INVESTIGACIÓN FORMATIVA

I. Protein and hematological evaluations

of infant formulated from cooking
banana fruits (Musa spp, ABB genome)
and fermented bambara groundnut
(Vigna subterranean L. Verdc) seeds
Nutr Res Pract. 2008 Autumn; 2(3): 165–170.
Published online 2008 Sep 30. doi: 10.4162/nrp.2008.2.3.165
Oluwole Steve Ijarotimi
Author information ► Article notes ► Copyright and License information ►

Abstract
Protein-energy malnutrition is regarded as one of the public health problems in
developing countries as a result of poor feeding practices due to poverty. This study,
therefore, aimed at evaluating nutritional quality of a potential weaning food formulated
from locally available food materials. The cooking banana fruit (CB) and bambara
groundnut seeds (BG) were purchased from local market in Akure, Ondo State, Nigeria.
The CB and BG were processed into flours, mixed in ratios of 90:10, 80:20, 70:30 and
60:40 and subjected into proximate, sensory and biochemical analyses using standard
procedures. Nutrend (a commercial formula) and ogi (corn gruel, a traditional weaning
food) were used as control. The nutritient composition (g/100 g) of the food samples
were ranged as follows: moisture 2.94-6.94, protein 7.02-16.0, ash 1.76-2.99, fat 0.76-
8.45, fibre 1.52-3.75, carbohydrate 63.84-88.43 and energy 1569.8-1665.7 kcal. The
biological value (BV), net protein retention (NPR), protein efficiency ratio (PER) and
feed efficiency ratio (FER) of the experimental food samples were significantly
(p<0.05) lower than nutrend, but higher than ogi. The haematological variables of rats
fed with formulated food samples, commercial formula (nutrend) and traditional
weaning food (ogi) were not significantly (p>0.05) influenced by the dietary treatment.
However, the values obtained for red blood cell (RBC), white blood cell (WBC), pack
cell volume (PCV) and erythrocyte sedimentation rate (ESR) were higher in the
experimental food samples than the commercial food. The growth rate of animals fed
with experimental food samples were lower than those fed with the nutrend, but higher
than those fed with ogi. In conclusion, the nutritional quality of CB and fermented BG
mix of 60:40 ratio was better than ogi; and comparable to the nutrend. This implies that
it can be used to replace low quality traditional weaning food and the expensive
commercial weaning formula.

Keywords: Nutritional quality, cooking banana, bambara groundnut, infant diets

Introduction
Protein-energy malnutrition continues to be a major public health problem among
children throughout the developing world, Nigeria inclusive (Brabin & Coulter, 2003;
FAO, 2004; Schofield & Ashworth, 1996). Poverty and poor feeding practices have
been attributed as the major factors responsible for this nutrition problem (Duncan,
2001; Sachs & McArthur, 2005). Evidence has shown that the real income of a
household is indeed an important determinant of its access to food which, in turn, is a
major determinant of the nutritional status of its members (Khan, 1997; Osmani, 1997).

A number of studies have reported that the nutritional qualities of traditional weaning
foods in developing countries, particularly in Nigeria, are low in protein content and
also devoid of vital nutrients that required for normal child growth and development
(FAO, 2004). For instance, the corn gruel (ogi) is the traditional weaning food of infants
in many parts of West Africa countries. Quite a number of studies have shown that corn
gruel (ogi) is bulky and devoid of essential nutrients, such as protein and vital
micronutrients that is needed for normal child growth and development (Levin et al.,
1993; Millward & Jackson, 2004; Pinstrup-Andersen et al., 1993)

In order to alleviate the problem of protein-energy malnutrition (PEM) among the

infants, several studies have been carried out on the formulation and processing of
weaning foods, such as soy-ogi (maize and soybean), crayfish-ogi (maize and crayfish),
maize-cowpea (maize and cowpea), etc., from locally available food materials
(Akpapunam & Sefa-Dedeh 1995; Ijarotimi, 2006; Ijarotimi & Aroge 2005; Ijarotimi &
Bakare, 2006; Ikujenlola & Fashakin, 2005; Onilude et al., 1999). However, it is evident
that most of these formulated complementary foods are still not accessible to many
nursing mothers, as a result of the high cost of food materials and production processes.
In view of this, this study is aimed at producing weaning foods from cooking banana
fruits and bambara groundnut seeds that are locally available.

Cooking banana (Musa aab species) plays a dual role as an important commercial crop
and a starchy food in the West and Central Africa where 50% of the world's plantain
crop is produced. Cooking bananas are consumed in a wide variety of manners in
Africa; and the fruits are consumed raw as snacks or desserts. Cooking banana is high in
carbohydrate and some vital minerals, but low in protein and fat (Swennen, 1990).
Bambara groundnut is essentially grown for human consumption. The seed makes a
complete food, as it contains 14-24% protein, 60% carbohydrate and 6-12% oil, which
is less than half the amount found in peanuts, making them not useful as an oilseed crop
(Mbata et al., 2007). The protein is reported to be higher in the essential amino acid
methionine than other grain legumes (Oliveira 1976; Oluyemi et al. 1976). The seeds
are used to prepare various fried or steamed products, such as 'akara', 'moin-moin' and
'milk' in Nigeria (Brough et al., 1993; Obizoba, 1983).

Materials and Methods

Materials

The materials, that is, cooking banana (Musa spp, ABB genome), Bambara groundnut
(Vigna subterranean L. Verdc), nutrend (commercial weaning food) and ogi (a corn
gruel, a traditional weaning food), were purchased from a reputable local market and
supermarket in Akure town, Ondo State, Nigeria. The nutrend was purposely selected as
the control, because it was formulated from maize and soybean a plant based food
materials.

Preparation of materials

Cooking banana fruit flour: The cooking bananas were peeled manually and sliced
into pieces lengthwise. The sliced chips were oven-dried at 60℃ for 24 hours. The dried
product was milled and sieved through 0.4 mm wire mesh screen. The cooking banana
flour was stored in sealed cellophane bag pending the time of analysis.

Fermented bambara groundnut flour: The seeds were sorted, cleaned and soaked for
48 hours in hot water. The soaked seeds were drained, hulled manually by hand rubbing,
boiled for one hour, cooled and tightly wrapped in a plantain leaf for four days to
ferment. The fermented seeds were oven dried, milled, sieved through 0.4 mm wire
mesh screen and stored in a cellophane bag for future used.

Food Formulation: The cooking banana fruit flour and fermented Bambara groundnut
flour were blended in ratios of 90:10, 80:20, 70:30 and 60:40 respectively.

Chemical analyses

Nutritional composition: The nutritional composition of the food samples was

determined using the standard procedures of Association of Official Analytical Chemists
(1990). Triplicate samples of each sample were determined for moisture content in a
hot-air circulating oven (Galenkamp, size 3, hot box, London, UK). Ash was determined
by incineration of known weights of the samples in a muffle furnace (Gallenkamp, size
3, hot box, London, UK). Crude fat was determined by exhaustively extracting a known
weight of sample in petroleum ether (boiling point, 40℃ to 60℃) in a soxhlet extractor.
The ether was volatilized and the dried residue was quantified gravimetrically and
calculated as percentage of fat. Protein (N × 6.25) was determined by the Kjeldahl
method. Crude fiber was determined after digesting a known weight of fat-free sample
in refluxing 1.25% sulfuric acid and 1.25% sodium hydroxide. The carbohydrate
content was determined by subtracting the total crude protein, crude fiber, ash, and fat
from the total dry weight (100 g) of the food sample differences. The gross energy was
determined using a Gallenkamp, Autobomb automatic adiabatic bomb calorimeter
(London, UK).

Experimental design

The experimental layout was of completely randomized design. Twenty-five (15 males
and 10 females) weanling albino rats of the Wistar strain at approximately 4 weeks of
age of an average weight of 50.5 g were used for the study. The albino rats were divided
into five groups of five rats per group. The rats were individually housed in separate
cubicles in a metabolic cage with facilities for separate collection of urine and feacal.
The animals were subjected to five days acclimatization. The animals were administered
with the experimental diets (containing 70:30 and 60:40 of cooking banana and bambara
groundnut flour respectively; based on nutritional composition and overall acceptablity,
control diets (nutrend and ogi, a commercial and traditional weaning food respectively)
and water ad libitum for 21 days. The body weights of the animals were measured at
two days intervals. The total feaces and urine voided during the last 5 days of
experiment were collected, weighed and preserved. The urine collected was preserved
by adding a few drops of H2S04 to prevent any loss of ammonia and also to serve as a
preservative agent, while the corresponding feed consumed was also recorded for
nitrogen determination.

Hematological analysis and organ measurements

At the end of the experiment, all the rats were starved for 3 hours and weighed. Before
sacrifice, each rat was anaesthetized with chloroform inside a desiccator. Blood samples
from each rat were collected into sample bottles containing a few milligrams of EDTA
prior to hematological analysis. The packed cell volume (PCV) was estimated by
spinning about 75 µl of each blood sample in heparinised capillary tubes in a
haematocrit microcentrifuge for 5 min, and the total red blood cell (RBC) and white
blood cell (WBC) counts were determined (Lamb, 1981). The heart, lungs, kidneys,
liver, intestine and carcass were separated, blotted free of blood oven dried and
weighed. The values were subsequently expressed in g/kg body weight.

Nutritional evaluations

The nutritional values of the diets in rats were evaluated using various parameters. For
protein, the following parameters were computed:

Where

A = Desired % protein level

Y = Weight of the sample to produced

w = % protein level of the sample

X = Expected weigh to be mixed with band diet

Ni = Nitrogen of animal feed with test diet

NF1 = Nitrogen executed in faeces of animal fed -test diet

NF2 = Nitrogen excreted in faeces of animal fed protein free diet

NU1 Nitrogen excreted in urine of animal fed test diet

NU2 = Nitrogen excreted in urine of fed-free diet

Sensory evaluation

The food samples obtained from the different fractions of cooking banana and bambara
groundnut mixes were made into light gruels, using about 20 g and 60 ml of water. The
reconstituted blends were then evaluated along with two references infant food (i.e
nutrend, a commercial weaning food and ogi, a traditional weaning food) for their
sensory characteristics. Sensory evaluation was conducted on the reconstituted samples
which were coded and presented to 10 panelist members familiar with the product. The
samples were rated on the following attributes namely: color, aroma, taste, mouth feel
and overall acceptability using 9 point hedonic scale ranked between dislike extremely
(1) and like extremely (9).

Statistical analysis

The data were analysed using SPSS version 13.0. The mean and standard deviations of
the triplicate analyses were calculated. The analysis of variance (ANOVA) was
performed to determine significant differences between the means using Duncan.

Results
The nutritional composition of cooking banana fruit and fermented bambara groundnut
seed flour (CBG) blend are presented in Table 1. The energy and nutrient values of the
formulated and control food samples ranged as follows: energy value 1569.82-1665.68
kcal., moisture content 2.94-6.94 g; protein 7.02-16.0 g, ash content 1.76-2.99 g, fat
0.76-8.45 g, while the remaining nutrient composition, that is, fibre and carbohydrate
ranged between 1.52-3.75 g and 63.84 -88.43 g respectively.

Table 1Proximate composition of formulated weaning foods, ogi (a traditional weaning

food) and nutrend (a commercial weaning food) (g/100 g)

Mean values followed by different superscript within column are significantly different
at p<0.05.
CBF1-Cooking Banana + Fermentation B.G (70:30)

CBF2-Cooking Banana + Fermentation B.G (60:40)

Nutrend-Commercial Diet (Nutrend)

BD-Basal Diet

Casein-Casein

The sensory attributes of the formulated weaning foods, ogi (corn gruel, a traditional
weaning food) and nutrend (a commercial weaning food) are presented in Table 2. The
commercial formula was significantly rated high in terms of color, aroma, taste, mouth
feel and overall acceptability compared to ogi and formulated food samples (p<0.05).
Of all the formulated food samples CBF3 (i.e., 70% cooking banana and 30% bambara
groundnut flour) was rated highest compared to others food samples.

Table 2Sensory attributes of reconstituted formulated weaning foods (cooking banana

and bambara groundnut flour mixed), ogi (traditional weaning food) and nutrend
(commercial weaning food)

Means with similar alphabets belong to the same homogenous subset are not
significantly different from each other at the 5% statistical level.

The nutritional quality of the food samples are presented in Table 3. The biological
value of the experimental food samples, that is, CBF3, (72.3%) and CBF4, (74.3%), were
significantly higher than the ogi (52.1%), but lower than that of the nutrend (93.8%)
(p<0.05). For the true digestibility (CBF 3, 59.3%; CBF4, 62.0%), net protein utilization
(CBF3, 55.4%; CBF4, 45.0%) and nitrogen retention (CBF3, 098; CBF4, 1.03) of the
experimental food samples were also significantly higher compared with ogi (that is,
BV 52.1%, TD 41.7%, NPU 31.3% and NR 0.09) and also that of the nutrend (that is,
TD 41.9%, NPU 42.9% and NR 1.03) respectively (p<0.05). For the net protein ratio
(NPR), protein efficiency ratio (PER) and feed efficiency ratio (FER) the values for the
experimental food samples were lower compared with the ogi and nutrend. The growth
patterns of animals fed with the formulated food samples and control food samples
(nutrend and ogi) are shown in Fig. 1. The result showed that the growth rate of animals
fed with commercial weaning formula (nutrend) was significantly higher compared to
the formulated food samples. But the growth rates of formulated food samples were
higher than those of animals fed with ogi.
Fig. 1Growth rate of albino rats fed with cooking banana-bambara groundnut mixed
compared to nutrend, ogi and casein diets

Table 3Nutritional Quality of Cooking Banana and fermented Bambara groundnut blend

Mean values followed by different superscript within column are significantly different
at p<0.05.

Table 4 shows various organ weights of rats fed with formulated food, nutrend and
ogi food samples. The weight range of the animal organs were as follows: heart was
between 0.95 g of animals fed with CBF 1 and 1.35 g of ogi; liver, 2.75 g of CBF 1 and
5.23 g of commercial formula; kidney, 1.53 g of CBF 1 and 2.1 g of ogi; intestine,
8.13 g of ogi and 13.93 g of CBF1 and carcass, 48.4 g of ogi and 60.17 g of
commercial formula.

Table 4Relative Organ Measurement of albino rats fed with experimental and control
diets
Mean values followed by different superscript within column are significantly different
at p<0.05.

The hematological variables of rats fed with formulated food samples, commercial
formula and traditional weaning food sample are presented in Table 5. The values of red
blood cell (RBC) (3.033), white blood cell (WBC) (3633.33) and pack cell volume
(PCV) (33.67) of commercial formula were not significantly high compared to 2.20,
2950.0 and 33.67 of red blood cells, white blood cells and pack cell volumes for CBF 2
and 2.75, 3560.0 and 31.0 were for RBC, WBC and PCV for CBF1 respectively.

Table 5Hematological variables of rats fed with cooking banana-bambara groundnuts

mixed, nutrend, casein and basal diet

Mean values followed by different superscript within column are significantly different
at p<0.05.

Discussion
Infants after 6 months of life need to consume adequate energy and nutrient densities
weaning foods to supplement breast milk (Dewey & Brown, 2003; WHO/UNICEF,
1998). However, in many parts of developing countries scientific studies have reported
that most of the traditional weaning foods were formulated from cereals and tubers that
are low in protein and other essential micronutrients which are important for the normal
physical growth and cognitive development of a child (Dewey & Brown, 2003). In this
present study, the protein content of the formulated food samples, particularly CBF 3 and
CBF4, were two- to three-fold higher than that of the traditional weaning foods (ogi);
but there were no significant differences between the protein contents of the
experimental food samples compared with that of commercial weaning formula
(nutrend) (p<0.05). The energy values of the formulated food samples (CBF4) were
almost the same with that of the ogi and commercial weaning formula. It is evident that
the nutritional qualities of most plant based food materials improved particularly when
combined (Ijarotimi & Aroge, 2005; Ijarotimi & Ashipa, 2006; Ijarotimi & Bakare,
2006; Mensa-Wilmot & Sefa-Dedeh, 2001; Uwaegbute & Nnanyelugo, 1987). The
significant difference between the nutritive values of the formulated food samples in
this present study and that of commercial formula and the traditional weaning food (ogi)
had been early on reported by several researchers (Guiro et al., 1987; Ikujenlola &
Fashakin, 2005). For instance, studies have shown that the ogi is bulky and devoid of
many vital nutrients; hence, it has been implicated in the eatiology of protein-energy
malnutrition in children, particularly in the areas where it is being used as sole infant
diets (Guiro et al., 1987; Ikujenlola & Fashakin, 2005).

The overall acceptability of the formulated food samples was significantly rated low
compared with commercial formula (p<0.05); however, there was no significant
difference between the overall acceptability of the formula and that of traditional
weaning food (ogi). The rating of commercial formula (nutrend) above the formulated
food samples in term of color, aroma, taste, mouth feel and overall acceptability could
be attributed to the incorporation of flavoring, sweetening and other sensory enhancing
agents to the formula during its formulation.

The biological value, net protein retention, protein efficiency ratio and feed efficiency
ratio of the formulated food samples were significantly higher than the traditional
weaning food (ogi); but lower compared to the commercial weaning formula. This
observation may be as a result of the fortification of the commercial formula with
quality protein like casein or other growth promoting nutrients. The growth pattern of
animals fed with experimental food samples was higher compared to those fed with ogi,
but lower when compared with the animals fed with the nutrend. Several findings have
established that traditional infant foods made of cereals or tubers were low in several
nutrients including protein, vitamin A, zinc and iron; these nutrients are of special
importance due to their impact on physical and cognitive development (Krebs &
Westcott, 2002; Levin et al., 1993; Michaelsen & Friis, 1998; Millward & Jackson,
2004; Neumann et al., 2002; Pinstrup-Andersen et al., 1993). The poor nutritive value of
traditional weaning foods and improper feeding practices have implicated as the main
factors that are responsible for the growth faltering in children belonging to low-income
families (WHO, 2001).

It was observed in this study that there were no significant differences (p>0.05) between
the weight of heart, kidney and intestine of the rats fed with formulated diets compared
to those fed with nutrend and ogi; however, the weight of the liver of animals fed with
formulated diets were significantly (p<0.05) lower than those fed with the nutrend and
ogi. This showed that the formulated food samples did not influence the weight of these
organs. Also, the haematological variables of rats fed with formulated food samples,
commercial formula and the traditional weaning food sample were not significantly
(p>0.05) influenced by the dietary treatment. However, the values obtained for red
blood cell (RBC), white blood cell (WBC), pack cell volume (PCV) and erythrocyte
sedimentation rate (ESR) were generally high and comparable to the commercial food,
thus indicating the adequacy of the diets in blood formation.

In conclussion, the results of this study established that the food sample containing 60%
cooking banana and 40% bambara groundnut, contains an appreciable amount of
proteins of comparable biological value with the commercial weaning formula;
therefore, it may be used as a substitute for traditional weaning food (ogi) and
commercial weaning fromula, particularly for the children belonging to low-income
families. However, further study is needed in the area of improving the sensory attribute
of the diet and also biological evaluation using human subjects.

References
1. Akpapunam MA, Sefa-Dedeh S. Traditional lactic acid fermentation, malt addition
and quality development in maize-cowpea weaning blends. Food Nutr Bull.
1995;16:75–80.
2. Hortwitz W, editor. AOAC. The Association of official analytical chemists. 13th
Edition. Washington, D.C. USA: AOAC; 1990. p. 858.
3. Brabin BJ, Coulter JBS. Nutrition-associated disease. In: Cook GC, Zumla AI,
editors. Manson's tropical diseases. London. UK: Saunders; 2003. pp. 561–580.
4. Brough SH, Azam-Ali SN, Taylor AJ. The potential of bambara groundnut (Vigna
subterranea) in vegetable milk production and basic protein functionality systems. Food
Chem. 1993;47:277–283.
5. Dewey KG, Brown KH. Update on Technical Issues Concerning Complementary
Feeding of Young Children in Developing Countries and Implications for Intervention
Programs. Food Nutr Bull. 2003;24:5–28. [PubMed]
6. Duncan T. Commission on Macroeconomics and Health. Health, nutrition and
economic prosperity: a microeconomic perspective. Geneva. Switzerland: World Health
Organization; 2001. CMH working papers no WG1:7.
7. FAO. The state of food insecurity in the World 2004. Rome: 2004.
Undernourishmentaround the world.
8. Guiro AT, Sail MG, Kane O, Ndiaye AM, Diarra D, Sy MTA. Protein-calorie
malnutrition in Senegalese children. Effects of rehabilitation with a pearl millet weaning
food. Nutr Rep Int. 1987;36:1071–1079.
9. Ijarotimi OS. Evaluation of nutritional quality and sensory attributes of home made
processed complementary diet from locally available food materials (Sorghum biclor
and Sphenostylis stenocarpa) Journal of Food Technology. 2006;4:334–338.
10. Ijarotimi OS, Aroge F. Evaluation of nutritional composition, sensory and physical
properties of a potential weaning food from locally available food materials-Breadfruit
(Artocarpus altilis) and soybeans (Glycine max) Polish Journal of Food and Nutritional
Sciences. 2005;14:411–415.
11. Ijarotimi OS, Bakare SS. Evaluation of proximate, mineral and antinutritional factor
of homemade processed complementary diet from locally available food materials
(Sorghum biclor and Sphenostylis stenocarpa) Journal of Food Technology.
2006;4:339–344.
12. Ijarotimi OS, Ashipa F. Evaluation of nutritional composition, sensory and physical
property of home processed weaning food based on low cost locally available food
materilas. Nutrition and Food Science. 2006;36:6–17.
13. Ikujenlola VA, Fashakin JB. The physicochemical properties of a complementary
diet prepared from vegetable proteins. Journal of Food Agriculture and Environment.
2005;3:23–26.
14. Khan AR. Macroeconomic Policies and Poverty: An Analysis based on the
Experience in Ten Asian Countries; Paper presented at the ILO Asian Regional Policy
Workshop on Poverty Alleviation, 5-7; 1997.
15. Krebs NF, Westcott J. Zinc and Breastfed Infants: If and When Is There a Risk of
Deficiency? Adv Exp Med Biol. 2002;503:69–75. [PubMed]
16. Lamb GN. Manual of Veterinary Laboratory Techniques. CIBAGEIGY: Nairobi.
Kenya; 1981. pp. 96–97.
17. Levin HM, Pollitt E, Galloway R, McGuire J. Micronutrient deficiency disorders.
In: Jamison DT, Mosley WH, Measham AR, Bobadilla JL, editors. Disease control
priorities in developing countries. 2nd ed. Oxford. UK: Oxford University Press; 1993.
pp. 421–451.
18. Mbata TI, kenebomeh MJ, Ahonkhai I. Nutritional status of maize fermented meal
by fortification with bambara-nut. African Journal of Food Agriculture Nutrition and
Development. 2007;7
19. Mensa-Wilmot Y, Phillips RD, Sefa-Dedeh S. Acceptability of extrusion cooked
cereal/legume weaning food supplements to Ghanaian mothers. Int J Food Sci Nutr.
2001;52:83–90. [PubMed]
20. Michaelsen KF, Friis H. Complementary feeding: a global perspective. Nutr.
1998;14:763–766. [PubMed]
21. Millward DJ, Jackson AA. Protein/energy ratios of current diets in developed and
developing countries compared with a safe protein/energy ratio: implications for
recommended protein and amino acid intakes. Public Health Nutr. 2004;7:387–405.
[PubMed]
22. Neumann C, Harris DM, Rogers LM. Contribution of Animal Source Foods in
Improving Diet Quality and Function in Children in the Developing World. Nutr Res.
2002;22:193–220.
23. Obizoba IC. Nutritive value of cowpea-bambara groundnut-rice mixtures in adult
rats. Nutr Rep Int. 1983;27:709–712.
24. Oliveira JS. Grain legumes of Mozambique. Tropical Grain Legume Bulletin.
1976;3:13–15.
25. Oluyemi JA, Fetuga BL, Endeley HNL. The metabolizable energy value of some
feed ingredients for young chicks. Poult Sci. 1976;55:611–618.
26. Onilude AA, Sanni AI, Ighalo MI. Effect of process improvement on the physico-
chemical properties of infant weaning food from fermented composite blends of cereal
and soybeans. Plant Foods Hum Nutr. 1999;54:239–250. [PubMed]
27. Osmani SR. Poverty and Nutrition in South Asia; Abraham Horwitz lecture
delivered at the Symposium on Nutrition and Poverty held on the occasion of UN
ACC/SCN 24th Session; Kathmandu. Nepal: 1997. pp. 17–21.
28. Pinstrup-Andersen P, Burger S, Habicht JP, Peterson K. Protein-energy malnutrition.
In: Jamison DT, Mosley WH, Measham AR, Bobadilla JL, editors. Disease control
priorities in developing countries. 2nd ed. Oxford. UK: Oxford University Press; 1993.
pp. 391–420.
29. Sachs JD, McArthur JW. The Millennium Project: a plan for meeting the
Millennium Development Goals. Lancet. 2005;365:347–353. [PubMed]
30. Schofield C, Ashworth A. Why have mortality rates for severe malnutrition
remained so high? Bull World Health Organ. 1996;74:223–229. [PMC free article]
[PubMed]
31. Swennen R. International Institute of Tropical Agriculture (IITA) Ibadan. Nigeria:
1990. Plantain Cultivation under West African Condition. A Reference Manual; pp. 1–
24.
32. Uwaegbute AC, Nnanyelugo DO. Nutritive value and biological evaluation of
processed cowpea diets compared with local weaning foods in Nigeria. Nutr Rep Int.
1987;36:119–129.
33. WHO/UNICEF. Complementary Feeding of Young Children in Developing
Countries: A Review of Current Scientific Knowledge. Geneva. Switzerland: World
Health Organization; 1998. WHO/NUT/98.1; pp. 1–228.
34. WHO. Global Strategy for Infant and Young Child Feeding (A54/INF.DOC./4)
Geneva. Switzerland: World Health Organization; 2001. pp. 1–5.

 Journal List

 J Food Sci Technol

 v.51(9); 2014 Sep

J Food Sci Technol. 2014 Sep; 51(9): 1827–1836.

Published online 2012 May 12. doi: 10.1007/s13197-012-0725-9

II. Optimum extrusion-cooking

conditions for improving physical
properties of fish-cereal based snacks by
response surface methodology
R. K. Ratankumar Singh, Ranendra K. Majumdar, and G. Venkateshwarlu
Author information ► Article notes ► Copyright and License information ►

Abstract
To establish the effect of barrel temperature, screw speed, total moisture and fish flour
content on the expansion ratio and bulk density of the fish based extrudates, response
surface methodology was adopted in this study. The experiments were optimized using
five-levels, four factors central composite design. Analysis of Variance was carried to
study the effects of main factors and interaction effects of various factors and regression
analysis was carried out to explain the variability. The fitting was done to a second order
model with the coded variables for each response. The response surface plots were
developed as a function of two independent variables while keeping the other two
independent variables at optimal values. Based on the ANOVA, the fitted model
confirmed the model fitness for both the dependent variables. Organoleptically highest
score was obtained with the combination of temperature—110 0 C, screw speed—
480 rpm, moisture—18 % and fish flour—20 %.

Keywords: Twin-screw extrusion, Extrusion variables, Fish snacks, Response surface

methodology, Central composite design, Quadratic polynomial model equation

Introduction
Extrusion cooking is a high temperature short residence time (HTST) process by which
moistened starchy and proteinaceous materials are plasticized and cooked in a tube by
combination of high pressure, intense mechanical shear and heat to create fabricated,
shaped products of varying texture. Extruded snack like products are mostly cereal
based and developed mainly from corn, wheat and rice. However, rice has relatively low
protein content (6–8 g/100 g db) and an amino acid profile that is high in glutamic and
aspartic acid, while lysine is the limiting amino acid. Thus, proteinaceous additives are
needed to ensure nutritional diets. Guha and Ali (2006) reported that the glutinous rice
was suitable material to produce the expanded extrudate rice products such as ready-to-
eat snacks, breakfast cereal with low bulk density, high expansion and low shear stress.
Research on extrusion processing of fish muscle started in the 1980s (Choudhury and
Gogoi 1995). Fish are not only excellent sources of high nutritional value protein but
also excellent sources of lipid that contains omega-3 fatty acids, especially,
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Kris-Etherton et al.
2000, 2002). The omega-3 fatty acids are essential for normal growth and development
and may prevent or moderate coronary artery disease, hypertension, diabetes, arthritis,
others inflammatory and autoimmune disorders, as well as cancer (Simopoulos 2000). A
number of studies have reported successful incorporation of fish flesh or fish powder
into starch-based materials by extrusion processes to produce nutritious extruded
products that were acceptable by consumers (Gogoi et al. 1996; Suknark et al. 2001).
Studies have been undertaken to develop dry expanded snack food products from fish
mince and starchy ingredients using single- and twin-screw extruders (Choudhury et al.
1998). The incorporation of fish hydrolysates along with cereals has been attempted to
improve the nutritional quality of extrudates (Choudhury et al. 1998). Rhee et al. (2004)
successfully developed a snack food by extrusion of minced catfish with corn and
defatted soya flour. The advantages of developing fish-based extruded products will
help in supplying nutritious and balanced diets to undernourished people in developing
countries (Venugopal and Shahidi 1995).

Product characteristics of extrudates made from rice and other starchy ingredients
depend on physicochemical changes that occur during extrusion due to the effects of
extrusion variables (Pansawat et al. 2008). Typically, the degree of expansion achieved
during high temperature extrusion is proportional to starch concentration (Linko and
Linko 1981). During the extrusion process, heat and shear facilitate hydration of
starches and proteins, both classified as structure-forming materials (Guy 2001). Starch
and protein are turned into a melt where droplets of water are entrapped. The physical
quality of extrudate is strongly affected by ingredient selection (Banerjee and
Chakraborty 1998; Rolf et al. 2000). The independent process variables such as screw
speed, barrel temperature and feed moisture content are likely to have a direct bearing
on the product quality.

Product density and expansion ratio are closely related. Bulk density has been reported
to be linked with the expansion ratio in describing the degree of puffing in extrudates
(Asare et al. 2004). Meng et al. (2010) used Response Surface Methodology to study the
effects of feed moisture content (16–18 %), screw speed (250–320 rpm) and barrel
temperature (150–1700 C) on extruder system parameters and physical properties
(expansion, bulk density, hardness etc.) of a chickpea flour-based snack. They observed
all three variables to affect product responses significantly. In their study, desirable
products characteristics of high expansion ratio and low bulk density and hardness were
obtained at low feed moisture, high screw speed and medium to high barrel temperature.
Extrusion of corn grits with fish flesh/fish protein can be used to produce high-protein
products that would be an option to provide nutrient snacks for consumers and to
increase fish consumption (Shaviklo et al. 2011a). Shaviklo et al. (2011b) studied the
acceptability of corn snack fortified with fish protein powder at different rates and
reported that snacks fortified with 9 % fish protein powder showed significantly lower
liking in respect of odour, texture, flavour and overall acceptability, whereas, snacks
with 3 %, 5 % and 7 % fish powder showed similar sensory attributes with better
acceptability.

Response surface methodology (RSM) is an important tool in process and product

improvement. RSM is a collection of experimental design and optimization techniques
that enables the experimenter to determine the relationship between the response and the
independent variables. RSM is typically used for mapping a response surface over a
particular region of interest, optimizing the response, or for selecting operating
conditions to achieve target specifications or customer requirements (Myers and
Montgomery 2002). Extrusion processes can be optimized by the use of RSM (Milan-
Carrillo et al. 2002).

The objective of this research was to investigate the effect of extrusion conditions like
barrel temperature, screw speed, total moisture content and fish flour on the physical
properties (expansion ratio and bulk density) and sensory characteristics of snack-like
extruded products from fish-rice-corn blends when die diameter, preconditioning and
feeding rate were kept constant.

Materials and methods

Extrusion

Extrusion trials were performed with a laboratory-scale co-rotating twin-screw extruder

(Model BTPL-1, Basic Technology Pvt. Ltd. Kolkata, India). The screws of the extruder
were 29.7 mm in diameter and 350 mm in length. The pitch of the screw was 75 mm
with a flight depth of 3.5 mm. The die diameter and feeding rate were kept constant for
all the experiment as 3.0 mm and 10 kg/h respectively. The extrudates were at 50 ± 50 C
for three hours in a hot air drier before analysis.

Raw materials

Indian major carp (Labeo rohita) was brought under ice from the local market. The fish
were de-scaled, beheaded, eviscerated and washed with potable water. The dressed fish
was cooked by boiling in water for 10–12 min under normal atmospheric pressure. The
cooked fish was cooled, de-skinned and de-boned manually. The separated cooked meat
was dried in an electrically heated cabinet drier at 43–45 0 C. The dried fish muscle was
powdered in a domestic mixer. The fish flour was packed in polythene pack after
sieving and stored in refrigerator till preparation of blend. Other ingredients such as rice
flour and corn flour were procured from the market. Different composition for extrusion
was prepared with predetermined level of fish flour alongwith other ingredients such as
rice and corn flour at 1:1 ratio. Total moisture content was calculated considering the
moisture level of the ingredients. The ingredients were equilibrated to room temperature
and weighed according to the formulation before mixing. All the ingredients except fish
flour were mixed in mixer with required quantity of water (predetermined) and salt
(2 %), packed in polythene bag and kept at room temperature (27–310 C) for three hours.
The fish flour was mixed thoroughly and kept further at room temperature for one hour.
This preconditioning procedure was employed to ensure uniform mixing and hydration
and to minimize variability in the state of the feed material. The mixtures were sieved
using 0.5 mm mesh screen before extrusion.

Determination of product responses

Expansion ratio (ER)

For determination of expansion ratio, the cross sectional diameter of the extrudates was
measured with a Vernier caliper. The expansion ratio was calculated as the cross-
sectional diameter of the extrudate divided by the diameter of the die opening (Ding et
al. 2005). The ER values were obtained from 15 random samples with 3 locations in
each for each extrusion condition.

Bulk density (BD)

Bulk density (g/cm3) of extrudates was calculated by measuring the actual dimensions
of the extrudates (Thymi et al. 2005). The diameter and length of the extrudates were
measured using Vernier caliper. The weight per unit length of extrudate was determined
by weighing measured lengths. The bulk density was then calculated using the
following formula, assuming a cylindrical shape of extrudate.

, where M is mass (g) and V is the volume

in cm3. Five pieces of extrudate were

randomly selected and average taken.

Compositional analysis

Moisture, ash, protein and fat analysis of raw materials and extrudates was carried out
using standard procedures of AOAC (2000). Carbohydrates were calculated by
difference. All the experiments were replicated, so that the data in the paper are
expressed as the mean (± SD) of triplicate analysis.

Optimization of experimental design

The optimum conditions for extrusion of fish based snack products were determined by
response surface methodology (RSM). Based on the results of preliminary studies, the
experiments were optimized using five-levels, four factors central composite design
(CCD). The range and center point values of the four independent variables are shown
in Table 1. The CCD in the experimental design consisted of 29 treatments with five
replicates of the central point. The physical quality of extrudate is strongly affected by
several process variables besides ingredient selection. Out of many processing factors,
barrel temperature, screw speed, total moisture content and fish flour content were
chosen as the independent variables. Quality extruded snacks have two important
attributes, expansion and density. Extrudates’ expansion ratio and bulk density were
selected as dependent variables.
Table 1Composition of ingredients used in the experiments
Levels

Code values
Independent variables Symbol
−2 −1 0 +1 −2

Real values

Barrel temperature (˚c) A 90 100 110 120 130

Screw speed (rpm) B 320 360 400 440 480

Total moisture (%) C 14 16 18 20 22

Fish flour (%) D 10 15 20 25 30

Statistical analysis

The experimental data for expansion ratio and bulk density from different treatments
was analyzed using multiple regression analysis using statistical software (The
Unscrambler, Version 9.5, CAMO Process AS, Oslo, Norway). The fitting was done to a
second order model for each response. This model can be expressed with the coded
variables (A, B, C, D) with the following equation.

where Y represented the estimated response, β0 represented the equation parameters for
the constant term, βi represented the linear terms, βii represented the quadratic terms for
a single variable, βij represented the interaction terms (i = 1–4; and j  = 1–4), and ε
represented the random error. The response surface plots were developed as a function
of two independent variables while keeping the other two independent variables at
optimal values.

Sensory analysis of extruded snacks

Sensory evaluations were conducted using a ten-member trained panel. Panelists were
trained to evaluate the extrudates for general appearance, surface texture, flavour,
crispiness and overall acceptability on 0–5 scale (Larmond 1977) and the results were
averaged. The data obtained from biochemical and sensory analysis was further
analyzed and interpreted by using suitable statistical method with SPSS windows 16.0
software and ANOVA.

Results and discussions

Experimental results

The composition of ingredients used in the experiments is presented in Table 2. Table 3

shows the central composite design and responses of dependent variables (expansion
ratio and bulk density of the extrudates) together with the predicted value according to
the second order response surface models. Regression analysis was employed to fit a
full response surface model for every response investigated including all linear (A, B, C,
D), interaction (AB, AC, AD, BC, BD, CD), and quadratic terms (A 2, B2, C2, D2). The
regression coefficients for the response surface model in terms of coded units are shown
in Table 4.

Table 2Experimental design range and values of the independent variables in the central
composite design for production of fish based extruded snack
Composition of ingredients (g/100 g)
Ingredients
Moisture Protein Fat Carbohydrate Ash
Rice flour 8.7 ± 0.58 6.8 ± 0.72 0.33 ± 0.11 83.1 ± 1.37 0.71 ± 0.18
Corn flour 8.2 ± 0.46 5.9 ± 0.57 0.56 ± 0.09 84.7 ± 2.76 0.37 ± 0.08
Fish flour 7.1 ± 0.28 82.5 ± 0.39 5.5 ± 0.28 0.4 ± 0.07 3.2 ± 0.43

Table 3Central composite design for optimizing the extrusion condition for expansion
ratio and bulk density of the extrudates in coded units together with experimental data
and their predicted value according to the 2nd order response surface models
Responses
Coded level of variables
Treatment Expansion Ratio (Y1) Bulk Density (Y2)
A B C D Experimental Predicted Experimental Predicted
1 −2 0 0 0 3.18 2.81 0.15 0.19
2 +2 0 0 0 2.86 2.81 0.19 0.19
3 0 −2 0 0 3.35 3.14 0.16 0.19
4 0 +2 0 0 3.46 3.14 0.10 0.19
5 0 0 −2 0 3.42 2.65 0.10 0.15
6 0 0 +2 0 2.05 2.17 0.30 0.23
7 0 0 0 −2 2.50 2.48 0.13 0.19
8 0 0 0 +2 1.97 2.34 0.18 0.19
9 −1 −1 −1 −1 3.48 3.20 0.08 0.19
10 +1 −1 −1 −1 2.93 3.00 0.08 0.14
11 −1 +1 −1 −1 3.45 2.99 0.09 0.14
12 +1 +1 −1 −1 2.97 2.79 0.16 0.19
13 −1 −1 +1 −1 2.72 2.96 0.24 0.23
14 +1 −1 +1 −1 2.81 2.69 0.09 0.18
15 −1 +1 +1 −1 2.25 2.46 0.28 0.18
16 +1 +1 +1 −1 2.18 2.26 0.28 0.23
17 −1 −1 −1 +1 2.42 2.43 0.24 0.19
18 +1 −1 −1 +1 2.43 2.63 0.07 0.14
19 0 +1 −1 +1 2.42 2.64 0.25 0.17
20 +1 +1 −1 +1 2.56 2.84 0.21 0.19
21 −1 −2 +1 +1 2.07 2.91 0.30 0.26
22 +1 −2 +1 +1 2.36 3.12 0.10 0.16
23 −1 +1 +1 +1 2.38 2.68 0.27 0.18
24 +1 +1 +1 +1 2.81 2.46 0.20 0.23
25 0 0 0 0 2.39 2.41 0.13 0.19
26 0 0 0 0 2.48 2.41 0.13 0.19
27 0 0 0 0 2.46 2.41 0.24 0.19
 Responses
Coded level of variables
Treatment Expansion Ratio (Y1) Bulk Density (Y2)
A B C D Experimental Predicted Experimental Predicted
28 0 0 0 0 2.27 2.41 0.26 0.19
29 0 0 0 0 2.46 2.41 0.19 0.19

Table 4Regression coefficients for the 2nd order response surface models in terms of
expansion ratio and bulk density of the extrudates

Responses
Parameter Term Expansion Ratio (Y1) Bulk Density (Y2)
Coefficient P-value Coefficient P-value
β0 Intercept 2.412 0.0000 0.188 0.0000
β1 Barrel temp. (A) −0.003250 0.4778 −0.002 0.0829
β2 Screw speed (B) .000002083 0.9853 0.0004375 0.1253
β3 Total moisture (C) −0.121 0.0001 0.02042 0.0016
β4 Fish flour (D) −0.03667 0.0011 0.003667 0.1095
β 12 Barrel temp. × screw speed (AB) 0.009643 0.8397 0.02571 0.0375
β 13 Barrel temp. × Total moisture (AC) 0.8679 0.0848 −0.015 0.2055
β 14 Barrel temp. × fish flour (AD) 0.101 0.0493 −0.02143 0.0773
β 23 Screw speed × Total moisture (BC) −0.02571 0.5912 0.000 __
β 24 Screw speed × fish flour (BD) 0.106 0.0397 0.000 __
β 34 Total moisture × fish flour ( CD) 0.142 0.0087 −0.02036 0.0919
β 11 Barrel temp. × barrel temp. (A²) 0.100 0.0161 0.000 __
β 22 Screw speed × screw speed (B²) 0.183 0.0002 −0.00937 0.2836
β 33 Total moisture × total moisture (C³) 0.03939 0.3017 0.00527 0.5566
β 44 Fish flour × fish flour (D²) 0.06775 0.0864 −0.004370 0.6251

The examination of the fitted model was always necessary to ensure that it provided an
adequate approximation to the true system (Zhou and Regenstein 2004). To develop the
fitted response surface model equations, all insignificant terms (P > 0.05) were
eliminated and the fitted models are shown in Table 5. Coefficient of determination (R2)
is defined to be the ratio of the explained variation to the total variation and is a
measurement of the degree of fitness (Nath and Chattopadhyay 2007). A small value of
R2 indicates a poor relevance of the dependent variables in the model. The model fit
well with the actual data when R2 approaches unity (Sin et al. 2006).

Table 5Response surface model for expansion ratio and bulk density of fish based
extrudates
P-
Response Quadratic polynomial model* R²
value
Expansion
Y1 = 2.412 − 0.121 C − 0.03667D + 0.101 AD + 0.106BD + 0.142CD + 0.1A² + 0.183B² 0.885 0.0002
ratio
Bulk
Y2 = 0.188 + 0.02042 C + 0.02571AB 0.694 0.01
density
*Y1 (expansion ratio), Y2 (bulk density, g/cm3), A (barrel temperature, 0 C), B (screw
speed, rpm), C (total moisture, %), D (fish flour, %)

The analysis of variance (ANOVA) was used to evaluate the significance of the
quadratic polynomial model equation. Any terms in the models with a large F-value and
a small P-value would indicate a more significant effect on the respective response
variables (Yuan et al. 2008). Table 6 shows the ANOVA for the models that explains the
response of the dependent variables. Both the models were highly significant (P ≤ 0.01)
at the 99 % probability level.

Table 6Sensory scores of selected extrudates produced at different process conditions

General Surface Overall
Treatment Flavour Crispness
Appearance Texture Acceptability
1 3.8 ± 0.63 ab 3.7 ± 0.67 ab 3.7 ± 0.67 abcd 3.5 ± 0.53 abc 3.7 ± 0.82 abc
2 3.8 ± 0.63 ab 3.8 ± 0.63 ab 3.4 ± 0.52 abcd 3.8 ± 0.63 abc 3.7 ± 0.48 abc
3 4.0 ± 0.47 b 3.7 ± 0.48 ab 3.5 ± 0.53 abcd 3.8 ± 0.92 abc 3.9 ± 0.74 abcd
4 3.9 ± 0.74 ab 3.8 ± 0.79 ab 3.8 ± 0.42 bcd 4.0 ± 0.67 c 4.4 ± 0.70 d
5 3.7 ± 0.67 ab 3.8 ± 0.63 ab 3.5 ± 0.71 abcd 3.6 ± 0.52 abc 3.7 ± 0.48 abc
7 3.8 ± 0.92 ab 3.6 ± 0.52 ab 4.0 ± 0.82 d 3.5 ± 0.85 abc 3.8 ± 0.79 abcd
9 3.9 ± 0.57 ab 3.8 ± 0.42 ab 3.9 ± 0.74 cd 3.9 ± 0.74 bc 4.2 ± 0.63 bcd
10 3.8 ± 0.63 ab 3.9 ± 0.57 b 3.6 ± 0.70 abcd 3.9 ± 0.74 bc 3.8 ± 0.92 abcd
11 3.8 ± 0.63 ab 3.9 ± 0.57 b 3.9 ± 0.74 cd 3.7 ± 0.67 abc 4.3 ± 0.48 cd
12 3.8 ± 0.63 ab 3.6 ± 0.52 ab 3.8 0 ± .63 bcd 3.9 ± 0.57 bc 3.6 ± 0.52 abc
13 3.5 ± 0.53 ab 3.5 ± 0.53 ab 3.1 ± 0.32 a 3.4 ± 0.52 abc 3.4 ± 0.52 a
14 3.7 ± 0.67 ab 3.8 ± 0.79 ab 3.3 ± 0.48 abc 3.5 ± 0.71 abc 3.6 ± 0.84 abc
20 3.4 ± 0.52 ab 3.8 ± 0.63 ab 3.2 ± 0.42 ab 3.3 ± 0.48 ab 3.3 ± 0.48 a
24 3.3 ± 0.48 a 3.2 ± 0.42 a 3.3 ± 0.48 abc 3.2 ± 0.42 a 3.5 ± 0.71 ab

Mean values in the column for all the samples with different superscripts are
significantly different (P < 0.05). Values are mean ± S.D. (n = 10)

In the present work, the optimum extrusion conditions for maximum expansion and
minimum bulk density were established by fixing two variables at coded zero level
(Table 1) while varying the other two variables. The estimated response function and the
effects of the independent variables (A, B, C and D) on the dependent variables
(expansion ratio and bulk density) are shown in Figs. 1 and and22.
Fig. 1Response surface plots showing the effects of (a) temperature and screw speed,
(b) moisture and fish flour, (c) temperature and fish flour, (d) temperature and moisture,
(e) screw speed and moisture and (f) screw speed and fish flour on expansion ratio
Fig. 2Response surface plot showing the effects of (a) temperature and moisture, (b)
temperature and fish flour, (c) moisture and fish flour and (d) screw speed and moisture
on bulk density

Results from sensory evaluations are presented in Table 6. Since expansion and bulk
density are considered as two important physical qualities of the extruded snacks,
extrudates only from 14 out of 29 treatments were subjected to sensory evaluation. The
overall acceptability varied between 3.3 to 4.4. General appearance, surface texture,
flavour and crispness scored 3.3–4.0, 3.2–3.9, 3.1–4.0 and 3.2–4.0 respectively.

Product’s expansion ratio

The amount of expansion in food depends on the difference between the vapor pressure
of water and the atmospheric pressure, as well as the ability of the exiting product to
sustain expansion. The expansion ratio (ER) of the products ranged from 1.97 to 3.48.
From the regression analysis, it was revealed that the A and B coefficients were not
significant (P > 0.05) whereas the C and D were highly significant (P ≤ 0.01). Further,
the interaction effect of temperature x fish flour (P ≤ 0.05), screw speed x fish flour (P ≤ 
0.05), total moisture x fish flour (P ≤ 0.01) and square of temperature (P ≤ 0.05) and
screw speed (P ≤ 0.01) were found significant.

A high co-efficient of determination (R2) for expansion in this experiment was 0.885
which indicates that the model (Table 5) was suitable to represent the real relationships
among the selected process parameters. The experimental values agreed well with the
predicted ones that are obtained from the model (Table 3). Any terms in the models with
a large F-value and a small P-value would indicate a more significant effect on the
respective response variables (Yuan et al. 2008). ANOVA for the model as fitted shows
significance (P < 0.01) with a moderate lack of fit which indicated the optimum models
for this experiment.

Figure 1a shows the effect of temperature and screw speed on expansion ratio and the
maximum predicted expansion ratio can be found with the screw speed of 480 rpm and
the temperature of 1100 C. Regression analysis showed that ER was significantly (P ≤ 
0.01) affected by the quadratic effect of both temperature (A 2) and screw speed (B2).
Launay and Lisch (1983) proposed that the longitudinal and sectional expansions are
dependent on the melt viscosity and elasticity. The increased temperature would yield a
lower melt viscosity and increased longitudinal expansion, while the melt viscosity
would be lowered and cause a decrease in sectional expansion. Similar observations of
the effect of temperature on product expansion were reported for corn starch, corn grits
and rice flour (Chinnaswamy and Hanna 1988; Ali et al. 1996; Hagenimana et al. 2006).
The expansion ratio decreases with the decrease of screw speed. This behaviour could
be explained by the development of less shear force and less pressure within the barrel.

The maximum predicted expansion can be found with the total moisture of 14 % and
fish flour of 20 % when the barrel temperature and screw speed are at zero level
(Fig. 1b). An inverse relationship was found between the expansion ratio with both the
total moisture content and fish flour. According to the regression analysis, both total
moisture and fish flour had a significant negative linear effect (P ≤ 0.001) on the ER
whereas the interaction effect of total moisture x fish flour (CD) showed significant
positive linear effect (P < 0.01). Since the expansion ratio and bulk density are inversely
related, this is similar to the observation of Pansawat et al. (2008) who reported that the
extrusion conditions that produced the lowest and the highest product density were very
similar to those producing the lowest and highest product moisture since they are
closely related.

Figure 1c and d shows the effect of temperature along with fish flour and total moisture
on expansion ratio. All the three independent variables showed inverse relationship with
the expansion ratio when two out of the three variables along with the screw speed are
kept constant at zero level. The interaction effect of temperature x fish flour (AD)
showed a significant positive linear effect (P < 0.05) on the expansion whereas the
interaction effect of temperature x total moisture (AC) was not significant (P > 0.05).
The maximum ER can be found with the combination of temperature and fish flour as
900 C and 20 % and temperature and total moisture as 110 0 C and 14 %. Shannon and
Robert (2010) studied the effect of protein and moisture content on the physico-
chemical characteristics of pea flour extrudates. They reported that EI decreased and
bulk density, particle density and hardness increased with increase in protein or
moisture content. The protein decreases shear force within the barrel, thereby lowering
the die pressure and create a lower pressure differential between the die and the
atmosphere resulting reduced amount of superheated water flashed off, thus expansion
is decreased (Shannon and Robert 2010).

The effect of screw speed alongwith fish flour and total moisture on expansion ratio is
shown in Fig. 1e and f. Regression analysis showed that the interaction effect of screw
speed x fish flour (BD) had a significant positive linear effect (P < 0.05) on the
extrudates’ expansion.

Product’s bulk density

The bulk density, which considers expansion in all directions, is an index of the extent
of puffing. Bulk density, expressed as g cm − 3, measures expansion too. The bulk
density (BD) of the products ranged from 0.07 to 0.3. From the regression analysis, it
was revealed that only coefficient C was found highly significant (P ≤ 0.01). Further, the
interaction effect of only temperature x screw speed (AB) was found significant (P ≤ 
0.05).

A reasonably good co-efficient of determination (R2) for bulk density in this experiment
was 0.694 which indicates that 69.4 % of the variability in bulk density could be
explained by this model (Table 4) and suitable to represent the real relationships among
the selected process parameters. The predicted values agreed well with the experimental
ones that are obtained from the model (Table 3). Based on the ANOVA, the P-value of
the model showed significance (P  = 0.01). Meanwhile the lack of fit value was 0.72
which was not significant. These two values confirmed that the model fitness was good.

The response surface plot for the effect of temperature and moisture on the bulk density
is presented in Fig. 2a. Both temperature and moisture showed positive relationship
with the bulk density and minimum bulk density can be found with the temperature of
1100 C and moisture of 14 %. Further, fish flour too showed positive relationship with
the bulk density when three other independent variables were kept constant and the
minimum BD can be found with the combination of temperature of 110 0 C and fish
flour of 10 % (Fig. 2b). Similar effect of total moisture and fish flour on the bulk
density was found (Fig. 2c). Only screw speed showed inverse relationship with the
bulk density when three other independent variables were kept constant (Fig. 2d).

Altan et al. (2008) investigated the physical characteristics of barley flour extrudates
and reported that temperature had a dominant effect on the bulk density and in respect
of screw speed they found a minor effect. The positive relationship between temperature
and bulk density could be explained by the decrease of expansion with increase of
temperature. The reduction of expansion caused an increase in bulk density. A high
barrel temperature reduce the melt viscosity to a larger extent and this could facilitate
bubble growth; however, the bubble walls become too thin to contain the vapor
pressure, resulting in more bubble fracture, thus increasing the rate of collapse and
resulting in an overall reduction in expansion (Yuliani et al. 2006). According to Meng
et al. (2010) product temperature or melt temperature plays an important role in
changing the rheological properties of the extruded melts, which in turn affect the
degree of expansion. Bulk density also describes the degree of expansion undergone by
the melt as it exits the extruder (Meng et al. 2010).

Product’s acceptability

The minimum and maximum overall acceptability corresponded to the treatment no. 20
and 4 respectively (Table 6). The general appearance of the extrudates evaluated were
found satisfactory and scored between moderate to good. Surface texture scored lowest
when fish flour and moisture content was 25 % and 20 % respectively. The flavour
scored highest when fish flour content was low. The crispness, a perception of the
human being and is associated with the expansion of the product and also one of the
most important quality attribute to the consumer, varied between 3.2–4.0. Higher
crispness was found for the extrudates produced with moderate level of fish flour and
feed moisture content. Probably, in case of higher fish flour, the cross linkage of protein
and development of a protein network as a result of starch protein interaction have
increased the maximum force of hardness with reduced crispness (Jeyakumari and
Rathnakumar 2006). Decrease of hardness with decreasing feed moisture content was
reported by Meng et al. (2010) while studying the effects of extrusion conditions on the
physical properties of chickpea flour based snack. Generally, incorporation of fish
proteins reduced extrudate expansion and increased hardness resulting reduced
crispness (Choudhury and Gautam 2003).

The overall acceptability of the extruded snacks was concerned; highest (4.4) was
scored by the snacks from the treatment 4, where the process variables were barrel
temperature −110°C, screw speed −480 rpm, moisture −18 % and fish flour −20 %.
Extrudates from two other treatments, namely, treatment-9 and treatment-11 scored 4.2
and 4.3 respectively. The overall acceptability of the extrudates from these three
treatments were not significantly different (P < 0.05). The process variables for
treatment-9 and treatment-11 were similar in respect of barrel temperature (1000 C),
moisture (16 %) and fish flour (15 %) except screw speed which were 360 and 440 rpm
respectively. Sharma and Basu (2003) observed that product prepared using fish flour
beyond 30 % became hard and the expansion ratio decreased. Dileep et al. (2010), based
on the sensory scores for rice flour-fish mince-based extruded products, reported
optimum process conditions as barrel temperature of 900 C and fish mince concentration
of 10 %, when extruded at a constant screw speed of 350 rpm using a 4 mm die
diameter.

Conclusion
Change of extrusion conditions especially, barrel temperature, screw speed, moisture
and fish flour are observed to have significant effect on the physical properties of
extruded fish based ready-to-eat snacks in the present experiment. The products with
high expansion ratio and low bulk density are generally considered as good
characteristics of extruded snacks. An inverse relationship was found between the
expansion ratio with both the total moisture content and fish flour. A desirable
expansion ratio and bulk density were obtained with fish flour 15–20 %, total moisture
content 14–18 %, barrel temperature 100–1100 C and screw speed 360–480 rpm. The
model developed could be used for designing of extrusion conditions for getting
extrudates with desirable physical characteristics.

Acknowledgments
This study is part of the thesis work to obtain the degree of Master of Fisheries Science,
which was developed in the Department of Fish Processing Technology of College of
Fisheries, Lembucherra, Tripura, India and supported by the Central Agricultural
University, Imphal, India. The authors gratefully acknowledge the staff of the
department of FPT for their technical assistance.
References
1. Ali Y, Hanna MA, Chinnaswamy R. Expansion characteristics of extruded corn
grits. Lebensm Wiss Technol. 1996;29:702–707. doi: 10.1006/fstl.1996.0109.
[Cross Ref]

2. Altan A, McCarthy KL, Maskan M. Extrusion cooking of barley flour and

process parameter optimization by using response surface methodology. J Sci
Food Agr. 2008;88:1648–1659. doi: 10.1002/jsfa.3262. [Cross Ref]

3. AOAC (2000) Official methods of analysis,15th edn. Association of Official

Analytical Chemists. Washington DC

4. Asare EK, Sefa-Dedeh S, Sakyi-Dawson E, Afoakwa EO. Application of

response surface methodology for studying the product characteristics of
extruded rice-cowpea-groundnut blends. Int J Food Sci Nutr. 2004;55:431–39.
doi: 10.1080/09637480400003238. [PubMed] [Cross Ref]

5. Banerjee S, Chakraborty P. Physico-chemical properties of extruded aquatic

feed. Ind J Fish. 1998;45:107–111.

6. Chinnaswamy R, Hanna MA. Optimum extrusion-cooking conditions for

maximum expansion of corn starch. J Food Sci. 1988;53:834–840. doi:
10.1111/j.1365-2621.1988.tb08965.x. [Cross Ref]

7. Choudhury GS, Gautam A. Effects of hydrolysed fish muscle on intermediate

process variables during twin-screw extrusion of rice flour. Lebensm Wiss
Technol. 2003;36:667–678. doi: 10.1016/S0023-6438(03)00087-2. [Cross Ref]

8. Choudhury GS, Gogoi BK. Extrusion processing of fish muscle: a review. J

Aquat Food Prod Tech. 1995;4(4):37–67. doi: 10.1300/J030v04n04_05. [Cross
Ref]

9. Choudhury GS, Gogoi BK, Oswalt AJ. Twin screw extrusion of pink salmon
muscle and rice flour blends: effects of kneading elements. J Aquat Food Prod
Tech. 1998;7(2):69–91. doi: 10.1300/J030v07n02_06. [Cross Ref]

10. Dileep AO, Shamasundar BA, Binsi PK, Howell NK. Composition and quality
of rice flour-fish mince based extruded products with emphasis on thermal
properties of rice flour. J Tex Stud. 2010;41:190–207. doi: 10.1111/j.1745-
4603.2010.00221.x. [Cross Ref]

11. Ding QB, Ainsworth P, Tucker G, Marson H. The effect of extrusion conditions
on the physicochemical conditions and sensory characteristics of rice-expanded
snacks. J Food Eng. 2005;66:283–289. doi: 10.1016/j.jfoodeng.2004.03.019.
[Cross Ref]

12. Gogoi BK, Oswalt AJ, Choudhury GS. Reverse screw element (s) and feed
composition effects during twin-screw extrusion of rice flour and fish muscle
 blends. J Food Sci. 1996;61:590–595. doi: 10.1111/j.1365-2621.1996.tb13165.x.
[Cross Ref]

13. Guha M, Ali SZ. Extrusion cooking of rice: effect of amylase content and barrel
temperature on product profile. J Food Process Pres. 2006;30:706–716. doi:
10.1111/j.1745-4549.2006.00099.x. [Cross Ref]

14. Guy RCE. Raw materials for extrusion cooking. In: Guy RCE, editor. Extrusion
cooking technologies and applications. Cambridge: Woodhead Publishing
Limited; 2001. pp. 5–28.

15. Hagenimana A, Ding X, Fang T. Evaluation of rice flour modified by extrusion

cooking. J Cer Sci. 2006;43:38–46. doi: 10.1016/j.jcs.2005.09.003. [Cross Ref]

16. Jeyakumari A, Rathnakumar K. Effect of incorporation of frozen stored minced

fish on the quality of gelatinized product from rice and maize flour. Fish
Technol. 2006;43(2):146–153.

17. Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V.

Polyunsaturated fatty acids in the food chain in the United States. Am J Clin
Nutr. 2000;7(Suppl):179–188. [PubMed]

18. Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3
fatty acids and cardiovascular disease. Circulation. 2002;106:2747–2757. doi:
10.1161/01.CIR.0000038493.65177.94. [PubMed] [Cross Ref]

19. Larmond E (1977) Method for sensory evaluation of food. Publ. No. 1286,
Canada Dept. of Agriculture, Canada

20. Launay B, Lisch LM. Twin-screw extrusion cooking of starches: flow behaviour
of starch pastes, expansion and mechanical properties of extrudates. J Food Eng.
1983;2:259–280. doi: 10.1016/0260-8774(83)90015-8. [Cross Ref]

21. Linko P, Linko YY. Bioconversion processes. In: Pomeranz Y, Munck L, editors.
Cereals: a renewable resource: theory and practice. St. Paul: American Assoc.
Cereal Chem Inc; 1981. pp. 339–494.

22. Meng X, Threinen D, Hansen M, Driedger D. Effects of extrusion conditions on

system parameters and physical properties of chickpea flour-based snack. Food
Res Int. 2010;43:650–658. doi: 10.1016/j.foodres.2009.07.016. [Cross Ref]

23. Milan-Carrillo J, Reyes-Moreno C, Camacho-Herńandez IL, Rouzaud-Sandez

O. Optimisation of extrusion process to transform hardened chickpeas (Cicer
arietinum L.) into a useful product. J Sci Food Agr. 2002;82:1718–1728. doi:
10.1002/jsfa.1242. [Cross Ref]

24. Myers R, Montgomery DC. Response surface methodology: process and

products optimization using designed experiments. 2. New York: Wiley; 2002.
25. Nath A, Chattopadhyay PK. Optimization of oven toasting for improving
crispness and other quality attributes or ready to eat potato-soy snack using
response surface methodology. J Food Eng. 2007;80:1282–1292. doi:
10.1016/j.jfoodeng.2006.09.023. [Cross Ref]

26. Pansawat N, Jangchud K, Jangchud A, Wuttijumnong P, Saalia RK, Eitenmiller

RR, Phillips RD. Effects of extrusion conditions on secondary extrusion
variables and physical properties of fish, rice-based snacks. J Food Sci Technol.
2008;41:632–641.

27. Rhee KS, Kim ES, Kim BK, Jung BM, Rhee C. Extrusion of minced catfish
with corn and defatted soya flours for snack foods. J Food Process Pres.
2004;28:288–301. doi: 10.1111/j.1745-4549.2004.23069.x. [Cross Ref]

28. Rolf LA, Huff HE, Hsieh F. The effect of processing conditions on the quality of
extruded catfish feed. Trans ASAE. 2000;43:1737–1743. doi:
10.13031/2013.3076. [Cross Ref]

29. Shannon DH, Robert TT. Effect of protein, moisture content and barrel
temperature on the physico-chemical characteristics of pea flour extrudates.
Food Res Int. 2010;43:659–663. doi: 10.1016/j.foodres.2009.09.033. [Cross
Ref]

30. Sharma SK, Basu S. Preparation of an extruded fish snack using twin screw
extruder and the storage characteristics of the product. J Ind Fish Assoc.
2003;30:149–156.

31. Shaviklo GR, Olafsdottir A, Sveinsdottir K, Thorkelsson G, Rafipour F. Quality

characteristics and consumer acceptance of a high fish protein puffed corn-fish
snack. J Food Sci Technol. 2011;48(6):668–676. doi: 10.1007/s13197-010-0191-
1. [PMC free article] [PubMed] [Cross Ref]

32. Shaviklo GR, Thorkelsson G, Rafipour F, Sigurgisladottir S. Quality and storage

stability of extruded puffed corn-fish snacks during 6-month storage at ambient
temperature. J Sci Food Agr. 2011;91(5):886–893. doi: 10.1002/jsfa.4261.
[PubMed] [Cross Ref]

33. Simopoulos AP. Symposium: Role of poultry products in enriching the human
diet with n-3 PUFA, human requirement for n-3 polyunsaturated fatty acids.
Poultry Sci. 2000;79:961–970. doi: 10.1093/ps/79.7.961. [PubMed] [Cross Ref]

34. Sin HN, Yusor S, Hamid NSA, Rahman RA. Optimixation of enzymatic
clarification of sapodilla juice using response surface methodology. J Food Eng.
2006;73:313–319. doi: 10.1016/j.jfoodeng.2005.01.031. [Cross Ref]

35. Suknark K, Lee J, Eitenmiller RR, Phillips RD. Stability of tocopherols and
retinyl palmitate in snack extrudates. J Food Sci. 2001;66:897–902. doi:
10.1111/j.1365-2621.2001.tb15193.x. [Cross Ref]
 36. Thymi S, Krokida MK, Papa A, Maroulis ZB. Structural properties of extruded
corn starch. J Food Eng. 2005;68:519–526. doi:
10.1016/j.jfoodeng.2004.07.002. [Cross Ref]

37. Venugopal V, Shahidi F. Value added products from underutilized fish species.
Crit Rev Food Sci Nutr. 1995;35(5):431–453. doi:
10.1080/10408399509527708. [PubMed] [Cross Ref]

38. Yuan Y, Gao Y, Mao L, Zhao J. Optimisation of conditions for the preparation of
b-carotene nanoemulsions using response surface methodology. Food Chem.
2008;107:1300–1306. doi: 10.1016/j.foodchem.2007.09.015. [Cross Ref]

39. Yuliani S, Torley PJ, D’Arcy B, Nicholson T, Bhandari B. Effect of extrusion

parameters on flavour retention, functional and physical properties of mixtures
of starch and D-limonene encapsulated in milk protein. Int J Food Sci Technol.
2006;41:83–94. doi: 10.1111/j.1365-2621.2006.01409.x. [Cross Ref]

40. Zhou P, Regenstein JM. Optimization of extraction condition for Pollock skin
gelatin. J Food Sci. 2004;69:393–398.

J Food Sci Technol. 2010 Dec; 47(6): 606–612.

Published online 2010 Oct 29. doi: 10.1007/s13197-010-0117-y

III: Optimization of extrusion conditions

for defatted soy-rice blend extrudates
Sanjeev Kumar Garg and Daya S. Singh
Author information ► Article notes ► Copyright and License information ►

Abstract
Processing parameters of feed including moisture content of feed (12, 15, 18, 21 and
24%), soy flour-rice blend ratio (10:90, 14:86, 18:82, 22:78 and 26:74), operational
parameters of extruder like barrel temperature (100, 110, 120, 130 and 140 °C), die head
temperature (160, 170, 180, 190 and 200 °C) and screw speed (100, 110, 120, 130 and
140 rpm) were optimized for physical and textural properties of soy-rice blend
extrudates. The minimum value of longitudinal expansion index lied near 185 °C die
head temperature at about 120 rpm. The minimum value of bulk density was observed
in between 18:82 blend ratio and 115 °C barrel temperature.

Keywords: Extrusion, Snacks, Response surface methodology

Introduction
Extruded snacks contribute as an important part for many consumers in daily nutrient
and calories intake (Teltweiler 1991). The defatted soybean (Glycine max L.) meal is the
by-product of solvent extraction plant and rice brokens (Oryza sativa L.) are the by-
product of rice milling industry. Rice flour prepared out of rice brokens can be used as
an important ingredient for many ready-to-eat breakfast cereals and snacks. Soybean,
the first vegetable proteinaceous feed material was used for making protein rich
extruded food (Harper 1981). Extrusion cooking is capable of converting soluble
globular legume protein into material having fibrous and chewy texture (Harper 1981).
Indian soybean industry produces about 3 million tons of meal every year (SOPA 1999).
Defatted soy flour contains about 50% protein and its protein digestible index is 60. In
the extrusion of snacks and other food products, proper control of the extrusion process
is important to quality of final product. In cereal-based products, the degree of starch
processing is important for major quality aspects such as taste, digestibility, texture and
appearance. Patil et al. (1990) studied on a dry extrusion cooker Insta Pro 2000 for
extrusion of soy-rice blend. It was observed that extrusion at lower level of moisture
(12% wb) is most suitable. The highly puffed product having bulk density of
266.23 kg/m3, water absorption index of 4.09 was obtained at this level of feed
moisture. Dahlin and Lorenz (1993) examined seven whole grain cereals to study
extrusion processing effects on in vitro protein digestibility. Eight extrusion conditions
were applied and effects of cereal variety, extrusion temperature and feed moisture on
protein digestibility were studied. The most protein-digested products were produced at
15% feed moisture, 100/150 °C product temperature and screw speed of 100 rpm.
Bhattacharya et al. (1999) observed that incorporation of chickpea into rice flour
decreased product expansion but increased bulk density and peak shear force of
extrudates. Boonyasinikool and Charunuch (2000) developed a nutritious soy fortified
snack with good texture and protein quality from 18% of soy flour (9% defatted soy
flour + 9% full fat soy flour) in rice broken and corn grits blend, with 2% soybean oil
fortified with vitamins, minerals and amino acid. The product obtained had expansion
ratio, bulk density and compression force of 3.9, 58 g/m3 and 60.17 N, respectively.
Protein content in the developed snack was 9.9%, which was 46.7–70.7% higher than
the market snacks. Chang and EI-Dash (2003) studied some physical characteristics of
extruded cassava starch using a single—screw extruder, by varying acid concentration,
feed moisture content and barrel temperature and evaluated using surface response
methodology. Singh et al. (2006a) optimized the processing parameters of extruded food
prepared from Bengalgram broken and sorghum blends. They optimized parameters for
maximum values of specific length, sectional expansion index and minimum value of
mass flow rate. They suggested that the best quality extruded snacks were found at
100 °C barrel temperature, 15:85 blend ratio of Bengalgram broken to sorghum and
15% moisture content. Singh et al. (2006b) found that the physical properties and
textural analysis of snacks prepared at 20% blending of soybean, 15% moisture content
and 85 °C temperature was found as the best quality extrudates. The present study was
undertaken to study the effect of moisture content, blend ratio and temperature (barrel
and die head) on the physical properties (moisture content of extrudates, specific length,
longitudinal expansion index, and bulk density) of rice and defatted soy flour based
ready-to-eat extrudates.

Materials and methods

The study was conducted at soy processing lab of Department of Post-harvest Process
and Food Engineering, College of Agricultural Engineering, Jabalpur, India. The single-
screw extruder (D47055 Duisburg, Brabender, Germany), consisted of grooved barrel
covered with heating and cooling elements. The constructional elements of the extruder
included motor and gear unit, coupling, loading unit, extruder barrel with screw and
control cabinet. The length to diameter ratio of the extruder was 20:1. During
experimentation the compression ratio (3:1) temperature of barrel zone I (90 °C) and
barrel zone II (120 °C) were kept constant. The defatted soy-flour and rice flour were
procured from local market. The experimental design is dependent on the symmetrical
selection of variation increments about the centre composition (Cochran and Cox 1957;
Myres 1976). These levels of variation were selected to be within the range of
reasonable formulations and the increments were carefully selected. The selected levels
were also based on the conclusions of previous studies (Chauhan and Bains 1988; Patil
et al. 1990; Singh et al. 2006b) and preliminary trails.

Results and discussion

Moisture Content of Extrudates (MCE)

The second degree polynomial equation obtained by response surface analysis of the
data showing the effect of MCE versus feed moisture content (MCF), blend ratio (BR),
barrel temperature (TBrl), die head temperature (TDie) and screw speed (SS) resulted in
following equation

The R2 value was 0.93 and F-value was 7.31 for the model. It is depicted from Fig. 1
that there is positive correlation between the moisture content of feed and MC E. At the
lower value of MCE and blend ratio, the value of moisture content of extrudate was
lowest and when the value of moisture content of feed increased keeping blend ratio at
constant level, the value of moisture content of extrudate also increased (Singh et al.
2005).

Fig. 1Effect of feed moisture content and blend ratio on moisture content of extrudates
Specific Length (SL) of extrudates

The second degree polynomial model for SL versus feed moisture content (MCF), blend
ratio (BR), barrel temperature (TBrl), die head temperature (TDie) and screw speed (Ss)
resulted into an equation

The R2 value was 0.922 for the model and F-value was 6.53, which implied that the
model is significant. In this case, linear term of moisture content of feed and square
terms of blending ratio, and screw speed were found highly influencing variables on the
SL of extrudates. Singh et al. (2006b) reported that SL was highly affected by moisture
content and temperature of feed. Figure 2 shows the maximum value of specific length
in the range of 18 to 20% blend ratio and 185 to 190 °C die head temperature.

Fig. 2Effect of blend ratio and die head temperature on specific length of extrudates

Longitudinal Expansion Index (LEI)

Multiple regression model of longitudinal expansion index LEI versus feed moisture
content (MCF), blend ratio (BR), barrel temperature (TBrl), die head temperature (TDie)
and screw speed (SS) resulted into the following equation

The R2 value was 0.80 and F-value was 2.22 for the model. The maximum value of LEI
lies at 110–115 °C barrel temperature and 18–20% blend ratio beyond that the value of
LEI decreased (Fig. 3). Manoharkumar et al. (1978) reported that blending with legume
flours decreased the expansion ratio. Figure 3 shows the barrel temperature has more
effect on LEI in comparison to blend ratio. The value of LEI was lowest at maximum
value of barrel temperature and blend ratio within the experimental range.

Fig. 3Effect of blend ratio and barrel temperature on longitudinal expansion index of
extrudates

Volumetric Expansion Index (VEI)

Regression analysis of VEI versus feed moisture content (MC F), blend ratio (BR), barrel
temperature (TBrl), die head temperature (TDie) and screw speed (SS) yielded following
polynomial model

The R2 was 0.895 and F-value was 4.70, which implies that the model is significant.
Figure 4 shows the look of umbrella shape where the contours are spreading in outward
direction which means the highest value of VEI lies near feed moisture content of 12%,
18:82 blend ratio, 120 °C barrel temperature, 180 °C die head temperature and 120 rpm
screw speed, nearly in the centre of contour graph and moving either side will increase
the VEI (Patil et al. 1990).
Fig. 4Effect of blend ratio and barrel temperature on volumetric expansion index of
extrudates

Bulk Density (BD)

The multiple regression analysis of BD versus feed moisture content (MC F), blend ratio
(BR), barrel temperature (TBrl), die head temperature (TDie) and screw speed (SS) yielded
following polynomial model

The R2 value was 0.93 and F-value was 7.4 for the model. In this case, the linear terms
of feed moisture content, interaction term of moisture content and die head temperature
and square terms of moisture content, blend ratio and die head temperature of feed were
highly influencing variable coefficients affected the model of bulk density of extrudates.
There was strong correlation between the feed moisture content and bulk density of
extrudates (Fig. 5). As the value of feed moisture content increased the value of bulk
density decreased and vice versa (Kulkarni and Joshi 1992). Hence the bulk density had
positive correlation with moisture content of feed.
Fig. 5Effect of feed moisture content and blend ratio on bulk density of extrudates

Crispness

The data showed that crispness was highest at 12% feed moisture content, 18:82 blend
ratio of defatted soy flour to rice, 120 °C barrel temperature, 180 °C die head
temperature and 120 rpm screw speed. The second degree polynomial was obtained and
the effect of crispness versus feed moisture content (MC F), blend ratio (BR), barrel
temperature (TBrl), die head temperature (TDie) and screw speed (SS) resulted in
following relationship

The R2 value was 0.932 for the model and F-value was 7.48, which implies that the
model is significant. Figure 6 showed the shape of umbrella where the contours are
spreading in outward direction, which means, the highest value of crispness lies nearly
in the range of 16 to 18% blend ratio and 120–125 °C barrel temperature, moving either
side will reduce the value. Singh et al. (2005) reported that crispness increased with
increase in temperature and decreased with increase in moisture content.

Fig. 6Effect of blend ratio and barrel temperature on crispness of extrudates

Hardness

The data showed that hardness was highest at 24% feed moisture content, 18:82 blend
ratio of defatted soy flour to rice, 120 °C barrel temperature, 180 °C die head
temperature and 120 rpm screw speed. The second degree polynomial for hardness
versus feed moisture content (MCF), blend ratio (BR), barrel temperature (TBrl), die head
temperature (TDie) and screw speed (SS) resulted in the following model

The R2 value was 0.877 with F-value was 3.93 for the model, which implied that the
model is significant. There is a positive correlation between these parameters on
hardness of the extrudate (Fig. 7). The lowest value of hardness lies nearly at 16% blend
ratio and middle of 15 to 16.5% moisture content of extrudates. When the value of
moisture content of feed increased by keeping blend ratio at constant level, the value of
hardness increased (Jha and Prasad 2003) and when the value of blend ratio increased
gradually by keeping moisture content of feed at constant level, the value of hardness
also increased up to 18% moisture content and beyond that it remained unchanged.

Fig. 7Effect of feed moisture content and blend ratio on hardness of extrudates

Cutting strength

The observed data showed that cutting strength was highest at 21% feed moisture
content, 22:78 blend ratio of defatted soy flour to rice, 110 °C barrel temperature,
170 °C die head temperature and 130 rpm screw speed. Regression analysis of cutting
strength versus feed moisture content (MCF), blend ratio (BR), barrel temperature (TBrl),
die head temperature (TDie) and screw speed (SS) yielded following polynomial model

8
The R2 gave a value of 0.786 and F-value was 3.45, which implied that the model is
significant. There was strong correlation between the feed moisture content and cutting
strength of extrudates (Fig. 8). As the value of feed moisture content increased, the
value of cutting strength increased which means product becomes harder at higher
moisture content. Hence, the cutting strength had positive correlation with moisture
content of feed because the linear, interactive and quadratic term had significant role
over the model (Singh et al. 2005).

Fig. 8Effect of feed moisture content and blend ratio on cutting strength of extrudates

Conclusion
After complete evaluation of all the attributes for physical parameters like moisture
content, specific length, longitudinal expansion index, volumetric expansion index and
bulk density and textural properties like crispness, hardness and cutting strength of
extrudates, it was found that there were strong positive correlations between the
moisture content of feed and blend ratio, while barrel temperature, and die head
temperature had significant influence and screw speed had the negligible influence on
the physical and textural properties of extrudates. The maximum value of specific
length existed at 18:82 blend ratio, 125 °C barrel temperature, 185 to 190 °C die head
temperature and 120–125 rpm screw speed. The minimum value of longitudinal
expansion index was near 185 °C die head temperature and about 120 rpm. The highest
value of volumetric expansion index was near 120 °C barrel temperature and 180 °C die
head temperature and near 120 rpm screw speed. The minimum value of bulk density
was observed between 18:82 blend ratio and 115 °C barrel temperature the maximum
value of crispness was found near higher side of die head temperature i.e. 185–190 °C
and 120 rpm screw speed. The lowest hardness was obtained near 16% blend ratio and
middle of 15 to 16.5% moisture content of extrudates. The minimum cutting strength
was observed at 170 °C die head temperature and 120–130 °C barrel temperature. The
optimum conditions for maximum acceptable extrudates were 16.0–16.5% moisture
content, 22:78 blend ratio, 130 °C barrel temperature, 190 °C die head temperature and
110 rpm screw speed followed by 171 moisture content, 22:78 blend ratio, 130 °C barrel
temperature, 190 °C die head temperature and 110 rpm screw speed.

References
1. Bhattacharya S, Sudha ML, Rahim A. Pasting characteristics of an extruded
blend of potato and wheat flours. J Food Eng. 1999;40:107–111. doi:
10.1016/S0260-8774(99)00046-1. [Cross Ref]

2. Boonyasinikool P, Charunuch C. Development of nutritious soy fortified snack

by extrusion cooking. J Kasetsart Nat Sci. 2000;34:279–288.

3. Chauhan GS, Bains GS. Effect of some extrudes variables on physico–chemical

properties of extended rice-legume blend. Food Chem. 1988;27:213–224. doi:
10.1016/0308-8146(88)90064-7. [Cross Ref]

4. Chang YK, EI-Dash AA. Effects of acid concentration and extrusion variables
on some physical characteristics and energy requirements of cassava starch.
Brazil J Chem Eng. 2003;20(2):129–137.

5. Cochran WG, Cox GW. Experimental design. New York: Wiley; 1957.

6. Dahlin K, Lorenz K. Protein digestibility of extruded cereal grains. Food Chem.

1993;48:13–18. doi: 10.1016/0308-8146(93)90214-Z. [Cross Ref]

7. Harper JM (1981) Starch-based extended foods. In: Extrusion of foods, Vol II,
CRC Press Inc. Florida, p 83–87

8. Jha SK, Prasad S. Studies on extrusion cooking of rice and mung blend with salt
and sugar. J Food Sci Technol. 2003;40:257–261.

9. Kulkarni SD, Joshi KC. Potato starch soy-blends: possible effects of starch
properties on few aspects of and products. Indian Food Pack. 1992;46:38–47.

10. Manoharkumar B, Seileer K, Gerstenkorn P. Pressure extrusion of Indian maize

and maize legume composite flours. J Food Sci Technol. 1978;15:173–174.

11. Myres HP. Response surface methodology. Boston: Altyn and Bacon; 1976.

12. Patil RT, Singh DS, Tribelhorn RE. Effect of processing conditions on extrusion
cooking of soy-rice blend with a dry extrusion cooker. J Food Sci Technol.
1990;27:376–378.

13. Singh DS, Garg SK, Singh M, Kalpana D. Optimization of processing

parameters of soy-sorghum blends extrudates. J Agric Eng. 2005;42(4):45–49.

14. Singh DS, Garg SK, Singh M, Goyal N. Optimization of the processing
parameters of extrudate prepared from Bengalgram brokens and sorghum blends
by using Wenger X-5 extruder. J Food Sci Technol. 2006;43:301–304.
 15. Singh DS, Garg SK, Singh M, Goyal N. Effect of major processing parameters
on the quality of extrudates made out of soy-kodo blends. J Food Sci Technol.
2006;43:434–437.

16. SOPA digest. Indore: Soybean Processors Association; 1999.

17. Teltweiler P. Snack food world wide. Food Technol. 1991;45(2):58–61.

Articles from Journal of Food Science and Technology are provided here courtesy of
Springer
|
Food Sci Nutr. 2014 Jan; 2(1): 39–45.

Published online 2013 Dec 8. doi: 10.1002/fsn3.80

IV.Hydration kinetics of direct expanded

tef flour breakfast cereals in water and
milk
W K Solomon1,2

Author information ► Article notes ► Copyright and License information ►

Abstract
Hydration kinetics of tef flour breakfast cereals extruded at barrel temperatures of 110,
130, and 150°C was investigated by hydrating them in water and whole milk at 25°C
(±1°C). The normalized Weibull model described the rehydration characteristics of the
extrudates in water and milk adequately (R2 = 0.98–0.99). Water absorption rate was
significantly (P  < 0.05) influenced by barrel temperature where extrudates
processed at 150°C barrel temperature exhibited high water absorption rate followed by
those extruded at 130 and 100°C, respectively. Hydration rate and equilibrium moisture
content were higher for samples hydrated in water than those in milk. In view of the
values of the shape parameter β, the hydration process is predominantly controlled by
diffusion (β = 0.40–0.51) for samples extruded at 110°C whereas external resistance
to mass transfer dominated (β  = 0.60–0.73) those extruded using 150 and 130°C.
Extrudates processed at 130 and 150°C exhibited better hydration characteristics. Thus,
these temperature ranges could be used to produce extruded products from tef.

Keywords: Breakfast cereals, Eragrostis tef, extrusion, hydration

Introduction
Tef (Eragrostis tef) is an important cereal crop indigenous to Ethiopia comprising about
20% of cereal production providing the major daily calorie for the vast majority of the
population (Umeta and Faulks 1988; Ketema 1997; Bultosa and Taylor 2004). Tef seed is a
millet-like, tiny, and prolate spheroid with average length and width of 1.01 and 0.59 
mm, respectively (Zewdu and Solomon 2007). The proximate composition (% d.b.) of tef
seed is reported to be 9.4–13.3 protein, 73 carbohydrate, 1.98–3.5 crude fiber, 2.0–3.1
fat, and 2.7–3.0 ash (Bultosa and Taylor 2004). Despite its major contribution, the
processing techniques and the types of food products made from tef remain traditional.
Recently, however, there is growing interest to develop new products from tef using
modern processing techniques like extrusion cooking to harness its potential.

Extrusion-cooked direct expanded breakfast cereals have cellular-like structures mainly

formed by air pockets surrounded by walls of gelatinized starch that contribute to
texture and hydration capacity (Bhattacharya 1997; Machado et al. 1998). Breakfast
cereals are commonly consumed by soaking in milk (Sacchetti et al. 2003, 2005) where the
product readily takes up moisture resulting in undesirable changes, especially in the
most desirable and primary quality attributes like crispiness, brittleness, and crunchiness
due to the plasticization effect of water (Fontanet et al. 1997; Machado et al. 1999;
Sacchetti et al. 2003; Lewicki 2004; Gondek and Lewicki 2006). In view of this, the
hydration characteristic of breakfast cereals and other dried food products is recognized
as an important quality attribute and recently has received increasing interest (Machado
et al. 1997, 1998, 1999; Sacchetti et al. 2003, 2005; Lucas et al. 2007).

Previous studies on the quality of extruded breakfast cereals and snacks focused mainly
on the effect of operating conditions on some physicochemical and functional properties
and sensory qualities. High extrusion temperature has been reported to result in less
compact, porous, and better expanded products (Anderson and Hedlund 1990; Ilo et al.
1996
; Lo et al. 1998; Sacchetti et al. 2004; De Pilli et al. 2005), which have better
hydration properties. Water absorption index (WAI) and water solubility index (WSI)
are regarded as measures of hydration capacity (power), and high extrusion
temperatures were found to generally increase WAI and WSI (Kirby et al. 1988; Ollett
et al. 1990; Sacchetti et al. 2004; Ding et al. 2006), thereby increasing the water
absorption. The degree of gelatinization of starch, which is also linked to the water
absorption capacity (Gambus et al. 1999; Cheyne et al. 2005), increased with increase in
extrusion temperature (Kirby et al. 1988; Ilo et al. 1996; Ding et al. 2005).

Describing and modeling the kinetics of moisture uptake by breakfast cereals could lead
to a better understanding and control over the mechanisms underlying the sorption
process and the ability of the product to rehydrate that could help in process
optimization (Machado et al. 1997, 1998, 1999; Sacchetti et al. 2003; Lucas et al. 2007).
Nonetheless, limited studies have been carried out in this regard. Previous hydration
kinetic studies on breakfast cereals indicated that water absorption rate increases with
increase in hydrating media temperature (Machado et al. 1998, 1999) and decreases with
increase in fat and total solid content of milk used as hydrating medium, and amount of
coating sugar (Machado et al. 1997, 1999; Sacchetti et al. 2003). These studies indicated
that the rate of moisture uptake depends on the physicochemical properties and
structural characteristics of products, which in turn are influenced by the extrusion
processing conditions. In view of few studies in the past, limited information is
available in the literature on the influence of extrusion operating conditions on the
kinetics of moisture uptake by breakfast cereals. Sacchetti et al. (2005) reported that the
hydration rate of cereal-based breakfast cereals increased with increase in extrusion
temperature from 100 to 110°C. However, studies over a wider span of processing
conditions covering the ranges in which breakfast cereals are produced are required.
Particularly, there is limited information on extruded products made from tef flour. The
effect of barrel temperature (110, 130, and 150°C) on the rehydration kinetics of tef
flour extrudates in water for DZ-01-196 cultivar was investigated (Solomon 2008).
However, different cultivars due to their proximate composition behave differently
during hydration. Moreover, breakfast cereals are commonly consumed after hydrating
in milk. Thus, hydration studies in milk are important. The objectives of this study were
to investigate the effect of hydration medium and barrel temperature on the hydration
characteristics and to model hydration kinetics of breakfast cereal made from tef flour
(DZ-01-99 cultivar) in water and milk.

Material and Methods

Sample preparation

Flour from tef grain (variety DZ-01-99) grown at the Debre Zeit Agricultural Research
Center, Ethiopia, during the 2005–2006 cropping season was used. The proximate
composition of the flour used in this study (g/100 g) was found to be 10.69, 2.50, and
2.52 for crude protein, crude fat, and ash, respectively (Kebede 2006).

Extrusion process

Extrusion was carried out using a pilot scale co-rotating twin screw extruder (model
Clextral, BC-21 No. 124, Firminy, France) having 25 mm screw diameter. The barrel
has a smooth 300 mm useful length and consists of three 100-mm-long modules. The
temperature of the modules is regulated by electrical heating and water cooling system,
and the temperature at each zone was controlled by a Eurotherm controller (Eurotherm
Ltd., Worthing, UK). The flour was fed to the extruder inlet using a twin screw
volumetric feeder (type KMV-KT20) and water at ambient temperature was injected
into the extruder via an inlet port by a positive displacement pump (DKM-Clextral). The
extruder die has four circular openings where the diameter tapers from 5 to 2 mm in a
length of 9 mm. Extrusion was carried out at 110, 130, and 150°C barrel temperature,
17% (w.b.) feed moisture content, 9 kg/h feed rate, and 140 rpm screw speed. The
operating conditions were selected based on a preliminary study and recommended
values in the literature (Bouvier 2001). The extruded samples, cut to specific length, were
dried in a hot air cabinet dryer at 150°C for 10 min to improve the crispiness (Bouvier
2001
; Kebede 2006). The initial moisture content of the extrudates was 3.8% (d.b.).

Hydration tests

About 2 g samples were kept in empty beakers and conditioned to the rehydration
temperature of 25°C in a hot air oven before each test (Hung et al. 1993; Maskan 2002).
After conditioning, 100 mL distilled water or whole milk at 25°C was poured into the
beakers and then placed in a constant temperature water bath (Model 25; Precision
Scientific Inc., Chicago, IL) set at 25°C (±1°C). The rehydration temperature falls in the
ranges used in earlier moisture uptake studies of breakfast cereals (Machado et al. 1997,
1998, 1999
; Sacchetti et al. 2003; Lucas et al. 2007). The chemical composition of milk used
was determined and was found to be 2.73, 3.92, 13.79 g/100 g for fat, protein, and
total solids, respectively. At time intervals ranging between 5 and 120 min (depending
on soaking duration), the beakers were removed from the water bath and drained, and
the samples were weighed using an analytical balance (CP 124S, Data Weighing
Systems, Inc., Gottingen, Germany) with 0.1 mg accuracy. The samples were then
dried in a hot air oven at 130°C for 4 h based on preliminary studies and in reference
to Gondek and Lewicki (2006) to determine the moisture content. Each test was repeated
four times to determine the mean values.

Data analysis

The probabilistic normalized Weibull model (eq. 1) has been found to adequately
describe the hydration characteristics of a variety of dry food products including
breakfast cereals (Ilincanu et al. 1995; Cunha et al. 1998; Machado et al. 1998, 1999;
Sacchetti et al. 2003, 2005; Marabi and Saguy 2004).

(1)

where M(t) is moisture content at time t, Me is equilibrium moisture content after

prolonged soaking, M0 is initial moisture content, α is the scale (rate) parameter, β is the
shape parameter, and t is soaking time. The scale (rate) parameter represents the time
needed to accomplish approximately 63% of the moisture absorption process whereas
the shape parameter is a behavior index which is linked to the mechanism controlling
the water absorption process (Machado et al. 1997; Cunha et al. 1998).

The parameters β, α, and Me in the model were determined by nonlinear regression

using JMPIN 5.0.1 statistics software (SAS 2002, JMP Version 5; SAS Institute Inc.,
Cary, NC). The degree of fit of the model was evaluated based on the values of
coefficient of determination (R2) and root mean square error (RMSE) (eq. 2) and by
examining the distribution of the residuals.

(2)

where Mexp and Mest are experimental and estimated moisture content, respectively, and
N is the number of data points.

Results and Discussion

Hydration phenomena

The rehydration data of extrudates processed using different barrel temperatures and
hydrated in water and whole milk are presented in Figure 1. Water absorption
occurred at a higher rate in the early stages (60–90 min) of the rehydration process
followed by decreasing rate and finally tending to cease at prolonged soaking time
indicating the rehydration approached equilibrium condition. This trend was similar for
samples extruded using 130 and 150°C barrel temperatures. For samples extruded using
barrel temperature of 110°C water absorption continued steadily till the end of the
hydration time and approaching equilibrium required more time. Water absorption rate
depends on the difference between the water content at a given time and at saturation,
which is the driving force. As hydration proceeds, the water content of the extrudates
increased, thereby decreasing the driving force and consequently the water absorption
rate. The process ceases when the extrudates attain the equilibrium moisture content.
Similar characteristic water absorption trends have been reported for ready-to-eat and
puffed breakfast cereals immersed in water or milk (Machado et al. 1997, 1998, 1999;
Sacchetti et al. 2003, 2005; Lucas et al. 2007; Solomon 2008).

Figure 1 Experimental and estimated rehydration curves in water and whole milk at different
barrel temperatures (m, milk; w, water).

Samples extruded using barrel temperature of 150°C exhibited higher water absorption
rate followed by those extruded at 130 and 110°C, which is apparent from the slope of
the fitted lines particularly in the early stages (about the first 60–90 min) of the
rehydration process (Fig. 1). This trend was similar while hydrating samples both in
milk and in water. The trend observed in this study is in agreement with Solomon (2008)
where tef extrudates processed at 150°C exhibited higher hydration rate than those
extruded at 130 and 110°C. Sacchetti et al. (2005) also reported that extrudates
processed at 110°C extrusion temperature exhibited higher hydration rate than those
extruded at 100°C. Gelatinization, which is the conversion of starch to a cooked and
digestible material by the application of heat and water, is one of the important effects
extrusion has on the starch component of foods (Ilo et al. 1996; Cheyne et al. 2005; Ding
et al. 2005, 2006). Water is absorbed and bound to the starch molecules inducing change in
the starch molecule structure. The fact is that high extrusion temperature increases the
degree of starch gelatinization (Ilo et al. 1996; Bhattacharya 1997; Ding et al. 2005; Singh
et al. 2007), and water absorption rate and water absorption capacity by gelatinized
starch are higher than those for raw starch (Gambus et al. 1999; Cheyne et al. 2005),
explaining the high water absorption rate exhibited by extrudates processed at barrel
temperature of 150°C followed by those extruded at 130 and 110°C, respectively.
Earlier studies also revealed that high extrusion temperature reduces melt viscosity
(Fletcher et al. 1985; Ilo et al. 1996; Lo et al. 1998; Ding et al. 2006), resulting in more
expanded, less compact, and more porous breakfast cereals and snacks having air cells
due to release of superheated steam (Fletcher et al. 1985; Anderson and Hedlund 1990; Ilo
et al. 1996, 1999; Bhattacharya 1997; Guha et al. 1997; Ding et al. 2005; De Pilli et al. 2005),
leading to less resistance to mass transfer and hence increased rate of water absorption.

It is also worth noting that water absorption rate in water is significantly higher than that
in milk. This is evident from the trends and slopes of the fitted lines (Fig. 1), which
were found to be significantly higher in water than those in milk. The lower water
absorption rate of extrudates hydrated in milk could be attributed to the solids in milk,
which block the pores of the extrudates, thereby reducing water absorption rate.
Moreover, the fat component in the milk could also influence water absorption by
accumulating on the surface of the extrudates (Machado et al. 1999). Hindrance to water
absorption could be aggravated further due to the hydrophobic nature of fat adhered to
the extrudates (Machado et al. 1998).

Modeling rehydration kinetics

The results of nonlinear regression including the scale (rate) parameter, α, the shape
parameter, β, and equilibrium moisture content, Me in equation 1, and the degree of fit
in terms of R2 and RMSE are presented in Table 1. The results revealed that the
normalized Weibull model adequately described the rehydration characteristics of tef
flour breakfast cereals in water and milk with high R2 (0.98–0.99) and small RMSE
values. Besides the quantified values of R2 and RMSE, the adequacy of the model was
evaluated by the residual plot (Fig. 2). The residuals were found to be randomly
distributed, demonstrating that the model adequately described the rehydration kinetics
of the extrudates. Earlier studies have also shown that the normalized Weibull model
adequately described the rehydration kinetics of puffed breakfast cereals and other dried
products (Ilincanu et al. 1995; Cunha et al. 1998; Machado et al. 1998, 1999; Sacchetti et 
al. 2003, 2005; Marabi and Saguy 2004; Solomon 2008).

Table 1 Parameters and degree of fit in normalized Weibull model at different barrel
temperatures.

In water In milk
Temperature (°C) 2
α (min) β Me R RMSE α (min) β Me R2 RMSE
110 2309.7 0.40 6.48 0.994 0.0424 381.65 0.51 3.22 0.981 0.084
130 150.59 0.73 5.88 0.986 0.1863 335.63 0.60 5.05 0.979 0.1605
150 123.47 0.73 6.05 0.992 0.1497 229.22 0.66 4.81 0.976 0.1684

Me, equilibrium moisture content; α, scale (rate) parameter; β, shape parameter; RMSE,
root mean square error.
Figure 2

Figure 2 Plot of predicted moisture contents versus residuals.

The value of α, which represents the time required to achieve 63% of the maximum
moisture uptake (1 − e−1), was determined to be 2309.7, 150.59, and 123.47 min
for samples extruded at 110, 130, and 150°C, respectively, indicating that the samples
hydrated in water and extruded at barrel temperature of 150°C had a high water
absorption rate followed by those extruded using 130 and 110°C barrel temperature. The
trend was similar for extrudates hydrated in milk (Table 1). Similar trends of the effect
of extrusion temperature on the hydration rate of breakfast cereals have been reported
(Sacchetti et al. 2005). The value of α was found to be higher in milk than that in water
(Table 1), demonstrating that the water absorption rate of samples hydrated in milk is
less than that in water. This is also evident from the trends of the fitted lines (Fig. 1).
Higher values of α indicate low water absorption rate or more time is required to reach
63% of the equilibrium moisture content (Ilincanu et al. 1995; Machado et al. 1998). The
solids and fat in the milk coupled with less amount of free water could hinder the mass
transfer process (Machado et al. 1997, 1999), resulting in lower degree of hydration of the
extrudates hydrated in milk than in water. It was also visually observed that extrudates
hydrated in water swell more and their structure was lost relatively easily, thereby
decreasing the resistance to mass transfer whereas those hydrated in milk had a better
retention of structure thus decreasing the water absorption rate. A similar phenomenon
was reported for puffed breakfast cereals hydrated in water and milk (Machado et al.
1997
). The water absorption rate of tef appeared to be less as compared to commercial
puffed corn breakfast cereals (α = 32 min) (Machado et al. 1997, 1999) hydrated in
water at 25°C, cereal-based breakfast cereals (α = 32.5 to 6.7 min) (Sacchetti et 
al. 2005), and corn flakes and flakes made from a mixture of cereals (α = 15.97 and
60.28 min) (Sacchetti et al. 2003) hydrated in semiskimmed milk. This difference
could be attributed to the high bulk density of tef extrudates, which was 1380, 610 and
490 kg/m3 at barrel temperature of 110, 130, and 150°C, respectively (Kebede 2006),
compared with most corn and rice-based commercial breakfast cereals where the bulk
density ranges from 90 to 320 kg/m3 (Ilo et al. 1996, 1999; Guha et al. 1997). However,
the hydration rate of tef extrudates processed at 130 and 150°C was higher than that of
commercial peanut butter breakfast cereals (α = 503 min) hydrated in water at
25°C (Machado et al. 1998), which could be attributed to the fat content of the peanut
butter breakfast cereals.

The value of the shape parameter, β, ranged between 0.40 and 0.73 (Table 1),
indicating that the process is predominantly controlled by diffusion for extrudates
processed at 110°C, whereas external resistance to mass transfer dominated for those
extruded at 130 and 150°C (Cunha et al. 1998). Higher values of β (preferably more
than 1) correspond to the lag time for moisture uptake (Machado et al. 1998) and are
desirable with regard to the maintenance of crispness during rehydration (Machado et 
al. 1997). Thus, extrudates processed at 130 and 150°C barrel temperature would maintain
better crispness during rehydration.

The equilibrium moisture content, Me, generally increased with an increase in barrel
temperature (Table 1), which is also apparent from the trends observed in Figure 1.
This phenomenon could be attributed to less expanded, less porous, and dense internal
structure of the extrudates extruded at low temperatures (Fletcher et al. 1985; Anderson
and Hedlund 1990; Ilo et al. 1996, 1999; Bhattacharya 1997; Guha et al. 1997; De Pilli et al.
2005
) that reduces the water-holding capacity. However, extrudates processed at 110°C
barrel temperature and hydrated in water appeared to have high moisture content
(Table 1). Despite its statistical reliability, this high Me value should be considered
cautiously. In view of the steadily continued water absorption observed during the
experimentation by samples extruded at 110°C and the associated α value (2309.7 
min), it was not possible to approach equilibrium condition. Therefore, it is possible to
arrive at such a high estimated equilibrium moisture content value. When the
experimental data depart from equilibrium, the precision of the parameter's estimate
decreases and Me tends to be overestimated (Cunha et al. 1998; Machado et al. 1998).

The equilibrium moisture content of extrudates hydrated in whole milk was found to be
slightly lower than those hydrated in water (Table 1). This could be attributed to the
role of the fat and solids, which might have blocked the pores, resulting in smaller
hydration or water-holding capacity. In earlier studies on rehydration of puffed breakfast
cereals, the equilibrium moisture content of samples hydrated in water was twice the
value of those hydrated in milk (Machado et al. 1997). Similarly, the increase in the
concentration of total solids and fat content of milk has been reported to decrease the
equilibrium moisture content of ready-to-eat breakfast cereals (Machado et al. 1999),
which further explains the influence of solids and fat content on the hydration (water-
holding) capacity.

Conclusions
The water absorption rate and water absorption capacity (equilibrium moisture content)
increased with an increase in barrel temperature from 110 to 150°C. The normalized
Weibull model adequately described the water absorption characteristic of tef flour
breakfast cereals hydrated in water and milk. Hence, the model could be used for
prediction and process optimization purposes, though it lacks theoretical basis. Water
absorption rate and equilibrium moisture content were higher for extrudates hydrated in
water than those hydrated in milk. In view of the values of the shape parameter β (0.40–
0.73), the rehydration process is predominantly controlled by diffusion for samples
extruded at 110°C, whereas external resistance to mass transfer dominated for those
extruded using 150 and 130°C. Extrudates processed at 130 and 150°C barrel
temperature could maintain better crispness during rehydration. Hence, this temperature
range might be preferred for producing tef flour breakfast cereals.

Acknowledgments
The author is grateful to Ashenafi Bahiru for his assistance in the course of the
experimentation. Laike Kebede is acknowledged for providing the extrudates.

Funding Information
No funding information provided.

References
1. Anderson Y, Hedlund B. Extruded wheat flour: correlation between processing and
product quality parameters. Food Qual. Prefer. 1990;2:201–216.

2. Bhattacharya S. Twin-screw extrusion of rice-green gram blend: extrusion and

extrudate characteristics. J. Food Eng. 1997;32:83–99.

3. Bouvier JM. Breakfast cereals. In: Guy R, editor. Extrusion cooking technologies and
applications. Cambridge, UK: Woodhead Publishing Ltd. and CRC Press; 2001. pp. 133–
160.

4. Bultosa G, Taylor JRN. Encyclopaedia of grain science. Amsterdam, the Netherlands:

Elsevier; 2004. Tef; pp. 253–262.

5. Cheyne A, Barnes J, Gedney S, Wilson DI. Extrusion behavior of cohesive potato starch
pastes: II. Microstructure–process interactions. J. Food Eng. 2005;66:13–24.

6. Cunha LM, Oliveira FAR, Ilincanu LA, Drumond MC. Application of the probabilistic
Weibull model to rehydration kinetics: relationship between the model parameters and
the underlying physical mechanisms. In: Oliveira JC, Oliveira FAR, editors. Proceeding of
the 3rd main meeting of the Copernicus Program Project “Process Optimization and
Minimal Processing of Foods, CIPA-Ct94-0195. Vol. 3: Drying”. Porto, Portugal: Escola
Superior de Biotechnologia; 1998. pp. 9–13.

7. De Pilli T, Severini C, Baiano A, Derossi A, Arhaliass A, Legrand J. Effects of operating

conditions on oil loss and properties of products obtained by co-rotating twin-
extrusion of fatty meal: preliminary study. J. Food Eng. 2005;70:109–116.
8. Ding QB, Ainsworth P, Plunkett A, Tucker G, Marson H. The effect of extrusion
conditions on the physicochemical properties and sensory characteristics of rice-based
expanded snacks. J. Food Eng. 2005;66:283–289.

9. Ding QB, Ainsworth P, Plunkett A, Tucker G, Marson H. The effect of extrusion

conditions on the functional and physical properties of what-based expanded snacks. J.
Food Eng. 2006;73:142–148.

10. Fletcher SI, Richmond P, Smith AC. An experimental study of twin-screw extrusion-
cooking of maize grits. J. Food Eng. 1985;4:291–312.

11. Fontanet I, Davidou S, Dacremont C, Le Meste M. Effect of water on the mechanical

behavior of extruded flat bread. J. Cereal Sci. 1997;25:303–311.

12. Gambus H, Golachowski A, Bala-Piasek A, Ziobro R, Nowotna A, Surowka K. Functional

properties of starch extrudates. Part I. Dependence of extrudates properties on starch
water content. J. Polish Agric. Univ. 1999;2:1–6.

13. Gondek E, Lewicki PP. Antiplasticization of cereal-based products by water. Part II.
Breakfast cereals. J. Food Eng. 2006;77:644–652.

14. Guha M, Ali SZ, Bhattacharya S. Twin-screw extrusion of rice flour without a die: effect
of barrel temperature and screw speed on extrusion and extrudate characteristics. J.
Food Eng. 1997;32:251–267.

15. Hung TV, Liu LH, Black RG, Trewhella MA. Water absorption in chickpea (C. arietinum)
cultivars using the Peleg model. J. Food Sci. 1993;58:848–852.

16. Ilincanu LA, Oliveira FAR, Drumond MC, Machado FM, Gekas V. Modeling moisture
uptake and soluble solids losses during rehydration of dried apple pieces. In: Oliveira
JC, editor. Proceeding of the 1st main meeting of the Copernicus Program Project
“Process Optimization and Minimal Processing of Foods, CIPA-Ct94-0195. Vol. 3:
Drying”. Porto, Portugal: Escola superior de Biotechnologia; 1995. pp. 64–69.

17. Ilo S, Tomschik U, Berghofer E, Mundigler N. The effect of extrusion operating

conditions on the apparent viscosity and the properties of extrudates in twin-screw
extrusion cooking of maize grits. Lebensm. Wiss. u. Technol. 1996;29:593–598.

18. Ilo S, Liu Y, Berghofer E. Extrusion cooking of rice flour and amaranth blends. Lebensm.
Wiss. u. Technol. 1999;32:79–88.

19. Kebede L. Effect of extrusion operating conditions on physico-chemical and sensory

attributes of Tef grain puffed products. Ethiopia: Haramaya University; 2006. M.Sc.
thesis.

20. Ketema S. Tef (Eragrostis tef (Zucc.) Trotter). Promoting the conservation and use of
under-utilised neglected crops. Italy: IPGRI; 1997. 12.

21. Kirby AR, Ollett AL, Parker R, Smith AC. An experimental study of screw configuration
effects in the twin-screw extrusion-cooking of maize grits. J. Food Eng. 1988;8:247–
272.
22. Lewicki PP. Water as the determinant of food engineering properties. A review. J. Food
Eng. 2004;61:483–495.

23. Lo TE, Moreira RG, Castell-Perez ME. Modeling product quality during twin-crew food
extrusion. Trans. ASAE. 1998;41:1729–1738.

24. Lucas T, Le Ray D, Mariette F. Kinetics of water absorption and solute leaching during
soaking of breakfast cereals. J. Food Eng. 2007;80:377–384.

25. Machado MF, Oliveira FAR, Gekas V. Modelling water uptake and soluble solid losses by
puffed breakfast cereal immersed in water or milk. In: Jowitt R, editor. Proceeding of
the Seventh International Congress on Engineering and Food. Part I. Sheffield, UK:
Sheffield Academic Press; 1997. pp. A65–A68.

26. Machado MF, Oliveira FAR, Gekas V, Singh RP. Kinetics of moisture uptake and soluble-
solid loss by puffed breakfast cereals immersed in water. Int. J. Food Sci. Technol.
1998;33:225–237.

27. Machado MF, Oliveira FAR, Cunha LM. Effect of milk fat and total solid concentration on
the kinetics of moisture uptake by ready-to-eat breakfast cereal. Int. J. Food Sci.
Technol. 1999;34:47–57.

28. Marabi A, Saguy IS. Effect of porosity on rehydration of dry food particulates. J. Sci.
Food Agric. 2004;84:1105–1110.

29. Maskan M. Effect of processing on hydration kinetics of three wheat products of same
variety. J. Food Eng. 2002;52:337–341.

30. Ollett AL, Parker R, Smith AC, Miles MJ, Morris VJ. Microstructural changes during the
twin-screw extrusion cooking of maize grits. Carbohydr. Polym. 1990;13:69–84.

31. Sacchetti G, Pittia P, Biserni M, Pinnavaia GG, Rosa MD. Kinetic modeling of textural
changes in ready-to-eat breakfast cereals during soaking in semi-skimmed milk. Int. J.
Food Sci. Technol. 2003;38:135–143.

32. Sacchetti G, Pinnavaia GG, Guidolin E, Rosa MD. Effects of extrusion temperature and
feed composition on the functional, physical and sensory properties of chestnut and
rice-based snack-like products. Food Res. Int. 2004;37:527–554.

33. Sacchetti G, Pittia P, Pinnavaia G. The effect of extrusion temperature and drying-
tempering on both the kinetics of hydration and the textural changes in extruded
ready-to-eat breakfast cereals during soaking in semi-skimmed milk. Int. J. Food Sci.
Technol. 2005;40:655–663.

34. Singh B, Sekhon KS, Singh N. Effects of moisture, temperature and level of pea grits on
extrusion behavior and product characteristics of rice. Food Chem. 2007;100:98–202.

35. Solomon WK. Effect of barrel temperature on rehydration kinetics of direct-expanded

tef flour breakfast cereal. J. Food Proc. Eng. 2008;31:469–487.
 36. Umeta M, Faulks RM. The effect of fermentation on the carbohydrates in Tef
(Eragrostis tef. Food Chem. 1988;27:181–189.

37. Zewdu AD, Solomon WK. Moisture dependent physical properties of tef. Biosyst. Eng.
2007;96:57–63

J Food Sci Technol. 2014 Oct; 51(10): 2803–2808.

Published online 2012 Jun 3. doi: 10.1007/s13197-012-0732-x

V. Effect of baking improvers on the

quality of whole cassava biscuits
A. O. Obadina, O. B. Oyewole, and G. Olaniyi

Author information ► Article notes ► Copyright and License information ►

Abstract
Improving agents such as ascorbic acid, sodium metabisulphite, sorbic acid and soyflour
were used in the production of whole cassava biscuits. Cassava flour and small amount
of soyflour were produced. Bulk density, water binding capacity and amylograph
viscosity of cassava flour were determined. Mixing and dough extrusion time were
recorded for each sample of biscuit dough developed with addition of improvers.
Length, width and thickness of cut-out dough were measured before and after baking to
evaluate biscuits flow. Proximate and sensory analysis of the biscuits samples was also
determined. The result showed that, there was a slight decrease in mixing time,
extrusion time, length and width of the biscuits samples prepared with improvers.
However, there was a considerable increase in biscuits thickness. Amylograph result
showed an improvement in flour stability and low retrogadation tendency, most
especially flour with inclusion of ascorbic acid. Crude protein and fat increased with
sample contained soyflour. Sensory evaluation result indicated no significant difference
among the samples except the texture of the biscuits.

Keywords: Cassava flour, Improver, Ascorbic acid, Sodium metabisulphite, Soyflour, Sorbic acid

Introduction
Cassava has until recently been undervalued as s crop of importance in Africa, this is
partly because cassava was argely processed for local traditional foods and its industrial
remain untapped (Hahn et al. 1989). This situation has changed with current efforts at
utilizing cassava for the industrial production of food grade starch, glue, beer and
alcohol (Eggleston et al. 1993) among others. One area in which cassava has been found
very useful is in the utilization of cassava flour in a composite flour programme.
The composite flour programme initiated by Food and Agriculture Organisation of the
United Nations in 1964 was conceived primarily to develop bakery products from
locally available raw material particularly from locally available raw material
particularly in the countries which could not meet their wheat requirements. Much
attention has been focussed on the development of composite flour technology for
confectionery production. Acceptable biscuits have been produced from mixture of
wheat flour and cassava flour. Work done at the International Institute of Tropical
Agriculture has led to the production of nutritious baked products such as cakes and
biscuits using cassava flour, cassava starch, margarine and eggs (Bokanga 1995).

However, the major challenge in cassava flour utilization is dough handling, which
lacks the functional viscoelastic properties which is the unique characteristics of wheat
flour. To overcome this, some workers have worked on gluten substitute. John suggested
that any ingredient capable of improving coherence between starch granules without
impairing the capacity of dough/batter to rise will be a suitable binder. He used the
surfaceactive emulsifier GMS (glycerol monostearate) to improve gas retention and
bread qualities of starches. Almazan put forward the idea that starch obtained from
cassava must be supplemented with an improver such as pentosan if used alone or at
very high concentration.

These substances (improving agents) give a whitened appearance to baked products

because of their beneficial effect on texture of the crumb. However, improving agents
do not increase the carbon dioxide production in fermented dough, but they improve gas
retention (because the dough is made more elastic) and this result in increased loaf
volume (Kent 1984). Today, small amount of oxidising agents (improvers) are added to
flour to accomplish the maturing process, and these oxidising agents are called flour
improvers or sulfhydryl oxidising agents. The influence of improver on dough rheology
and baking performance for the production of semi-sweet biscuits have been studied by
Olive et al. (1995). The use of soy-flour as an improver in the baking industry is well
known in improving dough handling characteristics particularly in products such as
cookies. This study was designed to investigate the effect of baking improvers on the
quality of whole cassava biscuits.

Materials and methods

All the materials for whole cassava biscuit production such as cassava flour, baking
powder, sugar, salt, baking powder, egg, bakery fat, powdered milk and Soya bean flour
except the baking improver were obtained from the retail market at kuto in Abeokuta,
Ogun State, Nigeria. Baking improvers were collected from the Food Science and
Technology laboratory in University of Agriculture, Abeokuta. Nigeria. These are
sodium metabisulphite, ascorbic acid, sorbic acid and Soya flour. Soy flour was
prepared in the laboratory.

Preparation of cassava flour

Manihot Palmata (low cyanide content) was used in the preparation of cassava flour.
Cassava tubers were peeled, washed with portable water and chipped with chipping
machine. The chips sizes from the machine are about 4 mm in thickness. After chipping,
it was dried with cabinet drier operated at 45 °C for about 12 h and finally dried at
65 °C for moisture removal. It was then milled with disc attrition mill. The resultant
product from milling was sieved using a sieve of size 70 mesh. The final product
(cassava flour) was packed in polyethylene bags at ambient temperature until needed for
biscuits production.

Preparation of soy flour

Cleaned soybean was washed and soaks for 6 h in water containing 0.2 % NaOHCO3, to
remove beany flavour. After this, it was decorticated and steamed for 20 min. After
steaming, it was drained, spread out inside a tray and placed in cabinet drier to dry at a
temperature of about 45–50 °C. It was dried mill into flour and sifted.

Production of cassava biscuit

Bakery fat (Breaden) produced by Lever Brother Nigeria Plc was weighed, and all other
ingredients according to the recipe. Bakery fat and sugar was weighed into a mixing
bowl and mixed until it becomes fluffy. Egg and baking improver dissolved in 40 ml of
water were added and mixed at medium speed, when thorough mixing was achieved.
Cassava flour, salt, milk powder, flavouring agent and baking powder were finally
added into the mixing bowl. The mixer was first operated at low speed to prevent
splashing of ingredients after which it was turned into high speed for thorough mixing
to form dough. The dough was rolled into a thin sheet, cut into shapes manually and
quickly baked at a temperature of 160 °C for 30 min in a forced air convection oven.

Physico-chemical analysis

Bulk density of the flour

The bulk density was determined by weighing 20 g of dried cassava into a 100 ml
measuring cylinder and drop the cylinder 150 mm onto a 80 ft pad 10 times, level off
powder and measure the bulk density in g/ml (Kirk and Ronald 1991).

Water binding capacity

The method of Medcalf was used for determining the water binding capacity.

Dough extrusion time and mixing time

A sample of the bulk dough (60 g) was rolled into a cylindrical shape (12 mm diameter)
and pressed firmly into a simple extruder. After 10 min a plunger with a weight
(2,370 g) was placed on top of the dough piece. The time taken for the plunger to travel
1 cm was recorded with stop watch (Olive et al. 1995). The mixing time during dough
development was determined by using stop clock.

Biscuit flow and break strength

The biscuit flow which is the increase in volume of unbaked stamped-out dough after
baking was determined by measuring the diameter and thickness of the cut-out biscuit
baked biscuit. Determination was carried out in duplicate and average values were
recorded.

For the biscuit strength, biscuit of known thickness (0.41 cm–0.51 cm) was placed
centrally between two parallel metal bars 5 cm apart. A wooden bar of thickness 2 cm
(0.368 kg) was then placed on the biscuit and weight added on the bar in increment of
0.20 kg until the biscuit snapped. The least weight that caused the breaking of the
biscuits was regarded as break strength of biscuit.

Biscuit hardness

Biscuit hardness was determined by using needle penetrometer, the speed of the needle
travelling in biscuit per seconds show the extent of hardness of the biscuits. The tests
were conducted in duplicate and the average was recorded.

Proximate analysis

The moisture, ash, crude fat, crude protein, crude fibre contents of the fortified tapioca
samples was determined by the method described by Association of Official Analytical
Chemists (AOAC) 1990.

Carbohydrate content

The carbohydrate content was determined by difference in food composition i.e. 100–
(% moisture + % Ash + % Fat + % Protein + % Crude fibre)

Cooking characteristics of cassava flour with improver

The cooking characteristics of the flour with improvers were evaluated using Barbender
Amylograph.

Sensory evaluation

The cassava biscuits prepared from the sample of cassava flour with different improvers
were subjected to sensory evaluation test based on colour, taste, texture and overall
acceptability. Twelve member consumer sensory panels were selected from the
University environment to evaluate the biscuits according to their likeness, using 9-
points hedonic scale, where 9 = like extremely and 1 = dislike extremely.

Statistical analysis

The experiments were carried out in triplicate and the statistical package for social
scientist (SPSS) version 17.0 was used in the analysis of data. Duncan Multiple Range
Test was used to separate the experimental data.

Results and discussion

Chemical composition of cassava biscuit
 Table 1 shows the chemical composition of cassava biscuit with different improvers
added to each sample at the point of dough development. Moisture contents of the
biscuit samples ranged from 6.39 % to 7.43 %, protein 1.91 % to 2.65 %, fat 20.2 % to
23.3 %, carbohydrate 63.1 % to 65.9 % and ash 2.35 to 2.9 %. Holland et al. (1991)
reported that 100 % wheat flour biscuits contained protein (6.2 %), carbohydrate
(62.0 %), fat (23.4 %) and moisture (2.6 %). The variation in chemical composition of
cassava biscuit from that of whole wheat biscuit may be traced to the raw materials.
High moisture content of cassava biscuit is due to excessive moisture contained in the
raw material. Low protein content of cassava biscuits as observed may also be ascribed
to the low protein content of cassava which is the principal raw material for cassava
biscuit production.

Table 1 Physical, chemical and sensory quality characteristics of whole cassava biscuits of
different recipe

Physical Chemical Sensory

Biscu
Doug
Level of it
h Dough Biscuit
additio flow Textur Widt
Recip Wat mixi extrusi s Lengt Thickn Prot Crud
n of or e h Moistu Crude Ash Colo Crispin
e er ng on break h ess ein e fat Taste Texture
improv sprea (mm/ (mm re (%) fiber (%) (%) ur ess
(ml) time time streng (mm) (mm) (%) (%)
ers d s) )
(Min (S) th (g)
(kg−1) ratio
)
(%)
CF + 22
0.2 40.0 7.0 10.0 69.9c 246.7c 2.1a 64.0c 41.4c 5.0a
a a
7.4e 1.5a 2.5c d 2.3a 7.3a 7.3c 7.3b 6.8d
AC .7
20
CF + 2.7
0.2 40.0 8.0a 10.0a 78.0d 246.6c 2.7b 39.1a 39.1a 5.0a 7.3c 1.6a 1.9a .2 b 6.9a 6.3 c 6.8 b 6.8de
SMB a

21
CF + 10.0 39.7a 39.0 2.3
0.1 40.0 b 10.0 a 64.9 a 240.0 b 2.9c b a 5.0 a 7.3c 1.5 a 2.2 b .1 a 6.7 a 6.8 c 6.7 b 6.7 e
SA b

CF + 11.0 40.0 40.0 23

0.05 40.0 b 10.0 a 69.6c 186.7 a 2.7 b ab ab 6.0 a 6.3 a 1.7 b 2.7d 2.9d 6.4 a 6.5 c 6.2 b 6.6 e
SF .2d
21
40.9b 2.8
CF Nil 40.0 13.0c 10.0 a 66.9 b 333.3d 2.7 b 40.1b c 6.0 a 6.9 b 1.5 a 2.2 b .2 c 6.8 a 7.2 c 6.6 b 6.6e
c

Values with different superscript on the same column are significantly different (p < 
0.05). (n = 3)

CF Cassava Flour, AC Ascorbic Acid, SMB Sodium metabisulphite, SA Sorbic acid, SF

Soya flour, CF Cassava flour

The sample that contain Soya bean flour has the highest protein content of 2.65 % due
to the protein contributed to the biscuit sample by the Soya bean flour which is very rich
in protein. The fat content was also high due to the addition of fat during processing.
The total carbohydrate content calculated by difference showed a slight decrease in
values. The crude fibre of the samples is within the same range with the exception of the
sample containing soy flour which shows highest percentage of crude fibre. The
difference may be caused by fractional amount of fibre contributed by soy flour to the
biscuit sample.
It was observed that mixing time of dough produced with addition of ascorbic acid was
lower than the mixing time of the other recipe formulation. Cassava biscuit dough
produced without improving agent had longer mixing time than the other recipe
formulations. These variations in the mixing time among the dough developed from
different improvers may be due to the improving action of each improver which varies
considerably. The mixing temperature was used to determine when the mixing should be
stopped (i.e. 35oc) during the dough development. The variations in mixing time
between dough developed from improvers and the one developed without improver was
also observed by Olive et al. (1995). Although, they reported longer mixing time for
dough prepared from wheat flour.

Variation in extrusion time of cassava dough prepared with the addition of improvers is
not so much. However, this is not in agreement with findings of Olive et al. (1995). The
reason is that the raw material used for the biscuit production is not the same because
cassava flour completely lacks gluten which is the unique functional characteristics of
wheat flour. The slight variations that were observed may be due to the effects of
different improving actions of each improver used in the dough development.

Cassava biscuits flow (spread) and break strength

The data of biscuit flow or increase in volume of biscuit dough after baking were
presented in Table 1. Cassava biscuit with improver increased in volume as follows:
Ascorbic acid 69.95, sodium metabisulphite 78 %, soy flour 69.6 % and ascorbic acid
64.9 %, while biscuit without improver (reference sample) had 66.9 %. The best
improving action was shown with biscuit that contains sodium methabisulphite as
shown above. Addo also observed similar spread or increase in volume in the biscuit
production, but the reason that was attributed to this is that has sugar content contributes
to increase in volume which in molten state flows within the biscuit matrix, and on
cooling, sets within the biscuit matrix and thus conferring rigidity to the much expanded
biscuit. Although, sugar contributes its own fractional increase in volume, the increased
volume in cassava biscuit as shown Table 1 could be due to the improvers added to
some samples because the same amount of sugar was used in the cassava biscuits
production which is considered to have similar effect throughout the biscuits samples.

Biscuit break strength is the weight required to break the biscuits and it shown in
Table 1. Cassava biscuit without improving agent exhibited the highest break strength
(333.30 g) while those with improvers showed a slight decrease from this value with the
exception of that contained soy flour. Action of soy flour in bakery products has been
described by Wetanbe et al., which improve dough handling and bleaching action.

Cassava biscuit hardness, thickness, length and width

The results of these parameters were presented in Table 1. Biscuits hardness (texture)
was determined by needle penetrometer, the texture that were presented on this table
shows that, the higher the value the softer the biscuit. Sorbic acid produced biscuit that
is softest in texture compared to other samples. Sodium metabisulphite exhibited similar
variation with soy containing biscuits and reference sample (sample without improver).
For all recipe formulations produced in this study, biscuits were shorter in length and
width but the thickness increased considerably compared to the cut dough piece. The
initial dough piece length (65 mm), width (42 mm) and thickness (4 mm) were altered
by the inclusion of improving agents. Olive et al. (1995) in their finding reported similar
contraction in dough piece length on processing, which results in a small increase in
width and increase in dough piece thickness. They also confirmed that an increase in
dough piece thickness occurs more readily than increase in dough piece width. The
result showed in Table 1 agreed with these findings, except the contraction in dough
pieces width that was observed in cassava biscuits. The reason for this occurrence may
be due to the raw materials used in biscuits production (i.e. cassava flour). However,
there is considerable increase in thickness of biscuits produced.

Sensory evaluation

Table 1 all shows the result of sensory evaluation by a panel of 12 judges carried out on
cassava biscuits prepared with different improving agents i.e. Ascorbic acid, sodium
metabisulphite, sorbic acid, soyflour (full fat soy flour) and one reference sample
without improving agent.

It showed that all samples presented for the sensory evaluation were not significantly
different in terms of colour, taste, crispiness, and overall acceptability. However,
significant difference were observed among the sample texture, in which biscuit
prepared from ascorbic acid differs in texture from other samples, but all other samples
are insignificantly different.

Kaputto and Chalwa, observed no significant difference in flavour and texture of

biscuits prepared from 40 % substitution with fermented cassava flour. The result of
sensory evaluation above is not in line with this and the differences may be ascribed to
the improving agents used in this research work in which different improving actions
were exercised on the texture of the biscuit which subsequently result in slight variation.

Bulk density and water binding capacity of cassava flour

The bulk density and water binding capacity of the cassava flour used in the production
of biscuits is given overleaf in Table 1. It was observed that the water binding capacity
is a room temperature measurement and does not indicate the behaviour of the starch
when heated. It increases as soon as heat starts disrupting the intragranular bonds during
the gelatinization process. Low water binding is attributed to a close association of
starch polymer in the native granule. While higher water binding capacity may be
attributed to a loose association of amylase and amylopectin molecules in the native
granule. According to Lorenz and Collins (1990), starches with low amylase content
had low water binding capacities.

Pasting characteristics of cassava flour with improving agents

Pasting temperature

The brabender amylograph of flour sample with addition of different improvers are
shown on Table 2. Swinkles (1985) express pasting temperature as the temperature at
which the viscosity of stirred starch suspension begin to rise. Pasting temperature
increase from 63 °C to 64.5 °C. Reference sample flour (flour without improver) and
flour with sodium metabisulphite inclusion showed the same pasting temperature or
gelatinization temperature. Although high pasting temperatures are associated with low
viscosities, the difference in pasting temperatures of the flour samples were not
significant enough to conclude on viscosity variations.

Table 2 Brabender amylograph pasting viscosity of cassava flour plus improvers

CF +
CF + AC CF + SA CF + SF CF
SMB
Pasting temperature (TP) °C 63.0 64.5 63.8 63.0 64.5
Gelatinization time (mg) minutes 22.0 23.0 22.5 22.0 23.0
Temperature at peak viscosity (TPV)°C 71.3 72.8 71.2 72.0 73.5
Peak viscosity during heating (Vp) B.U. 485.0 505.0 480.0 470.0 480.0
Time to reach peak viscosity (mn)
27.5 28.5 27.5 28.0 29.0
minutes
Viscosity at 95 °C B.U. 265.0 328.0 310.0 290.0 300.0
Viscosity after 30 min holding at 95 °C
80.0 100.0 90.0 110.0 110.0
B.U.
Viscosity on cooling to 50 °C (Ve) B.U. 150.0 210.0 200.0 180.0 180.0
Ease of cooking (mn-Mg) 5.5 5.5 5.0 6.0 6.0
Stability of flour (Vp-Vr) B.U 405.0 405.0 390.0 360.0 370.0
Setback value (Ve-Vp) B.U −335.0 −295.0 −280.0 −290.0 −300.0

(n = 3)

CF Cassava Flour, AC Ascorbic Acid, SMB Sodium metabisulphite, SA Sorbic acid, SF

Soya flour, CF Cassava flour

Peak viscosity

The maximum viscosity is obtained when the increase in the structural viscosity caused
by swollen starch aggregate is counter balanced by the decrease in viscosity resulting
from the disintegration of starch. When the heating is continued, the viscosity of the
starch increases from almost zero to a peak value.

The peak viscosity recorded for the samples were 480B. U. 485.U. 505B.U, 480B.u and
470B.U. From these peak viscosity sodium metabisulphite and Ascorbic acid had the
highest peak viscosity. This result is in contrary of what Faboya and Asagbra (1990) had
reported, that higher pasting temperature is associated with low viscosities. However,
sorbic acid addition and reference sample showed similar variation in peak viscosity,
while flour sample containing soyflour showed a significant different in peak viscosity.
The lower pasting temperature and reduced peak viscosity of “soyflour” containint flour
compared to others might be due to effect of fractional lipoxygenase present in soy-
flour.

But Numfor and Noubi (1995), has reported similar decrease in Brabender viscosity of
the composite flour with soyflour addition. However, the reason attributed to such
increase is that the decrease in viscosity of composite flour is due more to the decrease
in proportion of cassava flour than to the increase in proportion of soya bean flour.
With this finding, where composite flour is not used, the improving action of soyflour is
conceived to be the causes of such occurrence in the viscosity of the flour used for
biscuit production.

Ease of cooking

The difference between the time taken for the flour to reach pasting temperature during
heating and the time taken to reach the peak viscosity is described as the ease of
cooking of the flour, Adeyemi et al. (1986). The smaller the time taken, the greateris the
ease of cooking. The ease of cooking of the flour with addition of improvers are as
presented in Table 2. From the table, ascorbic acid, sodium metabisulphite and sorbic
acid addition give the greatest ease of cooking, while reference sample (sample without
improver) and “soy flour” containing flour showed similar ease of cooking.

Stability if the flour

Stability of the flour is the difference between the peak viscosity on heating and
viscosity on heating, and viscosity on holding at 95 °C for 15 min, Mazurus et al.
(1957). The lower the value, the stable the flour and vice-versa. The samples stabilities
were shown on Table 2. “Soyflour” contained flour has the highest stability (360 B.U)
Ascorbic acid and sodium metabisulphite and similar stability (405 B.U) while sorbic
acid contained flour had stability of 390 B.U., this implies that, it is the next stable to
“soyflour” contained flour that is used in biscuits production.

Set back value

The set back value of the flour is the difference between the viscosity of the paste on
cooling to 50 °C, and the maximum viscosity of the paste during heating. The extent of
increase viscosity on cooling reflects the retrogradation tendency of the flour, Rasper
(1969).

It has been reported by Halick and Kelly (1959) that the increase in viscosity on cooling
to 50 °C reflects the retrogradation tendency of the starch product. It also follows that
high amylase starches would show maximal set back on cooling. From the Table 2,
when all the samples cooled to 50 °C, all the samples showed maximum decrease in
viscosity, even the samples exhibited decrease in viscosity beyond their corresponding
peak viscosities recorded by the hot paste. From the result from the table it shows that
all of them have low tendency for retrogradation.

The flour sample with addition of ascorbic acid has the lowest tendency to retrograde
followed by the reference sample. The reason for this is that ascorbic contained flour
has the lowest set back value of −335B.U., and −300 B.U for reference sample (sample
without improver).

Conclusion
The improvers caused slight increase in mixing time, extrusion time, length, width of
the biscuits and considerable thickness in the biscuits. The flour with the inclusion of
ascorbic acid resulted in the improvement in flour stability and low retrogadation
tendency while crude protein and fat increased in the sample with the addition of
soyflour. The samples with the addition of all the improvers were liked by the
consumers in terms of colour, taste and crispiness but not for the texture. There is need
for extra studies on the storage stability of the biscuits.

References
1. Adeyemi IA, Fagades SO, Ayotade KA (1986) Some physico-chemical and cooking
qualities of Nigeria rice varieties. Nigerian Food Journal 4(1):26–33

2. AOAC (1990) Association of Official Analysis Chemists. Official methods of analysis

(15th Ed.). Virginian. Association Official Analytical Chemists, USA, 1990

3. Bokanga M (1995) Cassava: opportunities for the food, feed and other industries in
Africa. In: Egbe TA, Brauman A, Griffon D, Treche S (eds) Transformation alimentaire du
manioc cassava food processing. 1995. Paris, pp 557–569

4. Eggleston G, Omoaka PE, Arowshegbe AU. Flour, starch and composite bread-making
quality of various cassava clones. J Sci Food Agric. 1993;62:49–59. doi:
10.1002/jsfa.2740620107. [Cross Ref]

5. Faboya OOP, Asagbra AA. The physico-chemical properties of starches from some
Nigeria cultivars of white yam. Dioscorea rotundata poir. Trop Sci. 1990;30(1):51–57.

6. Hahn SK, Isoba JCG, Ikotun T (1989) Resistance breeding in root and tuber crops at the
IITA, Ibadan, Nigeria. Crop Prot 8:147–168

7. Halick JV, Kelly VJ (1959) Gelatinization and pasting characteristics of rice varieties as
related to cooking behaviour. Cereal Chem 36:91

8. Holland B, Welch AA, Unwin ID, Buss DH, Paul AA, Southgate DAT (1991) McCance and
Widdowson’s the composition of foods, 5th ed. Royal Society of Chemistry, Cambridge,
UK

9. Kent NL. Technology of cereals. New York: Pergamon; 1984. pp. 109–110.

10. Kirk R, Ronald S. Pearson’s composition and analysis of food. New York: Wiley; 1991.
pp. 300–327.

11. Lorenz K, Collins F. Quinoa (Chenopodium quinoa) starch—physico-chemical properties

and functional characteristics. Starch-Starke. 1990;42:81–86. doi:
10.1002/star.19900420302. [Cross Ref]

12. Mazurus EG, Schoch TJ, Francis K. Graphical analysis of the Branbender viscosity curve
of various starches. Cereal Chem. 1957;34(3):141–152.

13. Numfor AF, Noubi L. Effect of full–fat soya bean flour on the quality and acceptability of
fermented cassava flour. Food Nutr Bull. 1995;16(3):242–243.
 14. Olive G, Thacker D, wheeler RJ (1995) Semi- sweet biscuit. In: The influence of sodium
metabisulphite on dough rheology and baking performance. J Sci Food Agric 69: 141–
150

15. Rasper V. Starches from major starch crop grown in Ghana (I) hot paste viscosity and
gel formation power. J Sci Food Agric. 1969;20(3):165–167. doi:
10.1002/jsfa.2740200309. [Cross Ref]

16. Swinkles JJM (1985) Composition and properties of commercial native starches.
Starch/Stark 37:1–5

17. Appl Environ Microbiol. 1999 Oct; 65(10): 4464–4469.

18. PMCID: PMC91594

VI. Modeling Yeast Spoilage in Cold-Filled Ready-To-Drink

Beverages with Saccharomyces cerevisiae, Zygosaccharomyces
bailii, and Candida lipolytica
Alyce Stiles Battey, Siobain Duffy, and Donald W. Schaffner*

Author information ► Article notes ► Copyright and License information ► Disclaimer

ABSTRACT
The high water activity (Aw) of most ready-to-drink beverages typically allows microbial
growth. Hurdles such as pH, sugar content, and chemical preservatives prevent the
growth of most organisms in ready-to-drink beverages (1). Spoilage yeasts, such
as Saccharomyces cerevisiae, Candida lipolytica, and Zygosaccharomyces bailii, are
sometimes able to overcome these hurdles. These organisms tolerate acidic
environments and are resistant to chemical preservatives, such as potassium sorbate and
sodium benzoate (1, 12).
Challenge studies are conducted in order to assess the ability of organisms to grow in a
particular foodstuff. Challenge studies require considerable labor, time, and materials,
and the number of parameters that can be tested is often limited. Validated predictive
models, however, can provide rapid information about the microbial stability of a
product and can be used in conjunction with challenge studies to improve product
stability and reduce costs.
The focus of predictive microbiology has been in the creation of pathogen models with
polynomial regression, such as the Food MicroModel (25) and the U.S. Department of
Agriculture's Pathogen Modeling Program (40). Spoilage models are less prevalent in
the literature (28, 39) but include models for yeasts (31), molds (18), and bacteria
(2, 9, 22). Logistic-regression models are also less prevalent in predictive food
microbiology but are gaining importance (42).
Our research applies the techniques of predictive food microbiology to the spoilage of a
model cold-filled beverage system. Several models for Z. bailiihave been developed
(5, 7, 11), but the focus of our research was to create a single mathematical model to
describe spoilage by three common spoilage yeasts, in order to aid in the product
development of ready-to-drink beverages. The resulting model demonstrates which
factors most influence spoilage yeast growth and allows comparison of product
formulations (40) and the identification of possible alternative formulations with similar
or enhanced resistance to growth (6).

MATERIALS AND METHODS

Organisms and cocktail preparation.
Cultures of S. cerevisiae, C. lipolytica, and Z. bailii isolated from spoiled ready-to-drink
beverages were obtained from Kraft Foods, Inc., Microbiology Department, Tarrytown,
N.Y. These cultures were grown in malt extract broth solutions (Difco Laboratories,
Detroit, Mich.) for 2 days at 30°C at 140 rpm on a Lab-Line Orbit Environ-Shaker
(Melrose Park, Ill.). Malt extract broth was chosen because it is commonly used in the
beverage industry for spoilage microorganism enumeration (14). Organisms were
counted by plating 1.0 ml of decimal dilutions on malt extract agar (Oxoid Ltd.,
Basingstoke, Hampshire, United Kingdom) and incubating at 25°C for 5 days. Decimal
dilutions of the cultures were made using phosphate buffer solution (Butterfield's buffer;
Nutramax Products, Inc., Gloucester, Mass.). The solutions were held refrigerated (5°C)
until enumerated (5 days). Once the individual solutions had been quantified, they were
diluted with buffer to obtain a concentration of 5.0 × 104 CFU/ml. The individual
solutions were then blended together in equal amounts and mixed thoroughly to form
the yeast cocktail. The cocktail was held at 5°C until used.
Experimental design.
A Box-Behnken design with five variables at three levels was created using JMP
software (SAS Institute, Cary, N.C.). Two points at the center of the design were used
for a total of 42 experiments. The variables and levels were pH (2.8, 3.3, and 3.8),
titratable acidity (TA) (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 °Brix),
sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate
concentration (100, 225, and 350 ppm).
Preparation of beverages.
The beverages were prepared with bottled water (Poland Springs, Poland, Maine), high-
fructose corn syrup 42 (Cargill, Edyville, Iowa), granular citric acid (Cargill), potassium
sorbate (Sorbistat-K; Cultor Food Science, Inc., Ardsley, N.Y.), and sodium benzoate
(Cultor). Desired pH levels were obtained by buffering the beverages with potassium
citrate (Cultor). Samples were mixed thoroughly on a magnetic stirrer, filtered through
sterile, 0.20-μm-pore-size, disposable filter units (Nalgene Co., Rochester, N.Y.), and
cold filled into sterile, 50-ml centrifuge tubes (Corning Inc., Corning, N.Y.). The tubes
simulated the conditions of a sealed, bottled beverage.
The TA and pH were confirmed using a pH titroprocessor (Brinkmann, Herisau,
Switzerland). Sugar concentration (°Brix) was determined using a RFM 340
refractometer (Bellingham and Stanley Ltd., London, United Kingdom). For
consistency, °Brix reported here is that from high-fructose corn syrup alone. Actual
°Brix measurements were slightly higher due to the presence of other solids (citric acid,
potassium citrate, and preservatives). Because the resulting predictive model will be
used for product formulation, changes in °Brix and pH over the course of the
experiment were not measured.
Experimental methods.
Duplicate beverage samples were inoculated with the yeast cocktail (100 μl/50 ml) and
were immediately plated on malt extract agar medium. The inoculated samples were
stored at 25°C and were sampled after 1, 2, 4, 6, and 8 weeks. Decimal dilutions were
made using phosphate buffer solution, and colonies were enumerated after a 5-day
incubation at 25°C. The goal of this study was to determine if any spoilage yeast growth
occurred, so total plate counts rather than enumeration for specific species were used.
Model development.
Plate count data were transformed into positive and negative growth responses over
time where a response of 1 meant that yeast growth was observed and 0 meant that no
detectable counts were observed. Logistic-regression analysis was conducted on the
data using SAS software (SAS Institute). The equation for the full second-order logistic-
regression model that includes linear and quadratic terms for time is

(1)

where P is the probability of growth, β's are the parameter estimates for each term,
and X's are the five variables (pH, TA, °Brix, potassium sorbate concentration, and
sodium benzoate concentration) in the model.
The simplified model was generated using backward stepwise regression (P ≤ 0.10).
Simplified models were developed using both actual values and normalized terms (−1,
0, +1) to describe the three levels of each variable. The normalized terms were used to
facilitate comparison and to minimize correlation between terms (24).
A simplified logistic-regression model was generated using the terms from the
backward stepwise regression with the actual values for the variables. The parameter
estimates and the corresponding prediction equation for yeast growth were generated.
Model simplification is useful for several reasons. Models with many terms can have
very high correlation coefficients and predict the data used to create the model with
great accuracy. This prediction accuracy can come at the expense of postdiction
accuracy (where postdiction is the ability to predict responses in experiments not used
to create the model). This occurs because factors in these overparameterized models are
actually fitting noise in the data rather than the data themselves (17). Model
simplification can also aid in formulating hypotheses consistent with underlying
biological mechanisms.
Model validation.
Validation experiments with various pH, TA, °Brix, potassium sorbate, and sodium
benzoate levels were conducted in the model beverages using the same methods.
Fourteen sets of validation conditions were selected using a new combination of factors
based on typical levels found in ready-to-drink beverages, and each was tested in
duplicate.

RESULTS
The yeast growth responses for all of the 42 duplicated experiments are shown in Table
Table1.1. Yeast grew in only 12 samples despite being inoculated with 100 CFU/ml.
There were four instances where the duplicate samples did not exhibit the same growth
response, even though they were inoculated with the same level of yeast and had the
same formulation. No visual signs of growth were noted in any of the 84 samples
inoculated with the yeast cocktail; all beverages remained clear and colorless
throughout the 8 weeks of the experiments.

TABLE 1.
Experimental variables with logistic growth responsesa of spoilage yeast cocktail after 8
weeks in model cold-filled ready-to-drink beverages
TA Potassium sorbate concn Sodium benzoate concn Growth
pH (%) °Brix (ppm) (ppm) response
2.8 0.2 12 225 225 0.0
0.4 8 225 225 0.0
0.4 12 100 225 0.0
0.4 12 350 225 0.0
0.4 12 225 100 0.0
0.4 12 225 350 0.0
0.4 16 225 225 0.0
0.6 12 225 225 0.0
3.3 0.2 8 225 225 0.0
0.2 12 225 100 1.0
0.2 12 225 350 0.0
0.2 12 100 225 0.0
0.2 12 350 225 0.0
0.2 16 225 225 0.0
0.4 8 100 225 0.0
0.4 8 350 225 0.0
0.4 8 225 100 1.0
0.4 8 225 350 0.0
0.4 12 100 100 1.1
0.4 12 100 350 0.0
0.4 12 350 100 0.0
0.4 12 350 350 0.0
0.4 12 225 225 0.0
0.4 12 225 225 0.0
0.4 16 100 225 1.0
0.4 16 350 225 0.0
0.4 16 225 100 1.0
0.4 16 225 350 0.0
0.6 8 225 225 0.0
 TA Potassium sorbate concn Sodium benzoate concn Growth
pH (%) °Brix (ppm) (ppm) response
0.6 12 225 100 1.1
0.6 12 225 350 0.0
0.6 12 100 225 0.0
0.6 12 350 225 0.0
0.6 16 225 225 0.0
3.8 0.2 12 225 225 0.0
0.4 8 225 225 0.0
0.4 12 100 225 1.1
0.4 12 225 100 1.1
0.4 12 225 350 0.0
0.4 12 350 225 0.0
0.4 16 225 225 0.0
0.6 12 225 225 0.0
Open in a separate window

0 = no growth; 1 = growth.
a

S. cerevisiae, C. lipolytica, and/or Z. bailii were unable to grow in any of the beverages
with a pH of 2.8. At the highest pH (3.8), yeasts grew in the samples with pH 3.8, 0.4%
TA, 12 °Brix, and a combined preservative concentration of 325 ppm. The yeasts were
not able to grow at pH 3.8 under any other conditions tested, which all had higher total
preservative concentrations. Yeast did not grow at any pH when the maximum level
(350 ppm) of either preservative was used. At pH 3.3 several sets of conditions showed
yeast growth. Two sets of conditions (0.4% TA, 12 °Brix, and 100 ppm of both
preservatives; and 0.6% TA, 12 °Brix, 225 ppm of potassium sorbate, and 100 ppm of
sodium benzoate) evidenced growth in both samples. Other conditions at pH 3.3
supported yeast growth in one of the two replicates, including the two conditions of
0.4% TA and 16 °Brix and a total of 325 ppm of both preservatives.
Simplified logistic model.
The full second-order logistic-regression model utilizing all of the linear, quadratic, and
interaction terms along with the time and time2 terms was 97.8% concordant with the
data set. Backward stepwise regression eliminated 14 statistically insignificant terms
from the full second-order logistic model, including the linear terms for potassium
sorbate and sodium benzoate concentrations. The preservatives were significant as
quadratic terms and in interaction terms, and so the linear potassium sorbate and sodium
benzoate terms were returned to the simplified model. This simplified model consisted
of 11 terms: intercept, time, time2, three linear, two quadratic, and three interaction
terms (Table (Table2).2). This simplified model was 97.3% concordant with the data set,
and the equation is

(2)
where P is the probability of growth and PS and SB are potassium sorbate and sodium
benzoate concentrations (in parts per million), respectively.

TABLE 2.
Simplified model parameters for predicting the probability of growth of the S.
cerevisiae, C. lipolytica, and Z. bailii cocktail in model cold-filled ready-to-drink
beveragesa
Parameter estimate
Term P
Native terms Coded terms
Intercept 44.225919 −2.459741 0.0004
Time 3.876881 3.876880 <0.0001
Time 2
−0.366265 −0.366265 <0.0001
pH −24.530336 −2.005873 <0.0001
Potassium sorbate −0.035051 2.340813 0.3591
Sodium benzoate −0.007356 3.414137 0.8544
Potassium sorbate × pH 0.039128 2.445490 0.0002
Potassium sorbate 2
−0.000079 −1.241274 0.0038
Sodium benzoate × pH 0.052066 3.254118 <0.0001
Sodium benzoate × potassium sorbate −0.000176 −2.749729 0.0002
Sodium benzoate 2
−0.000217 −3.387250 <0.0001

Logistic-regression models predict the probability of growth between 0 and 1. Model

predictions are shown in Fig. Fig.11 and and2.2. In Fig. Fig.1,1, it is clear that sodium
benzoate has a greater inhibitory effect on spoilage yeast growth than does potassium
sorbate, as more potassium sorbate than sodium benzoate must be used to achieve the
same probability of spoilage yeast growth. The pH-dependent efficacy of sodium
benzoate is shown in Fig. Fig.2.2. At lower pH values, less sodium benzoate must be
used to achieve equal probability of yeast growth.

FIG. 1.
The effects of sodium benzoate and potassium sorbate concentrations on the probability of
growth of a C. lipolytica, S. cerevisiae, and Z. bailii cocktail after 8 weeks at initial pH of 3.3.

FIG. 2.

The effects of pH and sodium benzoate on probability of growth of a C. lipolytica, S. cerevisiae,

and Z. bailii cocktail after 8 weeks when potassium sorbate concentration was 100 ppm.

Predicted yeast growth responses from the simplified model were compared to the
growth responses of a new set of experiments, as detailed in Table Table3.3. The model
predicts growth if its output is greater than 0.5 (50 to 100% chance of growth). If the
output is lower than 0.5 (less than a 50% chance of growth), the model predicts no
growth at that condition. The model correctly predicted the observed growth responses
in all 28 samples of the 14 beverage formulations tested. It should be noted that if many
replicate experiments were performed under a single set of conditions, one would
expect the predicted probability to be equal to the fraction of observed cases (i.e., P =
0.25, one in four samples positive for growth).

TABLE 3.
Comparison of predicted and observed outcomes after 8 weeks to validate the simplified
model of the growth of S. cerevisiae, C. lipolytica, and Z. bailii in cold-filled ready-to-
drink beverages
Sodium
TA Potassium benzoate Observed Model Predicted
pH (%) °Brix sorbate (ppm) (ppm) outcomea prediction outcome
2.8 0.2 8 100 225 0 0.0025 0
2.8 0.3 12 350 225 0 0.0031 0
2.8 0.4 12 350 225 0 0.0031 0
2.9 0.2 12 100 225 0 0.0061 0
2.9 0.6 12 100 225 0 0.0061 0
 Sodium
TA Potassium benzoate Observed Model Predicted
pH (%) °Brix sorbate (ppm) (ppm) outcomea prediction outcome
3.0 0.3 16 100 350 0 0.0065 0
3.1 0.5 8 350 100 0 0.0360 0
3.2 0.4 12 100 100 1 0.9985 1
3.2 0.5 8 225 225 0 0.0040 0
3.3 0.4 16 225 100 1 0.8438 1
3.3 0.4 16 350 225 0 0.0020 0
3.4 0.4 8 100 100 1 0.9999 1
3.5 0.2 12 100 225 1 0.5618 1
3.5 0.6 12 100 225 1 0.5618 1
*, outcome of both replicates.
a

Comparison of full and simplified logistic models.

The accuracy of the logistic model decreased only slightly from the full to simplified
models, indicating that the simplified model has roughly the same postdictive and
predictive ability as the full second-order model. Neither could postdict the divergent
growth responses of the four sets of conditions where the two replicates did not agree
(one growth and one no-growth response were used in model development). For these
four discordant sets of conditions, both models postdicted one false negative
(occurrence where growth was observed but not postdicted) and three false positives
(occurrence where growth was postdicted but not observed). However, the simplified
logistic model predicted the growth responses of the validation experiments with
complete success, validating the model.

DISCUSSION
Explanation of parameters and interactions in simplified model.
The microbial stability of foods can be thought to be based upon a combination of
several factors (hurdles) which, when acting in concert, should inhibit the growth of
microorganisms (20). This “hurdle concept” can be applied to ready-to-drink beverages
by using the simplified model (Table (Table2).2). The terms in this model and their
degree of statistical significance can be employed to understand the possible
mechanisms affecting yeast spoilage of beverage products. Only three of the variables
(pH, potassium sorbate, and sodium benzoate) were found to be significant predictors
for the growth of the spoilage yeasts. TA and sugar content were not significant factors
in spoilage yeast growth. The magnitude of each term can be observed by comparing
the normalized parameter estimates (Table (Table22).
The pH values used in these experiments were highly acidic (2.8 to 3.8). Many yeasts
are able to survive but not grow at these low pH values (37), but S. cerevisiae, Z. bailii,
and C. lipolytica can grow at pH levels as low as 2.0 (31). The effects of external pH on
microbial homeostasis are well understood (4). As the pH of the beverage decreases, the
amount of undissociated citric acid increases and can permeate the cell wall, altering the
internal pH of the microorganisms (8).
The quadratic term for pH in the logistic model was not significant and was not
included in the simplified yeast model. This is consistent with several previously
published papers on the time to detection and time to growth of Z. bailii (11, 19).
Quadratic pH terms, however, are included in other models for Z. bailii (5, 7)
and Kluyveromyces marxianus (38), although the level of statistical significance of the
quadratic pH terms was not provided.
The sugar content of the beverages in this study (8 to 16 °Brix, corresponding
to Aw values from 0.95 to 0.99) was not a factor in predicting growth of these spoilage
yeasts. S. cerevisiae, C. lipolytica, and Z. bailii have shown the ability to grow at 50
°Brix (31, 37), and they have enzyme systems that produce compatible solutes that
allow them to grow at decreased Aw (21, 41). Due to the resistance of these spoilage
yeasts to low Aw, it is not surprising that the sugar concentrations in these experiments
did not affect the yeast growth. However, these results were inconsistent with Cole and
Keenan (7) and Cole et al. (5), who included the °Brix in their models predicting the
growth of Z. bailii in beverages. The sugar contents of the beverages in those studies
were 20 to 55 and 5 to 15 °Brix, respectively, but the models in these studies were not
simplified to exclude the statistically insignificant terms in the model. Fructose content
was determined to be a significant (P < 0.0001) factor for a Z. bailii time-to-growth
model (19), but the range of sugar content was higher (up to 32% [wt/vol] fructose) than
the range used in our experiments.
Both preservatives were significant in quadratic and interaction terms and were thus
included in the simplified model. Higher levels of potassium sorbate or sodium
benzoate decrease the likelihood of yeast growth. Sorbic acid affects yeast growth by
inhibiting the uptake of amino acids and the function of sulfhydral enzymes (10).
Benzoic acid uncouples the electron transport system and destroys the proton motor
force by increasing the internal proton level of the microorganism (10). This study
showed that sodium benzoate, when used in combination with potassium sorbate, was a
better inhibitor of spoilage yeasts than potassium sorbate (Fig. (Fig.1).1). This is
consistent with Beuchat (3), who reported that sodium benzoate was more lethal
towards S. cerevisiae than was potassium sorbate. Others (31), however, found that
sorbic acid is more inhibitory than benzoic acid against spoilage yeasts, including C.
lipolytica, S. cerevisiae, and Z. bailii.
There is a significant interaction between potassium sorbate and sodium benzoate in the
simplified model (P = 0.0002), indicating that the two preservatives do not act
independently of each other. The parameter estimate is very small and negative
(−0.000176), indicating that there could be a slight antagonism (27) between potassium
sorbate and sodium benzoate. This is consistent with Osman and El-Mariah (29), who
reported that higher combined concentrations of sorbic acid and benzoic acid were
required to prevent the growth of S. cerevisiae. It should be noted that other researchers
have found evidence of synergistic inhibition of spoilage yeasts when both preservatives
were used (6, 34).
There are also significant interactions between pH and potassium sorbate (P = 0.0002)
and pH and sodium benzoate (P < 0.0001). The synergy between pH and sodium
benzoate is clearly illustrated in Fig. Fig.2.2. This is consistent with the idea that
undissociated benzoic acid is more inhibitory than dissociated benzoic acid because
undissociated organic acids are more lipophilic and can therefore pass through the
cytoplasmic membrane and affect intracellular pH (10, 23).
Decreasing the pH of the beverage would allow a beverage developer to use less
potassium sorbate and/or sodium benzoate to achieve the same probability of yeast
growth. Conversely, increasing preservative levels provides microbial stability at
increased pH levels (31). Eklund has suggested that the increased microbial action of
sorbic and benzoic acid at low pH may be due to the increased susceptibility of the
organism and not due to increased activity of the undissociated forms (15, 16); however,
it is difficult to ascertain whether the stress of low pH or the increased level of the
undissociated acid causes the interaction between pH and organic acids. Regardless, our
simplified model reflects the synergy between pH and preservative efficacy and can be
used to show different beverage conditions that provide equal microbial stability.
Interactions between potassium sorbate and °Brix and sodium benzoate and °Brix were
not significant, although it has been shown that sugars can act synergistically with
organic acids to inhibit microbial growth (1, 36). As noted above, sugar content may
become a significant factor only at higher concentrations than those used in our study.
An interaction between pH and °Brix was expected but was not found to be significant
in the model. Cole and Keenan (7) and Cole et al. (5) reported synergy between pH and
°Brix when describing the influence of these two variables on the growth of Z. bailii in
model fruit drink systems, but these studies, as previously mentioned, did not use any
method to simplify their models. Also, Cole and Keenan used much higher °Brix values
than those used in this study, so it is possible that sugar content is more influential and
acts synergistically with pH at larger concentrations. Other studies have also found that
increasing the sugar level allows the effects of pH to be more pronounced (4).
Implications.
The growth of spoilage yeasts in ready-to-drink beverages can cause off-flavors to
develop in the beverage and can cause containers to explode from a buildup of carbon
dioxide (30, 33). Spoilage yeasts can also alter the environment of the beverage by
changing the pH or degrading preservatives, allowing other spoilage organisms to grow
(26). Candida, Saccharomyces, and Zygosaccharomyces also possess lipolytic enzymes
which can degrade the fatty acids of benzoate and sorbate (26). The control of any
growth of spoilage yeasts is, therefore, essential for quality assurance.
The pH, potassium sorbate, and sodium benzoate levels of a cold-filled ready-to- drink
beverage were all found to have an impact on yeast growth. However, it can be
challenging to formulate beverages of an acceptable sensory quality at very low pH
levels (4). Sodium benzoate was found to be a better inhibitor of spoilage yeast growth
than potassium sorbate. This is an advantage for beverage developers since sodium
benzoate tends to be less expensive than potassium sorbate (12) and is less prone to
oxidation and degradation (35). Benzoic acid, however, imparts a burning aftertaste
while sorbic acid tends to be neutral (13).
This predictive model provides an important tool for beverage product developers.
While the pH, sodium benzoate, and potassium sorbate levels can be adjusted to provide
desired cost and flavor profiles, the developers can consider each formulation's
microbial stability. While a predictive model cannot replace microbial testing or the
judgment of a trained and experienced microbiologist (39), this model should reduce the
need for time-consuming and invasive microbiological testing procedures (32).
Conclusion.
A mathematical model predicting the growth of S. cerevisiae, C. lipolytica, and Z.
bailii in cold-filled ready-to-drink beverages has been presented. The model includes
factors that can be controlled by a product developer, such as pH, sodium benzoate, and
potassium sorbate concentration. The TA and °Brix of a beverage did not significantly
impact spoilage yeast growth. This model can predict microbial stability of ready-to-
drink beverages while allowing beverage developers to consider the ingredient costs,
quality, and flavor implications of various preservation systems.

ACKNOWLEDGMENTS
We thank Kristin M. Jackson for her editorial assistance and Kraft for their assistance
and support of this project.

REFERENCES
1. Baird-Parker, A. C., R. L. Buchanan, F. F. Busta, M. B. Cole, M. P. Doyle, M. Eyles, J. Farkas, R.
S. Flowers, J. L. Jouve, S. Mendoza, et al.1998. Soft drinks, fruit juices, concentrates and fruit
preserves, p. 440-460. InT. A. Roberts, J. I. Pitt, J. Farkas, and F. H. Grau (ed.), Microorganisms in
foods 6: microbial ecology of food commodities. Blackie Academic & Professional, London,
United Kingdom.

2. Battey, A. S., and D. W. Schaffner. 2001. Modeling bacterial spoilage in cold-filled ready to
drink beverages by Acinetobacter calcoaceticus and Gluconobacter oxydans. J. Appl.
Microbiol. 91:237-247. [PubMed]

3. Beuchat, L. R. 1981. Effects of potassium sorbate and sodium benzoate on inactivating yeasts
heated in broths containing sodium chloride and sucrose. J. Food Protect. 44:765-769.

4. Brown, M. H., and I. R. Booth. 1991. Acidulants and low pH, p. 22-43. InN. J. Russell and G.
W. Gould (ed.), Food preservatives. Van Nostrand Reinhold, New York, N.Y.

5. Cole, M. B., J. G. Franklin, and M. H. J. Keenan. 1987. Probability of growth of the spoilage
yeast Zygosaccharomyces bailii in a model fruit drink system. Food Microbiol. 4:115-119.

6. Cole, M. B., and M. H. J. Keenan. 1986. Synergistic effects of weak-acid preservatives and pH
on the growth of Zygosaccharomyces bailii. Yeast 2:93-100. [PubMed]

7. Cole, M. B., and M. H. J. Keenan. 1987. A quantitative method for predicting shelf life of soft
drinks using a model system. J. Ind. Microbiol.2:59-62.

8. Cortlett, D. A., Jr., and M. H. Brown. 1980. Microbial ecology of foods, vol. 1. Factors
affecting life and death of micro-organisms, p. 92-111. Academic Press, New York, N.Y.

9. Cuppers, H. G. A. M., and J. P. P. M. Smelt. 1993. Time to turbidity measurement as a tool

for modeling spoilage by Lactobacillus. J. Ind. Microbiol. 12:168-171.

10. Davidson, P. M. 1997. Chemical preservatives and natural antimicrobial compounds, p. 520-
556. In M. P. Doyle, L. R. Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals
and frontiers. ASM Press, Washington, D.C.
11. Deak, T., and L. R. Beuchat. 1994. Use of indirect conductimetry to predict the growth of
spoilage yeasts, with special consideration of Zygosaccharomyces bailii. Int. J. Food
Micro. 23:405-417. [PubMed]

12. De Boer, E., and P. V. Nielsen. 1995. Food preservatives, p. 289-293. InR. A. Samson, E. S.
Hoekstra, J. C. Frisvad, and O. Filtenborg (ed.), Introduction of food-borne fungi.
Centraalbureau voor Schimmelcultures, Wageningen, The Netherlands.

13. Dryden, E. C., and C. H. Hills. 1959. Taste thresholds for sodium benzoate and sodium
sorbate in apple cider. Food Technol. 13:84-86.

14. Dworetsky, T. 1998. Still warning: cold-filled still beverage microbiology. Beverage
World 117:94-98.

15. Eklund, T. 1983. The antimicrobial effect of dissociated and undissociated sorbic acid at
different pH levels. J. Appl. Bacteriol. 54:383-389. [PubMed]

16. Eklund, T. 1985. Inhibition of microbial growth at different pH levels by benzoic and
propionic acids and esters of p-hydroxybenzoic acid. Int. J. Food Micro. 2:159-167.

17. Gauch, H. G. 1993. Prediction, parsimony and noise. Am. Sci. 81:468-478.

18. Gibson, A. M., J. Baranyi, J. I. Pitt, M. J. Eyles, and T. A. Roberts.1994. Predicting fungal
growth: the effect of water activity on Aspergillus flavus and related species. Int. J. Food
Micro. 23:419-431. [PubMed]

19. Jenkins, P., P. G. Poulos, M. B. Cole, M. H. Vandeven, and J. D. Legan.2000. The boundary
for growth of Zygosaccharomyces bailii in acidified products described by models for time to
growth and probability of growth. J. Food Protect. 63:222-230. [PubMed]

20. Leistner, L. 1995. Principles and applications of hurdle technology, p. 1-21. In G. W. Gould
(ed.), New methods of food preservation. Blackie Academic & Professional, New York, N.Y.

21. Leistner, L., and N. J. Russell. 1991. Solutes and low water activity, p. 111-134. In N. J.
Russell and G. W. Gould (ed.), Food preservatives. Avi Publishing Co., Inc., New York, N.Y.

22. Llaudes, M., L. Zhao, S. Duffy, and D. W. Schaffner. 2001. Simulation and modeling of the
effect of small inoculum size on the time to spoilage by Bacillus stearothermophilus. Food
Microbiol. 18:395-405.

23. Macris, B. J. 1975. Mechanism of benzoic acid uptake by Saccharomyces cerevisiae. Appl.
Microbiol. 30:503-506. [PMC free article] [PubMed]

24. Mantel, N. 1970. Why stepdown procedures in variable selection. Technometrics 12:621-
625.

25. McClure, P. J., C. D. Blackburn, M. B. Cole, P. S. Curtis, J. E. Jones, J. D. Legan, I. D. Ogden,

M. W. Peck, T. A. Roberts, J. P. Sutherland, and S. J. Walker. 1994. Modelling the growth,
survival and death of microorganisms in foods: the UK food micromodel approach. Int. J. Food
Micro. 23:265-275. [PubMed]
26. Mossel, D. A. A., J. E. L. Corry, C. B. Struijk, and R. M. Baird. 1995. Essentials of the
microbiology of foods: a textbook for advanced studies. John Wiley & Sons, New York, N.Y.

27. Neter, J., W. Wasserman, and M. H. Kutner. 1989. Applied linear regression models. Irwin,
Burr Ridge, Ill.

28. Ng, T. M., and D. W. Schaffner. 1997. Mathematical models for the effects of pH,
temperature, and sodium chloride on the growth of Bacillus stearothermophilus in salty
carrots. Appl. Environ. Microbiol. 63:1237-1243. [PMC free article] [PubMed]

29. Osman, H. G., and A. El-Mariah. 1960. Studies on the inhibitory effect of combined
chemical preservatives on Saccharomyces cerevisiae. J. Am. Pharm. Assoc. 49:231-
233. [PubMed]

30. Panezai, A. K. 1978. Microbiology, p. 209-227. In L. F. Green (ed.), Developments in soft

drinks technology. Applied Science Publishers Ltd., London, United Kingdom.

31. Praphailong, W., and G. H. Fleet. 1997. The effect of pH, sodium chloride, sucrose, sorbate
and benzoate on the growth of food spoilage yeasts. Food Microbiol. 14:459-468.

32. Ross, T., and T. A. McMeekin. 1994. Predictive microbiology. Int. J. Food Micro. 23:241-
264. [PubMed]

33. Rouseff, R. L., S. Nagy, M. Naim, and U. Zahavi. 1992. Off-flavor development in citrus juice
products, p. 211-227. In G. Charalambous (ed.), Off-flavors in foods and beverages. Elsevier,
New York, N.Y.

34. Rushing, N. B., and V. J. Senn. 1963. The effect of benzoic, sorbic, and dehydroacetic acids
on the growth of citrus products spoilage organisms. Fla. State Hortic. Soc. 76:271-276.

35. Sofos, J. 1989. Sorbate food preservatives. CRC Press, Boca Raton, Fla.

36. Sofos, J. N., and F. F. Busta. 1993. Sorbic acid and sorbates, p. 49-94. InG. A. Davidson
(ed.), Antimicrobials in foods. Marcel Dekker, Inc., New York, N.Y.

37. Thomas, D. S., and R. R. Davenport. 1985. Zygosaccharomyces bailii—a profile of

characteristics and spoilage activities. Food Microbiol. 2:157-169.

38. Vivier, D., R. Ratomahenina, G. Moulin, and P. Galzy. 1993. Study of physicochemical
factors limiting the growth of Kluyveromyces marxianus. J. Ind. Microbiol. 11:157-161.

39. Whiting, R. C. 1995. Microbial modeling in foods. Crit. Rev. Food Sci. Nutr. 35:467-
494. [PubMed]

40. Whiting, R. C., and R. L. Buchanan. 1997. Predictive modeling, p. 728-739. In M. P. Doyle, L.
R. Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals and frontiers. ASM
Press, Washington, D.C.
41. Witter, L. D., and C. B. Anderson. 1987. Osmoregulation by microorganisms at reduced
water activity, p. 1-34. In T. J. Montville (ed.), Food microbiology, vol. I. Concepts in physiology
and metabolism. CRC Press, Inc., Boca Raton, Fla.

42. Zhao, L., Y. Chen, and D. W. Schaffner. 2001. Comparison of logistic regression and linear
regression in modeling percentage data. Appl. Environ. Microbiol. 67:2129-2135. [PMC free
article] [PubMed]

VII. Processing maize flour and corn meal food products

Jeffrey A Gwirtz1 and Maria Nieves Garcia-Casal2

Author information ► Copyright and License information ► Disclaimer

Abstract

Introduction
Maize is a domesticated grass that originated approximately 7000 years ago in what is
now Mexico. It is also referred to as corn, and both words are used as synonyms in this
review, depending on the source of data or references consulted. Maize was spread
across the world shortly after the European discovery of the Americas. Regardless of
origin, corn has proven to be one of the most adaptable crops. Its evolution apparently
occurred mainly under domestication and resulted in biotypes with adaptation ranging
from the tropics to the north temperate zone, from sea level to 12,000 feet altitude, and
growing periods (planting to maturity) extending from 6 weeks to 13
months.1,2 Currently, the United States, Brazil, Mexico, Argentina, India, France,
Indonesia, South Africa, and Italy produce 79% of the world's maize
production.3 Between 1990 and 2011, the number of millions of maize hectares
harvested ranged from 129.1 to 163.9. During the same period the production of maize
in metric tons per hectare increased from 3.7 to 5.1, and total maize production
increased from 482.0 to 832.5 million metric tons. Worldwide, 60–70% of maize
production is used domestically as livestock feed, and the remaining 30–40% is used for
production of items for human consumption.4
Corn is the main cereal grain as measured by production but ranks third as a staple food,
after wheat and rice. The reasons for this fact are varied, but some of them are related to
cultural or social preferences and also because in some countries, corn is cultivated as
livestock feed. More recently, the use of corn as a biofuel has generated great concern
about rises in the market price of corn for consumption, the need to increase cultivable
areas, as well as water quality and other ecological damages. Some predictive models
project that large-scale corn ethanol production could lead to decreases in food exports,
higher prices, and a greater global deforestation.5,6

Maize kernel anatomy

The maize kernel is composed of four primary structures from a processing perspective.
They are endosperm, germ, pericarp, and tip cap, making up 83%, 11%, 5%, and 1% of
the maize kernel, respectively (Fig. (Fig.1).1). The endosperm is primarily starch
surrounded by a protein matrix. Two main types of starch include hard or vitreous, and
soft or opaque. Vitreous endosperm is negatively related to starch degradability and in
vivo starch digestibility in ruminants.7,8

Figure 1

Components of the corn kernel.

The germ or embryo of the maize kernel is high in fat (33.3%) in addition to enzymes
and nutrients for new maize plant growth and development. The germ also contains
vitamins from B complex and antioxidants such as vitamin E. Maize germ oil is
particularly high in polyunsaturated fatty acids (54.7%), which are subject to oxidative
and other forms of rancidity resulting in off or objectionable flavors from full-fat maize
products. Pericarp is a high-fiber (8.8% crude) semipermeable barrier surrounding the
endosperm and germ, covering all but the tip cap. The tip cap is the structure through
which all moisture and nutrients pass through during development and kernel drydown.
The black or hilar layer on the tip cap acts as a seal.9 The term bran is also used to refer
to the fiber-rich outer layer (pericarp) that contains B vitamins and minerals and the tip
cap.
Corn variations may be artificially defined according to kernel type as follows: dent,
flint, waxy, flour, sweet, pop, Indian, and pod corn. Except for pod corn, these divisions
are based on the quality, quantity, and pattern of endosperm composition, which defines
the size of the kernel, and are not indicative of natural relationships. Endosperm
composition may be changed by a single gene difference, as in the case of floury (fl)
versus flint (FI), sugary (su) versus starchy (Su), waxy (wx) versus nonwaxy (Wx), and
other single recessive gene modifiers that have been used in breeding special-purpose
types of corn.1,10

Maize kernel composition

TablesTables11 and and22 provide the vitamin and mineral analysis of corn, crude bran,
and cornstarch as available from the U.S. Department of Agriculture Nutritional Data
Base.11 As can be observed, the corn bran is a significant contributor to maize vitamin
and mineral content. The wet milling of maize separates much of its nutrient content
away from the starch component.

Table 1
Vitamin content of whole kernel, crude bran, and corn starch of yellow corn
Unit/ Corn, Corn, Corn,
Vitamin 100 g whol bran starch
e
Thiamin mg 0.39 0.01 0
Riboflavin mg 0.20 0.10 0
Niacin mg 3.63 2.74 0
Pantothenic acid mg 0.42 0.64 0
Vitamin B6 mg 0.62 0.15 0
Folate μg 19.00 4.00 0
Choline μg 18.10 0.40

NOTE: Data from U.S. Department of Agriculture.11

Table 2
Mineral content of whole kernel, crude bran, and corn starch of yellow corn
Unit/ Corn, Corn, Corn,
Mineral 100 g whole bran starch
Calcium, Ca mg 7.00 42.0 2.00
0
Iron, Fe mg 2.71 2.79 0.47
Magnesium, Mg mg 127.00 64.0 3.00
0
Phosphorus, P mg 210.00 72.0 13.00
0
Potassium, K mg 287.00 44.0 3.00
0
Sodium, Na mg 35.00 7.00 9.00
Zinc, Zn mg 2.21 1.56 0.06
Copper, Cu mg 0.31 0.25 0.05
Manganese, Mn mg 0.49 0.14 0.05
Selenium, Se μg 15.50 16.5 2.80

NOTE: Data from U.S. Department of Agriculture.11

In addition to chemical composition, physical characteristics of maize in the commercial

market place influence the value of the grain or the final product. Often, countries will
have grading standards for maize entering the supply chain to assist buyers and sellers
assessing maize value. Test weight, moisture content, foreign material, and damage are
among typical measures of maize quality and value.12
Maize pathways to the consumer
Maize food products can be processed at home on a small local scale as well as on a
larger industrial scale, transforming the raw material into food products (Fig. (Fig.2).2).
Some of the products are more suitable for commercial trade because they require
further processing or provide convenience and extended shelf life, while other products
should be consumed immediately after production. For example, degerminated corn
grits, meal, or flour has an extended shelf life and can be moved and traded easily. The
product itself has to be further processed, including some degree of cooking, in order
for it to be palatable as a food product.

Figure 2

Pathways of maize from field to consumer.

Nixtamalized maize, when prepared in the household or by a small-scale processor, is

typically used to form ready-to-eat finished masa products with a limited shelf life. On
an industrial scale, nixtamalized maize flour may be processed and sold as a shelf-stable
product that can be prepared for consumption in the home, reducing meal preparation
time and providing convenience. Industrial processors strive to offer maize products that
replicate the ones consumed in the target market region. Identifying the market share of
industrially produced maize product consumed in the market place is essential in
evaluating the potential for implementing a successful maize product fortification
program. The influence of a small-scale maize processing industry with local impact
may also play a significant role in some target populations, especially in some poor
countries.
Maize color is important to specific consumer groups in Central and South America as
well as in Africa, and white maize is preferred for food consumption. In some areas of
the world, such as North America, the desired color depends on the region or the food
use. For example, in the eastern United States, cornmeal, grits, and homily are white,
while in the northern part of the country, maize meal, and maize products used for
breakfast cereals and snack foods are expected to be manufactured with yellow maize.
Maize meal or flour composition can also be driven by regional preferences, with some
preferring whole ground maize rather than degerminated or partially degerminated
maize products. The nutrient composition, including vitamins, minerals, and antinutrient
factors, are influenced by local product preferences, which include not only the way the
corn product is consumed, but also what other food items or additives are part of a
complete meal. Local and regional standards for both the form and composition are to
be evaluated as part of planning a maize flour or cornmeal fortification program.
Maize products and processing methods are as diverse as the maize crop itself.
Processing of maize at the household or local industrial level may be accomplished with
wooden mortar and pestles, stone metates, and manos (stones).13 Particle reduction in
germ and/or fiber content may be accomplished with screening. Poor product stability,
especially due to fat content, results in the need for frequent processing of maize at the
household and small-scale industry level. Additionally, the number of nutrients removed
or altered through home or small-scale industry processing may vary widely. As a result,
fortification of maize processed at home or in small-scale industrial mills may not be
practical. On the other hand, in some countries, local or household production and/or
processing could account for the majority of maize consumption. In those countries,
central or nationwide maize fortification programs would not be a solution for
improving micronutrient intake.

Industrial maize processes

There are two basic categories of industrial processing employed for transforming
maize into products for human consumption. They are known as dry and wet milling. In
the wet milling process, maize is separated into relatively pure chemical compound
classes of starch, protein, oil, and fiber. The products and coproducts obtained from wet
maize milling are not typically directly used by the consumer and often require further
industrial processing before consumption. The products of wet maize milling are not
typically produced on a small scale commercially or in the home. The primary product,
starch, can be processed into a variety of starch products or further refined into a variety
of sweeteners sold in liquid and dry forms. Wet milling of maize will not be further
addressed in this article.
Industrial dry milling includes particle size reduction of clean whole maize with or
without screening separation, retaining all or some of the original maize germ and
fiber.14 Because of the high-fat content, these whole or partially degerminated maize
products are not particularly shelf stable. Degermination of maize involves mechanical
separation and processing, resulting in dry shelf-stable products with a majority of both
germ and fiber removed. Much of the particle size reduction and separation is
accomplished with equipment similar to that employed in wheat flour milling, including
hammer mills, stone mills, roller mills, screeners, sifters, specific gravity separators, and
aspirators. Specialized equipment, such as degerminators and de-hullers or peelers, may
be employed in maize processing.
Generally, whole, partially degerminated, and degerminated maize products require
additional processing before consumption. These processing steps may be accomplished
in a large-scale industrial setting, small-scale local processor, or in the home. These
secondary processes may include addition of other ingredients along with thermal
processing, including boiling, drying, frying, or baking, all of which can affect the
nutritional attributes of the finished product.
A second type of industrial dry maize processing is alkali processing or nixtamalization
in which whole maize is cooked with an excess of water treated with calcium
oxide.13 The maize kernel may be ground whole, fractionated, or have other corn
components added. Unlike wheat flour milling, processing equipment for alkali-treated
corn is specialized to handle the moisture, chemicals, and heat required for wet
processing. Conventional dry bulk material handling and processing equipment is
employed with raw maize and dry finished product. The resulting intermediate product
may be dried for commercial sales of further processed consumer food product. In
North America and Mexico, dry alkali-processed maize flour is known as masa flour, a
name that is not used in Spanish-speaking countries of Central and South America or
non-Spanish speaking countries of Africa. These products are referred herein as alkali
processed. The alkali process improves flavor, starch gelatinization, and water uptake.
The process partially removes some of the germ and most of the pericarp, but the
amount varies. In some cases, pericarp may be added into the process for visual product
enhancement. The heating in the process causes loss of thiamine, riboflavin, niacin, fat,
and fiber. As might be expected the calcium content increases owing to the alkali
processing.
In the nixtamalization process, there are several stages. First, dried maize is soaked in a
solution of water with lime, often with ashes mixed in. The grain is then cooked,
steeped, drained, and rinsed multiple times. The grain is then ground to make a wet
dough from which tortillas are formed or allowed to dry into flour. Currently, there is an
important diminution in production of homemade tortillas because they are now
prepared from commercial instantaneous flour or bought as packaged
tortillas.15,16 Nixtamalized maize has several benefits compared to unprocessed grains:
they are more easily ground and have a higher nutritional value (increased
bioavailability of niacin, improved protein quality, increased calcium) and reduced
mycotoxins content.17
A staple maize product in South America, particularly in Venezuela and Colombia, is
arepa, which is a fried or baked bread prepared from precooked refined corn flour.
Traditionally, arepas are made by dehulling and degerming previously soaked whole
kernels by manually grinding maize kernels in a pilon, a wooden mortar. The bran and
germ are removed by repeatedly rinsing the mixture containing the endosperm with
water. This fraction is then cooked and milled to prepare a dough that will be shaped
and cooked (baked or fried) to obtain the arepa.18 The traditional process for preparing
homemade arepas involves soaking, cooking, cooling, draining, grinding, and forming a
dough piece for additional grilling or baking. The process takes 18 or more hours to
complete in the home.
The traditional method has been modified with the introduction of precooked maize
flour.19 This process includes conditioning, cooking, flaking, drying, grinding, and
sifting to produce dry instant precooked refined arepa flour. The flour can be transported
and stored easily until used in the home. Preparation is reduced to less than an hour,
making it more convenient for consumers.20–23Although arepa is prepared from flour that
is 100% corn, there are commercial presentations of mixtures of corn with rice, corn
with wheat, and corn with oat and wheat bran.
Fermented maize products, such as ogi, are prepared by soaking the maize kernel for 1–
3 days until soft. It is then grinded with a stone and dehulled and degermed by repeated
washes with water. The filtered endosperm is fermented for 2 or 3 days, producing a
slurry that becomes the ogi porridge when boiled. Fermented products that are similar
but prepared with different maize varieties or minor preparation changes include uji in
Kenya, kenkey, banku, ogi, and koko from Ghana and Nigeria.24,25 Nixtamal is another
fermented maize product, prepared from dehulled kernels ground to a coarse dough and
wrapped in banana leaves to ferment for 2 or 3 days.20
Figure Figure33 provides a schematic of three of the maize dry-milling processes:
whole or refined, nixtamalized, and precooked corn flours. There is a common, similar
initial process between household and industrial preparation of nixtamalized and
precooked corn flours that is taken from the traditional way of household preparation.
At the point of product 1, the masa or dough is obtained and final products could be
prepared. Industrial processing from this point produces the commercial flour that only
needs added water and to be cooked at the household level, to obtain the traditional
product without repeating all the processing on a daily or regular basis.

Figure 3

Schematics of dry-milling maize processing.

Separation of maize constituents (e.g., dehulling or degerming) varies depending on

regional customs and consumer preference. These differences affect the vitamin and
mineral content of the finished product from primary processing and should be taken
into consideration when developing a maize fortification strategy. Yield, fat, and fiber
content of the maize product from the primary processes will be directly proportional to
nutrient content. Particle size will also be important to the fortification strategy as
presented later in the article. Fortification of products from the secondary processing of
maize becomes widely varied and inherently more difficult to manage.
The products derived from dry milling are numerous, with their variety depending to a
large extent on particle size. In Africa, ground maize is cooked into a paste accompanied
by a thick low-alcoholic beer. This maize paste could be fried or baked, depending on
the region of Africa. Many Africans depend on some variation of this mush, which is
made with water and ground maize. It can also be eaten as a porridge or a dumpling,
depending on the thickness of the batter and the cooking method.26 In Kenya, they
prepare uji, a porridge of maize flour cooked in water and sweetened with sugar.27
Other maize preparations include humitas prepared from precooked maize flour, mote
made from cooked maize and cheese, pupusas made from lime-treated maize and
cheese, and patasca, which is like a lime-treated maize kernel.20 Table Table33 shows a
variety of maize products of global interest.13

Table 3
Various maize products consumed globally
Bread
Flat, unleavened, Tortilla, arepa
unfermented
 Fermented and/or Pancakes, cornbread, hoe cake, blintzes
leavened
Porridges Atole, ogi, kenkei, ugali, ugi, edo, pap,
Fermented, unfermented  maizena, posho, asidah
Steamed products Tamales, couscous, rice-like products, Chinese breads, dumplings,
chengu
Beverages
 Alcoholic Koda, chicha, kafir beer, maize beer
 Nonalcoholic Mahewu, magou, chicha dulce
Snacks Empanadas, chips, tostadas, popped corn, fritters

NOTE: From Rooney and Serna-Saldavar13

Attempts have been made to classify and define products of maize processing; however,
there is not a globally recognized terminology for dry-milled maize products.9 Table
Table44 identifies the commonly accepted terms used according to ranges of particle
size for maize products. The fat values are for degerminated maize products.28 Some
have subdivided the definition of maize meal into smaller size categories to include
coarse meal (1190–730 μm), medium meal (730–420 μm), and fine meal or cones (420–
212 μm).29

Table 4
Degerminated maize products defined by particle size and fat content
Particle size
Less than (μ) Greater than (μ) Fat (%)
Grits 1400 600 0.8
Meal 600 300 1.8
Fine 300 212 2.5
meal
Flour 212 2.7

NOTE: Data from Baltenspreger.18

The U.S. Code of Federal Regulations, Title 21, provides standards for various maize
products, such as white corn flour (137.211), yellow corn flour (137.215), white corn
meal (137.250), enriched cornmeal (137.260), degerminated white cornmeal (137.265),
and self-rising white cornmeal (137.270).30 It is important to note that publicly available
standards do not always identify end-use properties. It is possible that some aspects of a
government-provided standard for a product may be less restrictive than the commercial
standard of the customer. In some countries, the government and consumer standard is
one and the same regulation.31,32
Many products of the industrial dry maize–milling processes may also be produced
locally on a small scale as well as in the home. High-moisture wet products from local
small-scale producers have limited shelf life and must be used in a short period.
However, the products are still interesting for users because they reduce preparation
time for the homemaker. When produced in the home the process is generally carried
out to a food product level ready for consumption.

Fortification of maize products

Fortification of maize flour or cornmeal with iron is a cost-effective, food-based
approach that should be regarded as part of a broader, integrated initiative to prevent
micronutrient malnutrition, complementing other efforts to improve micronutrient
status. Other initiatives include supplementation, change of food habits, food-to-food
fortification, point-of-use fortification, promotion of increased consumption and/or
production of food, improvement of health and sanitary conditions, biofortification,
genetically modified foods, and nanotechnologies.
Identification of maize consumption volume as well as product type and significant
maize product production capacity at the industrial levels is required for a successful
maize fortification program. Maize flour, masa, or dry maize products from the primary
processing identified in Figure Figure33 with less than 13% moisture content are stable
and may be fortified with a powdered premix composed of appropriate vitamins and
minerals that could be similar to those used in wheat flour fortification programs33,34 but
are always based on the needs of a particular population, country, or region and not on
flour capabilities of accepting a particular premix. The fine maize products (100% less
than 600 μm) are suitable for the addition of powdered premix and will not be subject to
significantly meaningful segregation. The selection of the adequate micronutrient
mixture is key for a program to be successful. It is important to select a mixture of
micronutrients, especially regarding iron, that is well absorbed and at the same time
does not change the organoleptic characteristics of the fortified maize flour, cornmeal,
or the meals that contain the fortified flour. A Flour Miller's Toolkit for flour
fortification has recently been updated to provide practical insights for flour fortification
within the context of a grain milling or processing facility.35
Table Table55 provides a proximate vitamin and mineral analysis for white maize,
whole-grain maize flour, degerminated meal (unenriched and enriched), alkali-
processed maize or masa flour (unenriched and enriched), and precooked corn flour
(unenriched and enriched).11,36 Degerminated maize products are clearly lower in fat,
fiber, and ash content when compared to whole maize or whole maize flour. The
influence of alkali processing on the calcium level of the finished product is readily
apparent given the higher values of calcium reported.
 Table 5Proximal analysis of vitamin and mineral content of white corn,
flour, meal, and alkali-processed masa, unenriched and enriched

Corn flour, Cornmeal, Cornmeal, Corn flour, Corn Precooked Precooked

flour,
Corn degermed, degermed, degermed, masa, masa, corn flour, corn flour,
flour,
Unit/100g Corn whole unenriched unenriched enriched unenriched enriched unenriched enriched
grain
Water g 10.4 10.9 11.2 11.2 9.0 9.0 11.2 11.2
Energy (Kcal) Kcal 365 361 370 370 365 365 354 354
Energy (KJ) KJ 1527 1510 1547 1547 1528 1528
Protein g 9.4 6.9 7.1 7.1 9.3 9.3 7.2 7.2
(N×6.25)
Total lipids g 4.7 3.9 1.8 1.8 3.9 3.9 1.1 1.1
Ash g 1.2 1.5 0.5 0.5 1.5 1.5 0.3 0.3
Carbohydrates g 74.3 76.9 79.5 79.5 76.3 76.3 80.2 80.2
Total fiber g 7.3 3.9 3.9 6.4 6.4 2.5 2.5
Sugars, total g 0.6 1.6 1.6 1.6 1.6
Starch g 73.3 73.3 66 66
Thiamin mg 0.39 0.25 0.07 0.14 0.55 0.22 1.48 0.06 0.31
Riboflavin mg 0.20 0.08 0.06 0.05 0.38 0.1 0.81 0.05 0.25
Niacin mg 3.63 1.9 2.66 1 4.97 1.63 9.93 0.6 5.1
Pantothenic mg 0.42 0.66 0.05 0.24 0.24 0.19 0.19
acid
Vitamin B6 mg 0.62 0.37 0.1 0.18 0.18 0.48 0.48
Folate, total μg 25 48 30 209 2.9 209
Folic acid μg 0 0 0 180 0 180
Folate, food μg 25 48 30 30 2.9 2.9
Folate, FDE μg 25 48 30 335 2.9 335
Choline mg 21.6 8.6 8.6 8.6 8.6
Calcium, Ca mg 7 7 2 3 3 136 136 12 12
Iron, Fe mg 2.7 2.4 0.9 1.1 4.4 1.5 7.5 0.9 5.0
Magnesium, mg 127 93 18 32 32 93 93
Mg
Phosphorus, P mg 210 272 60 99 99 214 214 64 64
Potassium, K mg 287 315 90 142 142 263 263
Sodium, Na mg 35 5 1 7 7 5 5
Zinc, Zn mg 2.2 1.7 0.4 0.7 0.7 1.8 1.8
Copper, Cu mg 0.3 0.2 0.1 0.1 0.1 0.2 0.2
Manganese, mg 0.5 0.5 0.06 0.2 0.2 0.4 0.4
Mn
 Corn flour, Cornmeal, Cornmeal, Corn flour, Corn Precooked Precooked
flour,
Corn degermed, degermed, degermed, masa, masa, corn flour, corn flour,
flour,
Unit/100g Corn whole unenriched unenriched enriched unenriched enriched unenriched enriched
grain
Selenium, Se μg 15 15.4 8 10.5 10.5 14 14
Vitamin A RE 270
Open in a separate window

NOTE: Data from U.S. Department of Agriculture11 and the Flour Fortification Initiative.35

A sustainable fortification program consists of numerous components and actors. In

each of these steps, there are many details to control and possible difficulties and
barriers to overcome. The components include the preliminary assessment of nutrient
deficiencies, the development of fortification standards and legislation, the acquisition
of equipment by industrials, communication strategies and social marketing activities,
food safety, quality assurance and control systems, and the assessment of the impact of
the fortification program on health.
The size, capacity, and number of maize processing facilities as well as process control
should be considered in selecting the threshold for maize product fortification
intervention. Attempts to fortify maize products at a small local level may not be cost-
effective and risk having a negative impact on fortification program perception if not
properly executed.37 It is beyond the goal of this article to make the maize processing
scale cut-off for a fortification program, as well as to discuss bioavailability of nutrients
remaining after different processes of corn milling.

Conclusion
Maize is a significant food source for much of the world's population and represents a
vehicle for vitamin and mineral deficiency intervention. There are several industrial
processes that generate a wide variety of maize products to fulfill consumers' habits and
preferences. Many products of the industrial dry maize–milling processes may also be
produced locally on a small scale as well as in the home. The materials, processes, and
equipment are readily available, but it is important to consider that the number of
nutrients removed or altered through home or small-scale industry processing may vary
widely. Proper assessment of population needs and understanding of industrial
capability, products, and losses are needed to determine the viability of maize product
fortification.

Acknowledgments
Financial support was provided by the Department of Nutrition for Health and
Development, Evidence and Programme Guidance Unit, World Health Organization
(Geneva, Switzerland). This manuscript was presented at the World Health Organization
consultation “Technical Considerations for Maize Flour and Corn Meal Fortification in
Public Health” in collaboration with the Sackler Institute for Nutrition Science at the
New York Academy of Sciences and the Flour Fortification Initiative (FFI) that
convened on 8 and 9 April 2013 at the New York Academy of Sciences in New York,
USA. This article is being published individually, but will be consolidated with other
manuscripts as a special issue of Annals of the New York Academy of Sciences, the
coordinators of which were Drs. Maria Nieves Garcia-Casal, Mireille McLean, Helena
Pachón, and Juan Pablo Peña-Rosas. The special issue is the responsibility of the
editorial staff of Annals of the New York Academy of Sciences, who delegated to the
coordinators preliminary supervision of both technical conformity to the publishing
requirements of Annals of the New York Academy of Sciences and general oversight of
the scientific merit of each article. The workshop was supported by the Sackler Institute
for Nutrition Science at the New York Academy of Sciences and the Flour Fortification
Initiative (FFI), United States of America. The authors alone are responsible for the
views expressed in this article; they do not necessarily represent the views, decisions, or
policies of the institutions with which they are affiliated or the decisions, policies, or
views of the World Health Organization. The opinions expressed in this publication are
those of the authors and are not attributable to the sponsors, publisher, or editorial staff
of Annals of the New York Academy of Sciences.

Conflicts of interest
The authors declare no conflicts of interest.

References
1. Brown W. Darrah L. Natl. Corn Handbook. Iowa State University. Iowa: Cooperative Extension
Service; 1985. Origin, adaptation, and types of corn; p. NCH-10.

2. Vollbrecht E. Sigmon B. Amazing grass: developmental genetics of maize

domestication. Bioch. Soc. Trans. 2005;33:1502–1506. [PubMed]

3. Zea Production Maps and Statistics. Maize Top 10 Production. 2013. Accessed April
1,http://www.gramene.org/species/zea/maize_maps_and_stats.html.

4. World Agricultural Production Foreign Agricultural Service Circular WAP 03–13 March 2013.
Accessed March 28, 2013. http://www.fas.usda.gov-WAP03-13 March 2013.

5. Secchi S, Grassman P, Jha M, et al. Potential water quality changes due to corn expansion in
the upper Mississippi river basin. Ecol. Appl. 2011;21:1068–1084. [PubMed]

6. Wallington T, Anderson J, Mueller S, et al. Corn ethanol production, food exports, ans
indirect land use change. Environ. Sci. Technol. 2012;46:6379–6384. [PubMed]

7. International Starch Institute. Technical memorandum on corn starch.Denmark: International

Starch Institute A/S, Agro Food Park 13, DK-8200 Aarhus N; www.starch.dk. Consulted July
2013.

8. Corn Refiners Association. Corn Starch. Washington, D.C: Corn Refiners Association;
2013. www.corn.org Consulted.

9. Eckhoff S. Correct terminology usage in corn processing. Cereal Foods World. 2010;55:144–
1478.

10. Doebley J. The genetics of maize evolution. Ann. Rev. Gen. 2004;38:37–59. [PubMed]
11. U.S. Department of Agriculture, Agricultural Research Service. USDA National Nutrient
Database for Standard Reference, Release 25. Nutrient Data Laboratory Home Page. 2012.
Accessed April 6, 2013,http://www.ars.usda.gov/ba/bhnrc/ndl.

12. USDA. United States Department of Agriculture. Grain Inspection, Packers & Stockyards
Administration. United States standards for corn. 2013. Accessed April 1,
2013.http://www.gipsa.usda.gov/fgis/standards/810corn.pdf.

13. Rooney L. Serna-Saldavar S. In: Food Uses of Whole Corn and Dry-Milled Fractions in Corn:
Chemistry and Technology. Ramstad PE, editor; Watson SA, editor. American Association of
Cereal Chemists; 1987.

14. Brubacher T. Dry corn milling: an introduction. Technical Bulletins, International Association
of Operative Millers. 2002:7857–7860.

15. Caballero-Briones F, Iribarren A, Pena J, et al. Recent advances on the understanding of the
nixtamalization process. Superficies y Vacío. 2000;10:20–24.

16. Ocheme O, Omobolanle O. Gladys M. Effect of lime soaking and cooking (nixtamalization)
on the proximate, functional and some anti-nutritional properties of millet
flour. AUJT. 2010;14:131–138.

17. Afoakwa E, Sefa-Dedeh S, Simposon A, et al. Influence of spontaneous fermentation on

some quality characteristicas of maize-based cowpea-fortified nixtamalized
foods. AJFAND. 2007;7:1–18.

18. Baltenspreger W. Technical Bulletins. Leawood, Kansas: International Association of

Operative Millers; 1996. Maize milling; pp. 6742–6748.

19. COVENIN Comision Venezolana de Normas Industriales. Covenin; 1996. Norma Venezolana.
Harina de Maiz Precocida; pp. 2135–1996.

20. FAO. Maize in human nutrition. FAO Food and Nutrition Series No. 25. 1992. ISBN 92-5-
103013-8.

21. Martinez-Torres C, Racca E, Rivero F, et al. Iron fortification of pre-cooked maize

flour. Interciencia. 1991;16:254–260.

22. Rivero F, Racca E, Martinez-Torres C, et al. Fortification of precooked maize flour with
coarse defatted maize germ. Arch. Lat. Nutr. 1994;44:129–132. [PubMed]

23. Gould M. Swartz D. Process for Preparing Precooked Corn Flour. United States Patent Office;
1965. 3,212,904.

24. Osungbaro T. Physical and nutritive properties of fermented cereal foods. Afr. J. Food
Sci. 2009;3:23–27.

25. Amoa A. Muller H. Studies on kenkey with particular reference to calcium and phytic
acid. Cereal Chem. 1979;53:365–375.
26. Sustain. Shared United States Technology to Aid in the Improvement of Nutrition.
Guidelines for iron fortification of cereal food staples. The Micronutrient Initiative.
Recommendations from the September 2000 Monterrey Workshop.2001.

27. Andang'o P, Osendarp S, Ayah R, et al. Efficacy of iron-fortified whole maize flour on iron
status of school children in Kenya: a randomised controlled trial. Lancet. 2007;369:1799–
806. [PubMed]

28. Stiver T. Technical Bulletins. Leawood, Kansas: International Association of Operative

Millers; 1955. American corn milling systems for de-germed products; pp. 2168–2180.

29. Wingfield J. Dictionary of Milling Terms and Equipment. Leawood, Kansas: International
Association of Operative Millers; 1989.

30. U.S. Code of Federal Regulations. Title 21:Food and Drugs. Part 137 Cereal Flours and
Related Products

31. Codex Alimentarius. CODEX standard for whole maize (corn) meal (CODEX STAN 154–1985)
Accessed July 2013.http://www.codexalimentarius.org.

32. Codex Alimentarius. CODEX standard for degermed maize (corn) meal and maize (corn)
grits (CODEX STAN 155–1985) Accessed July 2013.http://www.codexalimentarius.org/

33. WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on Wheat and Maize Flour
Fortification. Meeting Report: Interim Consensus Statement.Geneva: World Health
Organization; 2009. Accessed July 2013.http://www.who.int/nutrition/publications/micro-
nutrients/wheat_maize_fort.pdf. [PubMed]

34. Dary O. Staple food fortification: a multifactorial decision. Nutr Rev. 2002;60:S34–
S41. [PubMed]

35. FFI. Flour Fortification Initiative. Flour millers tool kit, flour fortification initiative. 2013.
Accessed March 13, 2013.http://www.ffinetwork.org/implement/toolkit.html.

36. Instituto Nacional de Nutrición. Instituto Nacional de Nutrición, División de Investigaciones

en Alimentos. Venezuela: Caracas; 1999. Tabla de composición de Alimentos para uso práctico.
Serie Cuadernos azules Num 54; pp. 18–19.

37. Dary O, Freire W. Kim S. Iron compounds for food fortification: guidelines for Latin America
and the Caribbean 2002. Nutr. Rev. 2002;60:S50–S61. [PubMed]

. Enzyme Res. 2010; 2010: 918761.

Published online 2010 Sep 30. doi: 10.4061/2010/918761

XVIII. Uses of Laccases in the Food Industry
Johann F. Osma, 1 José L. Toca-Herrera, 2 and Susana Rodríguez-Couto 3, 4 , *

Author information ► Article notes ► Copyright and License information ► Disclaimer

Abstract

1. Introduction
Laccases (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) are particularly abundant
in white-rot fungi, which are the only organisms able to degrade the whole wood
components [1]. Fungal laccases are secreted, glycosylated proteins with two disulphide
bonds and four copper atoms distributed in one mononuclear termed T1 (where the
reducing substrate place is) and one trinuclear cluster T2/T3 (where oxygen binds and is
reduced to water) [2]. Thus, electrons are transferred from substrate molecules through
the T1 copper to the trinuclear T2/T3 centre. After the transfer of four electrons, the
dioxygen in the trinuclear centre is reduced to two molecules of water [3, 4] (Figure 1).

Figure 1

Reactions on phenolic compounds catalysed by laccases (extracted from [10]).

From a mechanistic point of view, the reactions catalysed by laccases can be represented
by one of the schemes shown in Figure 2. The simplest case (Figure 2(a)) is the one in
which the substrate molecules are oxidised to the corresponding radicals by direct
interaction with the copper cluster. Frequently, however, the substrates of interest cannot
be oxidised directly by laccases, either because they are too large to penetrate into the
enzyme active site or because they have a particularly high redox potential. By
mimicking nature, it is possible to overcome this limitation with the addition of so-
called “redox mediators”, which are low-weight molecular compounds that act as
intermediate substrates for laccases, whose oxidised radical forms are able to interact
with the bulky or high redox potential substrate targets (Figure 2(b)).
Figure 2

Schematic representation of laccase-catalysed redox cycles for substrates oxidation in the

absence (a) or in the presence (b) of redox mediators (extracted from [11], with kind
permission of Elsevier Ltd.)

In nature, the role of laccases is to degrade lignin in order to gain access to the other
carbohydrates in wood (cellulose and hemicellulose). Their low substrate specificity
allows laccases to degrade compounds with a structure similar to lignin, such as
polyaromatic hydrocarbons (PAHs), textile dyes, and other xenobiotic compounds [2].
This together with the simple requirements of laccase catalysis (presence of substrate
and O2) makes laccases both suitable and attractive for industrial applications.
Typical fungal laccases are extracellular proteins of approximately 60–70 kDa with
acidic isoelectric point around pH 4.0 [5]. They are generally glycosylated, with an
extent of glycosylation ranging between 10 and 25% and only in a few cases higher than
30% [6, 7]. This feature may contribute to the high stability of the enzyme [8].
A few laccases are at present in the market for textile, food and other industries (Table
1), and more candidates are being actively developed for future commercialisation [9].
A vast amount of industrial applications for laccases have been proposed which include
pulp and paper, textile, organic synthesis, environmental, food, pharmaceutical, and
nano-biotechnology. Being energy-saving and biodegradable, laccase-based biocatalysts
fit well with the development of highly efficient, sustainable, and eco-friendly
industries.

Table 1
Commercial preparations based on laccases for industrial processes.
Main application Brand name Manufacturer
Food Brewing Flavourstar Advanced Enzyme Technologies
industry Ltd. (India)
Colour enhancement in LACCASE Y120 Amano Enzyme USA Co. Ltd.
tea, etc.
Cork modification Suberase Novozymes (Denmark)
 Main application Brand name Manufacturer
Paper Pulp bleaching Lignozym-process Lignozym GmbH (Germany)
industry Paper pulp delignification Novozym 51003 Novozymes (Denmark)

Textile Denim bleaching Bleach Cut 3-S Season Chemicals (China)

Industry Denim finishing Cololacc BB Colotex Biotechnology Co. Ltd.
(Hong Kong)
Denim bleaching DeniLite Novozymes (Denmark)
Denim finishing Ecostone LC10 AB Enzymes GmbH (Germany)
Denim finishing IndiStar Genencor Inc. (Rochester, USA)
Denim finishing Novoprime Base 268 Novozymes (Denmark)
Denim bleaching and Primagreen Ecofade Genencor Inc. (Rochester, USA)
shading LT100
Denim bleaching ZyLite Zytex Pvt. Ltd. (India)

This paper reviews the potential application of laccases in the food industry. The
utilisation of whole laccase-producing microorganisms is not considered in the present
paper.

2. Application of Laccases in the Food Industry

Many laccase substrates, such as carbohydrates, unsaturated fatty acids, phenols, and
thiol-containing proteins, are important components of various foods and beverages.
Their modification by laccase may lead to new functionality, quality improvement, or
cost reduction [12, 13].

2.1. As Additives in Food and Beverage Processing

Laccases can be applied to certain processes that enhance or modify the colour
appearance of food or beverage.

2.1.1. Wine Stabilisation

Wine stabilisation is one of the main applications of laccase in the food industry as
alternative to physical-chemical adsorbents [12]. Musts and wines are complex mixtures
of different chemical compounds such as ethanol, organic acids (aroma), salts, and
phenolic compounds (colour and taste). Polyphenol removal must be selective to avoid
an undesirable alteration in the wine's organoleptic characteristics. Laccase presents
some important requirements when used for the treatment of polyphenol removal in
wines such as stability in acid medium and reversible inhibition with sulphite [14].
Additionally, a laccase has been commercialised for preparing cork stoppers for wine
bottles [15]. The enzyme oxidatively reduces the characteristic cork taint and/or
astringency, which is frequently imparted to aged bottled wine.

2.1.2. Beer Stabilisation

The storage life of beer depends on different factors such us haze formation, oxygen
content, and temperature. The former is produced by small quantities of naturally-
occurring proanthocyanidins, polyphenols that generate protein precipitation and,
therefore, the formation of haze [16]. This type of complex is commonly found as chill-
haze and appears during cooling processes but may re-dissolve at room temperature or
above [12]. Even products that are haze-free at the time of packing can develop this type
of complex during long-term storage. Thus, the formation of haze has been a persistent
problem in the brewing industry [17]. The use of laccases for the oxidation of
polyphenols as an alternative to the traditional treatment has been tested by different
authors [16, 18, 19]. However, laccases have also been used for the removal of oxygen
at the end of the beer production process. According to Mathiasen [16], laccase could be
added at the end of the process in order to remove the unwanted oxygen in the finished
beer, and thereby the storage life of beer is enhanced. Also, a commercialised laccase
preparation named “Flavourstar”, manufactured by Novozymes A/S, is marketed for
using in brewing beer to prevent the formation of off-flavour compounds (e.g., trans-2-
nonenal) by scavenging the oxygen, which otherwise would react with fatty acids,
amino acids, proteins and alcohol to form off-flavour precursors [20] (Table 1).

2.1.3. Fruit Juice Processing

Enzymatic preparations have been studied since the decade of the 1930s for juice
clarification [21]. The interaction between proteins and polyphenols results in the
formation of haze or sediment in clear fruit juices. Therefore, clear fruit juices are
typically stabilised to delay the onset of protein-polyphenol haze formation [22].
Several authors have proposed the use of laccase for the stabilisation of fruit juices [23–
30]; however, results are contradictory. On one hand, Sammartino et al. [24] compared
the treatment of apple juice with a conventional method (SO2 added as metabisulfite,
polyvinylpolypyrrolidone (PVPP), bentonite) with the use of free and immobilised
laccase. They showed that the enzymatically treated juice was less stable than the one
conventionally treated. Also, Giovanelli and Ravasini [25] and Gökmen et al. [31]
showed by stability tests of ultrafiltrated samples that laccase treatment increased the
susceptibility of browning during storage. On the other hand, Cantarelli [30] used a
mutant laccase from Polyporus versicolor to treat black grape juice. He showed a
removal of 50% of total polyphenols and higher stabilisation than the physical-chemical
treatment.
The use of laccase in conjunction with a filtration process has shown better results.
Thus, Ritter et al. [27] and Maier et al. [29] obtained a stable and clear apple juice by
applying laccase in conjunction with cross-flow-filtration (ultrafiltration) in a
continuous process without the addition of finishing agents. Cantarelli and Giovanelli
[28] reported that the use of laccase followed by ‘‘active” filtration or ultrafiltration, by
the addition of ascorbic acid and sulphites, improved colour and flavour stability in
comparison to conventional treatments. Also, Stutz [26] used laccase and ultrafiltration
to produce clear and stable juice concentrates with a light colour.
Artik et al. [32] studied the effect of laccase application on clarity stability of sour
cherry juice. They found that high clarity was obtained by adding laccase in case of
heating to 50°C for 6 h and filtering through 20 kDa membrane after 1 h of oxidation.
Also, the phenolic content decreased by around 70%.
More recently, Neifar et al. [23] used a combined laccase-ultrafiltration process for
controlling the haze formation and browning of the pomegranate juice. The optimised
treatment with laccase (laccase concentration 5 U/mL; incubation time 300 min;
incubation temperature 20°C) followed by ultrafiltration led to a clear and stable
pomegranate juice.
2.1.4. Baking
Laccases are currently of interest in baking due to their ability to cross-link
biopolymers. The use of laccase in baking is reported to result in an increased strength,
stability, and reduced stickiness and thereby improved machinability of the dough; in
addition, an increased volume and an improved crumb structure and softness of the
baked product were observed [33, 34].
Selinheimo et al. [35] showed that a laccase from the white-rot fungus Trametes
hirsuta increased the maximum resistance of dough and decreased the dough
extensibility in both flour and gluten doughs. It was concluded that the effect of laccase
was mainly due to the cross-linking of the esterified ferulic acid (FA) on the
arabinoxylan (AX) fraction of dough resulting in a strong AX network. Gluten dough
treated with laccase also showed some hardening suggesting that laccase can also act to
some extent on the gluten protein matrix. The hardening effect of laccase was, however,
clearly weaker in gluten dough. Thus, the AX fraction in flour dough is the predominant
substrate for laccase, and its activity caused the hardening effect. Interestingly, laccase-
treated flour dough softened as a result of prolonged incubation: the extent of softening
increasing as a function of laccase dosage. It is proposed that softening phenomenon is
due to radical catalysed breakdown of the cross-linked AX network.
Renzetti et al. [36] showed that a commercial laccase preparation significantly improved
the bread-making performances of oat flour and the textural quality of oat bread by
increasing specific volume and lowering crumb hardness and chewiness. The improved
bread-making performances could be related to the increased softness, deformability
and elasticity of oat batters with laccase supplementation.

2.1.5. Improving of Food Sensory Parameters

The physico-chemical deterioration of food products is a major problem related to the
evolution of storing and distribution systems and influences the consumer's perception
of the product quality. Thus, different uses of laccase have promoted odour control, taste
enhancement, or reduction of undesired products in several food products.
Takemori et al. [37] used crude laccase from Coriolus versicolor to improve the flavour
and taste of cacao nib and its products. Bitterness and other unpleasant tastes were
removed by the laccase treatment, and the chocolate manufactured from the cacao mass
tasted better than the control.
Another type of food products that may use laccase to improve sensory parameters is
oil. Oil products may be deoxygenated by adding an effective amount of laccase [38].
Oils, especially vegetable oils (e.g., soybean oil), are present in many food items such
us dressings, salads, mayonnaise, and other sauces. Soybean oil contains a large amount
of linoleic and linolenic acids that can react with dissolved oxygen in the product
producing undesirable volatile compounds. Therefore, the flavour quality of some oils
may be improved by eliminating the oxygen present in the oils. Other food products
(e.g., juices, soups, concentrates, puree, pastes, and sauces) can also be deoxygenated by
the mean of laccase [39].
Bouwens et al. [40, 41] reported that the colour of tea-based products could be enhanced
when treated with laccase from a Pleurotus species. In the same way, chopped olives in
an olive-water mixture were treated with laccase from Trametes villosa. In this case, the
bitterness was considerably reduced while the colour turned darker compared to the
controls (Novo Nordisk A/S, 1995).
Tsuchiya et al. [42] used a recombinant laccase from Myceliophthora thermophilum and
chlorogenic acid to control the malodour of cysteine. They showed that enzymatically
treated cysteine presented a very weak odour while the nontreated cysteine presented a
strong characteristic H2S odour. HPLC analysis showed the reduction of more than 50%
of cysteine.

2.1.6. Sugar Beet Pectin Gelation

The sugar beet pectin is a functional food ingredient that can form thermo-irreversible
gels. These types of gels are very interesting for the food industry as can be heated
while maintaining the gel structure.
Norsker et al. [43] analysed the gelling effect of two laccases and a peroxidase in food
products. They found that laccases were more efficient as gelling agents in luncheon
meat and milk than peroxidase. In addition, in many countries it is prohibited to add
hydrogen peroxide to food products making it impossible to use peroxidases as gelling
agents. Hence, it is more realistic to add laccase to food products.
Kuuva et al. [44] reported that by using laccases as cross-linking agents together with
calcium, the ratio of covalent and electrostatic cross-links of sugar beet pectin gels can
be varied and it can be possible to tailor different types of gel structures.
Littoz and McClements [45] showed that laccase could be used to covalently cross-link
beet pectin molecules adsorbed to the surfaces of protein-coated lipid droplets at pH 4.5,
thus suggesting that emulsions with improved functional performance could be prepared
using a biomimetic approach that utilised enzymes (laccases) to cross-link adsorbed
biopolymers.

2.2. Determination of Certain Compounds in Beverages

The use of laccases for improving the sensing parameters of food products is not limited
to treatment processes but also to diagnosis systems. In this regard, different
amperometric biosensors based on laccases have been developed to measure
polyphenols in different food products (e.g., wine, beer, and tea). Thus, Ghindilis et al.
[46] showed the practical validity of a biosensor based on immobilised laccase in
analysing tannin in tea of different brands.
Montereali et al. [47] reported the detection of polyphenols present in musts and wines
from Imola (Italy) through an amperometric biosensor based on the utilisation of
tyrosinase and laccase from Trametes versicolor. Both enzymes were immobilised on
graphite screen-printed electrodes modified with ferrocene. Biosensors exhibited a good
sampling behaviour compared to that obtained from spectrophotometric analysis;
however, the presence of SO2clearly inhibited the enzymatic activity, and, thus, the
measurements on musts and wines recently bottled were seriously affected.
Di Fusco et al. [48] reported the development of an amperometric biosensor based on
laccases from T. versicolor and T. hirsuta for the determination of polyphenol index in
wines. Enzymes were immobilised on carbon nanotubes screen-printed electrodes using
polyazetidine prepolymer (PAP). They showed that biosensor performance depended on
the laccase source. Thus, values obtained by using T. hirsuta laccase were close to those
determined by Folin-Ciocalteu method whereas polyphenol index measured with T.
versicolorlaccase was discordant to that found with the reference assay.
Prasetyo et al. [49] studied the use of tetramethoxy azobismethylene quinone
(TMAMQ) for measuring the antioxidant activity of a wide range of structurally diverse
molecules present in food and humans. TMAMQ was generated by the oxidation of
syringaldazine with laccases and used to detect the antioxidant activity present in
different food products.
Ibarra-Escutia et al. [50] developed and optimised an amperometric biosensor based on
laccase from T. versicolor for monitoring the phenolic compounds content in tea
infusions. The biosensor developed showed an excellent stability and exhibited good
performance in terms of response time, sensitivity, operational stability, and
manufacturing process simplicity and can be used for accurate determination of the
phenolic content without any pretreatment of the sample.

2.3. Bioremediation of Food Industry Wastewater

The presence of phenols in agroindustrial effluents has attracted interest for the
application of laccase-based processes in wastewater treatment and bioremediation. The
presence of phenolic compounds in drinking and irrigation water or in cultivated land
represents a significant health and/or environmental hazard. With government policies
on pollution control becoming more and more stringent, industries have been forced to
look for more effective treatment technologies for their wastewater.
Some fraction of beer factory wastewater represents an important environmental
concern due to its high content in polyphenols and dark brown colour.
Distillery wastewater is generated during ethanol production from fermentation of
sugarcane molasses (vinasses). It produces a serious ecological impact due to its high
content in soluble organic matter and its intense dark brown colour. In fact, vinasses
represent a major environmental problem for the ethanol production industry and they
are considered as the most aggressive by-product generated by sugar-cane factories.
Most of the organic matter present in the vinasses can be diminished by conventional
anaerobic-aerobic digestion, but the colour is hardly removed by these treatments [51]
making this effluent a potential water pollutant blocking out light from rivers and
streams thereby preventing oxygenation by photosynthesis and provoking their
eutrophication.
Strong and Burgess [52] studied the fungal (Trametes pubescens) and enzymatic
(laccase from T. pubescens) remediation of different distillery wastewater and found
that the fungal culture displayed much better properties than laccase alone in removing
both the total phenolic compounds and colour.
Olive mill wastewater (OMW) is a characteristic by-product of olive oil production and
a major environmental problem in the Mediterranean area. Thus, 30 million m3 of OMW
is produced in the Mediterranean area [53] which generate 2.5 litres of waste per litre of
oil produced [54]. OMW contains large concentrations of phenol compounds (up to 10 
g/L) [54, 55], which are highly toxic [52, 56]. Also, it has high chemical and
biochemical oxygen demands (COD and BOD, resp.) [57].
OMW is characterised by a colour variable from dark red to black depending on the age
and type of olive processed [58], low pH value (~5), high salt content and high organic
load with elevated concentrations of aromatic compounds [59], fatty acids, pectins,
sugar, tannins and phenolic compounds, in particular polyphenols [58]. The presence of
a large number of compounds, many with polluting, phytotoxic, and antimicrobial
properties [60], renders OMW a waste with high harmful effects towards humans and
environment and makes its disposal one of the main environmental concerns in all
producing countries.
Martirani et al. [61] reported that the treatment of an OMW effluent collected at an olive
oil factory in Abruzzo (Italy) with a purified laccase from Pleurotus
ostreatus significantly decreased its phenolic content (up to 90%) but no reduction of its
toxicity was observed when tested on Bacillus cereus.
Gianfreda et al. [62] showed that laccase from Cerrena unicolor was able to oxidise
different phenolic substances usually present in OMW with oxidation percentages
ranging from 60 to 100% after 24 h of laccase incubation.
D'Annibale et al. [63] used a laccase from the white-rot fungus Lentinula
edodes immobilised on chitosan to treat OMV from an olive oil mill located in Viterbo
(Italy). They found that the treatment of the OMW with immobilised laccase led to a
partial decolouration as well as to significant abatements in its content in polyphenols,
and orthodiphenols combined with a decreased toxicity of the effluent. They also
showed that an oxirane-immobilised laccase from L. edodes efficiently removed the
OMW phenolics [64].
Casa et al. [65] investigated the potential of a laccase from L. edodes in removing
OMW phytotoxicity. For this, they performed germinability experiments on durum
wheat (Triticum durum) in the presence of different dilutions of raw or laccase-treated
OMW. The treatment with laccase resulted in a 65% and an 86% reduction in total
phenols and orthodiphenols, respectively, due to their polymerisation as revealed by
size-exclusion chromatography. In addition, germinability of durum wheat seeds was
increased by 57% at a 1 : 8 dilution and by 94% at a 1 : 2 dilution, as compared to the
same dilutions using untreated OMW.
Attanasio et al. [66] studied the application of a non-isothermal bioreactor with laccases
from T. versicolor immobilised on a nylon membrane to detoxify OMW and showed
that the technology of non-isothermal bioreactors was very useful in the treatment of
OMW.
Jaouani et al. [67] studied the role of a purified laccase from Pycnoporus coccineus in
the degradation of aromatic compounds in OMW. They found that the treatment of
OMW with laccase showed similar results to those reported with the fungus indicating
that laccase plays an important role in the degradative process. Berrio et al. [68] studied
the treatment of OMW with a laccase from P. coccineus immobilised on Eupergit C
250L. Gel filtration profiles of the OMW treated with the immobilised enzyme (for 8 h
at room temperature) showed both degradation and polymerisation of the phenolic
compounds.
Quaratino et al. [69] reported that phenols were the main determinants for OMW
phytotoxicity and showed that the use of a commercial laccase preparation (DeniLite,
Novo Nordisk, Denmark) might be very promising for a safer agronomic use of the
wastewater.
Iamarino et al. [70] studied the capability of a laccase from Rhus vernicifera to degrade
and detoxify two OMW samples of different complexity and composition.
Pant and Adholeya [71] used a concentrated enzymatic extract from solid-state
fermentation (SSF) cultures of different fungi on wheat straw to decolourise a distillery
effluent. They reported a maximum decolouration of 37% in the undiluted distillery
effluent using the extract of Pleurotus florida EM1303 which was attributed to its high
laccase production.

3. Future Trends and Perspectives

This paper shows that laccase has a great potential application in several areas of food
industry. However, one of the limitations to the large-scale application of laccases is the
lack of capacity to produce large volumes of highly active enzyme at an affordable cost
(Table 2). The use of inexpensive sources for laccase production is being explored in
recent times. In this regard, an emerging field in management of industrial wastewater is
exploiting its nutritive potential for production of laccase enzymes. Besides solid
wastes, wastewater from the food processing industry is particularly promising for that.

Table 2
Some prices of commercially available laccases (extracted from [12], with kind
permission of Elsevier Ltd).
Quantity (Units)a Price
 From Agaricus bisporus 10.000 305.00
(US$)
100.000 1.560.00
(US$)
 From Coriolus versicolor 10.000 250.00
(US$)
100.000 1.290.00
(US$)

 From Pleurotus ostreatus 10.000 150.00

(concentrate) (US$)
10.000 (purified) 400.00
(US$)
100.000 650.00
(concentrate) (US$)
100.000 (purified) 1,600.00
(US$)

USBiological
(www.usbio.net/)
 From heterologus expression of Trametes versicolor  laccase 100 (purified) 169 (US$)
in Saccharomyces cerevisiae
 Quantity (Units)a Price
Sigma-Aldrich
  From Rhus vernicfiera 10,000 72.30 (US$)
  From Agaricus bisporus (≥1.5 U/mg) 1 g 30.50 (US$)
  From Coriolus versicolor (≥1 U/mg) 5 g 120.90
(US$)
  1 g 44.00 (US$)
10 g 358.20
(US$)

Jena BioScience
 From Trametes versicolor, Coprinus cinereus and Pycnoporus 100 U 15.00 (EUR)
cinnabarinus 1000 U 75.00 (EUR)
Open in a separate window
a
The methodology and expression of laccase activity (Units) are different among the companies.

Acknowledgment
This paper was financed by the Spanish Ministry of Science and Innovation (Project
CTM2008-02453/TECNO).

References
1. Kirk TK, Fenn P. Formation and Action of the Ligninolytic System in Basidiomycetes: Their
Biology and Ecology. Cambridge, UK: Cambridge University Press; 1982.

2. Mayer AM, Staples RC. Laccase: new functions for an old

enzyme. Phytochemistry. 2002;60(6):551–565. [PubMed]

3. Bertrand T, Jolivalt C, Briozzo P, et al. Crystal structure of a four-copper laccase complexed

with an arylamine: insights into substrate recognition and correlation with
kinetics. Biochemistry. 2002;41(23):7325–7333. [PubMed]

4. Bento I, Arménia Carrondo M, Lindley PF. Reduction of dioxygen by enzymes containing

copper. Journal of Biological Inorganic Chemistry. 2006;11(5):539–547. [PubMed]

5. Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiology

Reviews. 2006;30(2):215–242. [PubMed]

6. Shleev SV, Morozova OV, Nikitina OV, et al. Comparison of physico-chemical characteristics of
four laccases from different basidiomycetes. Biochimie. 2004;86(9-10):693–703. [PubMed]

7. De Souza CGM, Peralta RM. Purification and characterization of the main laccase produced
by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium. Journal of
Basic Microbiology. 2003;43(4):278–286.[PubMed]
8. Durán N, Rosa MA, D’Annibale A, Gianfreda L. Applications of laccases and tyrosinases
(phenoloxidases) immobilized on different supports: a review. Enzyme and Microbial
Technology. 2002;31(7):907–931.

9. Kunamneni A, Plou FJ, Ballesteros A, Alcalde M. Laccases and their applications: a patent
review. Recent Patents on Biotechnology. 2008;2(1):10–24. [PubMed]

10. Selinheimo E. Tyrosinase and laccase as novel crosslinking tools for food
biopolymers. Helsinki, Finland: Helsinki University of Technology; 2008. Ph.D. thesis.

11. Riva S. Laccases: blue enzymes for green chemistry. Trends in

Biotechnology. 2006;24(5):219–226. [PubMed]

12. Minussi RC, Pastore GM, Durán N. Potential applications of laccase in the food
industry. Trends in Food Science and Technology. 2002;13(6-7):205–216.

13. Kirk O, Borchert TV, Fuglsang CC. Industrial enzyme applications. Current Opinion in
Biotechnology. 2002;13(4):345–351. [PubMed]

14. Tanriöven D, Ekşi A. Phenolic compounds in pear juice from different cultivars. Food
Chemistry. 2005;93(1):89–93.

15. Conrad LS, Sponholz WR, Berker O. Treatment of cork with a phenol oxidizing enzyme. US
patent 6152966, 2000.

16. Mathiasen TE. Laccase and beer storage. PCT international application, WO 9521240 A2,
1995.

17. McMurrough I, Madigan D, Kelly R, O’Rourke T. Haze formation shelf-life prediction for lager
beer. Food Technology. 1999;53(1):58–63.

18. Giovanelli G. Enzymic treatment of malt polyphenols for beer stabilization. Industrie delle
Bevande. 1989;18:497–502.

19. Rossi M, Giovanelli G, Cantarelli C, Brenna O. Effects of laccase and other enzymes on
barley wort phenolics as a possible treatment to prevent haze in beer. Bulletin de Liaison-
Groupe Polyphenols. 1988;14:85–88.

20. FAO. Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae . Chemical
and Technical Assessment (Cta), 2004.

21. Minussi RC, Rossi M, Bologna L, Rotilio D, Pastore GM, Durán N. Phenols removal in musts:
strategy for wine stabilization by laccase. Journal of Molecular Catalysis B. 2007;45(3-4):102–
107.

22. Siebert KJ. Protein-polyphenol haze in beverages. Food Technology. 1999;53(1):54–57.

23. Neifar M, Elleouze-Ghorbel R, Kamoun A, et al. Effective clarification of pomegranate juice

using laccase treatment optimized by response surface methodology followed by
ultrafiltration. Journal of Food Process Engineering. In press.
24. Sammartino M, Piacquadio P, De Stefano G, Sciancalepore V. Apple juice stabilization by
conventional and innovative methods. Industrie delle Bevande. 1998;27:367–369.

25. Giovanelli G, Ravasini G. Apple juice stabilization by combined enzyme-membrane filtration

process. Lebensmittel-Wissenschaft und Technologie. 1993;26(1):1–7.

26. Stutz C. The use of enzymes in ultrafiltration. Fruit Processing. 1993;3:248–252.

27. Ritter G, Maier G, Schoepplein E, Dietrich H. The application of polyphenoloxidase in the

processing of apple juice. Bulletin de Liaison-Groupe Polyphenols. 1992;16:209–212.

28. Cantarelli C, Giovanelli G. Stabilization of pome and grape juice against phenolic
deterioration by enzymic treatments. Internationale Fruchtsaft-Union, Wissenschaftlich-
Technische Commission. 1990;21:35–57.

29. Maier G, Dietrich H, Wucherpfenning K. Winemaking without SO 2-with the aid of

enzymes? Weineirtschaft-Technik. 1990;126:18–22.

30. Cantarelli C. Trattamenti enzimatici sui costituenti fenolici dei mosti come prevenzione della
maderizzazione. Vini d’Italia. 1986;3:87–98.

31. Gökmen V, Borneman Z, Nijhuis HH. Improved ultrafiltration for color reduction and
stabilization of apple juice. Journal of Food Science. 1998;63(3):504–507.

32. Artik N, Karhan M, Aydar G. Effects of polyphenoloxidase (LACCASE) application on clarity

stability of sour cherry juice. Journal of Food Technology. 2004;2(4):237–243.

33. Labat E, Morel MH, Rouau X. Effects of laccase and ferulic acid on wheat flour
doughs. Cereal Chemistry. 2000;77(6):823–828.

34. Si JQ. Use of laccase in baking industry. International patent application PCT/DK94/00232,
1994.

35. Selinheimo E, Kruus K, Buchert J, Hopia A, Autio K. Effects of laccase, xylanase and their
combination on the rheological properties of wheat doughs. Journal of Cereal
Science. 2006;43(2):152–159.

36. Renzetti S, Courtin CM, Delcour JA, Arendt EK. Oxidative and proteolytic enzyme
preparations as promising improvers for oat bread formulations: rheological, biochemical and
microstructural background. Food Chemistry. 2010;119(4):1465–1473.

37. Takemori T, Ito Y, Ito M, Yoshama M. Flavor and taste improvement of cacao nib by
enzymatic treatment. Japan Kokai Tokkyo Koho JP 04126037 A2, 1992.

38. Petersen BR, Mathiasen TE, Peelen B, Andersen H. Use of laccase for deoxygenation of oil-
containing product such as salad dressing. PCT international application, WO 9635768 A1,
1996.

39. Petersen BR, Mathiasen TE. Deoxygenation of a food item using a laccase. PCT international
application, WO 9631133 A1, 1996.
40. Bouwens EM, Trivedi K, Van Vliet C, Winkel C. Method of enhancing color in a tea based
foodstuff. US 5879730 A, 1999.

41. Bouwens EM, Trivedi K, Van Vliet C, Winkel C. Method of enhancing color in a tea based
foodstuff. European patent application; EP 760213 A1, 1997.

42. Tsuchiya R, Petersen BR, Christensen S. Oxidoreductases for reduction of malodor. US

6074631 A, 2000.

43. Norsker M, Jensen M, Adler-Nissen J. Enzymatic gelation of sugar beet pectin in food
products. Food Hydrocolloids. 2000;14(3):237–243.

44. Kuuva T, Lantto R, Reinikainen T, Buchert J, Autio K. Rheological properties of laccase-

induced sugar beet pectin gels. Food Hydrocolloids. 2003;17(5):679–684.

45. Littoz F, McClements DJ. Bio-mimetic approach to improving emulsion stability: cross-
linking adsorbed beet pectin layers using laccase. Food Hydrocolloids. 2008;22(7):1203–1211.

46. Ghindilis AL, Gavrilova VP, Yaropolov AI. Laccase-based biosensor for determination of
polyphenols: determination of catechols in tea. Biosensors and Bioelectronics. 1992;7(2):127–
131. [PubMed]

47. Montereali MR, Della Seta L, Vastarella W, Pilloton R. A disposable Laccase-Tyrosinase

based biosensor for amperometric detection of phenolic compounds in must and wine. Journal
of Molecular Catalysis B. 2010;64(3-4):189–194.

48. Di Fusco M, Tortolini C, Deriu D, Mazzei F. Laccase-based biosensor for the determination of
polyphenol index in wine. Talanta. 2010;81(1-2):235–240. [PubMed]

49. Prasetyo EN, Kudanga T, Steiner W, Murkovic M, Nyanhongo GS, Guebitz GM. Laccase-
generated tetramethoxy azobismethylene quinone (TMAMQ) as a tool for antioxidant activity
measurement. Food Chemistry. 2010;118(2):437–444.

50. Ibarra-Escutia P, Juarez Gómez J, Calas-Blanchard C, Marty JL, Ramírez-Silva MT.

Amperometric biosensor based on a high resolution photopolymer deposited onto a screen-
printed electrode for phenolic compounds monitoring in tea infusions. Talanta. 2010;81(4-
5):1636–1642. [PubMed]

51. Valdez E, Obaya MC. Estudio del tratamiento anaerobico de los residuales de la industria
alcoholera. Revista ICIDCA. 1985;19(1):7–10.

52. Strong PJ, Burgess JE. Treatment methods for wine-related and distillery wastewaters: a
review. Bioremediation Journal. 2008;12(2):70–87.

53. Fiestas Ros de Ursinos JA. Differentes utilisations des margines. In: Proceedings of the
Seminaire International sur la Valorisation des Sous Produits de l’Olivier; 1981; Monastir,
Tunisia. Organisation des Nations Unies pour l’Alimentation et l’Agriculture; pp. 93–110.
54. Borja R, Martin A, Maestro R, Alba J, Fiestas JA. Enhancement of the anaerobic digestion of
olive mill wastewater by the removal of phenolic inhibitors. Process
Biochemistry. 1992;27(4):231–237.

55. Klibanov AM, Tu TM, Scott KP. Peroxidase-catalyzed removal of phenols from coal-
conversion waste waters. Science. 1983;221(4607):259–261.[PubMed]

56. Fedorak PM, Hrudey SE. The effects of phenol and some alkyl phenolics on batch anaerobic
methanogenesis. Water Research. 1984;18(3):361–367.

57. Hamdi M. Future prospects and constraints of olive mill wastewaters use and treatment: a
review. Bioprocess Engineering. 1993;8(5-6):209–214.

58. Jaouani A, Sayadi S, Vanthournhout M, Penninckx MJ. Potent fungi for decolourisation of
olive oil mill wastewaters. Enzyme and Microbial Technology. 2003;33(6):802–809.

59. D’Annibale A, Casa R, Pieruccetti F, Ricci M, Marabottini R. Lentinula edodes removes

phenols from olive-mill wastewater: impact on durum wheat (Triticum durum Desf.)
germinability. Chemosphere. 2004;54(7):887–894.[PubMed]

60. Paredes C, Cegarra J, Roig A, Sánchez-Monedero MA, Bernal MP. Characterization of olive
mill wastewater (alpechin) and its sludge for agricultural purposes. Bioresource
Technology. 1999;67(2):111–115.

61. Martirani L, Giardina P, Marzullo L, Sannia G. Reduction of phenol content and toxicity in
olive oil mill waste waters with the ligninolytic fungus Pleurotus ostreatus . Water
Research. 1996;30(8):1914–1918.

62. Gianfreda L, Sannino F, Filazzola MT, Leonowicz A. Catalytic behavior and detoxifying ability
of a laccase from the fungal strain Cerrena unicolor . Journal of Molecular Catalysis B. 1998;4(1-
2):13–23.

63. D’Annibale A, Stazi SR, Vinciguerra V, Di Mattia E, Giovannozzi Sermanni G. Characterization

of immobilized laccase from Lentinula edodesand its use in olive-mill wastewater
treatment. Process Biochemistry. 1999;34(6-7):697–706.

64. D’Annibale A, Stazi SR, Vinciguerra V, Giovannozzi Sermanni G. Oxirane-

immobilized Lentinula edodes laccase: stability and phenolics removal efficiency in olive mill
wastewater. Journal of Biotechnology. 2000;77(2-3):265–273. [PubMed]

65. Casa R, D’Annibale A, Pieruccetti F, Stazi SR, Sermanni GG, Lo Cascio B. Reduction of the
phenolic components in olive-mill wastewater by an enzymatic treatment and its impact on
durum wheat (Triticum durum Desf.) germinability. Chemosphere. 2003;50(8):959–
966. [PubMed]

66. Attanasio A, Diano N, Grano V, et al. Nonisothermal bioreactors in the treatment of

vegetation waters from olive oil: laccase versus syringic acid as bioremediation
model. Biotechnology Progress. 2005;21(3):806–815.[PubMed]
67. Jaouani A, Guillén F, Penninckx MJ, Martínez AT, Martínez MJ. Role of Pycnoporus
coccineus laccase in the degradation of aromatic compounds in olive oil mill
wastewater. Enzyme and Microbial Technology. 2005;36(4):478–486.

68. Berrio J, Plou FJ, Ballesteros A, Martínez ÁT, Martínez MJ. Immobilization of Pycnoporus
coccineus laccase on Eupergit C: stabilization and treatment of olive oil mill
wastewaters. Biocatalysis and Biotransformation. 2007;25(2–4):130–134.

69. Quaratino D, D’Annibale A, Federici F, Cereti CF, Rossini F, Fenice M. Enzyme and fungal
treatments and a combination thereof reduce olive mill wastewater phytotoxicity on Zea
mays L. seeds. Chemosphere. 2007;66(9):1627–1633. [PubMed]

70. Iamarino G, Rao MA, Gianfreda L. Dephenolization and detoxification of olive-mill

wastewater (OMW) by purified biotic and abiotic oxidative
catalysts. Chemosphere. 2009;74(2):216–223. [PubMed]

71. Pant D, Adholeya A. Concentration of fungal ligninolytic enzymes by ultrafiltration and their
use in distillery effluent decolorization. World Journal of Microbiology and
Biotechnology. 2009;25(10):1793–1800.