega-3 PUFA results in the increased
amounts of EPA and DHA in plasma lipids, platelets, erythro-
cytes, leukocytes, colon tissue, cardiac tissue, etc. Several stud-
ies have reported an almost linear relationship between EPA
and DHA intake and the resulting EPA and DHA contents of
plasma phospholipids and of platelet phospholipids.
Health Effects
Mechanisms of Action
This section will discuss the health beneficial effects of EPA and
DHA in the different stages of human life. As an introduction,
the different mechanisms through which these fatty acids may
exert their effect will be reviewed. Figure 2 provides an over-
view of the most well-understood mechanisms, but more stud-
ies are needed to fully understand the mechanisms behind all
the known health beneficial effects of EPA and DHA.
Several studies suggest that the beneficial role of EPA on
inflammatory response and platelet aggregation is related to
eicosanoids produced in the body from EPA. Eicosanoids are
potent chemical messengers that have well-established roles in
the regulation of inflammation, immunity, platelet aggrega-
tion, smooth muscle contraction, and renal function. EPA is a
precursor of the 3-series prostanoids TXA3 and PGI3, whereas
the omega-6 fatty acid arachidonic acid is a precursor of TXA2
and PGI2. TXA2 and TXA3 are both prothrombotic, but TXA3 is
less prothrombotic than TXA2. In contrast, PGI2 and PGI3 are
equally antithrombotic. Hence, an improper balance between
omega-3 eicosanoids and omega-6 eicosanoids can lead to an
imbalance in inflammatory response and thrombosis.
EPA and DHA are also precursors for another group of
metabolites, namely, resolvins. These lipid mediators have
been demonstrated in cell culture and animal feeding studies
to have potent anti-inflammatory, inflammation resolving,
and immunomodulating effects. A higher intake of EPA and
DHA will lead to increased synthesis of resolvins.
The EPA and DHA content of membrane phospholipids
influences physical structure and fluidity of the membrane
and the structure and composition of important regions of
the membranes, which are called rafts. These rafts play a role
for receptor action and for the initiation of intracellular signal-
ing pathways. Together, the fluidity of the membrane and raft
structure influences the activity of membrane proteins includ-
ing receptors, transporters, ion channels, and signaling
enzymes. In turn, intracellular signal transduction and tran-
scription factor activation may be altered and gene expression
modified. This may affect the regulation of inflammatory

Increased oral intake of

EPA and DHA

Increased content of EPA and DHA

In cell membrane phospholipids

Altered physical state of Altered signal

membranes and altered transduction
Altered
raft formation and function Altered pattern of pathways
lipid mediators produced gene expression
(eicosanoids, resolvins)

Altered cell phenotype & function

Altered physiology and improved health

Figure 2 Overview of underlying mechanisms behind health beneficial effects of EPA and DHA. Reproduced from Calder, P. C. (2012). Mechanisms of
action of (n-3) fatty acids. The Journal of Nutrition 142, 592S–599S.
690 Fish Oils: Composition and Health Effects
processes, the regulation of lipid metabolism, and insulin
sensitivity.
The anti-inflammatory effects of EPA and DHA may also be
related to their binding to the membrane G protein-coupled
receptor called GPR120, which is a lipid-sensing receptor
highly expressed in proinflammatory macrophages. When
macrophages are stimulated to activate their proinflammatory
pathways, this can be inhibited by pretreatment with EPA and
DHA via GPR120. A similar receptor dependency for the
insulin-sensitizing, anti-inflammatory effects of EPA and
DHA was observed in mice deficient in the GPR120 gene.
This suggests that GPR120 is the functional receptor/sensor
for these effects of omega-3 PUFA. The same signaling system
functions in the hypothalamus. This results in decreased food
intake, weight reduction, and improved insulin sensitivity.
Effects of EPA and DHA during Pregnancy and Lactation
PUFAs are preferentially transferred to the fetus at the expense
of the mother’s own physiological stores. This preferential
transfer is termed biomagnification. Maternal arachidonic
acid is preferentially consumed by the fetus primarily in early
pregnancy followed by DHA in the third trimester and post-
partum during lactation. DHA is important for the develop-
ment of the nervous system including the brain of the fetus.
The primary rationale for supplying sufficient intakes of
omega-3 PUFA for mothers during pregnancy is therefore to
support the optimal health and brain development of the baby
during the neonatal period as well as providing for the health
and physiological needs of the mother. Maternal omega-3
PUFA intake under the present dietary conditions seems to be
inadequate to keep up with the increased demand for omega-3
PUFA during pregnancy. Therefore, it has been suggested that
pregnant women should increase their intake of DHA and that
infant formulas for both preterm and term infants should
contain DHA. Infant formulas with DHA are now available in
several countries.
As previously mentioned, high levels of DHA are needed in
the brain and retina (eye) for optimal neuronal functioning
(learning ability and memory) and visual acuity. Therefore,
high levels of DHA in breast milk are necessary in order to
ensure proper development of these functions in the infant.
The amount of DHA in breast milk is dependent on the intake
of the mother during lactation. Hence, consumption of DHA
provides a fairly rapid and marked improvement in the DHA
level found in breast milk.
Effects of EPA and DHA during Infancy
The body of literature suggests that long-chain PUFAs are
important to the growth and development of infants. Fleith
and Clandinin concluded in a review that a target DHA level of
0.4% of total fat for preterm infants is desirable and that
slightly higher levels than necessary for term infants may be
needed. Several studies have demonstrated that addition of
omega-3 PUFA to infant formula results in better performances
in various tests for visual acuity, mental development, and
motor activity including body control, large muscle coordina-
tion, manipulatory skills of the hands and fingers, and ability
to recognize objects by touch.
Effects of EPA and DHA during Childhood
Unfortunately, there are limited reports on the relationship
between fatty acid consumption and health outcomes in chil-
dren >2 years. One study found that toddlers consuming
formula with 130 mg DHA per day for 60 days had fewer
adverse events (e.g., strep throat, cough, and upper respiratory
infection) and a lower incidence of respiratory illness com-
pared with toddlers consuming the formula without DHA.
Other studies of the impact of maternal fish or omega-3 con-
sumption on childhood outcomes have not been able to dem-
onstrate any effects. The relationship between cognitive
development of 4-year-old children and their DHA levels has
recently been studied, and no association between umbilical
venous plasma or red blood cell phospholipid DHA and ara-
chidonic acid levels and cognitive outcomes was observed.
There are methodological limitations of the existing random-
ized controlled trials and epidemiological studies. These limi-
tations include the challenges in quantifying fatty acid
exposure and measuring neurodevelopmental outcomes.
Therefore, more studies are required to understand the impor-
tance of omega-3 intake during childhood.
Effects of EPA and DHA during Adulthood
In adults, low intake of omega-3 PUFAs has been associated
with many disease states, including cardiovascular, inflamma-
tory/autoimmune, neurodevelopmental, and psychiatric
disorders.
Several studies have demonstrated a beneficial effect of
omega-3 PUFA intake on fatal and nonfatal myocardial infarc-
tion. An overall decrease of risk of suffering a cardiovascular
event of any kind of 10%, a decrease of risk of cardiac death of
9%, and a decrease of coronary events (fatal or nonfatal) of
18% upon intake of omega-3 PUFA have been reported, and it
was concluded that marine omega-3 PUFAs are effective in
preventing cardiovascular events, cardiac death, and coronary
events, particularly in persons with high cardiovascular risk.
Moreover, marine omega-3 PUFA may positively affect coro-
nary disease risk factors by decreasing plasma TAG levels,
improving platelet function, and lowering blood viscosity.
Omega-3 PUFA has also been found to reduce incidence of
joint tenderness and morning stiffness in patients suffering
from rheumatoid arthritis. Intake of omega-3 PUFA also
appears to offer some protection against asthma, cystic fibrosis,
and Crohn’s disease. These diseases are all related to a mal-
functioning immune system.
The ability of omega-3 PUFA to prevent and treat cancer has
been much discussed. A recent review concluded that omega-3
fatty acids are likely to have multifaceted roles in both preven-
tion and treatment of colorectal cancer and other cancers. A
review by Gerber was less conclusive. Thus, it concluded that
there is only limited evidence from observational studies on
colorectal, prostate, and breast cancers on a possible role of
omega-3 fatty acids in cancer prevention. Nonetheless, the
author stated that there is an increasing biological plausibility
of the beneficial anti-inflammatory effect of omega-3 fatty
acids on cancers.
Several studies have investigated the role of DHA in
Alzheimer’s, schizophrenia, and mood disorders. A review by

Hegarty and Parker found that studies investigating the efficacy
of omega-3 fatty acid supplementation for mood disorders
have provided inconsistent results. They concluded that more
research is required before omega-3 supplementation can be
firmly recommended as an effective treatment for mood
disorders. It has been reported that both arachidonic acid
and DHA levels were low in the brain of patients with
schizophrenia but that there is only limited evidence that
supplements may reduce symptoms. Despite the limited level
of evidence for the potential preventative effects of omega-3
fatty acids in mental diseases, the role of DHA in brain func-
tion is recognized and a cause and effect relationship has been
established as concluded by the EFSA. This also means that
health claims on the role of DHA in brain function are allowed.
Foods bearing such a claim should contain 250 mg DHA in
one or more servings. EFSA has recognized a similar role of
DHA in maintaining normal vision, and also, in this case,
foods must contain 250 mg DHA in one or more servings to
bear the claim (EFSA).
Negative effects of omega-3 PUFA have also been reported.
In a recent case–cohort study on 834 men diagnosed with
prostate cancer, of which 156 had high-grade cancer, it was
found that when compared with men in the lowest quartiles of
blood plasma EPA, DPA, and DHA, men in the highest quartile
had increased risk of low-grade and high-grade prostate cancer.
The reported increased risk of low-grade prostate cancer was
44%, and the reported increased risk for high-grade prostate
cancer was 71% for the highest quartile blood plasma omega-3
PUFA group. However, this study has been criticized for not
being specifically designed to look at the exact relationship
between omega-3 fatty acid intake and prostate cancer as it
was an observational study and not a cause/effect study.
Another criticism was that plasma phospholipid fatty acids,
as measured in this study, are not a good index for long-term
intake and can be influenced dramatically by a single meal or
even the timing of a fish oil supplement. Moreover, there was
no standardization of the omega-3 PUFA quality or quantity
consumed by the study subjects. Some were dietary and some
were supplementation. Therefore, more research is needed to
further study if high intakes of EPA and DHA can promote
prostate cancer.
Effects of EPA and DHA during Aging
Studies investigating the role of omega-3 PUFA in healthy
older adults are limited. However, there are studies showing
that elderly people who consume higher levels of fish as a
source of DHA/EPA exhibited a slower rate of decline in their
cognitive scoring with age. Other studies have shown that
aging individuals who utilized fish oil supplementation
appeared to show improved retention of their cognitive func-
tioning in old age. Studies in elderly individuals (65–98 years
of age), who were free of known cerebrovascular disease at
baseline, exhibited a significantly lower risk of developing a
stroke during 12 years of follow-up with increased fish con-
sumption as a source of DHA/EPA. The lowest risk (by 30%)
was found in those aging individuals who consumed fish at
least five times per week. Moreover, omega-3 PUFAs have been
demonstrated to inhibit hepatic triglyceride synthesis, decrease
inflammation, and inhibit platelet aggregation in the elderly.
Fish Oils: Composition and Health Effects 691
Another study suggested that elderly women may be more
sensitive to the immunologic properties of omega-3 fatty
acids. They found that fish oil supplementation (2.4 g day1
for 3 months) reduced interleukin (IL)-6 in young
women (aged 23–33 years) by 30% and in older women
(aged 51–68 years) by 60%. Total IL-1b synthesis was reduced
by 48% in young women in comparison to 90% in older
women, whereas tumor necrosis factor was reduced by 58%
in young and 70% in older women.
Conclusions and Future Trends
Consumer awareness about health beneficial effects of omega-
3 PUFA is increasing, and this is expected to lead to increasing
consumption and thereby increasing demands for marine oils
in the coming years. The production of fish oils from tradi-
tional resources has stagnated, and therefore, increasing supply
must come from new resources such as by-products and dis-
cards from the fish industry as well as from microalgae, bacte-
ria, yeast, and GMO plants.
The health benefits from omega-3 PUFA are many and in
several cases well documented. However, more studies are
needed to obtain more documentation for possible beneficial
effects on the prevention of diseases such as mental disorders
and cancer particularly in healthy subjects. Likewise, more
research is needed to understand the long-term effects of
omega-3 PUFA during pregnancy, infancy, and childhood.
See also: Fatty Acids: Determination and Requirements; Fatty Acids:
Essential Fatty Acids; Fatty Acids: Fatty Acids; Fatty Acids: Metabolism;
Fish: Fish in the Human Diet; Fish Oils: Production and Properties;
Functional Foods; Infants: Nutritional Requirements; Lipoproteins;
Shellfish: Characteristics of Crustaceans and Mollusks.
Further Reading
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Bajpai P, Bajpai PK, and Ward OP (1991) Production of docosahexaenoic acid by
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Polyunsaturated fatty acids and inflammatory bowel disease. The American Journal
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Bimbo A (2013) Sources of omega-3 PUFA. In: Jacobsen C, Horn AF, Sørensen A-DM,
and Nielsen NS (eds.) Food enrichment with omega-3 fatty acids, pp. 27–107.
Cambridge: Woodhead Publishing.
Birch EE, Garfield S, Hoffman DR, Uauy R, and Birch DG (2000) A randomized
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42: 174–181.
Birch EE, Castaneda YS, Wheaton DH, Birch DG, Uauy R, and Hoffman DR (2005)
Visual maturation of term infants fed long-chain polyunsaturated fatty acid-
supplemented or control formula for 12 mo. The American Journal of Clinical
Nutrition 81: 871–879.
692 Fish Oils: Composition and Health Effects
Blonk MC, Bilo HJ, Popp-Snijders C, Mulder C, and Donker AJ (1990) Dose–response
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Calder PC (2012) Mechanisms of action of (n-3) fatty acids. The Journal of Nutrition
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the treatment and prevention of colorectal cancer. Gut 61: 135–149.
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prenatal intake of n-3 polyunsaturated fatty acids and cognitive development.
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EFSA Panel on Biological Hazards (BIOHAZ) (2010) Scientific opinion on fish oil for
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Relevant Websites
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www.omega-3centre.com/ – Omega-3 Centre.
 Fish Oils: Production and Properties
AK Carvajal and R Mozuraityte, SINTEF Fisheries and Aquaculture, Trondheim, Norway
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
The total world fisheries and aquaculture production was 158
million tons in 2012, of which 136.2 tons were used for
human consumption and 21.7 million tons of the catch and
aquaculture production was used for fish oil and fish meal
production. The global fish oil production is estimated to be
about 1 million tons per year (0.980 tons in 2012 according to
FAO). Out of the estimated 1 million tons of fish oil, about
70 000 tons is estimated to be used for human consumption.
Peru and Chile are the largest producers of fish oil (around
295 000 tons in 2012), followed by Norway and Denmark
(140 000 tons in 2012) and Iceland (around 176 tons in
2012). Norway is the largest global importer of fish oil
(approximately 200 000 tons) followed by Chile, United
Kingdom, and Canada.
Marine Sources for Fish Oil Production
Marine sources used for fish oil and fish meal production can
be divided into four categories:
(1) Fish caught especially for the production of oil and meal.
This includes species such as anchovy, jack mackerel, cap-
elin, menhaden, and blue whiting. By-catches from other
fisheries can also be included in this category.
(2) By-products/rest raw material from the processing industry
(both fisheries and aquaculture).
(3) Cod liver (from Gadus morhua L. or other gadidae species).
(4) Other marine resources such as seal, krill, micro algae, etc.
Raw Material Quality
The quality of the fish oil depends on the sorting, storage, and
handling of the raw material. Fish rest raw material is especially
vulnerable to spoilage and degradation because it contains
fractions like viscera and blood with high amounts of endog-
enous enzymes. Lipases and phospholipases will lead to for-
mation of free fatty acids (FFAs) and reduction in quality and
stability of the lipids. Prolonged storage of the rest raw material
before processing has been shown to lead to an increased
concentration of FFA, thus lowering the quality of oil produced
from herring and pollock rest raw material. Proteolytic
enzymes will cause a reduction in the molecular weight of
protein, thus reducing the functional properties. Rest raw
materials are also susceptible to microbial spoilage and it is
important that they are handled as a raw material intended for
human consumption. To ensure high quality oil, it is impor-
tant to process the raw material within a short time.
There are some regulations for the raw material for fish oil if
it is going to be used for human consumption or feed. If the
whole fish is going to be used for fish oil production for
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00294-4
human consumption, the freshness criteria are based on the
total volatile basic nitrogen (TVB-N), which shall not exceed
60 mg of nitrogen/100 g.
Storage and transport must be carried out under hygienic
conditions by using tanks of stainless and mild steel construc-
tion. The catch has to be chilled within 24 h. Fishing of pelagic
species used for fish oil and meal production is mainly done by
purse seiners and the fish is stored in bulk in refrigerated sea
water (RSW) tanks at
1.5  C on-board. Whole fish being
processed within 36 h must have an acceptable freshness cri-
teria based on TVB-N. Rest raw material may be used directly
after production at the slaughter lines to produce oil or may be
chilled and transported in containers to the production site.
Organic acids can be added to the rest raw material to conserve
the raw material before further processing (production of
silage). However, the use of non-food-grade acids will result
in fish oils that can be used in feed and not for human
consumption.
Fish Oil Production
Fish oil and fish meal are traditionally produced by the wet
rendering method. The first step is mincing of the raw material
(whole fish or by-products), followed by cooking. The cooked
material is separated into different fractions, resulting in pro-
duction of crude fish oil and fish meal. A simplified scheme of
the oil and meal production is given in Figure 1.
Cooking
Minced whole fish or by-products are transferred to a contin-
uous cooker and steam heated to 90–95  C for approximately
10–20 min. The heating will cause protein coagulation and
rupture of fat depots, resulting in liberation of oil and water.
The cooker usually consists of a long, steam-jacket cylinder
where the raw material is moved by a heated rotary screw
conveyer. The procedure normally takes around 20 min; how-
ever, if a scraped surface heat exchanger is used, the heating
time can be reduced to less than 2 min.
Pressing
After cooking, the heated material is transferred to a screw press
where the liquid (‘press liquor’) is squeezed from the solid
phase (‘press cake’) in order to remove as much liquid as
possible. Optimal pressing will lead to a higher oil yield and
a lower amount of oil in the press cake; thus, a fish meal with
low oil content. The press cake liquid consists of water, dis-
solved material, and oil, while the press cake consists of
693
694 Fish Oils: Production and Properties
Whole fish or
cut-offs from fish
Cooking
Pressing Press cake
Press Liquor
Solids Removal Solids
Oil-water Seperation Stick water
Crude fish oil
Figure 1 Simplified scheme of oil and meal production.
60–80% of the oil free dry matter. The press cake is further
processed into fish meal.
Separation of Press Liquid
Before the oil and water is separated, the press liquid is passed
over a vibrating screen (with 5–6 mm perforation) to remove
unwanted coarse particles of fish and bones. Different proces-
sing solutions based on the fractions’ different specific gravity
can be used for separation of the press liquid (oil, water, and
remaining solids). First, a decanter (horizontal centrifuge) can
be used to remove fine suspended solids from the liquor in
order to obtain an oil and water phase suitable for further
separation. The oil and water phase is transferred to a centri-
fuge for separation into oil and stick water (water soluble
components). The oil is pumped to a polishing centrifuge
while the stick water is transferred to the evaporator.
An alternative separation technique is to use a tri-canter
which enables the separation of three fractions (sludge, oil, and
stick water) at the same time. However, industry experience
indicates that it is difficult to produce three high quality fractions
and in many cases one of the fractions has to be downgraded in
order to produce the other high quality fractions.
Oil Polishing
A polishing centrifuge can be used to remove the final traces of
moisture and impurities in the oil. This reduces the amount of
Dryer Cooler
Grinder
Fish meal
Evaporator
pro-oxidants in the oil, thus increasing the oil stability during
storage. After polishing, the oil is pumped into storage tanks.
The produced oil is called crude fish oil.
Drying of Press Cake
Sludge separated from the press liquor is mixed with the press
cake together with the concentrated stick water. The press cake
is then dried to reduce the moisture content to a level low
enough to hinder microbial growth. Two types of driers are
used in the fish meal industry: direct and indirect driers. In
direct driers, the press cake is in direct contact with the hot
gases, while in indirect driers the heat is transferred to the cake
through a retaining wall. Indirect driers are mainly used in the
fish oil industry.
Enzymatic Protein Hydrolysis
New processes for production of oil based on enzymatic
protein hydrolysis are being developed and applied in the
industry. Enzymatic protein hydrolysis is based on the use
of commercial proteases for proteolytical cleavage of peptide
bonds, facilitating the degradation of fish tissue and release of
oil. The advantages of using enzymatic hydrolysis compared
to the wet rendering method could be a protein product with
better biological, digestible, and functional properties. In
addition, the use of lower cooking temperature (50–60  C)
compared to the wet rendering method (90–95  C) can

give an oil of higher quality (low oxidation status) and
stability.
For processing of whole fish and by-products by enzy-
matic hydrolysis, a hydrolysis stage has to be implemented
after the material is minced. The reaction can take place in
either a tank reactor or a screw-mixing pipe reactor. Heated
minced raw material is transferred to a hydrolysis tank and
mixed with water (usually in a 1:1 ratio) and commercial
proteolytical enzymes (0.1–1 wt% of raw material weight).
The reaction proceeds for 30–90 min (hydrolysis time),
followed by enzyme inactivation at 90  C for several
minutes.
Several factors can influence the hydrolysis process: sub-
strate type and properties, enzyme type and properties, and
processing conditions including temperature, pH, hydrolysis
time, and amount of added water. These factors are important
for product yield and quality and need to be controlled during
processing.
Krill Oil Production
The krill oil industry has developed and optimized the produc-
tion process for krill, resulting in different processing steps and
end products (oil, meal, pasta, etc.). Processing details are
often confidential and little is published in the literature. The
most common process is to pump live krill onboard the factory
trawler were the krill is frozen or directly processed into krill
meal. The dried meal can be transported to shore and used to
extract krill oil by solvent extraction.
Production of Refined Fish Oil
Crude fish oils contain minor amounts of water and unwanted
substances as phospholipids, mono- and diacylglycerols,
FFAs, cholestereol, proteinaceous compounds, trace metals,
pigments, and primary and secondary oxidation products.
The crude oil needs to go through different refining steps
before it can be used for human consumption. The general
objective of refining of oils is the removal of impurities and
unwanted products which cause the crude oil to have an unat-
tractive color or taste or can cause harmful metabolic effects.
The refining process can include the following steps:
• Neutralization by addition of citric acid or caustic soda:
removal of mainly FFAs, but also oxidation products, phos-
pholipids, pigments, trace metals, and residual soaps
• Washing with hot water: removal of water-soluble material,
oxidation products, trace metals, and soaps
• Winterization
• Bleaching by using bleaching earth and activated carbon:
removal of pigments, peroxides, phospholipids, soaps,
trace metals, sulfur compounds, persistent organic pollut-
ants (POPs), polycyclic aromatic hydrocarbons (PAHs),
retinol, tocopherol, and astaxanthin
• Deodorization (steam stripping): removal of volatile com-
pounds, aldehydes, ketones, POPs, FFAs and pigment
decomposition products
Fish Oils: Production and Properties 695
• Short path distillation (SPD): further reduction of POPs,
removal of cholestereol, primary and secondary oxidation
products, FFAs, and polymeric materials
Which refining steps are included and in which order can vary
between the different fish oils processors.
Neutralization
Neutralization is carried out to remove FFAs (lowering the oils’
acid value) and reduce the content of phospholipids,
pigments, and trace metals. FFAs can cause darkening of the
oil, foaming, and possible smoke during heating. During
neutralization, the oil is mixed with caustic soda, resulting in
the formation of soaps that can be separated from the crude
oil. The oil is heated to the optimum processing temperature of
80–95  C before caustic soda is added. Hot water is then added
to the solution, mixed, and the soapy wash water is removed by
using a separator. Washing with water will also remove any
water-soluble components present in the oil such as decompo-
sition products of proteins, free amino acids, trimethyl amines
and histidine, oxidation products, and phospholipids.
Winterization
Winterization is carried out to reduce the amount of high
melting point triacylglycerols and waxes in the crude oil. The
process is carried out in tanks with inert conditions and the oil
is slowly chilled to 0–2  C allowing the formation of triacyl-
glycerol crystals with higher melting points to settle and be
removed by filtration. The winterization process prevents
clouding of the oil at refrigeration temperatures.
Bleaching
The bleaching step removes pigments, but will also further
reduce the content of FFAs, residual trace metals, and oxidation
products (peroxides) that are left in oil after neutralization. In
addition, the process will also remove soaps and sulfur-
containing materials. Bleaching by using activated carbon
reduces the residual traces of environmental pollutants such
as dioxins, furans, and PAHs. However, the process will also
reduce the content of desirable natural antioxidants present in
the oil as retinol, tocopherol, cholecalciferol, and astaxanthin.
The process is carried out by adding bleaching earth and/or
activated carbon to the oil through a dosing unit. It is per-
formed at 130–150  C for 30–60 min under vacuum.
Deodorization
The deodorization step is carried out to remove as much as
possible of the unwanted volatile compounds (secondary oxi-
dation products such as aldehydes, ketones, etc.) that are
responsible for the unwanted odor and taste of the oil. How-
ever, possible nonvolatile secondary and tertiary oxidation
products that are formed during processing will not be removed
696 Fish Oils: Production and Properties
by deodorization. The oil is steamed under vacuum at a high
temperature (150–250  C) to remove the volatiles. The process
is also called stripping, desorption, or steam distillation when
oil is mixed with stripping gas to facilitate the mass transfer of
the volatiles to the gas phase. The gas phase is continuously
removed from the liquid phase. Deodorization should be car-
ried out at a maximum temperature of 180  C to reduce the risk
for polymerization and geometrical isomerization.
Molecular (Short Path) Distillation
An additional step can be added to the refining line to reduce
trace amounts of POPs such as dioxins, PCBs, and furans. The
use of short path distillation will also lead to the reduction of
odor compounds, FFAs, oxidation products, and polymers
formed during decompostion/oxidation of the oil. Short path
distillation is also used in the production of omega-3-acid
ethyl esters.
Fish Oil Properties
Physical Properties
Many of the physical properties of fish oil are dependent on the
composition of fatty acids and on the nature of their incorpo-
ration into acylglycerols. Density is one of the characteristic
that determines quality of the oil and is usually in the range
920–930 kg m
3. Density increases as the mean molecular
weight decreases (i.e., with higher saponification values) and
the degree of unsaturation increases. Fish oil is a liquid above
10  C and can solidify below this temperature. The viscosity of
fish oils at 20  C ranges between 60 and 90 cP and decreases
exponentially with increasing temperature, the regression
equation being 121e
0.028T where T is in the range of
20–90  C.
Fish color can vary depending on the species of fish used
and processing conditions.
Chemical Properties
Fish oil is usually made up of triacylglycerols, which often
account for more than 95% of the lipids in the oil. The char-
acteristic of a specific triacylglycerol is dependent on the fatty
acids composition. However, some fish oils, for example, shark
oil, can comprise 40–50% ether linked glycerides. Some fish
species can have wax-esters as dominant storage lipids. Wax
ester–rich oils generally contain lower levels of omega-3 LC
PUFA.
Fatty Acid Composition
The fatty acid composition of fish oils is complex and variable.
The composition of fish oils in terms of fatty acids is usually
determined by chromatographic methods. The fatty acids tend
to contain an even number of carbon atoms and double bonds
are of the ‘cis’ configuration.
The most abundant fatty acids in fish oils are: 14:0 –
myristic; 16:0 – palmitic; 16:1 n
7 – palmitoleic; 18:1 –
oleic acid, 20:1 n
9 gondoic, 22:1 n
11 – cetoleic, 20:5
n
3 – eicosapentaenoic and 22:6 n
3 – docosahexaenoic
fatty acids. The triacylglycerols consist of fatty acids esterified
at three different positions on a glycerol molecule. This distri-
bution of fatty acids in the glycerol molecule is more or less
unique for different types of natural fat. An indication of
approximate composition of various fish oils are given in
Table 1. In most of the fish oils the omega-3 LC PUFAs, mostly
EPA and DHA, generally make up 10–35% of the total fatty
acids. Fatty acids such as 20:1 and 22:1 are present in signifi-
cant parts in oils produced from fish such as herring, mackerel,
and capelin, caught in northern waters.
Other Chemical Constituent
Fish body and liver oils contain other chemical components
such as vitamins. Vitamin D tends to concentrate in the liver of
fish. Therefore, body oils contain less vitamin D compared to
liver oils. The content of vitamin D depends on the species, age,
size, sex, nutritional conditions, and spawning state of the fish.
The liver oils also contain vitamin A. Vitamin E is not synthe-
sized by fish; therefore, vitamin E content is dependent on the
fish diet. The most abundant form of vitamin E is alpha
tocopherol with the concentration being higher in the liver
than body oils. The level of tocopherol in fish oil is reduced
during refining and deodorization and generally less than
100 ppm is found.
Oxidative Stability
Because fish oils usually contain long chain polyunsaturated
fatty acids, they are highly susceptible to oxidation. In addition
to fatty acid composition, the oxidative stability of oils also
depends on the amount of prooxidants and antioxidants in the
oils and storage conditions such as temperature, light, and
oxygen availability. Anoxic production and storage of oils pre-
vent against oxidation. Generally, each 10  C decrease in stor-
ing temperature doubles the shelf-life of the oil. Trace amounts
of prooxidants (catalysts of lipid oxidation) such as transition
metals are usually present in fish oils. Therefore, in order to
reduce oxidation, antioxidants that inactivate metals or lipid
radicals are added to the fish oils. Encapsulation of n-3 fatty
acids reduces the contact between lipids and oxygen and
contributes to protection against oxidation. Packaging in mate-
rial nonpermeable for light and oxygen also contributes to
protection against oxidation.
Lipid oxidation is a dynamic process creating a complex
mixture of primary, secondary, and tertiary products. Because
several products are intermediate; they are formed and then
broken down again during marine oil processing, and there is
not always a linear increase in the oxidation-products with
time of oxidation.
The oxidative state of oil is defined as the sum of all oxida-
tion products present. Traditionally, an estimate of primary
(peroxide value) and secondary products (anisidine value) is
applied.
PV analysis usually is performed using the iodometric titra-
tion method—that is, the one referred to both by Ph. Eur.
 Fish Oils: Production and Properties 697

Table 1 Composition of major fatty acids of selected fish

Fatty Percentage composition

acid
Farmed Atlantic Jack Atlantic cod Atlantic South American Sardine/
salmona Tunaa mackerela liverb menhadenb anchovyb pilchardc Capelinc Herringc

14:0 4.2 3.9 7.3 3.3 7.3 7.5 8 7 7

16:0 15.7 17.6 15.7 13.4 19 17.5 18 10 16
18:0 4.2 4.1 3.1 2.7 4.2 4 nd nd nd
16:1 5.1 5.4 5.1 9.6 9.1 9 10 10 6
n
7
18:1 16.5 12.4 9.9 23.4 13.2 11.6 11 14 13
n
9
18:1 3.5 2.4 2.9 nd nd nd nd nd nd
n
7
20:1 3.3 1.3 8.3 7.8 2 1.6 4 17 12
n
9
22:1 2.5 0.5 5.8 5.3 0.6 1.2 3 14 20
18:2 6.6 1.9 1.7 1.4 1.3 1.2 nd nd nd
n
6
20:5 7.1 12.4 10.9 11.5 11 17 18 8 5
n
3
22:6 15.7 27.8 11.5 12.6 9.1 8.8 9 6 6
n
3
22:5 3.9 1.7 2 1.6 1.9 1.6 nd nd nd
n
3
a
Nichols (2007).
b
Ackman (2006).
c
Bimbo (1990).

(Monograph 2.5.5 Method A) and by GOED (AOCS procedure Table 2 Chemical properties of crude oils
Cd 8–53).
Anisidine value (AV) determines the amount of aldehydes Quality guidelines for crude oils
(secondary oxidation products) formed as a result of break-
Moisture and impurities (%) Usual basis 0.5 up to maximum of 1
down of lipid peroxides in the oils. The method is based on the Fatty acids (% oleic acids) Range 1–7, usually 2–5%
color reaction between the aldehydic compound and the Peroxide value (mequiv. kg
1) 3–20
p-anisidine (AOCS procedure Cd 7–58, Ph.Eur. Monograph Anisidine value 4–60
2.5.36). TOTOX (2PV þ AV) 10–60
Oxidative stability of oils is usually difficult to determine. The Iodine value 95–200 (depending on fish source)
effect of different antioxidants on the oxidative stability of fish Color (Gardner scale) Up to 14
oils can be determined by accelerated oxidative stability methods Iron (ppm) 0.5–7.0
such as the oxidative stability index (OSI). The OSI method is an Copper (ppm) Less than 0.7
Phosphorus (ppm) 5–100
American Oil Chemist Society (AOCS) approved method that
determines the relative resistance of fat and oil samples to oxi-
dation. The instrument measures the conductivity increase in
deionized water as a result of the secondary products’ formation
during the oxidation of the oil. The Schaal oven test is another material. This will influence the properties of the oils in regard
accelerated method for determining the stability of lipids and the to edible properties and technical applications. No standard
effect of antioxidants. The test is based on the measurement of quality criteria are set for the crude oils, but an overview of the
weight gain of lipid samples over time at a chosen temperature. typical chemical and physical properties of the crude oils has
The increase in the weight is a result of oxygen binding to the been reported by Bimbo in ‘Guidelines for Characterizing
unsaturated fatty acids (i.e., peroxidation). Active antioxidants Food-grade Fish Oil’ shown in Table 2. Several tests are used
will delay the onset of peroxidation and, therefore, the weight to evaluate the quality, moisture content, and impurities. The
gain, compared to a control (no antioxidants). quality criteria for crude oils are mainly set by the buyer of the
oils depending on their range of application; feed, nutraceuti-
cals, functional foods, or pharmaceuticals.
According to the European Food Safety Authority (EFSA), it
Quality Criteria for Marine Oils for Human Consumption is difficult to give an exact recommendation for the maximum
tolerable PV and AV levels in crude oils because no literature is
The chemical composition and quality of crude oils depend on available on the relationship between oxidation values in the
both the production process and the quality of the raw crude oil and in the final oil. However, the quality of the crude
698 Fish Oils: Production and Properties

Table 3 Recommended criteria by the European Pharmacopeia (Ph.Eu) and the Global Organization for EPA and DHA (GOED) for oxidation status in
refined marine oils intended for human consumption
a b
Ph.Eur.7.0 (2011) (maximum values) GOED monograph (2006) (maximum values)
c d e f
Type of oil Peroxide value Anisidine value Peroxide value Anisidine value

Omega-3 rich fish oil (type 1) 10 30 5 20

Omega-3 rich fish oil (type 2)g 5 15
Omega-3-acid triglycerides 10 30
Omega-3-acid ethyl esters 60 10 20
Omega-3-acid ethyl esters 90 10 20 None None

oil will decide the range of application and how extensive the
refining that is required.
At present, no adequate international, European, or national
legislation standard for origin, quality, and/or composition of
marine oil for human consumption is available. The European
Pharmacopeia (Ph.Eur) has developed standards with quality
criteria for some types of refined marine oils intended for
human consumption; cod liver oil, farmed salmon oil, omega-
3 rich fish oils, omega-3-acid triglycerides, omega-3-acid ethyl
esters 60, and omega-3-acid ethyl esters 90. In addition, both the
Ph.Eur and the Global organization for EPA and DHA have
prepared recommendations for the evaluation of oxidation sta-
tus in refined fish oils and set maximum values for the quality
parameters. An overview is given in Table 3.
See also: Fish: Processing; Fish Oils: Composition and Health Effects.
Further Reading
Ackman RG (2006) Marin lipids and omega-3 fatty acids. In: Akoh CC (ed.) Handbook
of functional lipids, pp. 311–324. Boca Raton, FL: CRC Press, Taylor and Francis
Group.
Aidos I, Kreb N, Boonman M, Luten JB, Boom RM, and Padt A (2003) Influence of
production process parameters on fish oil quality in a pilot plant. Journal of Food
Science 68: 581–586.
Bimbo AP (2007) Processing of marine oils. In: Breivik H (ed.) Long chain omega-3
specialty oils, pp. 77–109. The Oily Press Bridgewater England.
Bimbo AP (1990) Guidelines for characterizing food-grade fish oil. INFORM 9(5):
473–483.
Carvajal, A. K., Slizyte, R., Storrø, I., Aursand, M. Production of high quality fish oil by
thermal treatment and enzymatic hydrolysis from fresh Norwegian spring spawning
herring by-products. Journal of Aquatic Food Product Technology, in press,
doi:10.1080/10498850.2013.814740.
Carvajal AK, Mozuraityte R, Standal IB, Storrø I, and Aursand M (2014) Antioxidants in
fish oil production for improved quality. JAOCS, Journal of the American Oil
Chemists’ Society 91: 1611–1621.
Dijkstra AJ and Segers JC (2007) Production and refining of oils and fats.
In: Harwood JL and Dijkstra AJ (eds.) The lipid handbook. Boca Raton: CRC Press.
FAO Globefish Quarterly Update April 2014 – Fishmeal and Fish Oil.
Hamm W (2009) Processing of fish oils. In: Rossell B (ed.) Fish oils, pp. 81–98.
Blackwell Publishing Ltd.
Jacobsen C, Rustad T, Nielsen NS, Falch E, Jansson S, and Storrø I (2009) Processing
of marine lipids and factors affecting their quality when used for functional foods.
In: Luten J (ed.) Marine functional foods, pp. 89–114. Wageningen, The
Netherlands: Wageningen Academic Publishers.
Nichols PD (2007) Fish oil sources. In: Brevik H (ed.) Long-chain omega-3 specialty
oils, pp. 23–42. England: Oily Press, PJ Barnes & Associates, Bridgewater.
Rubio-Rodrı́guez N, Beltrán S, Jaime I, de Diego SM, Sanz MT, and Carballido JR
(2010) Production of omega-3 polyunsaturated fatty acid concentrates: a review.
Innovative Food Science & Emerging Technologies 11: 1–12.
Shahidi F and Wanasundara UN (1998) Omega-3 fatty acid concentrates: nutritional
aspects and production technologies. Trends in Food Science & Technology
9: 230–240.
Xu X (2005) Short-path distillation for lipid processing. In: Akoh CC and Lai O-M (eds.)
Healthful lipids. Urbana, IL: AOCS Press.
Xu X, Kittikun AK, and Zhang H (2007) Enzymatic processing of omega-3 specialty oils.
In: Breivik H (ed.) Long chain omega-3 specialty oils, pp. 141–164. England: The
Oily Press Bridgewater.
Relevant Websites
http://www.fao.org/docrep/003/x6899e/x6899e04.htm.
http://lipidlibrary.aocs.org/processing/marine/index.htm.
 Fish: Dietary Importance and Health Effects
HK Mæhre, I-J Jensen, and K-E Eilertsen, UiT The Arctic University of Norway, Tromsø, Norway
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
According to the Food and Agriculture Organization (FAO) of
the United Nations, the worldwide production of seafood
reached 149 million metric tons (MMT) in 2010. This produc-
tion was divided between capture fisheries at 89 MMT and
aquaculture at 60 MMT, and 128 MMT was utilized for
human consumption.
Fish consumption varies greatly between countries and is
influenced by several factors. Availability in forms of produc-
tion (capture and aquaculture) and trade (imports and
exports) is often the main variable. Factors affecting availability
within different countries are geographic location and political
conditions. Production is also largely dependent on climatic
conditions, as extreme weather conditions such as El Niño
affect both capture fisheries and the production of aquaculture
feeds. The consumption pattern is also influenced by cultural
variables, such as religion and dietary traditions.
Statistics from the FAO show that the general fish consump-
tion in the world has increased over the last 50 years, from 9.0
to 17.8 kg capita
1 year
1. This increase is recognizable in
most countries. However, the variation between countries is
large. For instance, in countries with no coastline, such as
Mongolia, the average fish consumption is 0.4 kg capita
1
year
1, while island states, like Faroe Islands, have an average
yearly consumption of 87.7 kg capita
1.
Most of the literature concerning fish consumption does
not distinguish between fish and other seafood, such as crus-
taceans and mollusks. In the following, unless otherwise spec-
ified, fish and other seafoods are hence treated as one.
Composition and Nutritional Advantages
The biodiversity and compositional variety among species
make it difficult to conclude on uniform nutritional character-
istics valid for all types of fish. In the following, the nutrients
particularly associated with fish consumption will be
emphasized.
Lipids and Fatty Acids
Lipids are the main energy storage in the human body and also
important constituents of brain tissue and other biological
membranes. Fatty acids are built up of a carbon (C) chain of
typically 4–22 C-atoms, with a methyl group at the proximal
end and a carboxyl group at the distal end. The C-atoms are
bound together by single or double bonds and the fatty acids
are termed thereafter as saturated (zero double bonds), mono-
unsaturated (one double bond), or polyunsaturated (two or
more double bonds) fatty acids. The ‘omega’ indication refers
to the location of the first double bond counting from the
methyl end of the C-chain. If the first double bond is located
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00291-9
between C-atoms 3 and 4, the fatty acid is an omega-3 fatty
acid, and if the first double bond is located between C-atoms 6
and 7, it is an omega-6 FA. Two fatty acids are essential to
humans, that is, they cannot be synthesized de novo in the
body. These are linoleic acid (LA, 18:2n
6) and a-linolenic
acid (ALA, 18:3n
3). These fatty acids are not crucial per se
but are metabolized through a series of chain elongation and
desaturation to the very long arachidonic acid (ARA, 20:4n –
6), eicosapentaenoic acid (EPA, 20:5n – 3), and docosahexae-
noic acid (DHA, 22:6n
3). The two former act as precursors
to fatty acid hormones, called eicosanoids, while the latter has
been proven essential for brain development. Depending on
whether the eicosanoids originate from omega-3 or omega-6
FA, they have distinctly different qualities. In general, eicosa-
noids derived from ARA are proinflammatory and pro-
thrombotic, whereas eicosanoids derived from EPA are less
inflammatory and even anti-inflammatory. The optimal die-
tary ratio between omega-6 and omega-3 fatty acids is 2–5/1,
depending on the disease in question. The change in diet
towards more processed foods with high intake of omega-6
vegetable oils has shifted the omega-6/omega-3 ratio of the
current western diet to 15–17/1. LA and ALA are competing
substrates for the enzymatic endogenous synthesis of n
6 and
n
3 long-chain PUFAs. The high dietary content of LA results
in a more predominant endogenous production of ARA at the
sacrifice of EPA and DHA. Hence, the only way for humans to
achieve sufficient amount of EPA and DHA is directly through
the diet. Fish contain high amounts of EPA and DHA due to
direct transfer up the food chain from phytoplankton that
produce them de novo. A daily intake between 200 and
500 mg EPA and DHA is usually recommended. This may be
achieved by eating fish, preferably fatty fish twice a week. It is
also recommended that energy from fat should be around 30%
and that saturated fat should contribute to not more than 10%
of the total energy. Fish is divided into three groups depending
on their fat content: Lean fish, such as cod and saithe/pollock,
have fat content less than 2%; medium fatty fish, such as
catfish, redfish, and tuna, have a fat content between 2 and
8%; and fatty fish, such as salmon, herring, and mackerel, have
a fat content higher than 8%.
Proteins
Fish was initially regarded as a healthy food choice because it is
a low-fat protein source. Nonetheless, in the last half century,
the positive health effects of seafood have generally been asso-
ciated with the marine omega-3 fatty acids EPA and DHA,
leaving the benefits of proteins and peptides disregarded. Pro-
teins are molecules consisting of one or several polypeptide
chains composed of amino acids bound together by peptide
bounds. Twenty different amino acids constitute proteins, and
of these, nine are essential to humans. The essential amino
acids are histidine, isoleucine, leucine, lysine, methionine,
699
700 Fish: Dietary Importance and Health Effects
phenylalanine, threonine, tryptophan, and valine. The quality
of a protein source is determined from the extent it meets our
requirement of essential amino acids, regarding both the
amount of essential amino acids and the extent we can absorb
and utilize them. Fish contains 12–24% protein depending on
species. The proteins contain all essential amino acids in suffi-
cient amounts to cover our daily requirement, and fish is
therefore recognized as a complete protein source. The digest-
ibility of fish protein is excellent, and the lower content of
connective tissue indicates that fish protein is more easily
digestible compared to meat protein from terrestrial animals.
Carbohydrates
Together with fat and protein, carbohydrates are the third
dietary source of energy. Fish contains almost no carbohy-
drates, while crustaceans and mollusks contain only small
amounts. Seafood is therefore not a source of carbohydrates.
Vitamins and Minerals
Vitamins and minerals are also essential nutrients, although
not for energy. The daily requirements of these components are
hence substantially smaller than those of the energy-providing
nutrients. Herein, the vitamins and minerals abundant in fish
will be discussed. Fish is an important source of the water-
soluble vitamin B12. In addition, fatty fish is also rich in the
lipid-soluble vitamins A and D. Fish is a good source of several
minerals, and whereas only iodine and selenium are more
abundant in fish than in terrestrial animals, fish may also be
an adequate source of other minerals such as zinc, iron,
magnesium, and calcium.
Vitamin A
Vitamin A is a collective term including compounds with bio-
logical effect as retinol. Carotenoids are provitamins as they are
transformed into retinol in the body. Vitamin A is important in
fetal development and for several functions in the body such as
reproduction, vision, skin, immune system, and epithelial
integrity. In Western countries, vitamin A deficiency is rare,
whereas it is a major problem in developing countries. The
earliest sign of vitamin A deficiency is reduced growth and
night blindness. Recommended daily intakes are presented as
retinol activity equivalents (RAE), where 1 RAE equals 1 mg
retinol, and are 900 mg for men and 700 mg for nonpregnant
and nonlactating women. While eggs and carrots are the main
sources of vitamin A, fatty fish, particularly fish liver and fish
oils, are also good sources. Fish contain vitamin A in its retinol
form that is more easily absorbed than the carotenoids.
Vitamin D
Vitamin D is crucial in development and maintenance of the
bone tissue and skeleton by ensuring adequate concentrations
of calcium and phosphate in plasma and extracellular tissue.
Deficiency of vitamin D can lead to low calcium and phos-
phate concentration and softening of the bones known as
rickets in infants and osteomalacia in adults. Humans synthe-
size vitamin D when exposed to sunlight, but in northern
latitudes, the synthesis is inadequate and additional intake
through diet is necessary. The current daily recommendation
of vitamin D is 10 mg per day. Good dietary sources of vitamin
D are fish liver oil, fatty fish, and fortified foods. Vitamin D
content in fish varies greatly, but a portion of fatty fish such as
herring or mackerel will ensure the recommended daily intake.
Vitamin B12
Vitamin B12, or cobalamin, is one of the eight vitamins in the
B-group. It has a key role in normal brain functioning and
nervous system and in blood formation. The most common
deficiency symptom is hence anemia. A special variety of ane-
mia, called pernicious anemia, is the result of a hereditary
condition where lack of intrinsic factor leads to reduced uptake
of B12. The daily requirement of B12 is low, around 2 mg, and
one portion of 100 g fish will generally cover the recom-
mended daily intake. In addition, most fish also contain
some amounts of the other B vitamins.
Iodine
Iodine is a trace element essential in the synthesis of the
thyroid hormones detrimental for growth and metabolism.
Deficiency of iodine is the largest deficiency disease worldwide,
causing goiter (thyroid enlargement), growth inhibition, and
mental retardation (cretinism). The recommended daily intake
of iodine is 150 mg. Fish, especially marine fish, are good
sources of iodine and one portion of cod will cover the daily
recommendations. Furthermore, iodine is the simplest micro-
nutrient to fortify and is usually added to dietary salt.
Selenium
Selenium is another trace mineral essential for humans. Being
a constituent of the antioxidative enzyme class glutathione
peroxidases (GPx), it is regarded as one of the most important
endogenous antioxidants. In addition, selenium is central in
the thyroid hormone regulation, and a diet deficient in sele-
nium may therefore lead to, and even intensify, some of the
deficiency characteristics seen in iodine deficiencies. The
recommended daily intake is 40–50 mg. Seafood is recognized
as a good source of selenium, especially in areas where the soil
is depleted of this trace mineral.
Bioactive components
In recent years, an increased focus has been directed towards
food components with beneficial health effects beyond that of
nutritional value, namely, bioactive compounds. The bioactiv-
ity in question may be linked to a vast range of physiological
conditions, such as oxidative stress, hypertension, cancer,
obesity, and infections. Likewise, the compounds may possess
very different chemical properties. Some of the compounds
and bioactivities related to fish will be discussed further.
Peptides
Peptides are parts of proteins, ranging from two to several
amino acids. The bioactivity is based on the sequence and
composition of typically up to 30 amino acids occasionally
occurring as free peptide in foods, but commonly within their
parent proteins. Within proteins, side groups of amino acids
are buried and the peptides have no bioactivity. Hence, they
need to be released through fermentation, food processing,
proteolysis, or gastrointestinal digestion to exert any activity.
Several bioactivities have been linked to fish peptides,

although three main activities are well documented, namely,
antihypertensive effect, antioxidative activity, and antiobesity
effect.
Taurine
Taurine (2-aminoethanesulfonic acid) is the end product of
the sulfuric amino acid metabolism, and although it contains a
sulfonic group instead of a carboxyl group, it is considered as
an amino acid. However, it is not bound in proteins, but exists
as an exclusively free amino acid in most human tissues, the
concentrations being especially high in the brain, retina, skel-
etal muscles, heart, and leukocytes. Its participation in several
physiological processes, such as osmoregulation, neuro- and
immunomodulation, and bile salt formation, has been well
documented. In addition, together with its precursors methio-
nine and cysteine, it is involved in protection from oxidative
stress reactions. Lately, several reviews have discussed the rela-
tionship between taurine and several other diseases, such as
cardiovascular disease (CVD), hypertension, and diabetes.
The endogenous synthesis of taurine is dependent on a
series of enzymes and the efficiency of the production is
species-dependent. In humans, it is quite inefficient and in
certain situations, the endogenous production may be inade-
quate. Taurine is therefore characterized as a conditionally
essential amino acid that should be provided through dietary
intake. The main dietary source of taurine is seafood, levels
being higher in mollusks and crustaceans than in fish.
Other bioactive components
Other bioactive components frequently found in foods are
ubiquinone, carotenoids, polyphenols, and phytosterols. Of
these, carotenoids are considered important in relation to con-
sumption of seafood. Carotenoids are a class of antioxidative
compounds, with some chemical resemblance to vitamin A,
and some of them may be chemically converted to this vita-
min. The most abundant carotenoid in seafood, especially in
crustaceans, is astaxanthin. This compound is produced by
microalgae as a defense towards UV light and may be found
in decreasing concentrations upward in the marine food web.
Within the fish, it acts as an endogenous antioxidant, protect-
ing it from autoxidation, and it is also believed to possess
similar protective effects in humans. Phytosterols are
cholesterol-like compounds abundant in plant materials and
to a certain degree in mollusks. In fish, these compounds have
traditionally not been present. However, in line with the
increasing inclusion of plant materials in aquaculture feeds
during the last years, an increasing amount of phytosterols
has been detected in farmed fish.
Health Effects of Fish Consumption
Numerous studies have been performed in order to evaluate a
potential causal relationship between fish consumption and
chronic diseases. Such studies make up the foundation of
evidence-based decisions and are combined by the world’s
large health organizations in order to establish official dietary
advice for consumption of fish. There are two main approaches
when studying this field, epidemiological and experimental
studies, and there are advantages and disadvantages associated
Fish: Dietary Importance and Health Effects 701
with both study types. A combination of studies will probably
return the most solid foundation for advice.
In brief, in epidemiological studies, human populations are
studied in order to describe and explain health and disease.
The main advantages of these studies are that they range over a
long period of time, the data material is very large, there are few
restrictions in diet, and diseases can be included as end point.
The main drawbacks are that they are poorly controlled and
that the sources of error are many. Among epidemiological
studies, several subtypes are defined: descriptive studies, eco-
logical studies, migrant studies, case–control studies, cohort
studies, randomized controlled studies, and meta-analysis.
Experimental studies are performed in a more controlled
environment than are epidemiological studies. The test partic-
ipants can either be human volunteers, but most experimental
studies are performed in animals (in vivo) or in cell-based
systems using human or animal cells grown in the laboratory
(in vitro). There are also subtypes of experimental studies:
human feeding studies, live animal models, in vitro studies,
and studies of biological pathways.
The world’s large health organizations, such as the World
Health Organization (WHO), the World Cancer Research Fund
(WCRF), the American Heart Association, and the Academy of
Nutrition and Dietetics, have defined different categories
describing the causal relationship between exposure/intake
and morbidity, and each category has different demands of
the strength of documentation. There are slight differences in
the definitions of the criteria between the various organiza-
tions, but the strength of documentation is the same. The main
categories used by WHO and WCRF ‘Convincing relationship,’
‘Probable relationship,’ ‘Limited relationship – suggested,’
‘Limited relationship – no conclusion,’ and ‘Substantial effect
on risk unlikely.’
The evidence-based documentation is often presented in
matrices, one for each food type and disease group. Potential
harmful effects/risks on intake of food are also included. The
associations presented in the matrices are documented rela-
tionships and meet the demands of convincing relationship or
probable relationship. Inclusion of evidence from more than
one study type, no substantial unexplained heterogeneity
within or between studies and control of confounding factors,
and random or systematic errors are among the qualifications
to be met in order to end up in one of these two categories.
Cardiovascular Diseases
The vast majority of studies regarding fish consumption and
chronic diseases are related to CVDs. CVD is a collective term
encompassing a group of disorders of the heart and blood
vessels and is the largest cause of morbidity and a major
cause of premature death worldwide. The two most frequent
disorders are coronary heart disease and cerebrovascular dis-
ease (stroke), affecting blood vessels of the heart and brain,
respectively.
There are several risk factors associated with the develop-
ment of CVDs. Some are nonmodifiable, such as age, gender,
and heredity. Yet others are modifiable, such as smoking,
hypertension, unfavorable blood lipid composition, physical
inactivity, obesity/overweight, and diabetes mellitus. The latter
ones are recognized as lifestyle-related risk factors and may be
702 Fish: Dietary Importance and Health Effects
influenced by dietary habits. Concurrent presence of several
risk factors is common and certain risk factors may in combi-
nation increase the risk of CVD considerably. Although there
are several independent risk factors for the development of
CVDs, causal relationship between their presence and actual
disease has not been established for most of them.
CVD and Fish Intake
The vast majority of studies regarding fish intake and health
benefits have been focused on their content of long-chain
omega-3 fatty acids. An increased intake of these fatty acids,
especially EPA, will lead to replacement of ARA in cell mem-
branes and red blood cells. As previously mentioned, eicosa-
noids may be formed from both ARA and EPA. However, the
properties of different eicosanoids are substantially different
depending on the originating fatty acid. As mentioned previ-
ously, eicosanoids formed from ARA are proinflammatory and
prothrombotic, whereas eicosanoids formed from EPA are less
potent and show anti-inflammatory and antithrombotic prop-
erties. This may have impact on the atherosclerotic process. In
addition, an increased intake of LC-PUFAs has been shown to
improve the stability of plaques.
It is well documented that a high intake of fish reduces the
level of triglycerides in the blood and, hence, attenuate the
effects of dyslipidemia. The effects on blood cholesterol levels
are not as consistent.
The relationship between the intake of LC-PUFAs and
blood pressure has also been studied, but also here, the results
are inconclusive. Some studies imply that hypertensive patients
may benefit from a high intake of these fatty acids, while the
same effect is not seen in persons within the normal range of
blood pressure. Several fish species are also considered good
sources of potassium that is essential for water and electrolyte
balance and the normal functioning of neurons and other cells.
Dietary potassium may lead to loss of sodium, and increased
dietary intake of potassium has been associated with a decrease
in blood pressure.
However, intake of fish may affect blood pressure through
yet another mechanism, that is, via peptides released through
gastrointestinal digestion. Numerous in vitro studies and ani-
mal models have shown that fish peptides exhibit blood
pressure-reducing effects by inhibition of ACE. A few human
studies have confirmed these findings.
Fish Intake and Other Chronic Conditions
Although not as frequent as for CVDs, studies on the correla-
tion between fish intake and other positive health effects have
also been performed. Among the studied effects are cognitive
development and disorders in later life, cancer, allergy, and
other immunologic conditions, along with lifestyle-related
conditions, such as type 2 diabetes. Due to large heterogeneity
between studies, the conclusions regarding these conditions
are not as convincing as for CVDs, but some of the main
suggestions and findings will be presented here.
Cognitive disorders: Development and later life
The fatty acid DHA is thought to be of great importance for
neurodevelopment and most studies regarding this subject are
focused on this fatty acid. However, the number of potential
confounders in studies regarding cognitive development in
early life is substantial. This makes it extremely challenging to
draw conclusions from such studies. Despite this, most studies
conclude that a high maternal intake of fish is beneficial for
overall cognitive development of the offspring and that neither
maternal nor postnatal supplementation with long-chained
n
3 fatty acids provides additional effects.
When cognitive decline and dementia, including
Alzheimer’s disease, have been investigated in relation to fish
and n
3 intake, the results are also inconclusive. In some
studies, there seem to be a trend, although not significant,
that fish intake, but not n
3 supplementation, may offer a
certain protection against dementia. Other studies find no
correlation at all.
Cancer
Lifestyle and dietary habits have been related to several types of
cancer, in particular the cancers of the gastrointestinal system.
As a natural result of this, the relationship between fish intake
and these cancers has been examined. For most of these cancer
forms, there is no convincing or probable relationship associ-
ated with fish intake.
Allergy and immunologic reactions
Also asthma, eczema, and other atopic conditions in children
have been investigated in relation to both maternal fish intake
and fish intake during the first year. Although the results are
inconclusive, eczema seems to be the only condition where
fish intake may have positive effects. Both maternal fish intake
and early introduction of fish during the first year seem to
positively affect the risk of this condition.
Type 2 diabetes
Type 2 diabetes is recognized as a lifestyle-related disease. As
dietary habits are a part of the factors related to the develop-
ment of these diseases, the intake of several food items, includ-
ing fish, has been investigated in order to discover potential
correlations. So far, the number of studies regarding fish intake
and type 2 diabetes is small and the heterogeneity between
them is large, and hence, no correlation has yet been identified.
Adverse Health Effects
Increased fish consumption has long been recommended due
to the well-documented health benefits. Nevertheless, fish as
other foods can accumulate environmental pollutants and
heavy metals that may reduce the overall nutritional value
and alter consumer’s health. In order to lower the risk of such
detrimental effects, the Joint WHO/FAO Expert Committee on
Food Additives has compiled guidelines and set limit values for
assumed safe intakes of most contaminants. These limits,
termed tolerable weekly intakes (TWI), are based on experi-
mental data and equal the amount of a compound that may be
consumed weekly during the total life span of a human being
without risk of adverse health effects. In cases where experi-
mental data are insufficient or lacking, a provisional tolerable
weekly intake (PTWI) is set. The unit of both TWI and PTWI is
mg or mg kg
1 body weight (BW) per week.

The contaminants most relevant for fish consumers are
heavy metals and persistent organic pollutants (POPs). Patho-
gens and marine toxins can undoubtedly result in negative
health effects. These risks may however be eliminated through
proper cooking and are therefore not discussed here.
Heavy Metals
One definition of heavy metals is that they are elements with
atomic weights ranging from 63.5 to 200.5 and specific gravi-
ties greater than 4.0. Small amounts of heavy metals occur
naturally in the environment, but the vast amount of them
may be attributed to human activities such as industrial emis-
sions. Some of the heavy metals, such as iron, copper, and zinc,
are essential for the human body in low doses. Others, such as
arsenic, cadmium, lead, and mercury, have no known essential
function in man. Common for all of the heavy metals is that
they are toxic if consumed in excess, but the level of toxicity
varies greatly between metals and which chemical state they are
in. They have great affinity to ligands in cellular proteins and
bind more easily to these than do other metals present in the
body. A result of this may be inhibition of enzymatic reactions.
As the elimination rate is slower than the absorption rate,
heavy metals accumulate in the body.
Arsenic
Arsenic is used in a number of industrial processes including
processing of glass, pigments, textiles, and ammunition. Small
amounts are also used in pesticides and feed additives. High
levels of arsenic are found both in the groundwater in several
areas of the world and in the oceans. The toxicity of inorganic
arsenic is far greater than the organic form and the PTWI of
15 mg kg
1 BW is hence related to this form. Most of the
registered adverse health effects related to arsenic are also
related to the inorganic form and include peripheral vascular
disease and various cancers. Fish is recognized as the most
significant dietary source of arsenic, however, mainly in its
less toxic organic form.
Cadmium
The main sources of exposure of cadmium are through com-
mercial fertilizers and atmospheric pollution. Galvanizing pro-
cesses and batteries are other sources. The absorption of
cadmium is generally low but may increase in the presence of
calcium or proteins. The main target organs for cadmium are
the kidney and liver and toxic effects of it are related to these
organs. Such effects include increased excretion of glucose,
amino acids, and calcium, along with increased gluconeogen-
esis. The PTWI of cadmium is 7 mg kg
1 BW. Shellfish are
recognized as one of the food sources containing the highest
level of cadmium.
Lead
Lead is abundant in nature, but also environmental contami-
nation contributes to the total amount present. During the last
half of the twentieth century, addition of lead to gasoline led to
a sharp increase in contamination. Introduction of unleaded
gasoline has turned this trend and lead contamination is now
decreasing. The absorption of lead is around 5–15% in adults
but slightly higher in children. The toxicity of lead is among
Fish: Dietary Importance and Health Effects 703
other things linked to its capability of competing with essential
metals for binding seats on enzymes and for inhibiting the
transport of important ions such as calcium. Lead poisoning
may affect the central nervous system, kidneys, and blood-
producing organs. Lead is classified as a carcinogenic substance
and may affect fertility. There is currently no PTWI for lead. In
contaminated areas, seafood has been shown to contain high
levels of lead, shellfish more than finfish.
Mercury
Mercury exists naturally in the environment, but industrial
emissions are a significant contributor. It exists in elemental,
inorganic, and organic forms, where the organic form is the
more toxic. In aquatic environment, inorganic mercury is con-
verted by microorganisms to methyl mercury (MeHg), which
accumulate in the aquatic food chain. Humans are therefore
exposed to MeHg through consumption of large predatory fish,
of both marine and freshwater origins. The harmful effects of
MeHg were first discovered after large-scale industrial poison-
ings in Japan in the 1950s and in Iraq between 1955 and 1972.
These events demonstrated the harmful effects of MeHg
primarily on the nervous system and in particular neurodeve-
lopmental effects of fetuses. The PTWI for MeHg is 3.3 mg kg
1
BW. Due to its well-documented negative developmental
effects, specific dietary advice has been made for pregnant
and lactating women. These state that these groups should
not consume pike at all, perch above 25 cm, and trout and
char above 1 kg. Dietary advice for other population groups is
to not consume these species more than once a month.
All groups are advised to minimize consumption of other
large predatory fish such as swordfish, shark, king mackerel,
and tuna.
Persistent Organic Pollutants
POPs are a large group of organic substances in which one or
more hydrogen atoms have been substituted with halogens.
The most important substances regarding fish consumption
and adverse health effects are polychlorinated dibenzo-p-
dioxins and dibenzofurans (PCDD/Fs), polychlorinated
biphenyls (PCBs), and polybrominated diphenyl ethers
(flame retardants). None of these substances occur naturally,
but are formed when hydrocarbons and halogens are exposed
to high temperatures in incineration or other industrial pro-
cesses. The decomposition rate is very slow due to the lack of
enzyme systems able to break carbon–halogen bindings. They
are lipophilic in general and the lipophilicity increases with
increasing degree of substitution. The combination of these
two factors leads to bioaccumulation in the marine food web,
especially in fatty fish.
Dioxins and PCBs
The chemical structures of PCDD, PCDF, and PCBs are quite
similar, as all of them contain two phenol rings. Hydrogen
atoms are attached to these phenol rings, eight in PCDD and
PCDF and ten in PCB. All of the hydrogen atoms may be
substituted with chlorine atoms, making a total of 75 possible
variants of PCDDs, 135 different PCDFs, and 209 PCBs.
Twelve of the 209 PCBs, those without chlorine substitution
in the ortho-position or those containing only one chlorine in
704 Fish: Dietary Importance and Health Effects
ortho-position, are characterized as dioxin-like PCBs. These act
on the same toxicological pathway as dioxins, that is, via the
aryl hydrocarbon receptor, and share more or less the same
toxic properties as the dioxins. The dioxin-like PCBs are
included in the term ‘dioxins’ in the following section. The
non-dioxin-like PCB will not be discussed further here.
Some of the common adverse health effects that may be
caused by these compounds are skin reactions (chloracne),
damage of the neurological and immunologic systems, organ
damage, enzyme induction, carcinogenic effects, and terato-
genic effects.
A common system for evaluation of total toxicity of these
compounds has been prepared, in which each compound has
been given a toxic equivalency factor (TEF) related to the most
toxic substance 2,3,7,8-tetrachlorodibenxodioxin, whose TEF is
1. In general, PCBs are less toxic than PCDDs and PCDFs. The
total amount of dioxins in a sample is reported as toxic equiv-
alents (TEQ) and is found by multiplying the concentration of
each compound with its respective TEF and finally adding them
up. The TWI for dioxins is currently 14 pg TEQ kg
1 BW.
Due to the bioaccumulation of dioxins in the aquatic food
chain, seafood is recognized as a significant dietary source of
dioxins. However, dioxins are also found in most other foods,
and studies of food consumption patterns show that the daily
intake of dioxins is approximately equal in most populations
regardless of the seafood intake.
From time to time, the issue of high levels of dioxins in
farmed fish is brought forward. The assumption is that levels of
these compounds may be high in farmed fish due to the use of
unrefined fish oil in the aquaculture feeds. However, the sub-
stitution of fish oil with plant oils during the last years has
actually led to a decrease of dioxins in the farmed fish, and
currently, the levels of these compounds are actually higher in
wild species.
The prevalence of dioxins in the environment has decreased
during the last decades, partly because of changes in industrial
processes and partly because of a ban introduced in the 1980s
on the usage of PCBs in new products. However, due to their
slow decomposition rates, these compounds will be present in
the environment for several years to come.
Polybrominated diphenyl ethers (flame retardants)
This is a group of compounds with similar chemical structures
as PCBs, the difference being that the substituting atoms are
bromine instead of chlorine. Their primary function is to serve
as flame retardants, and as opposed to PCBs, their use and
presence in the environment are increasing. Their toxicological
properties have not been studied to the same extent as the
dioxins, but several recent reviews have suggested that their
use should be restricted.
Risk versus Benefit Analysis
Fish and seafoods have been extensively evaluated for their
risks and benefits. Balancing risks and benefits is important,
particularly in countries with high per capita seafood consump-
tion such as Japan, Portugal, Iceland, and Maldives. Several
meta-analyses have been published evaluating the risks and
benefits associated with seafood consumption. An example of
a risk–benefit analysis is the one performed by the Joint WHO/
FAO Expert Committee in 2010 where the increased risk of
dying from cancer caused by dioxins versus the reduced risk of
CVD mortality due to EPA þ DHA by consumption of equal
amounts of fish was evaluated. The evaluation criteria were
changes in total mortality per million people by intake of one
fish meal per week for a lifetime. An example of such an
analysis is for farmed Atlantic salmon, 600 lost lives to cancer
versus 39 800 avoided deaths due to CVD, corresponding to a
ratio between benefits and risks of approximately 66:1.
Conclusions
Fish and other seafoods are excellent sources of several impor-
tant nutrients, and it is concluded that a high intake of these
food items lowers the risk of CVD. Although not as consistent,
positive health effects related to fish consumption and other
chronic diseases have been confirmed. Despite being a source
of several contaminants, the benefits of seafood consumption
outweigh the potential risks, and hence, the conclusion is clear;
the general seafood consumption should be increased.
See also: Antioxidants: Role on Health and Prevention; Bioactive
Peptides in Foods; Cadmium: Properties and Determination; Fatty
Acids: Essential Fatty Acids; Fatty Acids: Fatty Acids; Fatty Acids:
Metabolism; Fish: Fish in the Human Diet; Fish: Processing; Fish Oils:
Production and Properties; Food and Agriculture Organization of the
United Nations; Hypertension and Diet; Infants: Nutritional
Requirements; Iodine: Physiology; Low-fat Foods: Types and
Manufacture; Mediterranean Diet; Oxidation of Food Components;
Protein: Digestion, Absorption and Metabolism; Protein: Food Sources;
Protein: Requirements; Protein Quality and Amino Acids in Maternal
and Child Nutrition and Health; Proteins: Chemistry, Characterization,
and Quality; Shellfish: Role in the diet; Trace Minerals and Trace
Elements.
Further Reading
Bouckenooghe T, Remacle C, and Reusens B (2006) Is taurine a functional nutrient?
Current Opinion in Clinical Nutrition and Metabolic Care 9: 728–733.
Calder PC (2009) Polyunsaturated fatty acids and inflammatory processes: new twists in
an old tale. Biochimie 91: 791–795.
Costa LG, Giordano G, Tagliaferri S, Caglieri A, and Mutti A (2008) Polybrominated
diphenyl ether (PBDE) flame retardants: environmental contamination, human body
burden and potential adverse health effects. Acta Bio-Medica: Atenei Parmensis
79: 172–183.
Falco G, Llobet JM, Bocio A, and Domingo JL (2006) Daily intake of arsenic, cadmium,
mercury, and lead by consumption of edible marine species. Journal of Agricultural
and Food Chemistry 54: 6106–6112.
FAO (2011) Fish and aquaculture statistics – food balance sheets. Rome: Food and
Agriculture Organization of the United Nations. http://faostat.fao.org/site/610/
default.aspx#ancor, Accessed 08.10.13.
FAO (2012) FAO yearbook, fishery and aquaculture statistics 2010. Rome: Food and
Agriculture Organization of the United Nations.
FAO/WHO (2011) Report of the joint FAO/WHO expert consultation on the risks and
benefits of fish consumption. Rome/Geneva: Food and Agriculture Organization of
the United Nations/World Health Organization, p. 46.
Kim SK and Wijesekara I (2010) Development and biological activities of marine-
derived bioactive peptides: a review. Journal of Functional Foods 2: 1–9.

Larsen R, Eilertsen K-E, and Elvevoll EO (2011) Health benefits of marine foods and
ingredients. Biotechnology Advances 29: 508–518.
Mozaffarian D and Rimm EB (2006) Fish intake, contaminants, and human health:
evaluating the risks and benefits. JAMA, the Journal of the American Medical
Association 296: 1885–1899.
Simopoulos AP (2008) The importance of the omega-6/omega-3 fatty acid ratio in
cardiovascular disease and other chronic diseases. Experimental Biology and
Medicine 233: 674–688.
Fish: Dietary Importance and Health Effects 705
Weichselbaum E, Coe S, Buttriss J, and Stanner S (2013) Fish in the diet: a review.
Nutrition Bulletin 38: 128–177.
WHO (2013) Fact sheet no. 317: cardiovascular diseases (CVDs). Geneva: World Health
Organization. http://www.who.int/mediacentre/factsheets/fs317/en/index.html,
Accessed 12.08.13.
Yu HY, Guo Y, and Zeng EY (2010) Dietary intake of persistent organic pollutants and
potential health risks via consumption of global aquatic products. Environmental
Toxicology and Chemistry 29: 2135–2142.
 Fish: Fish in the Human Diet
B Blakistone, R Kleiner, and J McGuire, National Fisheries Institute, McLean, VA, USA
ã 2016 Elsevier Ltd. All rights reserved.
Seafood Sources and Production
Countries of Origin for Seafood
According to the Food and Agriculture Organization of the
United Nations, in 2012, China was the major seafood exporting
nation to the world (14% by value), followed by Norway at 7%
by value, Thailand (6% by value), and Vietnam (5% by value). In
2012, the FAO ranked China as the top fishing country by
quantity and value, followed by Indonesia, the United States,
India, and Peru (primarily due to anchoveta). China is also the
leading producer of aquacultured species in the world at 41.1
million tonnes, and its production is nearly 2.5 times that of its
wild capture. In fact, in 2008, Asia started producing more
farmed fish than wild catch, contributing 54% of the world’s
production in 2012, with Europe at a distant second at 18% and
other continents below 15%. Other countries important to aqua-
culture are India (4.2 million tonnes), Vietnam (3.1 million
tonnes), Indonesia (3.1 million tonnes), and Bangladesh,
Norway, Thailand, Chile, Egypt, and Myanmar, each supplying
less than 2 million tonnes. All these countries supply 88% of the
world’s production.
The most captured species is anchoveta, followed by Alaska
pollock, skipjack tuna, Atlantic herring, and chub mackerel.
The FAO monitors 1600 harvested marine species, but 40% of
the total marine catch comes from 23 species.
In 2012, shrimp continued to be the most important com-
modity traded in world commerce in terms of value, accounting
for about 15% of the total value of internationally traded fish
proteins. Other main groups of exports are salmon and trout
(more than 14%), groundfish at 9% (e.g., hake, cod, haddock,
and Alaska pollock), and tuna (8%).
In 2012, about 73% of the total fishery imports in value
were in developed countries, with the United States and Japan
accounting for 27% of the total. The European Union repre-
sented 36% of the total world imports, which include intrare-
gional trade among its members, and thus is the largest market
in the world.
The total global capture fisheries production for 2012 is
91.3 million tonnes, while aquaculture was 66.6 million
tonnes for a total of 157.9 million tonnes.
Patterns of Consumption
According to the FAO, in 2012, about 86%, or 136.1 million
tonnes, of the total fishery production was used for direct human
consumption. The remaining 14%, or 21.7 million tonnes, goes
primarily for fish meal and fish oil. About 46% of seafood for
human consumption is eaten in live or fresh form.
In 2011, the global per capita annual consumption of sea-
food was estimated by the FAO at 18.9 kg, with fish accounting
for 16.7% of the global population’s intake of animal protein
and 6.5% of all proteins consumed. Globally, fish provides 3.0
billion people with almost 20% of their average per capita
706 Encyclopedia of Food and Health
intake of animal protein and 4.9 billion people with 10% of
such protein. Table 1, based on FAO data, is a look at the role of
seafood in supplying protein to the diet. Interesting and some-
what predictable from Table 1 are the countries and continents
that rely on land sources of animal protein. Fourteen countries
have a consumption of fish/animal proteins greater than 50%
(i.e., the British Virgin Islands, Ghana, French Guiana, Guade-
loupe, Martinique, Bangladesh, Cambodia, Indonesia, Mal-
dives, Sri Lanka, Kiribati, the Solomon Islands, Tokelau, and
the Wallis and Futuna Islands). Seafood contribution to the diet
is reduced by consideration of other nonanimal proteins as
food sources, but the countries of the British Virgin Islands,
Guadeloupe, Maldives, Kiribati, Tokelau, and the Wallis and
Futuna Islands remain strong at >30% of fish/total proteins.
Health Effects
Omega-3 Fatty Acids in the Diet
Omega-3 fatty acids (omega-3s) are long-chain fatty acids (LCFAs)
that contain two or more double bonds and are also known as
polyunsaturated fatty acids (PUFAs). The beneficial effects of
PUFAs vary depending on the length and structure of the fatty
acid. The two most recognized PUFAs are omega-3 and omega-6,
both of which are important for optimal growth, development,
and health, and these are essential in the diet because the human
body does not make omega-3 and omega-6 fatty acids.
Three of the most well-researched omega-3s include DHA
(22 carbons in length with 6 double bonds) and EPA (20
carbons in length and 5 double bonds) and alpha-linolenic
acid (ALA; 18 carbons in length and 3 double bonds). Both
EPA and DHA are LCFAs primarily found in salmon, sardines,
trout, tuna, and herring. ALA is a short-chain fatty acid (SCFA)
and is found in plant sources, such as nuts, hempseeds, flax,
and canola and soybean oils.
While all of these PUFAs are important for optimal health,
the human body can utilize DHA and EPA much more effi-
ciently than ALA. Because ALA is a SCFA, enzymes are necessary
to convert ALA to a more bioavailable LCFA. While a small
percentage of ALA can be converted to EPA (approximately a
5–15% conversion rate depending on the individual’s body
efficiency), there is little to no conversion of ALA to DHA.
Omega-3 PUFAs are important for heart health and brain
development (discussed in the succeeding text). EPA and DHA
help to lower inflammation, improve blood cholesterol levels,
and reduce the risk of death from heart attacks and stroke.
The dietary recommendations for omega-3s from global
organizations for the general adult population are as follows:
• World Health Organization (WHO) and FAO: Aim for
250 mg of EPA and DHA each day.
• International Society for the Study of Fatty Acids and Lipids
(ISSFAL): To reduce the risk of cardiovascular disease, con-
sume a minimum of 500 mg of DHA þ EPA (combined)
each day.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00289-0
 Fish: Fish in the Human Diet 707

Table 1 Seafood contribution to protein supply by continents and by selected nations, particularly those with % fish/animal protein greater
than 50%

Per capita Fish proteins (g per Animal proteins (g per Fish/animal Fish/total
Nations by continent supply (kg) capita per day) capita per day) proteins (%) proteins (%)

Africa 10.4 3.0 15.4 19.4 4.6

Ghana 22.9 9.0 17.3 51.9 14.5
Morocco 12.5 4.0 24.2 16.7 4.2
Nigeria 17.1 4.4 10.3 43.1 7.1
The Seychelles 59.3 17.0 35.8 47.6 22.2
North America 21.7 5.3 69.4 7.6 4.8
Canada 22.3 5.8 57.9 1.0 5.6
The United States 21.7 5.2 70.7 7.4 4.8
Latin America/the Caribbean 9.9 2.7 41.8 6.5 3.3
Brazil 10.6 2.7 51.0 5.4 2.9
The British Virgin Islands 28.6 7.4 11.9 61.9 59.8
French Guiana 17.0 4.0 6.2 64.7 20.7
Guadeloupe 21.7 6.2 11.1 55.6 38.9
Martinique 12.6 3.8 7.5 50.3 25.7
Asia 21.4 5.8 25.5 22.9 7.7
Bangladesh 19.7 5.5 9.8 56.2 10.0
Cambodia 40.5 12.8 19.3 66.3 19.8
China 33.5 8.1 36.2 22.4 8.6
Indonesia 28.9 9.6 17.4 54.8 15.6
Japan 51.7 17.9 48.1 37.3 20.4
Korea, the Democratic People’s 9.4 2.6 10.0 25.8 4.6
Republic of (North Korea)
Korea, the Republic of (South Korea) 60.4 16.7 43.1 38.7 17.5
Maldives 164.0 52.2 72.1 72.4 48.4
Sri Lanka 26.3 9.2 16.3 56.1 15.5
Thailand 26.3 8.5 24.2 35.3 14.4
Vietnam 33.6 8.7 31.8 27.3 11.2
Europe 22.0 6.6 57.5 11.6 6.5
The Faroe Islands 85.5 22.4 51.9 43.2 28.0
Iceland 89.9 28.0 96.2 29.1 21.3
Norway 53.4 15.1 64.4 23.4 13.8
Portugal 57.1 15.3 68.9 22.1 13.7
Spain 43.0 13.0 65.2 19.9 12.6
Oceania 25.1 6.4 61.9 10.3 6.7
Kiribati 74.1 22.5 37.5 59.9 30.0
The Solomon Islands 35.0 10.8 18.7 57.5 18.6
Tokelau 54.0 15.5 25.7 60.2 43.2
The Wallis and Futuna Islands 61.5 17.4 28.9 60.0 43.8

Source: FAO, 2012. ftp://ftp.fao.org/FI/STAT/summary/FBS_bycontinent.pdf -FAO (2012). Statistical collections, Summary tables of fishery statistics: capture, aquaculture,
commodity and food balance sheets.

• NATO workshop on omega-3 and omega-6 fatty acids:
300–400 mg EPA þ DHA each day.
• World Gastroenterology Organization: 3–5 servings of sea-
food each week.
Table 1 documents the variable importance of seafood per
capita, as a percent of animal protein and as a percent of total
protein in the world, including those countries where the
percent of fish/animal proteins is greater than 50%. In a Har-
vard study of dietary fats and oils, the global mean intake of
omega-3s is about 163 mg per day, far lower than the recom-
mendations in the preceding text. This study considered 113
countries and a population of 1 630 069 or 82% of the global
population. Worldwide, omega-3 intake from seafood has
increased in the past 20 years by 24 mg per day. By 2020,
nearly 75% of all deaths and 60% of all disability-adjusted
worldwide will be attributable to noncommunicable diseases
including cardiovascular diseases, type 2 diabetes, obesity, and
cancers. Cardiovascular disease particularly can be better con-
trolled by omegas.
Seafood is the premiere dietary source of omega-3s DHA
and EPA. According to the FAO, Oceania boasts the highest per
capita seafood consumption in the world, followed very
closely by Europe (Table 1). Countries who consume the
least amount of seafood include Africa and the Near East.
With the exception of islands with large fishing communities,
seafood consumption tends to be greater in developed coun-
tries as compared with developing countries.
708 Fish: Fish in the Human Diet

Beneficial Nutrients of Seafood limited evidence that omega-3s from seafood intake during
later adulthood may help reduce the risk of dementia. In a
Seafood is a nutrient-dense food naturally rich in protein,
study that looked at nearly 15 000 older adults (aged 65 years
vitamins (especially B and D), minerals, and heart-healthy
or older) in low- to moderate-income areas in 11 countries
omegas. Seafood is grouped with meat, poultry, eggs, nuts,
(including China, India, Cuba, Venezuela, Mexico, Peru, and
legumes, and seeds as major contributors (greater than 50%)
the Dominican Republic), there was a beneficial effect of sea-
of protein, niacin, zinc, and vitamin B6 to the diet by the
food on dementia in this population in all countries except
Institute of Medicine (IOM) and as an important source of
India. More research is needed, but the effect of omega-3s on
greater than 10% of vitamins E and B12, thiamine, and ribo-
brain health throughout the lifetime is promising.
flavin and phosphorus, magnesium, iron, copper, potassium,
and linoleic acid. Higher levels of the antioxidant selenium
and the omegas EPA and DHA and generally lower levels of
saturated fats compared with meat, poultry, eggs, etc., are Mercury in Seafood
unique to seafood, notes the IOM. In fact, says the IOM,
Essentially, all seafood includes traces of mercury, a compound
depending on the preparation method, seafood is lower in
that originates primarily from natural volcanic activity and
total and saturated fats and cholesterol than some more fre-
thermal events in oceans. Although some mercury has been
quently selected animal protein sources such as lean and fatty
contributed by human activity, studies show that mercury in
cuts of beef, pork, and poultry. Certain species of seafood (e.g.,
the oceans has remained virtually unchanged over time.
canned salmon with bones) may also be a good source of
The amount of mercury equated with serious illness has
calcium.
only been recorded in two international industrial accidents
and poisonings in Japan and Iraq in the 1950s–1970s. While
Heart Health Benefits of Eating Seafood the levels of mercury present in those instances are on a scale
dramatically different than the levels seen in commercial sea-
Omega-3 fatty acids have been shown to be cardioprotective.
food, the devastating effects of the poisonings, specifically on
While the exact mechanism of how these fatty acids benefit
developing babies, piqued the scientific community’s interest
heart health is largely unknown, omega-3s decrease triglyceride
in studying cultures who eat seafood-rich diets. People from
levels and risk of arrhythmias (abnormal heartbeats), slow the
two island areas, the Faroes and Seychelles, were followed to
growth rate of atherosclerotic plaque, and slightly lower blood
determine the health effects of regular, high-level seafood con-
pressure, according to the American Heart Association (Wash-
sumption. Subclinical cognitive deficits were found among
ington, DC). Omega-3s from seafood may also reduce the risk
children of women in the Faroes who ate pilot whale, a mam-
of sudden cardiac death.
mal with a nutrient and contaminant makeup unlike seafood,
Recent studies by the FAO/WHO have shown that modest
during pregnancy. No pattern of harm was found among chil-
intake of omega-3s (approximately 250–500 mg EPA þ DHA
dren of women who ate seafood during pregnancy in the
per day) in various populations in the United States, Europe,
Seychelles. Subsequent adjustment of the Faroes data to isolate
Asia, and Australia lowers the risk of dying from coronary heart
the effects of seafood from whale meat also showed no harm
disease. The magnitude of the protective effect of eating sea-
from eating fish.
food about twice each week is a 36% reduced risk of heart
disease death. For this reason, public health organizations and
experts around the world advise adults to consume at least 2–3
servings of seafood each week to help meet heart health rec- See also: Fatty Acids: Essential Fatty Acids; Fatty Acids: Fatty Acids;
ommendations for omega-3s. Fish: Dietary Importance and Health Effects; Fish: Processing; Fish
The Mediterranean-style diet has long been touted as a Oils: Composition and Health Effects; Fish Oils: Production and
heart-healthy diet. To determine whether seafood in the Med- Properties; Food and Agriculture Organization of the United Nations.
iterranean diet is beneficial to heart health, researchers exam-
ined the seafood intake of more than 3000 men and women in
the Attica region of Greece. In this region, about 88% of men
and 91% of women consumed fish or shellfish at least once a Further Reading
week. Fish intake was positively correlated to reduced levels of
inflammatory markers related to cardiovascular disease. Berry M and Ralston N (2008) Mercury toxicity and the mitigating role of selenium.
EcoHealth 5: 456–459.
Clemens R, Hayes A, Hicks D, et al. (2014) Deconstructing the methylmercury myth.
Food Technology 05: 34–39.
Brain Benefits of Eating Seafood Harris M, Bruhn C, Schor D, et al. (2009) Communicating the net benefits of seafood
consumption. Food Technology 11: 38–44.
The need for the nutrients in fish is especially high during Mozaffarian D and Rimm E (2006) Fish intake, contaminants, and human health:
pregnancy. During the last trimester, a fetus’ brain and nervous evaluating the risks and the benefits. JAMA 296: 1885–1899.
system rapidly develops, requiring about 65 mg per day of Mozaffarian D and Wu J (2011) Omega-3 fatty acids and cardiovascular disease: effects
DHA. The heightened demand for DHA continues to 2 years on risk factors, molecular pathways, and clinical events. Journal of the American
College of Cardiology 58: 2047–2067.
of age. Nielsen S, Kit B, Aoki Y, et al. (2014) Seafood consumption and blood mercury
While the benefits of omega-3s on brain development dur- concentrations in adults  20 y, 2007–2010. American Journal of Clinical Nutrition
ing infancy and early childhood are well documented, there is 99: 1066–1070.

Relevant Websites
www.fao.org/fishery/sofia/en – FAO (2014). The State of World Fisheries and
Aquaculture.
http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/Metals/
UCM396785.pdf – US Food and Drug Administration (FDA) (2014). A quantitative
assessment of the net effects on fetal neurodevelopment from eating commercial
fish.
Fish: Fish in the Human Diet 709
http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/Metals/
UCM396785.pdf – US Food and Drug Administration (FDA) (2014). Section V,
Modeling results for exposure and for IQ. A quantitative assessment of the net
effects on fetal neurodevelopment from eating commercial fish, Table V-12.
http://www.goedomega3.com/healthcare – Global Organization for EPA and DHA
(GOED) Omega-3s (2014). Resources for healthcare professionals.
 Fish: Processing
SP Aubourg, Marine Research Institute (CSIC), Vigo, Spain
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Marine species are known to provide high proportions of
important constituents for the human diet such as: (i) nutri-
tional and digestive proteins including high levels on essential
amino acids; (ii) lipid soluble vitamins (namely, A and D); (iii)
microelements (I, F, Ca, Cu, Zn, Fe and others); and (iv) highly
unsaturated fatty acids. Among these components, the lipid
fraction is now the subject of a great deal of attention due to
its high content on o-3 polyunsaturated fatty acids (PUFA).
In spite of this profitable and valuable composition, wild
and farmed marine species are known to be highly perishable
products. In this sense, it should be stressed that such food
products arise from poikilothermic organisms with a high
water and non-protein-nitrogen content, a soft muscle and
skin structure, and low collagen content. Further, the highly
unsaturated lipid composition has been shown to be especially
prone to lipid oxidation, which leads to important sensory and
nutritional quality losses.
Marine products can be obtained through a wide range of
technological processes. In order to minimize the above-
mentioned deteriorative behavior, different strategies have
been applied for years such as cooling (chilling, freezing and
frozen storage), heating (cooking, canning, smoking), and
water activity reduction (salting, drying). Among marine prod-
ucts, there is an increased demand for fresh products and
represent very high proportions in fish production and
human consumption. In order to commercialize fresh seafood,
refrigerated and chilled storage have been widely employed. In
spite of the reduction in temperature, it has been shown that
quality loss could be produced by the following damage path-
ways: (a) endogenous enzyme activity; (b) microbial decom-
position; (c) lipid oxidation. Consequently, significant
deterioration of sensory acceptance and loss of nutritional
value have been detected which lead to a strong effect on the
commercial value. The rate of alteration depends on factors
such as the nature of the species, size, lipid content, feeding
state at the moment of capture, importance and nature of the
microbial load, and storage temperature.
With a view to retarding quality deterioration and improv-
ing the storage stability of seafood, refrigeration and chilling
technologies have constantly been performed. In some cases, a
combination of strategies has been applied, so that each one
provides different and complementary advantages (or barriers
to spoilage) to ensure quality retention. In connection with the
above-mentioned trend of fresh seafood consumption, public
health concerns have become an issue requiring careful atten-
tion, not only to ensure sensory acceptability and nutritional
value, but also safety, as the major challenges faced by marine
food trade and technologists. The potential health risks associ-
ated with seafood, together with the high demand far from
local fishing ports or farms, entails the need for advanced
preservation techniques to be addressed continually.
710 Encyclopedia of Food and Health
The present article provides an overview on commercial
technologies suitable for application in the manufacture of
fresh marine products. In it, traditional and advanced strategies
are overlooked.
Preliminary Steps to Storage
It is well known that prior to any technological treatment, steps
like catching, harvesting, slaughtering and handling can
strongly affect the quality of marine species. Such steps, carried
out either in farm, on vessel or ashore, are intended to: (i)
prevent physical damage; (ii) remove blood, digestive juices,
slime and feces, and (iii) avoid bacterial contamination.
Soon after catching/harvesting, the fish begins to stiffen,
giving rise to the rigor mortis period. This state in marine
species generally has a shorter duration than in mammals. It
starts 1–7 h after death; its peak in slaughtered individuals,
kept in ice, lies between 5 and 22 h after death. In this period,
various physiological reactions in the animals, as the use of
energy reserves in the muscle and liver, and change in the
acid–base balance are produced. These reactions, by changing
the conditions in the tissues, affect the enzyme activity in
postmortem fish and the rate of freshness degradation. Physi-
cal activity at or before slaughter affects the flesh quality neg-
atively; as the muscle metabolism is predominantly anaerobic,
rapid reduction in ATP and pH is produced, resulting in for-
mation of lactic acid. Stressed individuals may develop shorter
rigor mortis periods, and a softer muscle texture which is more
susceptible to the undesirable separation of muscle blocks
(gaping phenomenon). In connection to farm handling, dif-
ferent commercial methods of slaughtering have shown to
cause varying stress and aversive behavior. Whatever method
is used, the important point is to carry it out efficiently and fast.
Killing requires special consideration in terms of both the
welfare of the species and the quality of the final product. A
prolongation of the rigor mortis period is of great economic
importance.
The precise nature and speed of deteriorative changes dur-
ing the on-board or ashore handling may vary with marine
species, and within one species according to endogenous (ana-
tomical, physiological, genetic, chemical composition) and
exogenous (previous feeding availability, temperature, season)
factors. Fish corresponding to the ‘top of the catch’ should be
of higher quality than those caught during the beginning of the
trip. Handling of fish with gaff hooks picks or forks should be
avoided. Breaks in the skin and unsightly holes in the flesh
quickly introduce spoilage bacteria and accelerate quality dete-
rioration. Fish should not be exposed to direct sunlight or to
the drying effect of wind, but should be carefully and safely
cleaned and washed, and then cooled down as quickly as
possible. Any careless treatment or delay in reducing the
http://dx.doi.org/10.1016/B978-0-12-384947-2.00290-7

temperature of fish will have a marked effect on their potential
keeping time.
A common on-board treatment for white fish is gutting.
Guts are known to be a reservoir of powerful digestive enzymes
and bacteria that can spoil the flesh, resulting in unacceptable
odors and flavors. As a result of gutting, many studies report an
extension of shelf life during marine species refrigeration. For
small-sized fatty species however, the process of gutting has
shown to be impracticable and may create new cut surfaces for
potential bacterial contamination and lipid oxidation develop-
ment and would not benefit the shelf life.
Traditional Refrigeration Systems
Common Flake Ice
Refrigeration to about 0  C by using common flake ice (chilled
storage) is the preservation method most extensively used to
cool marine species rapidly and to extend their shelf life. Under
such conditions, the normal storage life of cold water species
immediately after post mortem is 1–2 weeks, while species
from warm tropical waters keep somewhat longer. Ideally,
aquatic species should be in contact only with ice and not
with each other. Finally, much of the care exercised by fisher-
men in handling and stowing the catch may be wasted unless
good standards of cleanliness, hygiene and sanitation are
maintained at all times.
The rate at which heat can be removed from marine foods
during flake ice chilling depends on several parameters. In the
case of packaged products, size and shape will affect the rate of
heat transfer while in the case of unpacked products special
attention should be paid to the relative humidity conditions,
with a view to prevent dehydration of the food material.
Although pure ice melts at 0  C, the temperature of material
stored in crushed ice may be slightly lower. This effect is due to
the salts present in the flesh, and to the seawater on the surface
of the carcasses, which cause some of the ice to melt and extract
heat from the surroundings.
Quick and efficient flake ice chilling of the seafood to the
lowest temperature practicable without actually freezing the
flesh is essential if spoilage is to be kept to a minimum.
Rapid chilling also aids bleeding and results in more attractive
fillets. Besides, contributing to the cooling, the flow or melt-
water also washes away bacterial slime, spoilage products and
any residual blood and thus helps to preserve the fresh appear-
ance and smell. Room temperature should be kept above 0  C,
so that a good flow of melting water is maintained; however, if
this temperature is too high, the weight of necessary ice will be
larger.
Chilled and Refrigerated Seawater
Chilled (CSW) or refrigerated (RSW) seawater are the terms
commonly used to describe seawater which has been cooled
just below 0  C. RSW holding of marine species is accom-
plished by filling a hole with seawater and then recirculating
it through refrigerated coils. As RSW requires complicated
pumping and filtering systems, it may be replaced by CSW, a
system where sufficient fresh seawater ice is taken and eventu-
ally mixed first with water and then with fish.
Fish: Processing 711
Both RSW and CSW are considered to be able to extend the
shelf life of fish and shellfish due to the lower storage temper-
atures achieved. Both hydro-cooling systems have led to a wide
range of commercial applications for both on-board and the
in-land use, being excellent media for refrigerating and trans-
porting whole carcasses while still at sea and for the road
transport of bulk quantities. Thus, the following advantages
compared to traditional flake icing have been described: (i)
faster cooling; (ii) less stress on food pieces; (iii) the possibility
of a lower temperature; (iv) faster handling of large quantities
of aquatic material; (v) a longer storage time in a few cases.
However, some negative effects on sensory and nutritional
qualities have been encountered, while its on-board handling
(pumping, filtering, and so on) has also found some economical
and technical difficulties. Such inconveniences when compared
with flake ice employment can be resumed as follows: (i) excess
of salt intake, (ii) water absorption by species of low lipid
content; (iii) partial water-soluble protein loss; (iv) presence of
anaerobic bacteria responsible for putrefaction; (v) appearance
modification in parameters such as gills, skin and eyes.
Addition of Chemical Preservatives
One previous or simultaneous handling to refrigerated storage
has been salt treatment. Such step consists of direct addition of
NaCl to marine species or to the ice used to cool them or
immersion of seafood material in a brine solution that is
followed by the cooling treatment. The preservative effect of
salt is due to a decrease in water activity, which leads to lesser
potential for microbial attack, and enhancement of functional
properties, leading to an increase of the shelf life time. As a
result, salt is absorbed by the flesh and imparts a desired flavor
to the finished product. Salting has an additional beneficial
effect as it toughens the skin and prevents its adhesion to any
surface. Salting also brightens the appearance of marine indi-
viduals by removing any remaining slime. Salt treatment has
mostly been targeted to species that are further processed by
any other technological treatment.
However, some bacteria have shown to be able to multiply
in a high concentration of salt, especially when the solution
carries substantial quantities of oil, blood and pieces of gut.
Another problem is that salt contact with fish has been
reported to enhance lipid oxidation of the highly unsaturated
lipids, protein denaturation, and texture changes. NaCl is said
to act as prooxidant by enhancement of the prooxidant effect
of chelatable iron ions widely present in fish muscle, especially
in the dark one.
Among factors limiting acceptability of crustaceans, mela-
nosis development plays a very important role. In the presence
of oxygen and polyphenoloxidase, the monophenolic com-
pounds are first hydroxylated to o-diphenols and then oxi-
dized to o-quinones, which react non-enzymatically with
other compounds, giving rise to pigments of high molecular
weight and dark color. Sulfites and SO2 have been applied as
dipping solutions in the crustacean industry as being very
effective as inhibitors of such enzymatic browning by interfer-
ing with the polymerization of quinones. Thus, such preserva-
tives have successfully been applied to delay melanosis
reactions in crustaceans, thereby increasing shelf life.
712 Fish: Processing
Ozone is a powerful antimicrobial agent that is suitable for
application in seafood in the gaseous and aqueous states.
Literature resumes most research where its employment at the
same time as flake ice has led to significant increases in sensory
quality and shelf life of marine species. As an explanation,
molecular ozone or its decomposition products rapidly inacti-
vate microorganisms by reacting with intracellular enzymes,
nucleic material and other components. In contrast to these
positive effects, ozone is also reported to be a powerful oxi-
dant, and has accordingly been said to negatively affect phos-
pholipids, PUFA and membrane proteins. Its employment is
recommended only under optimized conditions.
Advanced Strategies in Refrigeration
Super-chilling Systems: Slurry Ice
Various types of cooling systems have been used for ‘super-
chilling’ ( 4  C to 0  C) of seafood products. Due to the low
storage temperatures achieved, such systems have permitted a
significant bacterial growth delay and accordingly extend the
seafood shelf life. Super-chilling techniques have been
described as being especially useful when the fishing banks
are so distant that storage in flake ice does not guarantee an
accurate preservation of the fish quality at landing; it can either
be used prior to traditional chilled distribution or throughout
the storage and distribution process. However, super-chilling
of muscle food can result in partial freezing, which may lead to
negative changes such as drip loss and decreased water-holding
capacity; in addition, enzyme activity may increase due to
higher concentrations of soluble matter in unfrozen water
and improved access of enzymes to substrate.
Newer chilling systems have enabled the storage at subzero
temperatures of fish products through the addition of salts and
other compounds to ice-water mixtures. These are called ‘slurry
ice systems,’ ‘water-binary systems’ or ‘liquid ice.’ Slurry ice can
be defined as a mixture of ice particles finely dispersed in an
aqueous solution composed of water and other solid compo-
nents (namely, NaCl) used to decrease its freezing point and
achieve temperatures lower than 0  C, but always above the
initial freezing point of fish material. Two phases co-exist in
slurry ice: 25–30% of the mixture consists of small spherical ice
crystals, although this percentage can be modified according to
specific applications, and the remaining 70–75% is water.
Compared with traditional flake icing, new slurry systems
show a wide number of advantages which can be summarized
as follows: (a) the lower temperature slows down degradative
biochemical and microbiological reactions, (b) faster heat
exchange that leads to faster cooling, (c) more efficient cooling
by complete coverage of external surface, (d) its flow exerts a
surface-washing effect, (e) size and geometry of slurry crystals
limit the physical damage, (f) its fluid nature allows automa-
tion of handling and distribution, (g) versatile technique
which can be combined with other preservative strategies.
Meanwhile, a main disadvantage has been identified. Thus,
the temperature employed should not decrease until partial
freezing of the product takes place; otherwise, sensory quality
(cloudy eyes, development of dull color, etc.) could be dimin-
ished. Consequently, it is specially recommended that the
slurry ice users take good advice from manufacturers in order
to apply, in each case, the best combination of parameters
involved (optimization pre-step): salt content, storage temper-
ature, liquid–solid ratio and marine species size.
In the scientific literature, reports referring to the applica-
tion of slurry ice systems to marine products have been exten-
sive in the last decade. In such research, the need to recognize
the main degradation processes involved in quality loss is
essential. Thus, slurry ice has been applied satisfactorily at
different stages: on-board storage and pre-cooling, in-land
storage system, slaughtering and storage in farming production
and as a treatment prior to other technological processing
(chilling storage under flake ice in retail, frozen storage, cook-
ing and canning). In such studies, comparative analysis with
traditional flake ice employment was undergone, taking into
account changes produced in sensory, microbiological, physi-
cal and chemical properties of marine foods.
Packaging Technologies
Different kinds of advanced packaging technologies have been
proposed and applied successfully in the latest decades.
The modified atmosphere packaging (MAP) is an increas-
ingly popular food preservation technique that involves the
total or partial removal of air contained in the package fol-
lowed by reintroduction of an altered or modified atmosphere
consisting of other gasses, usually carbon dioxide, nitrogen,
oxygen alone or in combination. It is specially recommended
to preserve quality and to maintain hygienic, sanitary and
sensory characteristics of perishable products such as chilled
marine products. The carbon dioxide is the most important gas
because of its bacteriostatic and fungistatic properties, this
property being increased by increasing its concentration in
the surrounding atmosphere. The extension of shelf life by
using MAP has shown to depend on species, fat content, initial
microbiological quality, handling, slaughtering, storage tem-
perature, gas nature and mixture composition, packaging
materials, gas-to-product volume ratio, presence of additives,
and combination with other hurdle technologies.
It must be taken into account that gas level in MAP could
change with time due to dissolution of gasses into the muscle
tissue and/or microbiological and biochemical reactions;
therefore, experiments are often conducted under controlled
atmosphere packaging (CAP) condition for studying the real
effect of the different gas constituents. Throughout the storage
in CAP, a more stable gas composition is ensured and the
accumulation of off-odors is avoided. These advantages make
the use of CAP of great interest for marine species preservation
on deep-sea fishing boats.
Vacuum skin packaging (VSP) is a technique which was
developed to overcome some of the disadvantages of the tra-
ditional vacuum packaging. It is characterized by the instanta-
neous heating of the upper packaging film immediately before
its descent over the food surface. The VSP concept relies upon a
highly ductile plastic barrier laminate which is gently draped
over a food product, thereby molding itself to the actual con-
tours of the product to form a second skin, which is discarded
when consuming the seafood. The product’s natural shape,
color and texture are highlighted and, since no mechanical
pressure is applied while drawing the vacuum, soft or delicate
products are not crushed or deformed.

Active packaging is an innovative concept in which the
package, the product, and the environment interact to extend
the shelf life or enhance the safety or sensory properties. This
technology includes packaging materials and edible films and
coatings which contain preservative substances and also tech-
niques modifying the atmosphere within the package. Addi-
tives can be incorporated in different ways, according to the
marine food encountered. It must be stressed that incorpora-
tion of preservatives into the packaging instead of its inclusion
into the bulk has been made for preventing surface microbial
growth or lipid oxidation. Moreover, a controlled release of the
agent from the surface to the inner of the food can assure the
presence of the agent in the food throughout storage. On the
contrary, when the preservative is added into the food, it could
be bounded to food components or react with other additives
or food components losing its activity. All forms of active
packaging have received remarkable attention in recent years,
improving due to the important role involved in extending
food shelf life.
Most of the films used are manufactured with synthetic
plastic materials but for environmental reasons, an increasing
attention is being paid to active edible films and coating as a
result of their biodegradable properties. Proteins, lipids and
polysaccharides are the main biopolymers used to produce
edible films. Two materials and their combination have
recently attracted most interest. On one side, gelatin from
marine fish species as a renewable material; on the other side,
chitosan obtained from natural chitin not only because of its
film-forming capabilities but also because of its antioxidant
and antimicrobial properties. It has been shown that films
composed of proteins or polysaccharides are sensitive to mois-
ture; however, films composed of lipids are more moisture
resistant but are opaque and susceptible of oxidizing. Accord-
ingly, actual trend is to use an optimized combination of
biopolymers.
Addition of Natural Preservatives
Sodium and potassium salts of low molecular weight organic
acids such as acetic, lactic, citric and sorbic have been used to
control microbial growth, improve sensory quality and extend
shelf life of many food systems including marine ones. These
preservatives are able to control the development of spoilage
bacteria and also possess antibacterial activity against various
foodborne pathogens such as Staphylococcus aureus, Yersinia
enterocolitica, Listeria monocytogenes, Escherichia coli and Clostrid-
ium botulinum. Moreover, these compounds represent a rele-
vant choice because of their easy availability, low commercial
cost and wide range of permitted concentration for their use.
Since ancient times, spices and herbs have been added to
foods as seasoning additives due to their aromatic properties.
They inhibit microbial growth of Gram-positive and Gram-
negative bacteria, yeasts and molds and also exhibit useful
antioxidant activity. Herbs of the Lamiaceae family, mainly
oregano (Origanum vulgare), rosemary (Rosmarinus officinalis)
and sage (Salvia officinalis), have been reported as having sig-
nificant preservative capacities. The active compounds respon-
sible for such activity in spices are primarily phenolic
components of the essential oil fraction. However, their use
in foods as preservatives is limited because of flavor
Fish: Processing 713
considerations, since effective doses may exceed sensorial
acceptable levels.
As already mentioned in the previous section, chitosan is a
modified, natural carbohydrate polymer derived by deacetyla-
tion of chitin. As one of the most abundant natural polymers
present in insects, fungi and crustaceans, it is produced from
shell wastes with different deacetylation grades and molecular
weights and therefore, different functional properties and bio-
logical activities. In particular, antimicrobial and antioxidant
activities have shown to depend on its molecular weight and
concentration. It was demonstrated that chitosan inhibited the
growth of many spoilage and pathogenic bacteria and also
yeast and molds; additionally, it may retard lipid oxidation
by chelating ferrous ions released during storage by proteins
such as myoglobin, hemoglobin and ferritin, thus eliminating
pro-oxidant activity of ferrous ions or preventing their conver-
sion into ferric ions.
Live tissues contain a complex oxidation/reduction system
which controls the oxidative reactions needed for life. How-
ever, during the post-mortem stage of marine species a signif-
icant number of reactions occur affecting the oxidative
equilibrium of fish muscle, so that the endogenous reducing
system (alpha-tocopherol, glutathione, coenzyme-Q, ascorbic
acid) is consumed. It is therefore necessary to provide exoge-
nous antioxidants during feeding of cultivated fish and/or
during processing/storage of seafood. Such antioxidants have
been classified as chelators, reductants or free radical scaven-
gers and represent an effective and profitable methodology for
stabilizing seafood products or o-3 PUFA rich products.
Recently, consumers have rejected synthetic antioxidants
because of their possible carcinogenicity, so that their replace-
ment by natural ones has been widely developed; the disad-
vantages are their higher cost and lower effectiveness.
In recent years, the inclusion of plant extracts as a source of
natural antioxidants has been a common practice in the food
industry. The radical scavenging capacity of some natural com-
pounds such as polyphenols which are known to be present in
many fruits and vegetables, as well as their corresponding by-
products, has resulted in their use as bioactive antioxidant
compounds in food material susceptible to lipid oxidation.
Purified natural compounds such as procyanidins, flavonoids
and terpenoids, have shown higher antioxidant activity than
their corresponding crude extracts. Hydrophilic compounds
offer advantages when compared with lipophilic ones as food
additives since the former can easily be incorporated into food
systems by application into aqueous solutions.
Biopreservation
The term biopreservation is defined as the use of a natural or
controlled microflora and/or its antimicrobial metabolites to
extend the shelf life and improve the safety of food. This term is
linked to lactic acid bacteria (LAB), a group of Gram-positive
bacteria which are often used in food to ensure its safety,
preserve its quality and develop new flavors. However, and
unlike starter cultures, protective cultures used for biopreserva-
tion purposes should modify the sensory features of the sea-
food product as little as possible. Certain LAB species and
strains have been shown to exert strong antagonistic activity
against spoilage and pathogenic microorganisms such as
714 Fish: Processing

Listeria, Clostridium, Staphylococcus and Bacillus spp. Such antag- High Hydrostatic Pressure Processing
onistic effect is caused by a decrease in the pH of the food –
Since the nineties of the last century, high hydrostatic pressure
mainly motivated by the production of lactic acid and other
(HHP) processing has been recognized as a powerful strategy
organic acids – the competition for nutrients, and/or the pro-
to provide attractive, nutritive and safety foods. HHP technol-
duction of antimicrobial metabolites.
ogy has been shown to retain the sensory and nutritional
Due to these benefits, LAB can be used as protective cultures
properties of foods while inactivating microbial populations
to outgrow competitor microorganisms such as certain spoil-
and leading to shelf-life extension and food safety enhance-
age and pathogenic bacteria, with the subsequent benefits in
ment. Among different food sources, seafood have proven to
terms of food safety. In addition, several LAB species and
be easily compressed and accordingly, and be well suited for
strains are able to produce bacteriocins. These molecules are
HHP processing, normally applied in the 100–600 MPa range.
synthesized by the ribosomes, released extracellularly as bioac-
From a physical point of view, an increase in pressure has
tive peptides or peptide complexes with a bactericidal or bac-
an important effect on the molecules, for they get closer to each
teriostatic effect on other closely and non-closely related
other. HHP treatment has been reported to follow the Le
species, while the producer bacterial cell exhibits specific
Châtelier principle; consequently, any reaction or phenome-
immunity to the action of its own bacteriocin. Members of
non in equilibrium accompanied by a decrease in volume can
the genera Lactococcus, Lactobacillus, Leuconostoc, Pediococcus,
be enhanced by the HHP treatment. Another interesting aspect
Carnobacterium and Enterococcus are known to secrete bacterio-
is that this treatment operates according to the isostatic rule; in
cins, being especially relevant for the biopreservation of raw
compliance with this, pressure is instantaneously and uni-
and processed seafood products the two latest genera.
formly transmitted throughout the whole food matter. From
Bacteriocins as food preservatives may be used for biopre-
a chemical point of view, HHP treatment can lead similar
servation purposes either by using the bacteriocinogenic strain
changes than does the thermal processing. Thus, the covalent
as a protective culture or by using the extracellular extracts of
bonds are not broken but the weak energy bonds like hydrogen
the producing bacteria in the food formulation. LAB bacterio-
and hydrophobic bonds can be irreversibly modified. This
cins or bacteriocin-producing strains can be utilized either
means that low-molecular-weight food components (respon-
alone, in combination with one another, or in combination
sible for nutritional and sensory characteristics) would not be
with other antimicrobials to improve the preservative effect.
affected, whereas high molecular weight components (proteins
Among bacteriocins, nisin is the most studied. It has been
whose tertiary structure is important for functionality determi-
found efficient for the inhibition of Listeria and other spoilage
nation) are sensitive.
and pathogenic bacteria in marine foods. Some bacteriocins
Concerning HHP application to seafood, previous research
(e.g., lactocin S) are much more slower acting and have different
accounts for a wide range of studies focused on protein dena-
inhibition spectra against spoilage and pathogenic bacteria.
turation analysis, specially concerning the activity inhibition of
Thus, combining fast- and slow-acting bacteriocins, and broad
deteriorative endogenous (namely, calpains, cathepsins, lipases,
and narrow inhibition spectra, the food remains safe longer.
phospholipases) and bacterial enzymes. This research topic has
Bacteriocin-producing Enterococci are widespread in nature, and
been related to all kinds of marine products, both from wild and
they have also been isolated from a variety of seafood products.
cultivated sources, and assessing the straight relationship with
One of the principal bacteriocins produced by Enterococci iso-
sensory and nutritional value. HHP conditions applied have
lated from fish is enterocin P, which shows a good antimicrobial
been reported to lead to reversible or irreversible damages in
activity against L. monocytogenes, S. aureus and other spoilage and
proteases, this leading to a marked inhibition of their action; on
pathogenic bacteria such as Bacillus spp.
the contrary, HHP treatment can also disrupt lysosomal mem-
An alternative to antimicrobial agents in the prevention and
branes and cause the enzyme release into the fish muscle and
management of fish diseases in aquaculture facilities is the use
provoking a subsequent hydrolytic activity increase. The need
of probiotics added to the animal feeds. Some protective cul-
for HHP conditions optimization is reported to be imperative to
tures of LAB have been reported to exhibit probiotic activity
attain important shelf life increases in fish products.
due to their positive contribution to the healthy microflora of
Concerning the lipid fraction, changes as a result of HHP
the human gut. Moreover, these probiotic LAB have also been
treatment of marine foods have also been studied, although
introduced into animal feeds due both to their contribution to
not in a so exhaustive way as for the protein fraction. Thus,
the health of farmed animals as well as for their activity for the
lipid oxidation has shown to increase as a result of HHP
biological control of fish pathogens in aquaculture facilities.
treatment; however, the possible prooxidant effect of HHP
The possible mechanisms of action of probiotics are the pro-
treatment on muscle lipids has proved to be eliminated if a
duction of compounds that are inhibitory towards pathogens,
previous water washing of the muscle was applied or if a
competition with targets microorganisms for nutrients and
complexation compound (EDTA, e.g.) was added. Conse-
energy, competition with undesirable species for adhesion
quently, iron-bound protein denaturation during HHP treat-
sites, enhancement of the immune response of the animal,
ment has been reported to facilitate a free metal ion content
improvement of water quality, and interaction with phyto-
increase which would be responsible for lipid oxidation in fish
plankton. It should be highlighted that the probiotic LAB
meat after HHP treatment. The effect of the HHP treatment on
strain introduced in the food chain through the animal feed
other kinds of marine chemical components (vitamins and
must also be safe for humans. Accordingly, all microbial strains
minerals) can be considered very scarce.
intended to be used as probiotics in animal feeds must be
Finally, it is worth to point out that this technology has
evaluated and authorized before being used as ingredients in
shown potential application in the seafood industry when
feedstuffs.

combined with other processing strategies. This accounts for
surimi and kamaboko production, cold-smoked fish prepara-
tion and when assisting freezing, thawing and thermal proces-
sing. A beneficial effect on quality retention has also been
observed when an HHP treatment is used prior to refrigerated,
chilled or frozen storage of fish products; as above mentioned,
deteriorative hydrolytic and oxidative (namely, peroxidases,
lipoxygenases) endogenous enzymes would be HHP-
inactivated for a further stabilization of the marine product.
See also: Acids: Natural Acids and Acidulants; Antioxidants:
Characterization and Analysis; Bacteriocins; Biofilms; Chilled Foods:
Effects on Shelf-life and Sensory Quality; Chilled Foods: Modified
Atmosphere Packaging; Chilled Foods: Packaging Under Vacuum;
Chilled Foods: Principles; Controlled Atmosphere Storage:
Applications for Bulk Storage of Foodstuffs; Essential Oils: Isolation,
Production and Uses; Lactic Acid Bacteria; Oxidation of Food
Components; Preservation of Foods; Preservatives: Food Use;
Probiotics; Quality Control in Food Processing; Spoilage: Bacterial
Spoilage; Storage Stability: Mechanisms of Degradation; Storage
Stability: Shelf Life Testing.
Further Reading
Aubourg S (2008) Practices and processing from catching or harvesting till packaging:
effect on canned product quality. In: Cabado A and Vieites J (eds.) Quality
parameters in canned seafoods, pp. 1–24. New York: Nova Science Publishers, Inc.
Böhme K, Barros-Velázquez J, Calo-Mata P, and Aubourg S (2014) Antibacterial,
antiviral and antifungal activity of essential oils: mechanisms and applications.
In: Villa T and Veiga-Crespo P (eds.) Antimicrobial compounds: actual strategies
and new alternatives, pp. 51–81. Heidelberg, Germany: Springer, Science and
Business, Inc.
Campos C, Castro M, Aubourg S, and Barros-Velázquez J (2011) Use of natural
preservatives in seafood. In: McElhatton A and do Amaral P (eds.) Novel
technologies in food science. Their impact on products, consumer trends and
Fish: Processing 715
environment, pp. 325–360. Heidelberg, Germany: Springer, Science and Business,
Inc, Chapter 14.
Campos C, Gliemmo M, Aubourg S, and Barros-Velázquez J (2011) Novel technologies
for the preservation of chilled aquatic food products. In: McElhatton A and do
Amaral P (eds.) Novel technologies in food science. Their impact on products,
consumer trends and environment, pp. 299–324. Heidelberg, Germany: Springer,
Science and Business, Inc, Chapter 13.
Campus M (2010) High pressure processing of meat, meat products and seafood. Food
Engineering Reviews 2: 256–273.
Egolf P and Kauffeld M (2005) From physical properties of ice slurries to industrial ice
slurry application. International Journal of Refrigeration 28: 4–12.
Khadre M, Yousef A, and Kim J (2001) Microbial aspects of ozone applications in food:
a review. Journal of Food Science 66: 1242–1252.
López-Rubio A, Gavara R, and Lagaron J (2006) Bioactive packaging: turning foods into
healthier foods through biomaterials. Trends in Food Science and Technology
17: 567–575.
Medina I, Gallardo JM, and Aubourg S (2009) Quality preservation in chilled and frozen
fish products by employment of slurry ice and natural antioxidants. International
Journal of Food Science and Technology 44: 1467–1479.
Schnürer J and Magnusson J (2005) Antifungal lactic acid bacteria as biopreservatives.
Trends in Food Science and Technology 16: 70–78.
Torres A and Velázquez G (2005) Commercial opportunities and research
challenges in the high pressure processing of foods. Journal of Food Engineering
67: 95–112.
Yanishlieva N, Marinova E, and Pokorný J (2006) Natural antioxidants from herbs and
spices. European Journal of Lipid Science and Technology 108: 776–793.
Relevant Websites
www.afsc.noaa.gov/history/tech/pubs/refrigeration-fish – Alaska Fisheries Science
Center. National Oceanic and Atmospheric Administration.
www.fao.org/fishery – Food and Agricultural Organization (Rome, Italy).
www.ictan.csic.es – Instituto de Ciencia y Tecnologı́a de Alimentos y Nutrición (CSIC;
Madrid, Spain).
www.iim.csic.es – Instituto de Investigaciones Marinas (CSIC; Vigo, Spain).
www.ior.org.uk – Institute of Refrigeration (UK).
www.matis.is – Matı́s Ltd. Icelandic Food and Biotech R and D.
www.mri.bund.de – MRI. Max Rubner Institut (Germany).
www.researchgate.net/institution/Instituto_Portugues_do_Mar_e_da_Atmosfera –
Instituto Português do Mar e da Atmosfera (Lisbon, Portugal).
www.wefta.org – West European Fish Technologists Association.
 Flavor Enhancers: Characteristics and Uses
D Baines, Baines Food Consultancy, Bristol, UK
M Brown, Biophase Ltd, London, UK
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
What is a Flavor Enhancer?
A flavor enhancer is a chemical compound that amplifies and
intensifies the flavor impact of other flavor compounds in the
mouth when food is consumed. Flavor enhancers are added to
food to potentiate its flavor and taste; they are regarded as food
additives and not flavorings in many countries. They are
defined in European legislation as “substances which enhance
the existing taste and/or odor of a foodstuff.” It is often thought
that flavor enhancers are tasteless; this is not the case as those in
common use in the food supply have their own gustatory
sensations, which are specific and recognizable as discreet tastes
in their own right.
The most commonly used flavor enhancers are the sodium
salt of the amino acid glutamic acid, monosodium glutamate
(MSG), and the nucleotides disodium-50 -inosinate (IMP) and
disodium-50 -guanylate (GMP). The use of these compounds is
regulated in various parts of the world, for example, in Europe,
they have been assigned E-numbers. Flavor enhancers permit-
ted for use in food are detailed in Table 1.
MSG, IMP, GMP, and other flavor enhancers initiate the
‘umami’ effect on the palate, which is associated with a savory
taste; umami is the Japanese word for delicious or succulent
and was the fifth taste sense to be discovered. The functions of
the five taste sensations recognized today are considered to be
• sweet (detection of carbohydrate for energy balance),
• sour (protection from acidic and potentially harmful
substances),
• bitter (protection from toxic substances),
• salty (regulation of blood salt levels),
• umami (detection of the nutritional/protein content of food,
which serves as a marker for adequate protein nutrition).
The umami taste produces salivation releasing gastric juices
containing enzymes that help to digest protein to release essen-
tial amino acids in the gut. MSG taste also induces insulin
secretion in anticipation of an increase in blood amino acids.
Historical Background
Glutamic acid was first isolated in 1866 by the German chemist
Karl Ritthausen from an acid hydrolysate of wheat gluten, and
he named it after its source. Forty-two years later, in 1908, MSG
was discovered by the Japanese chemist Kikunae Ikeda in the
broth derived from kombu, Laminaria japonica, a kelp-like sea-
weed used for centuries as a flavorful broth in Japanese cuisine.
He patented the discovery and marketed MSG as a table condi-
ment and flavor enhancer through a newly founded company,
Ajinomoto, which roughly translated means ‘essence of taste.’
The Ajinomoto company remains a major force in the market
for MSG today.
716 Encyclopedia of Food and Health
IMP, the disodium salt of inosinic acid, was discovered in
1913 by Shintaro Kodama a Japanese chemist and student of
Kikunae Ikeda, in bonito flakes, traditionally used in Japan as a
flavoring agent. Bonito is a sardine-like fish of the Scombridae
family, common in the seas around Japan. Inosinic acid had
been known for a long time since Justus van Liebig’s mid-
nineteenth century work on beef broth, but its significance as
a flavor enhancer had not been recognized.
GMP, the disodium salt of guanylic acid, was discovered by
the Japanese chemist Akira Kuninaka in 1960 in the broth
derived from shiitake mushrooms used by the Japanese for
hundreds of years to flavor foods. Guanylic acid had been
known since 1898 when the Norwegian medical chemist, Ivar
Christian Bang, elucidated the structure during the course of
his work on pancreatic nucleic acid.
Japanese researchers have been instrumental in identifying
the significance of MSG, IMP, and GMP as flavor enhancers
through their study of the flavorings properties of the tradi-
tional Japanese products kombu, bonito, and shiitake mush-
room. It is not surprising therefore that the Japanese word
‘umami’ is used to describe the taste sensation, which was
recognized as a taste sense long before its discovery by workers
at University Of Miami School Of Medicine in 2000. The team,
led by Nirupa Chaudhari, discovered a receptor that interacted
with glutamate in a manner consistent with the umami taste
sensation and established that umami is a fundamental taste
quality similar to sweet, sour, bitter, and salty.
Characteristics of Flavor Enhancers
Chemical Properties of MSG
Glutamic acid is an a,g-amino dicarboxylate with the structure
depicted in Figure 1(a). The monosodium salt (MSG) can
form an a-salt or a g-salt; the a-salt is shown in Figure 1(b).
At the correct pH, the compound has the potential to form a
loosely bound cyclic tautomer between the two carboxylate
functions Figure 1(c).
Compounds with related chemical structures have also
been found to exhibit an umami effect such as monosodium
D,L-threo-ß-hydroxy glutamate Figure 1(d), monosodium D,L-
homocystate (e), monosodium L-aspartate (f), and the salts of
two unusual amino acids, monosodium L-tricholomate (g) and
monosodium L-ibotenate (h).
The relative umami contributions measured from these
compounds is shown in Table 2.
Chemical Properties of Nucleotides
A nucleotide combines a nucleobase, a pentose sugar (ribose or
deoxyribose), and at least one phosphate group. IMP is the
disodium salt of inosinic acid (Figure 2), which has an
http://dx.doi.org/10.1016/B978-0-12-384947-2.00297-X
 Flavor Enhancers: Characteristics and Uses 717

Table 1 Flavor enhancers permitted for use in food

EU E-number Flavor enhancer Australasian number US FEMA GRAS numbera

Glutamic acid and its salts

E620 Glutamic acid 620 FEMA 3285
E621 Monosodium glutamate (MSG) 621 FEMA 2756
E622 Monopotassium glutamate 622
E623 Calcium diglutamate 623
E624 Monoammonium glutamate 624
E625 Magnesium diglutamate 625
Nucleotides
E626 Guanylic acid (guanosine monophosphate)
E627 Disodium guanylate (disodium guanosine-50 -monophosphate GMP) 627 FEMA 3668
E628 Dipotassium guanylate
E629 Calcium guanylate
E630 Inosinic acid (inosine monophosphate)
E631 Disodium inosinate (disodium inosine-50 -monophosphate IMP) 631 FEMA 3669
E632 Dipotassium inosinate
E633 Calcium inosinate
E634 Calcium-50 -ribonucleotides (mixtures of guanylates and inosinates)
E635 Disodium-50 -ribonucleotides (mixtures of IMP and GMP) 635
a
FEMA, flavor and extract manufacturers; GRAS, generally recognized as safe.

H2N
H2N
O
COOH
HOOC COOH
(a) (b) O−Na+

H2N

O
H2N OH

O−
O HOOC COOH
(c) Na + −O
(d)

H2N
H2N O
COOH
HOOC SO3Na
(e) (f) O−Na+

NH2 NH2

COONa COONa

O O
O N HO N
H (h)
(g)

Figure 1 Chemical structures of glutamic acid and other amino acid salts producing umami flavor.
718 Flavor Enhancers: Characteristics and Uses

important role in metabolism. It is formed by the deamination
of adenosine monophosphate (AMP). GMP is the disodium
salt of guanosine monophosphate and consists of a phosphate
group, the pentose sugar ribose, and the nucleobase guanine
(Figure 2).
When the nucleobase is a purine moiety with a hydroxyl
function on the 6-carbon of the purine ring and there is a
phosphate ester on the 50 -carbon of the ribose moiety, an
umami taste is imparted. Both IMP and GMP possess these
chemical features (Figure 2). If the ribose moiety is phosphor-
ylated on the 20 or 30 positions, the compounds are tasteless.
Phosphorylation on the 50 carbon of the ribose ring is essential
for umami taste.
lost. Phosphatase enzymes, occurring naturally in plant and
animal materials, remove the phosphate group from IMP and
GMP. Consequently, during food processing, IMP and GMP
should be added after deactivation of these enzymes by heat.
Measurement of Flavor Enhancers in Foods
A range of methods has been used for the analysis of glutamate
in food products: spectrophotometric, derivative HPLC,
HPLC with UV detection, fluorescence detection, GC, paper
chromatography, and potentiometric methods.
Standard methods for analysis of nucleotides are based
on liquid chromatography with UV, fluorescence, and mass

Physical Properties Table 3 Glutamate content of various foods

MSG, IMP, and GMP are all crystalline solids and are non- Glutamate content
hygroscopic; consequently, they can be stored for long periods Food (mg 100g1)
of time at ambient temperature without their appearance or
quality changing. The solubility of flavor enhancers is impor- Kombu (kelp) 1608
tant when they are blended into food formulations. MSG is Parmesan cheese 1680
much more soluble in water than IMP or GMP. However, IMP Emmentaler cheese 308
has a much higher solubility in vinegar than either MSG or Cheddar cheese 182
Chicken 22
GMP; GMP becomes gelatinous in acidic solutions, such as
Beef 10
vinegar.
Pork 9
Glutamate is not decomposed during normal cooking Alaskan king crab 72
processes. IMP and GMP are stable in aqueous solution, but Shrimp 20
under acidic conditions (pH 3.5 and below) and at high Scallop 140
temperatures, decomposition takes place and umami taste is Sweet corn 106
Tomato 246
Table 2 The relative umami contributions measured from the Potato 10
compounds shown in Figure 1 Spinach 48
Cabbage 50
Umami substance Relative umami intensity Green peas 106
Shiitake mushroom (fresh) 71
MSG 1.00 Soy sauce (Korea) 1246
Monosodium D,L-threo-ß-hydroxy glutamate 0.86 Fish sauce (Vietnam) 1370
Monosodium D,L-homocystate 0.77 Natto/soybeans (Japan) 136
Monosodium L-aspartate 0.077 Douche/soybeans (China) 476
Monosodium L-tricholomate 5–30 Soumnara/locust beans (West 1700
Monosodium L-ibotenate 5–30 Africa)

Source: Yamaguchi, S., (1979). The umami taste. In: Boudreau, J. L. (ed.) Food taste Source: Ninomiya, K., (1998). Natural occurrence. Food Review International 14,
chemistry, ACS Symposium Series 115. Washington, DC. 177–212.

N
7 6 N
5 1
8
4 2
ONa 9 3
N
N Y
O P O
5'
O
ONa X is OH and Y is H = IMP
4' H H 1'

3' 2' X is OH and Y is NH2 = GMP

H H
OH OH
Figure 2 Chemical structures of nucleotides disodium inosinate (IMP) and disodium guanylate (GMP).
 Flavor Enhancers: Characteristics and Uses 719

spectrometer detection. Other specialist techniques, such as
reverse-phase HPLC and ion-pairing HPLC, are also available.
Threshold Levels and Synergy
Studies by a number of groups of researchers into the threshold
levels of the three major umami inducers have produced
broadly similar results; the average detection threshold levels
are shown in Table 4. When IMP and GMP are combined with
MSG, there is a synergistic enhancement such that the ‘umami’
taste is increased to an extent that would not be expected from
the sum of their individual effects; food manufacturers use
combinations of MSG and nucleotides to increase the
‘umami’ taste of savory products.
There is also a synergistic effect between the nucleotides; a
50:50 mixture of IMP and GMP reduces the threshold of the
two flavor enhancers by roughly 50%. However, the combina-
tion of low levels of MSG with the individual nucleotides pro-
duces a dramatic change to their threshold detection levels.
Conversely, the addition of a small quantity of IMP to MSG
lowers the detection threshold of MSG, but the effect is less
dramatic (Table 5).
The nucleotides have lower thresholds than MSG; when
used commercially, the prices of the nucleotides are approxi-
mately ten times higher than MSG, so exploiting the synergistic
effect in food products has been aimed at lowering cost by
adding small amounts of nucleotides to MSG. Table 4 shows
that low concentrations of IMP and GMP can significantly
reduce the amount of MSG applied to a food to produce an
equivalent umami effect (Table 6).
In practice, manufacturing methods for the two 50 -
nucleotides results in equal quantities of IMP and GMP being
produced, which has led suppliers to offer 50:50 mixtures of
IMP and GMP, and it is this mixture that is used in combina-
tion with MSG to produce the synergistic flavor enhancement
of food products. Nucleotides were commercially introduced
in 1960, and due to their higher flavor potency, they have
increasingly been replacing MSG or reducing the amount
used in foods by exploiting the synergy phenomena.
The Japanese have exploited synergy in their cuisine for
centuries. For example, miso soup is made by producing a
dashi stock to which miso paste is added. The dashi stock
usually consists of a combination of kombu (MSG source) and
bonito (IMP source). The miso paste is produced by fermenting
soybeans, barley, or rice with a koji fungus, Aspergillus oryzae,
followed by treatment with a lactic acid bacterium, which leads
to the formation of high levels of glutamic acid and its salts.
Flavor Enhancers and Salt Reduction
The intake of sodium in the diet has been linked to hypertension
and increased risk of cardiovascular disease. Currently, the daily
adult intake of sodium is approximately three times the recom-
mended daily allowance, and therefore, regulatory authorities
are recommending a reduction in sodium intake to 2.4 g (6 g
salt) per day. Food companies are investigating a range of strat-
egies for reducing sodium or salt content of foods, and some of
these involve the use of flavor enhancers to increase the salty
taste of foods so that lower concentrations of salt can be used to
achieve the same effect. For example, glutamic acid can be used
to mask the bitter taste of potassium chloride (added as a salt
replacer), and IMP and GMP can be used in conjunction with
sodium acid sulfate (US Patent 2013/0171327A1) to reduce salt
content of food by between 30% and 50%.

Table 4 Content of IMP, GMP, and AMP in various foods Table 5 Average detection threshold concentrations for nucleotides
in the presence of different MSG concentrations
IMP content GMP content AMP a content
Food (mg 100g1) (mg 100g1) (mg 100g1) Flavor Threshold levels In 0.1% MSG In 0.01% IMP
enhancer (water) (%) solution solution
Beef 70 4 8
Pork 200 2 9 IMP 0.012 0.0001% –
Chicken 200 5 13 GMP 0.0035 0.00003% –
MSG 0.03 – 0.002%
Squid ND ND 184
Tuna 286 ND 6
Source: Kuninaka, K., (1966). Recent studies of 50 -nucleotides as new flavor enhancers.
Bonito 285 – Trace
In: Advances in Chemistry Series, No 56, 261–274, American Chemical Society.
Snow crab 5 4 32
Scallop ND ND 172
Tomato ND ND 21 Table 6 The effect of increasing the ratio of MSG to IMP or GMP on
Green peas ND ND 2 relative umami intensity
Shiitake ND 150 –
mushroom Ratio of Ratio of
(dried) Relative umami Relative umami
Porcini ND 10 – MSG IMP intensity MSG GMP intensity
mushroom
Oyster ND 10 – 1 0 1.0 1 0 1.0
mushroom 1 1 7.0 1 1 30.0
Morel ND 40 – 10 1 5.0 10 1 18.8
mushroom 20 1 3.5 20 1 12.5
(dried) 50 1 2.5 50 1 6.4
100 1 2.0 100 1 5.4
a
AMP ¼ adenosine monophosphate.
Source: Yamaguchi, S. and Ninomiya, K., (2000). The use and utility of glutamates as Source: Matheis, G., (2007). Flavour Modifiers. In: Ziegler, E and Ziegler, H., (eds.),
flavouring agents in food. Journal of Nutrition 130, 921S–926S. Flavourings: production, composition, applications, regulations, (2nd ed.), Wiley.
720 Flavor Enhancers: Characteristics and Uses
Food Occurrence
Flavor enhancers are used widely in conjunction with flavoring
materials throughout the food supply chain for a number of
reasons:
• To improve the palatability and enjoyment of foods
• To standardize foods and reduce natural variations in flavor
• To mask bitterness in foods
• To create brand recognition
• To create variety across a brand such as the range of flavors
found in snack foods
• To create novelty and unique flavor delivery effects
• To create cultural identity
• To serve as a vehicle to deliver nutraceuticals (molecules
that benefit health)
Glutamic Acid and its Salts
Glutamate is naturally present in virtually all foods especially in
meat, fish, shellfish, cheese, and tomatoes. It is present not only
as its sodium salt but also as its potassium, calcium, and ammo-
nium salts depending on which cations are available to com-
bine with glutamic acid. The highest concentrations are found
in kombu, Parmesan cheese, Roquefort cheese, soy sauce, fish
sauce, yeast extract, and tomatoes. The concentration of gluta-
mates increases considerably after ripening of certain foods,
such as Parmesan cheese and dry-cured ham. Free glutamates
exist in certain cheeses and dry-cured meats, in tomato prod-
ucts, and in soy sauce. Glutamic acid in the bound form is a
major constituent of food proteins (plant and animal), and free
glutamic acid is naturally present in most foods providing a
mild umami taste compared to MSG (Table 3).
Patterns of consumption
It is estimated that the average person consumes around 17 g of
glutamate in free and bound forms (in protein) every day. It is
important in brain function, and there are considerable amounts
of both free and bound glutamates in our bodies at any one time –
the body produces around 40 g d1 to maintain normal physio-
logical functioning. Free glutamate consumed as MSG equals
around 1/1000th of the total glutamate present in the body.
Individual consumption ranges from around 0.5 to 1 g d1 in
Western countries, but in Japan and Korea, it is typically three
times this level rising to around 4–5 g d1 in China, where MSG is
widely used in cooking.
Nucleotides
Nucleotides are naturally occurring substances that are ubiqui-
tous in foods especially meat, fish, shellfish, and mushrooms.
GMP is present at the highest concentrations in shiitake mush-
rooms, while IMP is found at the highest concentration in
bonito flakes (Table 4).
Pressure on the food industry to remove E-number com-
pounds, such as flavor enhancers, from food products has led
to a search for alternatives. Yeast extracts contain significant
amounts of the nucleotides IMP and GMP as well as varying
amounts of glutamate salts. Other sources of MSG are hydroly-
zed vegetable proteins (HVPs), tomato powder or purees, and
soy sauce. These materials do not require E-numbers on the
label and therefore satisfy the ‘clean label, additive-free’ strat-
egy adopted many food companies.
Manufacturing
Fermentation
Ajinomoto has been producing amino acids by fermentation for
many years. It dominates the MSG market with a market share
of 34% (200 000 tons per annum). Cheil Jedang Group in
Indonesia and Daesang in Korea are also major producers of
MSG using fermentation technology. Production capacity
and demand for MSG are greater than for any other amino
acid. It is manufactured via fermentation of corn, sugarcane, or
tapioca using the bacterium, Corynebacterium glutamicum, which
is capable of producing high yields of L-glutamic acid from
sugar. Ammonia is used as the nitrogen source and oxygen is
provided by passing compressed air into the fermenting vessel.
The L-glutamic acid is released by the microorganism into
the fermentation medium and recovered by downstream
crystallization.
In addition to the amino acid MSG, these companies also
produce the nucleotides disodium inosine 50 -monophosphate
(IMP) and disodium guanosine 50 -monophosphate (GMP) by
fermentation. They have developed microbial strains, such as
Escherichia or Bacillus, capable of overproducing MSG and nucle-
otides. These overproducing strains have been developed using
molecular biological techniques and tools, such as gene
disruption and amplification and efficient gene expression sys-
tems. IMP and GMP can also be produced by the enzymatic
degradation of RNA with 50 -phosphodiesterase; however,
economically, this process does not compete with fermentation.
The use of fermentation to produce these nucleotides and
MSG can satisfy the vegetarian market with umami flavor
enhancers derived from plants. Fermentation also offers a
route to natural flavor enhancers; there is an enormous con-
sumer demand for natural foods and beverages in the United
States and Europe, and the requirement for natural flavorings
to satisfy this is increasing.
Health Effects
Absorption and Metabolism of Flavor Enhancers
Glutamate is absorbed from the gut by an active transport
system specific for amino acids. This process can be saturated
and competitively inhibited and is dependent on sodium ion
concentration. Glutamic acid is a nonessential amino acid, but
it is used in the synthesis of L-arginine, which is an essential
amino acid for protein synthesis in cells.
Evidence from human subjects, rats, and nonprimates indi-
cates that free glutamate acts as a signal to regulate protein
intake and nutritional status. Glutamate plays an important
role in the body’s disposal of excess or waste nitrogen. It
undergoes deamination, an oxidative reaction to produce
ammonia, which is then excreted predominantly as urea, and
transamination with pyruvate to yield oxaloacetic acid. This
effectively allows nitrogen from the amine groups of amino

acids to be removed, via glutamate as an intermediate, and
excreted from the body in the form of urea.
Glutamate is also an excitatory neurotransmitter in the
vertebrate nervous system; it is stored in vesicles at chemical
synapses. Nerve impulses trigger release of glutamate from the
presynaptic cell, and glutamate receptors in the postsynaptic
cell bind glutamate and are activated. Because of its role in
synaptic plasticity, glutamate is involved in cognitive functions
like learning and memory in the brain.
Nucleotides are synthesized within the cells of the body but
are also ingested in foods. They are the essential monomeric
building blocks of DNA and RNA for cells in the body, but they
also function as cofactors and coenzymes for cellular messen-
gers aiding with communication between the nucleus and the
cytoplasm.
Most dietary nucleotides are rapidly metabolized and often
excreted. However, at times of rapid growth, limited food
supply, disease, stress, and immunologic challenges, dietary
nucleotides become essential for healthy development and
are incorporated into tissues. Under normal conditions, suffi-
cient nucleotides are available through internal metabolic and
catabolic processes: either the de novo synthesis of nucleotides
or the reuse of nucleotides from dead cells (the ‘salvage path-
way’). Dietary nucleotides are reported to have significant
effects on lymphoid, intestinal and hepatic tissues, and lipid
metabolism. The nucleotides can serve as nucleic acid precur-
sors and exogenous sources of purine and pyrimidine bases.
Dietary purines are not significantly incorporated into hepatic
nucleic acids, but pyrimidines are. Both are taken up by intes-
tinal cells with excess purines converted to uric acid.
Health Effects of Glutamate
Chinese restaurant syndrome
Controversy regarding the use of MSG in foods stems from a
letter to the New England Journal of Medicine in 1968, which
speculated that MSG could be the cause of adverse reactions
(a burning sensation in the neck, chest tightness, nausea, and
sweating) following consumption of food in Chinese restau-
rants. This became known as Chinese restaurant syndrome
(CRS) and resulted in a backlash against the use of MSG and
its subsequent removal from many foods. After a considerable
period of safety evaluations, the Joint FAO/WHO Expert Com-
mittee on Food Additives, the EU Scientific Committee for
Food, and the US Food and Drug Administration all concluded
that the levels of MSG added to food do not represent a hazard
to health.
Investigation of the ‘syndrome’ itself has yielded no clear
causative agent to date. A double-blind controlled challenge of
individuals claiming to suffer from the syndrome failed to
confirm MSG as the causative agent. Other studies have
found that allergic-type reactions after Asian meals are more
often due to other ingredients, such as shrimp, peanuts, spices,
and herbs. More recently, CRS has been linked to the pathogen
B. cereus and the practice of reheating rice in Chinese restau-
rants. B. cereus is a spore former and a toxin producer; when
rice is heated, the vegetative B. cereus cells are destroyed but the
spores survive. On cooling, they germinate, grow, and produce
an emetic toxin in the product during storage. Reheating the
rice prior to serving does not inactivate the toxin, which is
Flavor Enhancers: Characteristics and Uses 721
Table 7 Concentrations of free glutamate in the milk of different
mammals
Free glutamate in milk (mg 100g1)
Human 21.6
Chimpanzee 38.9
Rhesus monkey 4.6
Cow 1.9
Sheep 1.4
Mouse 2.2
Cat 2.6
Rat 1.6
Rabbit 4.6
Source: Sweet, sour, bitter, salty, umami – It’s simply a matter of taste. Published by the
Umami Information Center, Tokyo, Japan.
insensitive to heat. Consuming rice containing a ‘trigger’ level
of the toxin will produce severe food poisoning, but lower
levels could cause CRS symptoms.
Glutamate in human infant milk
Human and primate milk is rich in free glutamate, about 20
times the amount found in cow’s milk. So glutamate is one of
the first tastes we experience in life (Table 7).
Glutamate is the most abundant free amino acid found in
human milk and that of other primates. It is not found at
significant levels in the milk of other species. Glutamate in
human milk is at its highest concentration straight after birth
and is important for inducing the newborn infant to feed.
Work in Japan with newborn infants has shown them to
produce ‘satisfied expressions’ when fed solutions of sugar and
vegetable soup containing MSG and ‘dissatisfied expressions’
when fed vegetable soup without MSG. Despite the clear
importance of MSG to infant nutrition, its addition to baby
milk is still banned following the CRS fallout. The synergistic
effect between the nucleotides IMP and GMP with MSG could
be important for the health of neonates.
Health Effects of Nucleotides
Although they do not fit the exact definition of a prebiotic,
dietary nucleotides act like prebiotic agents, demonstrating
immunomodulating and direct beneficial effects on the com-
position of gut microflora. Nucleotides can also be described as
‘functional foods’ because they have a potentially positive
effect on health beyond basic nutrition.
Nucleotides in infant milk
Rapidly growing infants require nucleotides for protein syn-
thesis and cell growth. This is particularly important in the gut,
where cell turnover is high and nucleotides are constantly
required to replace cells. The gut also plays a key role in the
immune system: some 70–85% of the body’s immune system
is located there. Nucleotides are increasingly considered by
nutrition experts to be essential nutrients at times when the
endogenous supply is insufficient to meet elevated demands,
such as under conditions of rapid growth.
A large number of studies have found that nucleotide sup-
plementation of infant formula milk has beneficial effects on
722 Flavor Enhancers: Characteristics and Uses
health. Human milk contains more nucleotides than cow’s
milk; thus, many infant formula manufacturers add small
amounts of nucleotides as an ingredient to make the formula
match maternal milk more closely. A high nucleotide intake
has been suggested as an explanation of some of the benefits of
breastfeeding compared with formula feeding. The European
Society for Paediatric Gastroenterology, Hepatology and Nutri-
tion coordinated a study with an International Expert Group,
which produced a medical position paper in 2005 on Global
Standard for the Composition of Infant Formula. The paper
recommends the addition of nucleotides to infant formulas at
a maximum total content of 5 mg 100 kcal1 (as well as max-
imal levels of 2.5 mg 100 kcal1 cytidine 50 monophosphate
(CMP), 1.75 mg 100 kcal1 uridine 50 monophosphate
(UMP), 1.5 mg 100 kcal1 AMP, 0.5 mg 100 kcal1 GMP,
and 1.0 mg 100 kcal1 IMP). No adverse effects have been
reported to date in healthy infants fed nucleotide-
supplemented formula milk.
The beneficial effects of nucleotide addition to infant for-
mula include improved composition of gut microbiota,
decreased risk of diarrhea, increased weight gain and head
growth, and increased immunity following vaccinations
against H. Influenzae type b, polio, and diphtheria toxoid in
formula-fed infants. For example, one study carried out in
2005 by a team including the Institute of Child Health in
London and the Universities of Nottingham and Dundee
found that supplementation of infant formula with 31 mg
nucleotides l1 had a prebiotic effect on colonic microbiology
by improving the ratio of Bacteroides–Porphyromonas–Prevotella
group to Bifidobacterium spp. Bifidobacterium spp. predominate
in the large intestine of breastfed infants (where they are sug-
gested to confer health benefits by protecting against infection
from enteropathogenic organisms), whereas formula-fed
infants are more frequently colonized by Bacteroides–
Porphyromonas–Prevotella spp. The study concluded that the
addition of nucleotides to infant formula milk could be impor-
tant for optimizing the diet of formula-fed babies.
Studies in other mammals have revealed a similar picture.
The supplementation of the diet of weaning piglets with nucle-
otides has shown a variety of beneficial effects. When piglets
are weaned, they can suffer severe stress due to a change in diet,
a lack of immunity, a change in gut flora, and a marked
reduction in food consumption. This can lead to a reduction
in the length of the villi on the intestine wall and decreased
intestinal development. Enterocytes in the gut have a high level
of metabolic activity, and their renewal is constant, with an
average life span in the intestine of a piglet of around 4–5 days.
Piglets are born without a functional immune system, relying
on maternal antibodies present in colostrum and milk for
defense against disease. The internal immune system of the
piglets develops as the concentration of antibodies in the
maternal milk decreases. This development is not complete at
weaning, and consequently, farmers can be faced with health
problems, such as diarrhea, in postweaned piglets. This prob-
lem can be significantly reduced by supplementation with
nucleotides.
Nucleotides improve the production parameters of animals
by enhancing the development of the immune system and the
intestinal mucosa of piglets and reducing the stress that the
animals may suffer as a result of diseases or weaning. Animals
fed nucleotide-supplemented diets generally show enhanced
resistance to viral, bacterial, and parasitic infections and have
an increased number of lymphocytes per villus. The effect is
long-lasting and most pronounced when piglets receive nucle-
otides preweaning. Nucleotides fed at an early stage of life
enhance the development of intestinal structures and micro-
flora leading to an increase in the active surface of the intestinal
tract, facilitating the digestion of feed and the uptake of nutri-
ents. The feed conversion ratio is thus improved. The effects of
nucleotides are dose-dependent; the final weight, the daily
weight gain, and the improvement of the feed conversion
ratio are dependent on the dosage of the nucleotides in
the feed.
Sows can also benefit from a nucleotide-supplemented diet.
When nucleotides are added to sow feeds, there is an increase
in the number of litters per year, the litter size, and the number
of piglets weaned. The farrowing rate was increased and the
number of sows returning to service was reduced. The unifor-
mity of the litter was also improved.
Role of Nucleotides in Immune Response
Dietary nucleotides have been shown to be required for nor-
mal immune defense in adults. In two separate double-blind
clinical studies, the patients fed a diet containing nucleotides
had an improved immune function compared with patients
receiving a nucleotide-free diet. In addition, infectious compli-
cations and length of hospital stay were reduced by nucleotide
supplementation of postoperative cancer patients compared
with a control group.
T lymphocytes appear to require dietary nucleotides for
normal maturation and function. Host resistance to bacterial
and fungal infections was decreased in mice on nucleotide-free
diets; addition of RNA or uracil prevented this vulnerability to
infection. Dietary RNA is required to restore lost immune
function after protein deprivation. Adult rats receiving diets
containing nucleotides (yeast, RNA, and arginine) showed
accelerated healing of ulcers. Investigations in animal models
and clinical literature suggest that administering dietary nucle-
otides helps to minimize infectious complications, improve
clinical outcomes, and provide cost-effective nutritional
support.
Exogenous nucleotides have also shown promise as dietary
supplements to enhance immunity and disease resistance of
fish produced in aquaculture. Research indicates that they can
improve growth of fishes in early stages of development,
enhance larval quality via broodstock fortification, alter intes-
tinal structure, increase stress tolerance, and modulate innate
and adaptive immune responses. Fishes fed nucleotide-
supplemented diets generally have shown enhanced resistance
to viral, bacterial, and parasitic infection.
Role of Nucleotides in Brain Function
Studies indicate that nucleotides play an important role in cell-
to-cell communication through P2 purinoceptors. Some P2
purinoceptors are activated by nucleotides and couple to intra-
cellular second-messenger systems through specific proteins
(heteromeric G-proteins). This represents strong evidence for

extracellular nucleotide signaling and communications in glial
cells in the CNS.
Other studies have suggested that nucleotide supplements
can reduce the deterioration of brain tissue and memory and
prevents aging. Aging mice given a nucleotide-supplemented diet
performed better in a learning capability test than the control
group of mice. The nucleotides were thought to stimulate glial
cell proliferation, neurotubule endothelial cell proliferation, and
the growth of nerve axons by interaction with other growth
factors. They also suppressed the spongy degeneration phenom-
enon of the brain stem reticular formation, which is closely
related with learning capability and memory disorder. In an
earlier study, memory-deficient senescence-accelerated mice
and mice with dementia showed improved memory with dietary
nucleoside and nucleotide supplementation.
Effects of Nucleotides on Liver Function
A balanced mixture of nucleosides (molecules comprising a
pentose sugar and a base) and nucleotides may improve
hepatic function in rats and promote earlier restoration of
nitrogen balance following liver injury according to one recent
study. In another report, increased hepatic cholesterol and
lipid phosphorous and serum bilirubin levels and decreased
liver weight and glycogen were observed when feeding
nucleotide-free diets to weaning mice in comparison with
mice receiving nucleotide-enriched diets.
Effects of Nucleotides on Cholesterol
Feeding a nucleotide-supplemented formula diet to rats
resulted in increases in long chain polyunsaturated fatty acids
in their plasma. Furthermore, when rats were fed diets supple-
mented with nucleotides (AMP, CMP, GMP, IMP, and UMP),
the concentrations of long chain polyunsaturated fatty acids on
red blood cell membranes were enhanced compared to the
control group. Dietary nucleotides may have the potential to
modify the conversion of essential fatty acids into their long
chain derivatives, which have been shown to lower cholesterol
and reduce the risk of heart disease.
Evolutionary Significance of Nucleotide Flavor Enhancers
The fact that nucleotides and MSG have a strong umami flavor
as well as many health benefits may not be coincidental. It
would be a clear evolutionary advantage to favor foods that
guaranteed healthy development of infants as well as resistance
and immunity to disease.
Concluding Remarks
Flavor enhancers have had a bad press in recent years and as a
result food manufacturers have been removing them from
many food products. However, humans enjoy the flavor of
these compounds and have a specific taste for them; removing
flavor enhancers from food reduces its palatability as well as
the market share for the food companies concerned. Because
flavor enhancers are as essential to savory foods as sugar is to
sweet foods, alternatives, such as yeast extracts and HVPs, have
been developed that naturally contain high levels of MSG, IMP,
and GMP.
Flavor Enhancers: Characteristics and Uses 723
Over the past decade, many discoveries have been made
showing the importance of flavor enhancers, in particular the
nucleotides, for health. Benefits are especially significant for
young animals including human infants. It is probably not
surprising, therefore, that animals possess a specific taste recep-
tor forged by evolutionary processes to detect flavor enhancers
as part of a survival strategy. Education of the public about
the health benefits of these food additives may change atti-
tudes and lead to the more judicious and considered use of
flavor enhancers in food products to promote health as well
as flavor.
See also: Amino Acids: Determination; Appetite Control in Humans: A
Psychobiological Approach; Cheese: Types of Cheeses – Hard;
Fermented Foods: Composition and Health Effects; Fermented Foods:
Origins and Applications; Fish: Dietary Importance and Health Effects;
Fish: Fish in the Human Diet; Food Additives: Classification, Uses and
Regulation; Functional Foods; Meat: Role in the Diet; Milk: Role in the
Diet; Mushrooms and Truffles: Role in the Diet; Poultry: Processing;
Prebiotics; Shellfish: Role in the diet; Soy Beans: Dietary Importance.
Further Reading
Bellisle F (1999) Glutamate and the UMAMI taste: sensory, metabolic, nutritional and
behavioural considerations. A review of the literature published in the last 10 years.
Neuroscience and Biobehavioural Reviews 23: 423–438.
Institute of Food Technologists, (1987). Monosodium glutamate (MSG), Food
Technology, May, 143–154, Scientific Status Summary. A publication of the
Institute of Food Technologists, USA.
Jinap S and Hajeb P (2010) Glutamate. Its applications in food and contribution to
health. Appetite 55: 1–10.
Kurihara K (2009) Glutamate: from discovery as a food flavour to role as a basic taste
(umami). American Journal of Clinical Nutrition 90(3): 7195–7225.
Koletzko B, Baker S, Cleghorn G, et al. (2005) Standard for the composition of infant
formula: recommendations of an ESPGHAN coordinated international expert group.
Journal of Pediatric Gastroenterology and Nutrition 41: 584–599.
Methven L (2012) Natural food and beverage flavour enhancers. In: Baines D and Seal R
(eds.) Natural food additives, ingredients and flavourings. Oxford: Woodhead
Publishing.
Ringø E, Olsen RE, Gonzalez Vecino JL, et al. (2012) Use of immunostimulants and
nucleotides in aquaculture: a review. Journal of Marine Science: Research and
Development 1: 104. http://dx.doi.org/10.4172/2155-9910.1000104.
Singhal A, Kennedy K, Lanigan J, et al. (2010) Dietary nucleotides and early growth in
formula-fed infants: a randomized controlled trial. Pediatrics 126(4): 946–953.
http://dx.doi.org/10.1542/peds.2009-2609.
Sugita Y-H (2002) Flavor enhancers. In: Branen AL, Davidson PM, Salminen S, and
Thorngate III JH III (eds.) Food additives. New York: Marcel Dekker.
Takahashi T, Toda E, Singh RB, et al. (2011) Essential and non-essential amino
acids in relation to glutamate. The Open Nutraceuticals Journal 2011(4):
205–212.
Relevant Websites
http://www.foodinsight.org/Content/76/Glutamate-and-Monosodium-Glutamate.pdf –
International Food Information Council Foundation.
http://www.chemoforma.com/uploads/1269254132496.pdf – Hoffmann K. The Key
Role of Nucleotides in Pig Nutrition. Chemoforma Ltd, Switzerland.
http://www.mednet.ca/en/report/nucleotidefortified-formulas-can-boost-neonate-i.html
– Medical Education Network, Nucleotide-fortified Formulas Can Boost Neonate
Immunity, Gastrointestinal Tract Maturation.
Sauer N, Bauer E, and Mosenthin R (2005) Dietary nucleotides and early growth in
formula-fed infants: a randomized controlled trial. http://www.bioiberica.com/web/
pdf/DietaryNucleotides.pdf.
 Folic acid and Folates: Physiology and Health Effects
C Witthöft, Linnaeus University, Kalmar, Sweden
M Hefni, Mansoura University, Mansoura, Egypt
ã 2016 Elsevier Ltd. All rights reserved.
The Vitamin Folate and Folic Acid
The generic term folate refers to a large number of mainly
reduced vitamers (Figure 1) with similar chemical and nutri-
tional properties to pteroyl-L-(mono)glutamic acid (folic acid).
The term folic acid denotes the fully oxidized more stable
synthetic compound of this B vitamin, which is used for food
fortification and in pharmaceutical preparations. Most of the
naturally occurring folates in foods are polyglutamyl vitamers
of tetrahydrofolate, which are susceptible to interconversion
and oxidative degradation. Oxidative cleavage of the molecule
results in loss of the vitamin activity.
The vitamin participates, as a coenzyme, in one-carbon
transfer reactions in the body, for example, purine biosynthesis
and amino acid interconversions. A good folate status is, there-
fore, essential for normal cell division. Folates have a recogni-
zed role in the prevention of megaloblastic anemia and of
neural tube defects (NTDs) in the fetus, such as spina bifida.
A good status is also linked to several other health benefits, for
example, a reduced risk for certain cancers, cardiovascular
diseases (CVDs), and neuropsychiatric disorders. However, in
several European countries, the estimated dietary folate intake
is below nutritional recommendations. To bridge the gap
between these recommendations and actual folate intake,
mandatory folic acid fortification has been implemented in
many different countries, for example, the United States,
Canada, and Chile.
Food Sources for Folate
Folate is an essential micronutrient that has to be obtained
through diet. Typical folate sources are leafy green vegetables,
pulses, liver, yeast, citrus fruits, nuts, and some berries. Native
folate content in food is usually low, ranging from a few to
some hundred parts per million.
Traditionally, food folate composition data derive from the
microbiological assay using L. rhamnosus ATCC7469. In order
to differentiate and quantify individual folate forms, HPLC
methods are also used. Depending on the chosen quantifica-
tion method, sample pretreatment is more or less laborious but
always a crucial step. Thorough quality control and documen-
tation during all analytic steps is required. However, conflict-
ing folate data also arise through variation in folate content in
foods of different origins, because climate, growing area,
cultivar, and maturity also affect folate content.
Foods are grouped according to their folate content into
rich (>100 mg per serving), good (50–100 mg per serving), and
moderate (15–50 mg per serving) sources. Rich sources of folate
are dried legumes with a folate content up to 600 mg/100 g
(cowpeas and chickpeas) and green leafy vegetables, which
reach up to 100 mg/100 g (spinach). Other vegetables and
724 Encyclopedia of Food and Health
fruits, except strawberries, which are considered a good folate
source with 65–100 mg/100 g, are moderate sources. Cereals
are important sources since they are consumed frequently in
high amounts in many countries. A general rule is that whole-
grain products or products with a high extraction rate contain
more folate than the starchy endosperm. The cereal fractions
bran, germs, and aleurone flour are rich folate sources. Differ-
ent folate forms are found in food. In cereals and legumes,
both formyl and methyl vitamers are predominant, whereas in
vegetables and fruits, mainly 5-methyltetrahydrofolate is
found.
Effects from Food Processing
Storage and processing of foods, under both household and
industrial conditions, affect folate content. Most foods are
processed before consumption and common processing
methods include thermal processing, freezing, fermentation,
soaking, germination, and drying.
Since most native food folates are unstable, the folate con-
tent of many processed and stored foods is considerably lower
than in the raw material. Food processing techniques and
storage decrease the folate content to a variable extent
(Table 1), and vitamin retention is affected by the amount of
soluble oxygen and protective antioxidants (e.g., ascorbic acid)
present in the food. Data regarding folate retention are incon-
sistent and depend on processing conditions.
Food fortification is the addition of nutrients to food
above levels normally present. For folic acid, this practice is
governed differently as either mandatory or voluntary fortifi-
cation. In the United States and Canada, cereal-grain products
are fortified at 140 mg/100 g, aiming to provide an additional
100 mg folic acid on top of dietary folate intake. Other folic
acid-fortified products – offered in some countries through
voluntary fortification – are beverages (juices, soft drinks, and
mineral water), snacks, and table salt. Also, dairy products
were considered as vehicles for folic acid fortification. In
retention trials with model food systems, synthetic folic acid
exhibited retention several-fold higher than native food
folates. However, processing techniques can also enhance
folate content (Tables 2 and 3). For example, milling seques-
ters cereal fractions rich in folate and bioprocessing
techniques; fermentation and germination can also lead to
enhanced vitamin content.
Germination is a biological process used to obtain a typical
flavor and texture in foods, during which folate is synthesized
de novo for cell differentiation and growth. Germination of
cereal seeds and legumes results in up to a sixfold increased
folate content, depending on germination conditions (temper-
ature and time) and plant variety (Figure 2). Soaking for just a
few hours or overnight increases folate content by up to half.
http://dx.doi.org/10.1016/B978-0-12-384947-2.00300-7
 Folic acid and Folates: Physiology and Health Effects 725

pteridine p-aminobenzoic acid (poly-) glutamic acid

R2 O H COOH O H COOH
N C [ N-CH-CH -CH -C
2 2
[ N-CH-CH2-CH2-COOH
OH R1 n

N CH2
N

H
H2N N N

Folate vitamer R1 at N5 R2 at N10

Tetrahydrofolate, H4folate H H
5-methyltetrahydrofolate, 5-CH3-H4folate CH3 H
5-formyltetrahydrofolate, 5-HCO-H4folate HCO H
10-formyltetrahydrofolate, 10-HCO-H4folate H HCO
5,10-methylenetetrahydrofolate, 5,10-CH2-H4folate -CH2-
5,10-methenyltetrahydrofolate, 5,10-CH+-H4folate -CH=

Figure 1 The structure of polyglutamyl tetrahydrofolates.

Table 1 Processing that decreases folate content in foods Table 3 Novel functional foods with increased folate content by the
addition of germinated plant ingredients
Folate loss
Process Food (%) Folate
increase
Boiling Various vegetables 20–80 Food Description (%)
Steaming Various vegetables 0–40
Blanching Various vegetables 40–90 Bean stew A canned stew prepared from soaked 50
Canning Various vegetables 0–90 foul and boiled fava beans, consumed in
Pasteurization Tomato puree 15 Arabic countries. The novel product is
Milk 10 prepared from germinated fava beans
Oven-baking Bread 15–70 before canning
Fish 30–45 Baladi/pita A flat wheat bread consumed in Arabic 40
Meat 35 bread countries. Wheat grains are
Chill þ reheating Various vegetables 25 germinated, dried, and milled. 50% of
Frozen þ reheating Ready-to-eat meals 5–50 wheat flour is replaced by germ flour
Soaking þ boiling Legumes 30–70 Biscuits Wheat flour biscuits are baked replacing 570
Blanching þ freezing Legumes 30–85 25% of flour with germinated
Blanching þ tinning Various vegetables 80–90 chickpea flour
Storage, frozen Various vegetables and 0–20 Rye muesli Malted rye grain flakes are produced 250
fruits from germinated rye and subsequent
Storage, chilled Juice 0–5 freeze-drying
Fermented milk 0 Wheat bread Wheat bread was leavened with a 300–500
Bread 15 Saccharomyces cerevisiae strain
Storage, ambient Various fruits 0–30 (CBS7764) and cultured in defined
temperature Bread 30 medium
Fermented Several fermented milk products are 200
milk prepared by fermentation with
products specific starter cultures and addition
of fruits

Table 2 Processing that increases folate content in foods

Process Food Folate increase During fermentation, raw materials undergo changes in
composition, flavor, and texture. Fermentation of cereals and
Germination Rye grains 1.6–6-fold
legumes, spontaneous or with selected microorganisms, is a
Wheat grains 3–6-fold
Legumes 1.8–3.5-fold