Trends in Food Science & Technology 20 (2009) 344e354
Review
Probiotic cheese:
health benefits,
technological and
stability aspects
Adriano Gomes da Cruza,1,
Flávia Carolina Alonso Buritib,
Cı́nthia Hoch Batista de Souzab,
José Assis Fonseca Fariaa,1 and
Susana Marta Isay Saadb,*
a
Departamento de Tecnologia de Alimentos,
Faculdade de Engenharia de Alimentos, Universidade
Estadual de Campinas, Caixa Postal 6121, 13083-862,
Campinas, Brazil
b states that the minimum viable quantity of probiotic culture
Departamento de Tecnologia Bioquı́mico-
Farmacêutica, Faculdade de Ciências Farmacêuticas,
Universidade de S~ao Paulo, Av. Prof. Lineu Prestes,
580, 05508-000, S~ ao Paulo, Brazil (Tel.: D55 11 3091
2378; fax: D55 11 3815 6386; e-mail: susaad@usp.br)
This review presents the technological hurdles involved in
the development and stability of probiotic cheeses. Firstly,
the potential of cheese as a food probiotic carrier is dis-
cussed, emphasizing its advantages, when compared to fer-
mented milks and yogurts. Fresh cheese and ripened
cheeses are also discussed, and questions concerning the
viability of probiotic cultures in these foods are considered.
Overall, the manufacture of probiotic cheese should have
minimum changes when compared to traditional products.
In addition, the physico-chemical parameters that influence
the quality of these products must be measured, aiming at
process optimization.
* Corresponding author.
1
Tel.: þ55 19 35214016.
0924-2244/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2009.05.001
Introduction
Probiotics are defined as ‘live microorganisms that,
when administered in adequate amounts, confer a health
benefit on the host’ (Food and Agriculture Organization
of United Nations; World Health Organization e FAO/
WHO, 2001). Probiotic food is defined as a processed pro-
duct which contains viable probiotic microorganisms in
a suitable matrix and in sufficient concentration (Saxelin,
Korpela, & Mäyrä-Mäkinen, 2003). This means that their
viability and metabolite activity must be maintained in all
the steps of the food processing operations, from their
manufacture up to their ingestion by the consumer, and
also that they must be able to survive in the gastrointestinal
tract (Sanz, 2007).
In the scientific literature, populations of 106e107 CFU/
g in the final product are established as therapeutic quanti-
ties of probiotic cultures in processed foods (Talwalkar,
Miller, Kailasapathy, & Nguyen, 2004), reaching 108e109
CFU, provided by a daily consumption of 100 g or
100 mL of food, hence benefiting human health (Jaya-
manne & Adams, 2006). In Brazil, the present legislation
should be between 108 and 109 CFU per daily portion of
product and that the probiotic population should be stated
on the product label (ANVISA, 2008).
Countless benefits to health are provided by the inges-
tion of foods containing probiotic cultures, some with sci-
entific proof, and other ones still needing more human
studies. Firstly, these bacteria beneficially affect human
health by improving the balance of the intestinal microbiota
and improving mucosal defenses against pathogens (Boy-
lston, Vinderola, Ghoddusi, & Reinheimer, 2004). Some
of the main beneficial effects on health related to probiotic
consumption are: antimicrobial activity, prevention and
treatment of diarrhoeas, relief in the symptoms resulting
from lactose intolerance, antimutagenic and anticarcino-
genic activities, stimulation of the immunological system,
improvement of urogenital health, relief of constipation,
and optimization of vaccine effects (Bomba, Nemcová,
Mudronová, & Guba, 2002; Nagpal et al., 2007; Saad,
2006; Shah, 2007). Probiotic bacteria have been recom-
mended for the treatment of atopic dermatitis, necrotizing
enterocolitis, pseudomembranous colitis, chronic liver
disease, allergic disease and food allergy (Boyle & Tang,
2006; Candy, Heath, Lewis, & Thomas, 2008). Also, probi-
otic bacteria can be used to treat irritable bowel syndrome
 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
(Santosa, Farnworth, & Jones, 2006) and to reduce serum
cholesterol (Shah, 2007). It is important to mention that
the effects of health improvement are dependent on the
strain present in the formulation of the product, and that
there is not a probiotic strain which simultaneously
provides all the benefits previously reported (Shah, 2007).
Hence, the number of available products and the con-
sumer’s familiarity with the ‘probiotic’ concept has been
increasing, and, as a consequence, the research into these
products has also been increasing. More than 600 food
products launched by the dairy industry in 2006 used the
term probiotic (Sveje, 2007). The best known examples
of probiotic foods are fermented milks and yogurts, which
are generally consumed within days or weeks of manufac-
ture (Nagpal et al., 2007).
Cheese is one of the most versatile food products avail-
able nowadays, appealing to many palates and suitable for
all age groups. Its versatility offers opportunities for many
marketing strategies (Wilkinson, Meehan, Stanton, &
Cowan, 2001), as a probiotic food carrier. However, the de-
velopment of probiotic cheeses implies obligatory knowl-
edge of all their processing steps, as well as on their level
of influence e positive or negative e on the survival of
these microorganisms throughout their shelf life. In this
context, this review covers the main hurdle technologies in-
volved in the manufacture and stability of probiotic cheese.
Cheese
Regulatory and market aspects
Cheese is the generic name for a group of fermented
milk-based food products produced throughout the world
in a great diversity of flavours, textures, and forms (Fox,
Guinee, Cogan, & McSweeney, 2000). An essential part
of the cheese-making process is the conversion of a liquid,
milk, into a solid material, the curd, that contains casein
and fat of the milk, but has expelled the main part of the
water, and usually, the whey proteins. This is achieved by
the addition of rennet to coagulate the casein gel. The
cheese curd thus forms the basis of the cheese, which is
later modified by processes such as pressing, salting and
ripening (Lomholt & Qvist, 1999).
Consumption of cheese has grown in the past decade in
most countries, unrelated to the socio-economic level of the
country. A production of 17,778 million tonnes (t) in 2004
was reported, which corresponds to a growth of almost
3272 t in the last decade. Cheese from cow’s milk repre-
sents 95e96% of the total cheese production (International
Dairy Federation e IDF, 2005). In 2006, an increase of
550,000 t (or 2.3%) in relation to 2005 was reported. Euro-
pean countries, including Ireland, Belgium, Germany, the
Netherlands, and France registered the main production in-
crease, with a similar tendency observed in the USA (Inter-
national Dairy Federation e IDF, 2007). However, in
Brazil, a low cheese consumption per capita was reported
e almost 0.107 kg in 2005 e and although the total produc-
tion has decreased in five years e 17.96 thousands t in 2001
345
against 16.85 thousands t in 2005, a growth of 31.6% in
sales has been observed. The market share of cream cheese
increased from 7 to 17% in this period and that of Minas
fresh cheese (traditional Brazilian soft fresh cheese)
decreased from 84% to 75% (Bourroul, 2006).
Overview of cheesemaking
The manufacture of cheese is a form of milk preserva-
tion, as milk is highly perishable. All cheeses, whether
rennet or acid set, can be classified as soft, semi-soft
(semi-hard), hard, or very hard, depending on their mois-
ture content. Although this classification is arbitrary and
practical, it helps to systematically group together cheeses
that are alike in certain basic features or characteristics
(e.g., moisture content), as moisture determines the body,
consistency or compactness of cheese (Farke, 2004). The
production of cheese requires the coagulation of milk, in
most cases through the action of chymosin on the k-casein
steric stabilizing layer of the casein micelle. Here, cheese
manufacture is essentially a process of the dehydration of
milk in combination with other preservative effects, such
as culturing, acidification, salting, packaging, and refriger-
ation (Everett & Auty, 2008). The rennet-induced milk
coagulum is cut and homogenized to expel moisture in
a process called syneresis (Grundelius, Lodaite, Östergren,
Paulsson, & Dejmek, 2000). Curd is later drained, salted,
and packaged into fresh cheese. Many cheeses need an ad-
ditional time to achieve their own sensory features, partic-
ularly flavour and aroma. To achieve this purpose, they are
maintained in a special room, with controlled environmen-
tal conditions for a determined time: this process is called
ripening and the final product is called ripened cheese
(Everett & Auty, 2008).
Cheese as a probiotic food carrier
As in the case of any probiotic food, in order to exert
their health benefits on the consumer’s body, probiotic bac-
teria incorporated in cheese must be able to grow and/or
proliferate in the human intestine and therefore should be
able to survive during the passage through the gastrointes-
tinal tract (GIT), which involves exposure to hydrochloric
acid in the stomach and bile in the small intestine (Stanton
et al., 2003).
In fact, cheese provides a valuable alternative to fer-
mented milks and yogurts as a food vehicle for probiotic
delivery, due to certain potential advantages. It creates
a buffer against the high acidic environment in the gastro-
intestinal tract, and thus creates a more favourable environ-
ment for probiotic survival throughout the gastric transit,
due to higher pH. Moreover, the dense matrix and relatively
high fat content of cheese may offer additional protection to
probiotic bacteria in the stomach (Bergamini, Hynes, Qui-
beroni, Suárez, & Zalazar, 2005; Ross, Fitzgerald, Collins,
& Stanton, 2002). This finding was confirmed by Sharp,
McMahon, and Broadbent (2008). The authors used Lacto-
bacillus casei 334e, an erythromycin-resistant derivative of
346 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
the strain ATCC 334, that was recently confirmed as mem-
ber of taxon Lactobacillus paracasei (Judicial Commission
of the International Committee on Systematics of Bacteria,
2008), as model to compare its viability in yogurt and in
low-fat Cheddar cheese during refrigerated storage, as
well as the influence of each food product submitted to gas-
tric conditions at pH 2. The authors concluded that the
viable cells presented good stability in both products
(107 CFU/g), with negligible population changes observed
during 3 months of storage for the latter and 3 weeks for
the former. In terms of exposure to acidic conditions, Ched-
dar cheese presented a superior performance, as the viable
count decreased to 104 CFU/g after 120 min of exposure,
whereas for yogurt the viable count was reduced to less
than 10 CFU/g within 30 min of exposure.
Whey Portuguese cheese (requeij~ ao) has been reported
as a food vector for environmental conditions prevailing
in the gastrointestinal tract. Nevertheless, the results were
strain-dependent, and loss of viability of different strains
(Bifidobacterium animalis strains e BLC-1, Bb-12 and
Bo, Lactobacillus acidophilus strains e LAC-1 and Ki, L.
paracasei LCS-1, and Lactobacillus brevis LMG6906)
was observed after passage through an acidic hydrochloric
(pH 2.5e3.0) and pepsin (1000 units/mL) solution at 37  C
and a bile salts 0.3% (w/v) solution (Madureira et al.,
2005). Argentinean fresco cheese showed the same behav-
iour. Different combinations of Bifidobacterium sp. (one
strain), L. acidophilus (two strains) and L. casei (two
strains) were tested as probiotic adjuncts for the preparation
of this cheese. In order to verify its suitability as a probiotic
vector under acidic conditions, environmental conditions
found in the stomach were tested (pH ¼ 3 and pH ¼ 2).
All cultures showed excellent viability, presenting suitable
populations for up to 3 h. For a pH 3 solution, L. casei C1
was the most resistant culture and presented a 2 log cycles
decrease. When a pH 2 solution was used, Bifidobacterium
bifidum B4 and L. casei C1 were, respectively, the most
sensitive and the most resistant microorganisms (Vinderola,
Prosello, Ghiberto, & Reinheimer, 2000).
The presence of the prebiotics inulin and oligofructose
was described to promote increased growth rates of bifido-
bacteria and lactobacilli, besides increased lactate and short
chain fatty acids production in petit-suisse cheese supple-
mented with these microorganisms and submitted to batch
culture fermentation with human faecal slurry (Cardarelli,
Saad, Gibson, & Vulevic, 2007).
Health benefits provided by the ingestion of probiotic
cheese
In addition to what has already been mentioned, probi-
otic food products must demonstrate efficacy in controlled
validated clinical trials to prove that the probiotic character-
istics were not altered or lost following subjection to the
technological processes involved in probiotic food manu-
facture (Stanton et al., 2003). Therefore, the development
of cheese supplemented with probiotic bacteria involves
previous clinical designs in vivo and verification of whether
these microorganisms maintain their viability in the product
at the time of administration. If multiple strains are clini-
cally tested, there must be a thorough understanding of
the properties of single strains, as well as tests for antago-
nistic and symbiotic effects between strains, in order to de-
termine whether a multiple strain product is effective
(Aimutis, 2001).
Clinical benefits both for animals and for humans have
been reported for the ingestion of probiotic cheeses. Ched-
dar cheese was reported as being as effective as yogurt to
deliver viable cells of Enterococcus faecium Fargo 688 dur-
ing its ripening period. Populations of 2.0  106 CFU/g
were observed in pigs faeces, while the same microorgan-
ism delivered through yogurt presented populations of
5.2  105 CFU/g of faeces, suggesting a positive effect
for Cheddar cheese as a delivery system for probiotic bac-
teria (Gardiner et al., 1999).
Probiotic fresh cheese (Argentinean fresh cheese) con-
taining L. acidophilus A9, B. bifidum A12 and L. paracasei
A13 demonstrated immunomodulating capacity in mice,
providing increased phagocytic activity in the small intes-
tine of peritoneal macrophages, after 2, 5 and 7 days of
its ingestion. Additionally, a significant increase in the
number of IgAþ producing cells in the large intestine after
5 days of administration was reported. Interaction of probi-
otic bacteria as bacterial antigens in the small (Peyer’s
patches) and large (lymphoid nodules) intestine were also
observed (Medici, Vinderola, & Perdigón, 2004). Phago-
cytic cells play a central role in protection against microbial
infections. In addition, macrophages are involved in antigen
presentation, tissue repair and also play an important role in
the regulation of immune responses. IgA antibodies pre-
dominate in mucosal surfaces and prevent adherence of
pathogens to the gut mucosa, being responsible for the
humoral immunity (Gill, 1998).
Ahola et al. (2002) studied the effect of probiotic Edam
cheese containing Lactobacillus rhamnosus LC705 and L.
rhamnosus GG ATCC53103 (LGG) on the risk of dental
caries. During the study, no significant difference was
found for Streptococcus mutans populations between the
control group and the group who ingested probiotic cheese.
However, a tendency for the probiotic intervention to re-
duce the high levels of this microorganism was observed:
S. mutans populations did not increase in any of the sub-
jects from the probiotic group, while they increased in three
subjects (8%) from the control group. During the 3 week
post-intervention, S. mutans populations decreased in
21% of the subjects who ingested probiotic cheese and
only in 8% of the control group. Additionally, there was
also a decrease in the number of subjects who presented
high yeast populations in the probiotic group. The probiotic
intervention also seemed to increase salivary lactobacilli
populations, originated from the cheese-making process.
The authors concluded that eating probiotic cheese could
reduce the risk of dental caries in general, although no
 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
significant statistical difference was observed in salivary
microbial populations.
Hatakka et al. (2007) investigated the effect of consump-
tion of probiotic cheese on the oral candidosis for elderly
people. Cheese containing a mixture of probiotic cultures
(L. rhamnosus GG, L. rhamnosus LC705, Propionibacte-
rium freundenreichii spp., and Shermani JS) was ingested
by 92 elderly people. Microbiological analyses of oral
yeasts were performed in saliva samples during 4 times
throughout the study. After 8 and 16 weeks, the prevalence
of high populations (>104 CFU/ml) of this microbial group
was, respectively, reduced to 25.0% and 20.7%. Moreover,
the probiotic treatment reduced the risk of high yeast pop-
ulations by 75.0%. On the other hand, for the control
cheese group (people who did not ingest probiotic cheese),
oral yeasts increased 31.0%, and prevalence of high popu-
lations was 34.0%. A positive tendency was also related to
the average unstimulated saliva flow. The authors hypothe-
sized that probiotic bacteria have somehow affected the
composition of saliva, the concentrations of mucins and sal-
ivary immunoglobulins. They suggested probiotic cheese
could be used as a prophylactic approach for decreasing
the risk of hyposalivation and the feeling of dry mouth,
and that it could be considered beneficial to oral health in
general.
Technological hurdles and the stability of probiotic
cheeses
The major challenge associated with the application of
probiotic cultures in the development of functional foods
is their viability maintenance during processing. Probiotic
microorganisms should also be technologically suitable
for the incorporation into food products so that they retain
both viability and efficacy in the food product (on a com-
mercial scale) and throughout consumption. Probiotics
should also be able to survive industrial applications (e.g.,
standard dairy processing or pharmaceutical manufacturing
protocols) and be able to grow/survive at high levels in the
products during their shelf life. Furthermore, from a food
processing perspective, it is desirable that such strains are
suitable for large-scale industrial production and should
withstand the processing conditions mentioned above,
such as freeze-drying or spray drying. Besides, probiotic
strains for incorporation into human foods should be con-
sidered as GRAS ( generally recognised as safe) and not
lead to undesirable changes from a sensorial point of
view as to their flavour, aroma, texture and other important
attributes (Stanton et al., 2003).
Probiotic bacteria used in food products, such as Lacto-
bacillus spp. and Bifidobacterium spp., present micro-
aerophile or anaerobic metabolism. Hence, the presence
of oxygen may represent a threat for their survival. In gen-
eral, Bifidobacterium spp. is more sensitive to oxygen than
L. acidophilus, due to its strict anaerobe nature, although
this sensibility varies according to the lineage of microor-
ganism (Talwalkar & Kailasapathy, 2004). Additional
347
features include degree of acidity, ability to grow well in
milk-based media and to rapidly acidify milk, thus reducing
the fermentation time and, consequently, the risk of con-
tamination during preparation of the inoculums (Gomes
& Malcata, 1999).
In order to use probiotic bacteria in the manufacture of
cheese products, the process may sometimes have to be
modified and adapted to the requirements of the stains em-
ployed. Where this is not possible, other probiotic strains
may be applied or new products may have to be developed
(Heller, Bockelmann, Schrezenmeir, & deVrese, 2003).
Overall, probiotic strains should be technologically com-
patible with the food manufacturing process of interest.
With regard to the development of probiotic cheese, this
means that such strains should be cultivable to high cell
density for inoculation into the cheese vat or be able to pro-
liferate during the manufacturing and/or ripening process
(Ross et al., 2002). In general, a probiotic cheese should
have the same performance as a conventional cheese: the
incorporation of probiotic bacteria should not imply
a loss of quality of the product. In this context, the level
of proteolysis and lipolysis must be the same or even better
than cheese which does not have this functional appeal.
Due to its manufacturing process, fresh cheese appears
to be ideally suited to serve as a carrier for probiotic bacte-
ria as it is an unripened cheese, during storage it is submit-
ted to refrigeration temperatures, and its shelf life is rather
limited (Heller et al., 2003). A number of scientific papers
reporting the development of fresh cheeses containing
recognized and potentially probiotic cultures have been
published, which described suitable viable counts and a pos-
itive influence on the texture and sensorial properties of
these cheeses. Having in mind that portions of around
100 g of cheese are usually consumed daily, populations
of about 106 CFU/g lead to an ingestion of 108 CFU/daily
portion.
Blanchette, Roy, Bélanger, and Gauthier (1996) devel-
oped a Cottage cheese containing Bifidobacterium infantis.
Cheeses presented populations as high as 1  106 CFU/g
during 10 days of refrigerated storage. Suárez-Solı́s, Car-
doso, Núñez de Villavicencio, Fernández, and Fragoso
(2002) manufactured fresh cheese supplemented with B. bi-
fidum and L. casei and obtained cheese with very good sen-
sory quality with viable populations of 1  107 CFU/g
during 15 days of storage.
Buriti, Rocha, Assis, and Saad (2005) tested the supple-
mentation of Minas fresh cheese with L. paracasei subsp.
paracasei LBC 82. The cheeses studied by the authors pre-
sented populations above 1  106 CFU/g during cheese
production, and population increased during the whole stor-
age, reaching 108 CFU/g after 21 days. In the same way,
Buriti, Rocha, and Saad (2005) studied the addition of L.
acidophilus La-5 solely and in co-culture with a mesophilic
type O lactic culture during Minas fresh cheese production
and observed levels above 1  106 CFU/g for probiotic
bacteria during the whole storage period. In both studies,
348 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
L. paracasei and L. acidophilus did not alter the texture and
sensorial characteristics of Minas fresh cheeses.
Masuda, Yamanari, and Itoh (2005) evaluated the viabil-
ity of three strains isolated from human intestine with high
tolerance for acid and bile, L. acidophilus JCN11047, L.
acidophilus 1132T and Lactobacillus gasseri JCM11657,
in fresh cheeses stored at 7  C during 4 weeks. Negligible
reductions in the viability were observed for all strains
throughout the shelf life of cheeses, which remained above
8  107 CFU/g up to the end of storage.
Buriti, Cardarelli, Filisetti, and Saad (2007) tested the
addition of L. paracasei subsp. paracasei LBC 82 in co-
culture with Streptococcus thermophilus to potentially
probiotic and synbiotic fresh cream cheeses (without and
with inulin, respectively). Viable counts of L. paracasei
remained above 1  107 CFU/g during the entire storage
period, 21 days, for both cheeses. In another study, Buriti,
Okazaki, Alegro, and Saad (2007) observed the viability
of L. acidophilus La-5 and B. animalis Bb-12 added to
Minas fresh cheese. Both probiotic cultures were present
in high levels throughout storage, above 1  106 CFU/g,
and resulted in cheeses with texture comparable to the tra-
ditional ones and with favourable sensorial features.
In a study carried out with potentially synbiotic petit-
suisse manufactured with inulin, oligofructose and/or
honey, Cardarelli, Buriti, Castro, and Saad (2008) obtained
L. acidophilus and B. animalis subsp. lactis populations
above 1  106 CFU/g and 1  107 CFU/g, respectively,
during the refrigerated storage at 4  1  C.
Souza, Buriti, Behrens, and Saad (2008) and Souza and
Saad (2009) studied the manufacture of Minas fresh cheese
supplemented with the probiotic strain of L. acidophilus
La-5 solely or in co-culture with S. thermophilus. Cheeses
manufactured with La-5 solely presented populations above
1  106 CFU/g, reaching 1  107 CFU/g on the 14th day of
storage. Also, the addition of La-5 strain resulted in good
acceptance of Minas fresh cheeses, improving the sensory
Table 1. Technological hurdles in the probiotic cheese processing.
Step Problem
Addition of U Interactions of the probiotic and
probiotic inoculum starter may cause negative impact;
U Loss of viable probiotic cells
in the whey during draining.
Salting U Probiotic bacteria are sensitive
to high salt concentrations.
Packaging U Probiotic bacteria are sensitive
to oxygen.
Ripening U Survival of probiotic bacteria through
the cheese ripening period.
Storage conditions U Inadequate storage conditions affect
the probiotic survival.
performance of these products during storage (Souza
et al., 2008).
However, even though cheese is likely to be one of the
best carriers for probiotics, the addition of high numbers
of viable and metabolically active cells can affect product
quality, especially sensory properties (Grattepanche,
Miescher-Schwenninger, Meile, & Lacroix, 2008). An
example is the observation reported by Modzelewska-Kapi-
tula, Klebukowska, and Kornacki (2007), who studied the
addition of the potentially probiotic culture of Lactobacillus
plantarum 14 to a soft cheese. Although suitable popula-
tions for a probiotic food, between 106 and 107 CFU/g,
were observed in the study, cheeses containing this strain
presented slightly lower scores in the sensory analysis.
Table 1 shows the main technological hurdles concern-
ing the development of probiotic cheeses. There are five
identified hurdles which directly influence the maintenance
of the functional activities of probiotic bacteria in cheese:
addition of the probiotic inoculum, salting, packaging,
ripening and storage conditions.
Addition of the probiotic inoculum
There are two options for the addition of probiotic bac-
teria during cheese processing which can directly affect the
survival rate of these microorganisms: probiotic bacteria
can be added before the fermentation (together with the
starter culture), or after it. Following the former option im-
plies making preliminary tests to know the amount of pro-
biotic cells which are lost in the whey during its drainage.
The ideal rate of probiotic inoculum to be added must be
checked according to the process. If the second option is
chosen, immediate cooling must be performed (below
8  C, preferentially), as metabolic activities of starters
and probiotics are drastically reduced at these temperatures.
For Cottage cheese, for e.g., the addition of probiotics to-
gether with cream and salt appears to be a desirable alter-
native, once the number of probiotics added can be
Possible solutions
U Preliminary tests to choose the most suitable probiotic and starter
combination;
U Use of strains from the same supplier;
U Check different moments of addition of the probiotic inoculum (observing
the impact on the final cost of the product and probiotic survival).
U Microencapsulation;
U Suitable strain selection (information from the strain supplier).
U Choose suitable packaging system: film plastic with low permeability to
oxygen, vacuum packaging or active packaging;
U Cell incubation under sub-lethal conditions to develop salt resistance;
U Suitable strain selection (information with the strain supplier).
U Microencapsulation;
U Optimize ripening conditions through preliminary tests.
U Strict control of storage temperature.
 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
exactly controlled and the adverse effects of the high scald-
ing temperature are avoided. It is also important to consider
their physiological state in order to have an idea of their
survival throughout ripening and/or storage (Heller et al.,
2003). In terms of the growth curve, microbial cells
between the late exponential and the stationary phase are
the favourite option, and, sometimes, the preparation of
a previous substrate to inoculate the strain might be
beneficial.
Daigle, Roy, Bélanger, and Vuillemard (1999) used
a cream fermented by B. infantis for the manufacture of pro-
biotic cheese (Cheddar like-cheese). Viable counts of this
microorganism were above 3.0  106 CFU/g through 84
days of storage at 4  C and there was no metabolic activity
which had impact on the sensory properties. This seemed
to be an effective way to obtain probiotic dairy foods. Berga-
mini et al. (2005) tested the effect of two methods for the ad-
dition of probiotic bacteria and their survival in a semi-hard
cheese: a freeze-dried powder dispersed in milk and a sub-
strate containing milk and milk fat. The second trial did
not improve the survival of probiotic bacteria during the
ripening period, although it increased the probiotic popula-
tion in the initial inoculum by one log cycle. This can be
a more economical option and will have an impact on the
final cost of the probiotic product.
Several other techniques have been adapted to enhance
the viability of probiotic bacteria to harsh conditions, typi-
cal to many cultured dairy products, and may also be used
in the production of probiotic cheeses, including the selec-
tion of oxygen-tolerant, acid-tolerant and bile-resistant
strains, the addition of amino acids, peptides and other mi-
cronutrients to supplement the growth of the probiotic bac-
teria (Boylston et al., 2004).
One strategy for enhancing bacterial tolerance to stress
such as temperature, pH or bile salts is prior exposure to
sub-lethal levels of the given stress. Stress responses may
be used to enhance the survival of probiotic bacteria in
stressful conditions and to improve their technological
properties (Roy, 2005; Saarela et al., 2004). The bacterial
responses can be adaptive (when an acid treatment at pH
3e4 protects the bacteria against pH 2.5 or a treatment at
47  C protects against 55  C) or cross-protective (when
heat protects against low pH or bile) (Saarela et al.,
2004). The exposure of early stationary phase bacterial
cells to combinations of reduced temperature, reduced pH
and starvation could enhance the probiotic cold- and/or
acid-tolerance and increase the number of viable probiotics
ingested via refrigerated dairy products (Maus & Ingham,
2003), which could be useful in the production and storage
of fresh cheeses, for e.g.
Moreover, an alternative for protecting bifidobacteria to
oxygen stress is the use of selected strains of S. thermophilus
with high oxygen consumption as starter for the production
of cheeses (Boylston et al., 2004).
In order to choose and adopt the best option for the ad-
dition of probiotic culture, it is important to analyze all the
349
steps involved in the traditional cheese processing, espe-
cially the scalding temperature normally used by producers,
which can have detrimental effect on the survival during
processing. It is also important to consider the interactions
between probiotic bacteria and starters cultures, which may
act in a negative manner on the processing and stability of
the product.
Searching for the interaction between probiotic and
starter strains, Vinderola, Mocchiutti, and Reinheimer
(2002) observed that probiotic bacteria proved to be more
inhibitory towards lactic acid bacteria than vice versa, since
the latter did not exert any effect on the growth of the for-
mer, except for cell-free supernatants of some strains of
Lactobacillus delbrueckii subsp. bulgaricus that weakly in-
hibited the growth of certain L. acidophilus strains in well-
diffusion agar assays. In addition, Bifidobacterium sp. and
L. casei strains did not show any effect on the growth of
L. delbrueckii subsp. bulgaricus strains. Moreover, strains
of S. thermophilus and Lactococcus lactis showed variable
results, depending on the strain considered. To avoid the
competition between the strains, it is advisable to use
starters and probiotic strains from the same supplier.
In addition, this inhibitory effect of probiotic and poten-
tially probiotic strains can be helpful for the use as biopre-
servatives in the production of cheeses. These strains,
besides contributing together with the starter culture for
the production of organic acids, may also produce other an-
timicrobial compounds, including hydrogen peroxide, alco-
holic compounds, diacetyl, and bacteriocins. This
inhibitory activity creates a hostile environment for patho-
gens and spoilage organisms. Lactobacilli species, in par-
ticular those from the L. casei group, are frequently
responsible for this kind of antagonistic action. The biopre-
servative effect can be enhanced by the combination of pro-
biotic strains with other lactic acid bacteria (Buriti,
Cardarelli, & Saad, 2007).
Salting
Apart from Domiati cheese, all cheeses are salted after
rennet coagulation and curd formation. Three methods are
available: dry salting, surface dry salting and brine salting
or brine immersion (Guinee, 2004). Salt exerts an effect
on the improvement of sensorial attributes of cheeses, as
well as on the biochemical reactions during the storage of
the product. It is well established that the survival of micro-
bial strains is restricted by the level of salt (Yilmaztekin,
Özer, & Atasoy, 2004). Gobbetti, Corsetti, Smacchi, Zoc-
chetti, and de Angelis (1998) reported that the viability of
probiotic bacteria is drastically reduced in cheese when
salt concentration is above 4%. Therefore, cheeses which
naturally present high salt content should have their pro-
cessing optimized to incorporate the functional status in or-
der to be probiotic bacteria carriers.
Another option is to find ways to protect the probiotic
bacteria from the hostile environment. One alternative is
microencapsulation or cell incubation under sub-lethal
350 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
conditions. The former is a promising alternative used in
the processing of probiotic cheese, being responsible for
improving the survival of probiotic bacteria, without nega-
tive effects on texture, aroma and acceptance by the con-
sumers (Dinakar & Mistry, 1994; Özer, Kirmaci, Sxenel,
Atamer, & Hayaloğlu, 2009; Özer, Uzun, & Kirmaci,
2008; Yilmaztekin et al., 2004). Sometimes, however, the
technique cannot offer the adequate protection. Kailasapa-
thy and Masondole (2005) reported that the use of microen-
capsulation of probiotic bacteria (L. acidophilus DD910
and Bifidobacterium lactis DD920) in calcium-induced al-
ginate-starch capsules did not improve their viability in
a Feta cheese matrix during storage in brine solution; losses
of 2e3 log cycles were observed in free and immobilized
cells during storage. Possible reasons given by the authors
were the open texture of the cheese and the high salt con-
centration, 7.3e8.4%, causing death of the cells.
Packaging
The packaging system ought to be considered as an im-
portant stage of the processing of probiotic dairy foods and
should be taken into consideration in order to improve the
stability of probiotic bacteria in foods. In general, probiotic
dairy foods, like cheese, are packaged in plastics films
which have different levels of permeability to oxygen.
This becomes a problem, because of the strain-dependence,
as most members of this microbial group are sensitive to
oxygen, due to anaerobic metabolism (Robertson, 2006).
Several factors affect the survival of L. acidophilus and
Bifidobacterium spp. when they are added to a food matrix.
These include strains of probiotic cultures, pH, hydrogen
peroxide, storage atmosphere, concentration of metabolites
such as lactic acid and acetic acids, dissolved oxygen and
buffers such as whey proteins. Among these factors, expo-
sure to dissolved oxygen during manufacture and storage is
considered highly significant. Both L. acidophilus and Bifi-
dobacterium spp. are human gut-derived microorganisms
and, as mentioned previously, they are microaerophilic
and anaerobic, respectively. Unlike aerobic bacteria, which
completely reduce oxygen to water, oxygen-scavenging
system in these probiotic bacteria is either reduced or com-
pletely absent. Consequently, accumulation of toxic oxygen
metabolites e superoxide anion (O-2), hydroxyl radical
(OH-), and hydrogen peroxide (H2O2) e in the cell, occurs,
eventually leading to its death (Champagne & Gardner,
2005; Talwalkar & Kailasapathy, 2004; Vasiljevic &
Shah, 2008). High levels of intracellular H2O2 block fruc-
tose-6-phosphofructoketolase, a key enzyme in the sugar
metabolism of bifidobacteria (Shah, 1997). A correlation
between the levels of two enzymes e NAD-oxidase and
NAD-peroxidase e and oxygen susceptibility of bifidobac-
teria has been reported. The first enzyme gives rise to H2O2,
prompting the second one to scavenge this compound and
prevent cell death (Roy, 2005).
Therefore, plastic films with low permeability to oxygen
should be chosen to pack these functional products;
alternatively, the practice of adopting other alternatives,
such as the use of vacuum packaging should be followed.
Kasımoğlu, Göncüoğlu, and Akgün (2004) investigated
the effect of a packaging system using vacuum and brine
on Turkish white cheese ripening. The authors observed
that vacuum packaged cheese presented best performance
in the sensorial evaluation (good flavour and texture) be-
sides a high level of proteolysis. Indeed, packaging systems
ought to be taken into consideration when developing
new probiotic food products. A review on this topic
was published elsewhere (Cruz, Faria, & Van Dender,
2007).
Ripening
The ripening process of cheese is very complex and in-
volves microbiological and biochemical changes in the
curd, resulting in the flavour and texture characteristic of
a particular variety. The biochemical changes occurring
during ripening may be grouped into primary events that in-
clude the metabolism of residual lactose, lactate, and citrate
(often, erroneously, referred as ‘glycolysis’), besides lipol-
ysis and proteolysis (McSweeney, 2004).
The presence of ripening stages during cheese process-
ing is an additional problem for the stability of a probiotic
culture, as its survival through this period cannot be pre-
dicted with accuracy. Biochemical changes occurring inside
the cheese environment, as water activity decreases, some-
times together with a decrease in pH, create a hostile and
stressful environment for the adjunct cultures.
An additional problem is the proliferation of a population
of non-pathogenic adventitious bacteria, usually lactobacilli
and pediococci, which often become the dominant micro-
biota in cheese. They are also called non-starter lactic acid
bacteria (NSLAB) (Hayes et al., 2006). This microbial group
competes for nutrients and sometimes creates a problem for
the quantitative determination of probiotic viability. Incorpo-
ration of probiotic bacteria in cheese does not generally
affect primary proteolysis, performed by a coagulant agent
and, to a less extent, by plasmin, residual coagulant and
enzymes from the starter microbiota (Sousa, Ardö, &
McSweeney, 2001). However, probiotic bacteria enzymes
act in the secondary proteolysis, increasing the total free
aminoacid content, which contributes decisively to cheese
flavour (sweet, bitter or malty) and can be precursors for
the synthesis of other flavours or volatile aroma, resulting
in off-flavours (Ardö, 2006).
Lipolysis is not influenced by probiotic bacteria, as their
enzymes have a lower lipolytic activity, when compared to
starters and NSLAB (Grattepanche et al., 2008). Despite
this, several ripened probiotic cheeses have been devel-
oped, without or with minimum changes in the proteolytic
and lipolytic profile, exerting a positive effect on the overall
quality of the cheese (Fernández, Delgado, Boris, Rodrı́-
guez, & Barbés, 2005; Kalavrouzioti, Hatzikamari, Lito-
poulou-Tzanetaki, & Tzanetakis, 2005; Kourkoutas et al.,
2006; Ong, Henriksson, & Shah, 2006; Ong, Henriksson,
 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
& Shah, 2007a; Ong, Henriksson, & Shah, 2007b; Songi-
sepp et al., 2004; Stanton et al., 1998), besides the produc-
tion of bioactive peptides (Ong & Shah, 2008).
Storage conditions
Consumers obviously demand that the product they pur-
chase contain the probiotic cultures at the time of consump-
tion. Thus, companies provide the required viable
population at the time the product is marketed, but are
also concerned with the evolution of the population
throughout storage. Typically, the ‘‘best before’’ date is
given in order to provide a period guaranteeing the desired
population (Champagne & Gardner, 2005).
The supplementation of cheeses with probiotic and/or
starters cultures can help to decrease growth of contami-
nant, since these cultures are able to produce natural anti-
microbial substances in order to inhibit undesirable
microorganisms in foods. Moreover, NaCl is widely used
in the food industry as a preservative agent (Vinderola,
Costa, Regenhardt, & Reinheimer, 2002). However, high
temperature found in most of sales points has a negative
impact on the survival of probiotic populations, and causes
undesirable changes in cheeses e texture, colour and fla-
vour e leading to the product to be rejected by the con-
sumer. Strict monitoring of this parameter is very
important, as failure may endanger the functional status
of the product and lead to the growth of contaminant and
pathogenic microorganisms.
Sensorial aspects of probiotic cheese
Probiotic cultures do not tend to strongly modify the
sensorial properties of the products to which they are
added (Champagne & Gardner, 2005). The main concern
is ripened cheeses containing bifidobacteria, since they
produce a high amount of acetic acid, as well as lactic
acid in a molar ratio of 2:3 from lactose fermentation
via the fructose-6-phosphate shunt pathway. In small
amounts, acetic acid exerts a positive influence on the
aroma of probiotic cheeses. However, excessive concentra-
tions are undesirable, causing off-flavours (Grattepanche
et al., 2008). It is therefore, important to verify the senso-
rial performance of probiotic cheese, compared to a pro-
duct without probiotic microorganisms, in order to
obtain more precise conclusions on this attribute of the
products. If possible, the sensorial profile should be inves-
tigated using appropriated sensorial techniques like
Descriptive Quantitative Analysis. A review on this topic
was published (Drake, 2007), and these principles can
be applied to development of probiotic cheeses.
Other challenges related to development of probiotic
cheeses
Nowadays, the current consumers’ interest towards
products that contribute to decreased risks of chronic-
degenerative diseases encourages the development of probi-
otic cheese with reduced contents of fat or sodium.
351
However, the reduction of these components may affect
the sensorial features of the final product. The proper
combination of probiotic strains used as adjunct cultures
can influence favourably the flavour and texture, particu-
larly for low-fat cheeses.
Compared to yogurt, the problem for cheese e especially
semi-hard and hard cheese e acting as carrier for probiotics
results from the high fat and salt content and the relatively
low recommended daily intake. It follows that the concentra-
tion of probiotics in cheese should be about four to five times
higher than in yogurt. However, this does not apply to fresh
cheese, such as Cottage cheese, which can easily be adjusted
to low fat and salt contents, and for which recommended
daily intake is rather high. Low-fat fresh cheese may thus
serve as a food with a high potential to be applied as a carrier
for probiotics (Heller et al., 2003).
Ryhänen, Pihlanto-Leppälä, and Pahkala (2001) studied
the behavior of L. acidophilus and Bifidobacterium sp.
added to Festivo cheese with reduced-fat content. The fat
content was reduced to approximately 11% and no adverse
effect on sensory quality was observed. Also, L. acidophi-
lus and Bifidobacterium sp. remained viable for up to seven
months having counts of over 106 CFU/g. Thage et al.
(2005) analyzed flavor profiles of reduced-fat and semi-
hard cheeses manufactured with L. paracasei subsp. para-
casei (strains CHCC 2115, 4256, and 5583). Additionally,
the authors observed that reduction in fat content did not af-
fect Lactobacillus strains populations, reaching
1  108 CFU/g during the whole storage period.
Because reduced and low-fat cheeses contain more
moisture and are generally produced using lower cooking
temperatures, lactic acid bacteria are able to grow to high
populations in these cheeses (Drake & Swanson, 1995).
So, low-fat probiotic cheese manufacture with suitable pro-
biotic populations is possible, so as to obtain cheeses with
added health benefits.
There are a limited number of studies available concern-
ing the probiotic response to salt concentration. Kasımoğlu
et al. (2004) studied the viability of L. acidophilus added to
Turkish white cheeses manufactured with and without the
addition of salt, and observed populations above
1  107 CFU/g and above 1  106 CFU/g, respectively.
It is, therefore, convenient to establish the effect of salt
on the growth and survival of probiotic bacteria, in order to
predict their potential utilization in a cheese containing dif-
ferent salt concentrations.
Perspectives
The use of cheese as probiotic food carrier presents po-
tential advantages and it is a valuable alternative for the
dairy industry. However, development on an industrial scale
requires knowledge of all technological steps involved in
the traditional process, and adaptations of the existing pro-
tocol are usually necessary.
As there are lots of cheese varieties available on the
market, is it important to conduct preliminary tests to verify
352 A. Gomes da Cruz et al. / Trends in Food Science & Technology 20 (2009) 344e354
the behaviour and the performance of the culture in the
cheese environment for traditional and low-fat processes.
Such tests must be completed with laboratorial analysis
testing parameters which are decisive for the marketing
of the product, like organic acid profile, and typical aroma
compounds of the product. Additionally, the use of senso-
rial techniques can help to determine the important attri-
butes which may influence consumers.
Acknowledgements
The authors would like to thank to Fundaç~ao de Amparo
à Pesquisa do Estado de S~ao Paulo (FAPESP), Coordenaç~ao
de Aperfeiçoamento de Pessoal de Nı́vel Superior
(CAPES), and Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico (CNPq) for financial support and
scholarships.
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