Trends in Food Science & Technology 20 (2009) 344e354

Review

* Corresponding author.1 Tel.: þ55 19 35214016.

0924-2244/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2009.05.001

Probiotic cheese:

health benefits,

technological and

stability aspects

Adriano Gomes da Cruza,1,

Flavia Carolina Alonso Buritib,

Cınthia Hoch Batista de Souzab,Jose Assis Fonseca Fariaa,1 and

Susana Marta Isay Saadb,*aDepartamento de Tecnologia de Alimentos,

Faculdade de Engenharia de Alimentos, Universidade

Estadual de Campinas, Caixa Postal 6121, 13083-862,

Campinas, BrazilbDepartamento de Tecnologia Bioquımico-

Farmaceutica, Faculdade de Ciencias Farmaceuticas,

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.

IntroductionProbiotics are defined as ‘live microorganisms that,

when administered in adequate amounts, confer a healthbenefit on the host’ (Food and Agriculture Organizationof United Nations; World Health Organization e FAO/WHO, 2001). Probiotic food is defined as a processed pro-duct which contains viable probiotic microorganisms ina suitable matrix and in sufficient concentration (Saxelin,Korpela, & Mayra-Makinen, 2003). This means that theirviability and metabolite activity must be maintained in allthe steps of the food processing operations, from theirmanufacture up to their ingestion by the consumer, andalso that they must be able to survive in the gastrointestinaltract (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 or100 mL of food, hence benefiting human health (Jaya-manne & Adams, 2006). In Brazil, the present legislationstates that the minimum viable quantity of probiotic cultureshould be between 108 and 109 CFU per daily portion ofproduct and that the probiotic population should be statedon 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 humanstudies. Firstly, these bacteria beneficially affect humanhealth by improving the balance of the intestinal microbiotaand improving mucosal defenses against pathogens (Boy-lston, Vinderola, Ghoddusi, & Reinheimer, 2004). Someof the main beneficial effects on health related to probioticconsumption are: antimicrobial activity, prevention andtreatment of diarrhoeas, relief in the symptoms resultingfrom 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, Nemcova,Mudronova, & Guba, 2002; Nagpal et al., 2007; Saad,2006; Shah, 2007). Probiotic bacteria have been recom-mended for the treatment of atopic dermatitis, necrotizingenterocolitis, pseudomembranous colitis, chronic liverdisease, 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

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(Santosa, Farnworth, & Jones, 2006) and to reduce serumcholesterol (Shah, 2007). It is important to mention thatthe effects of health improvement are dependent on thestrain present in the formulation of the product, and thatthere is not a probiotic strain which simultaneouslyprovides all the benefits previously reported (Shah, 2007).

Hence, the number of available products and the con-sumer’s familiarity with the ‘probiotic’ concept has beenincreasing, and, as a consequence, the research into theseproducts has also been increasing. More than 600 foodproducts launched by the dairy industry in 2006 used theterm probiotic (Sveje, 2007). The best known examplesof probiotic foods are fermented milks and yogurts, whichare 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 forall age groups. Its versatility offers opportunities for manymarketing 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 levelof influence e positive or negative e on the survival ofthese microorganisms throughout their shelf life. In thiscontext, this review covers the main hurdle technologies in-volved in the manufacture and stability of probiotic cheese.

CheeseRegulatory and market aspects

Cheese is the generic name for a group of fermentedmilk-based food products produced throughout the worldin a great diversity of flavours, textures, and forms (Fox,Guinee, Cogan, & McSweeney, 2000). An essential partof the cheese-making process is the conversion of a liquid,milk, into a solid material, the curd, that contains caseinand fat of the milk, but has expelled the main part of thewater, and usually, the whey proteins. This is achieved bythe addition of rennet to coagulate the casein gel. Thecheese curd thus forms the basis of the cheese, which islater modified by processes such as pressing, salting andripening (Lomholt & Qvist, 1999).

Consumption of cheese has grown in the past decade inmost countries, unrelated to the socio-economic level of thecountry. A production of 17,778 million tonnes (t) in 2004was reported, which corresponds to a growth of almost3272 t in the last decade. Cheese from cow’s milk repre-sents 95e96% of the total cheese production (InternationalDairy Federation e IDF, 2005). In 2006, an increase of550,000 t (or 2.3%) in relation to 2005 was reported. Euro-pean countries, including Ireland, Belgium, Germany, theNetherlands, and France registered the main production in-crease, with a similar tendency observed in the USA (Inter-national Dairy Federation e IDF, 2007). However, inBrazil, a low cheese consumption per capita was reportede 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

against 16.85 thousands t in 2005, a growth of 31.6% insales has been observed. The market share of cream cheeseincreased from 7 to 17% in this period and that of Minasfresh cheese (traditional Brazilian soft fresh cheese)decreased from 84% to 75% (Bourroul, 2006).

Overview of cheesemakingThe manufacture of cheese is a form of milk preserva-

tion, as milk is highly perishable. All cheeses, whetherrennet 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 andpractical, it helps to systematically group together cheesesthat are alike in certain basic features or characteristics(e.g., moisture content), as moisture determines the body,consistency or compactness of cheese (Farke, 2004). Theproduction of cheese requires the coagulation of milk, inmost cases through the action of chymosin on the k-caseinsteric stabilizing layer of the casein micelle. Here, cheesemanufacture is essentially a process of the dehydration ofmilk in combination with other preservative effects, suchas culturing, acidification, salting, packaging, and refriger-ation (Everett & Auty, 2008). The rennet-induced milkcoagulum is cut and homogenized to expel moisture ina process called syneresis (Grundelius, Lodaite, Ostergren,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 aremaintained in a special room, with controlled environmen-tal conditions for a determined time: this process is calledripening and the final product is called ripened cheese(Everett & Auty, 2008).

Cheese as a probiotic food carrierAs 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/orproliferate in the human intestine and therefore should beable to survive during the passage through the gastrointes-tinal tract (GIT), which involves exposure to hydrochloricacid in the stomach and bile in the small intestine (Stantonet al., 2003).

In fact, cheese provides a valuable alternative to fer-mented milks and yogurts as a food vehicle for probioticdelivery, due to certain potential advantages. It createsa 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 relativelyhigh fat content of cheese may offer additional protection toprobiotic bacteria in the stomach (Bergamini, Hynes, Qui-beroni, Suarez, & 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

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the strain ATCC 334, that was recently confirmed as mem-ber of taxon Lactobacillus paracasei (Judicial Commissionof the International Committee on Systematics of Bacteria,2008), as model to compare its viability in yogurt and inlow-fat Cheddar cheese during refrigerated storage, aswell as the influence of each food product submitted to gas-tric conditions at pH 2. The authors concluded that theviable cells presented good stability in both products(107 CFU/g), with negligible population changes observedduring 3 months of storage for the latter and 3 weeks forthe former. In terms of exposure to acidic conditions, Ched-dar cheese presented a superior performance, as the viablecount decreased to 104 CFU/g after 120 min of exposure,whereas for yogurt the viable count was reduced to lessthan 10 CFU/g within 30 min of exposure.

Whey Portuguese cheese (requeij~ao) has been reportedas a food vector for environmental conditions prevailingin the gastrointestinal tract. Nevertheless, the results werestrain-dependent, and loss of viability of different strains(Bifidobacterium animalis strains e BLC-1, Bb-12 andBo, 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 �Cand 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. (onestrain), L. acidophilus (two strains) and L. casei (twostrains) were tested as probiotic adjuncts for the preparationof this cheese. In order to verify its suitability as a probioticvector under acidic conditions, environmental conditionsfound in the stomach were tested (pH ¼ 3 and pH ¼ 2).All cultures showed excellent viability, presenting suitablepopulations for up to 3 h. For a pH 3 solution, L. casei C1was the most resistant culture and presented a 2 log cyclesdecrease. When a pH 2 solution was used, Bifidobacteriumbifidum B4 and L. casei C1 were, respectively, the mostsensitive and the most resistant microorganisms (Vinderola,Prosello, Ghiberto, & Reinheimer, 2000).

The presence of the prebiotics inulin and oligofructosewas described to promote increased growth rates of bifido-bacteria and lactobacilli, besides increased lactate and shortchain fatty acids production in petit-suisse cheese supple-mented with these microorganisms and submitted to batchculture fermentation with human faecal slurry (Cardarelli,Saad, Gibson, & Vulevic, 2007).

Health benefits provided by the ingestion of probioticcheese

In addition to what has already been mentioned, probi-otic food products must demonstrate efficacy in controlledvalidated clinical trials to prove that the probiotic character-istics were not altered or lost following subjection to thetechnological processes involved in probiotic food manu-facture (Stanton et al., 2003). Therefore, the developmentof cheese supplemented with probiotic bacteria involves

previous clinical designs in vivo and verification of whetherthese microorganisms maintain their viability in the productat the time of administration. If multiple strains are clini-cally tested, there must be a thorough understanding ofthe 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 havebeen reported for the ingestion of probiotic cheeses. Ched-dar cheese was reported as being as effective as yogurt todeliver viable cells of Enterococcus faecium Fargo 688 dur-ing its ripening period. Populations of 2.0� 106 CFU/gwere observed in pigs faeces, while the same microorgan-ism delivered through yogurt presented populations of5.2� 105 CFU/g of faeces, suggesting a positive effectfor 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. paracaseiA13 demonstrated immunomodulating capacity in mice,providing increased phagocytic activity in the small intes-tine of peritoneal macrophages, after 2, 5 and 7 days ofits ingestion. Additionally, a significant increase in thenumber of IgAþ producing cells in the large intestine after5 days of administration was reported. Interaction of probi-otic bacteria as bacterial antigens in the small (Peyer’spatches) and large (lymphoid nodules) intestine were alsoobserved (Medici, Vinderola, & Perdigon, 2004). Phago-cytic cells play a central role in protection against microbialinfections. In addition, macrophages are involved in antigenpresentation, tissue repair and also play an important role inthe regulation of immune responses. IgA antibodies pre-dominate in mucosal surfaces and prevent adherence ofpathogens to the gut mucosa, being responsible for thehumoral immunity (Gill, 1998).

Ahola et al. (2002) studied the effect of probiotic Edamcheese containing Lactobacillus rhamnosus LC705 and L.rhamnosus GG ATCC53103 (LGG) on the risk of dentalcaries. During the study, no significant difference wasfound for Streptococcus mutans populations between thecontrol 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 threesubjects (8%) from the control group. During the 3 weekpost-intervention, S. mutans populations decreased in21% of the subjects who ingested probiotic cheese andonly in 8% of the control group. Additionally, there wasalso a decrease in the number of subjects who presentedhigh yeast populations in the probiotic group. The probioticintervention also seemed to increase salivary lactobacillipopulations, originated from the cheese-making process.The authors concluded that eating probiotic cheese couldreduce the risk of dental caries in general, although no

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significant statistical difference was observed in salivarymicrobial populations.

Hatakka et al. (2007) investigated the effect of consump-tion of probiotic cheese on the oral candidosis for elderlypeople. Cheese containing a mixture of probiotic cultures(L. rhamnosus GG, L. rhamnosus LC705, Propionibacte-rium freundenreichii spp., and Shermani JS) was ingestedby 92 elderly people. Microbiological analyses of oralyeasts were performed in saliva samples during 4 timesthroughout the study. After 8 and 16 weeks, the prevalenceof high populations (>104 CFU/ml) of this microbial groupwas, 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 controlcheese 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 tothe average unstimulated saliva flow. The authors hypothe-sized that probiotic bacteria have somehow affected thecomposition of saliva, the concentrations of mucins and sal-ivary immunoglobulins. They suggested probiotic cheesecould be used as a prophylactic approach for decreasingthe risk of hyposalivation and the feeling of dry mouth,and that it could be considered beneficial to oral health ingeneral.

Technological hurdles and the stability of probioticcheeses

The major challenge associated with the application ofprobiotic cultures in the development of functional foodsis their viability maintenance during processing. Probioticmicroorganisms should also be technologically suitablefor the incorporation into food products so that they retainboth viability and efficacy in the food product (on a com-mercial scale) and throughout consumption. Probioticsshould also be able to survive industrial applications (e.g.,standard dairy processing or pharmaceutical manufacturingprotocols) and be able to grow/survive at high levels in theproducts during their shelf life. Furthermore, from a foodprocessing perspective, it is desirable that such strains aresuitable for large-scale industrial production and shouldwithstand the processing conditions mentioned above,such as freeze-drying or spray drying. Besides, probioticstrains for incorporation into human foods should be con-sidered as GRAS ( generally recognised as safe) and notlead to undesirable changes from a sensorial point ofview as to their flavour, aroma, texture and other importantattributes (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 presenceof oxygen may represent a threat for their survival. In gen-eral, Bifidobacterium spp. is more sensitive to oxygen thanL. acidophilus, due to its strict anaerobe nature, althoughthis sensibility varies according to the lineage of microor-ganism (Talwalkar & Kailasapathy, 2004). Additional

features include degree of acidity, ability to grow well inmilk-based media and to rapidly acidify milk, thus reducingthe 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 ofcheese products, the process may sometimes have to bemodified and adapted to the requirements of the stains em-ployed. Where this is not possible, other probiotic strainsmay 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, thismeans that such strains should be cultivable to high celldensity 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 shouldhave the same performance as a conventional cheese: theincorporation of probiotic bacteria should not implya loss of quality of the product. In this context, the levelof proteolysis and lipolysis must be the same or even betterthan cheese which does not have this functional appeal.

Due to its manufacturing process, fresh cheese appearsto 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 ratherlimited (Heller et al., 2003). A number of scientific papersreporting the development of fresh cheeses containingrecognized and potentially probiotic cultures have beenpublished, which described suitable viable counts and a pos-itive influence on the texture and sensorial properties ofthese cheeses. Having in mind that portions of around100 g of cheese are usually consumed daily, populationsof about 106 CFU/g lead to an ingestion of 108 CFU/dailyportion.

Blanchette, Roy, Belanger, and Gauthier (1996) devel-oped a Cottage cheese containing Bifidobacterium infantis.Cheeses presented populations as high as 1� 106 CFU/gduring 10 days of refrigerated storage. Suarez-Solıs, Car-doso, Nunez de Villavicencio, Fernandez, 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/gduring 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 cheeseproduction, 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 mesophilictype O lactic culture during Minas fresh cheese productionand observed levels above 1� 106 CFU/g for probioticbacteria during the whole storage period. In both studies,

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L. paracasei and L. acidophilus did not alter the texture andsensorial characteristics of Minas fresh cheeses.

Masuda, Yamanari, and Itoh (2005) evaluated the viabil-ity of three strains isolated from human intestine with hightolerance for acid and bile, L. acidophilus JCN11047, L.acidophilus 1132T and Lactobacillus gasseri JCM11657,in fresh cheeses stored at 7 �C during 4 weeks. Negligiblereductions in the viability were observed for all strainsthroughout the shelf life of cheeses, which remained above8� 107 CFU/g up to the end of storage.

Buriti, Cardarelli, Filisetti, and Saad (2007) tested theaddition of L. paracasei subsp. paracasei LBC 82 in co-culture with Streptococcus thermophilus to potentiallyprobiotic and synbiotic fresh cream cheeses (without andwith inulin, respectively). Viable counts of L. paracaseiremained above 1� 107 CFU/g during the entire storageperiod, 21 days, for both cheeses. In another study, Buriti,Okazaki, Alegro, and Saad (2007) observed the viabilityof L. acidophilus La-5 and B. animalis Bb-12 added toMinas fresh cheese. Both probiotic cultures were presentin 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/orhoney, Cardarelli, Buriti, Castro, and Saad (2008) obtainedL. acidophilus and B. animalis subsp. lactis populationsabove 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 andSaad (2009) studied the manufacture of Minas fresh cheesesupplemented with the probiotic strain of L. acidophilusLa-5 solely or in co-culture with S. thermophilus. Cheesesmanufactured with La-5 solely presented populations above1� 106 CFU/g, reaching 1� 107 CFU/g on the 14th day ofstorage. Also, the addition of La-5 strain resulted in goodacceptance of Minas fresh cheeses, improving the sensory

Table 1. Technological hurdles in the probiotic cheese processing.

Step Problem Possible s

Addition ofprobiotic inoculum

U Interactions of the probiotic andstarter may cause negative impact;

U Loss of viable probiotic cellsin the whey during draining.

U Prelimcombi

U Use oU Check

the im

Salting U Probiotic bacteria are sensitiveto high salt concentrations.

U MicroU Suitab

Packaging U Probiotic bacteria are sensitiveto oxygen.

U Choosoxyge

U Cell inU Suitab

Ripening U Survival of probiotic bacteria throughthe cheese ripening period.

U MicroU Optim

Storage conditions U Inadequate storage conditions affectthe probiotic survival.

U Strict

performance of these products during storage (Souzaet al., 2008).

However, even though cheese is likely to be one of thebest carriers for probiotics, the addition of high numbersof viable and metabolically active cells can affect productquality, especially sensory properties (Grattepanche,Miescher-Schwenninger, Meile, & Lacroix, 2008). Anexample is the observation reported by Modzelewska-Kapi-tula, Klebukowska, and Kornacki (2007), who studied theaddition of the potentially probiotic culture of Lactobacillusplantarum 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 strainpresented slightly lower scores in the sensory analysis.

Table 1 shows the main technological hurdles concern-ing the development of probiotic cheeses. There are fiveidentified hurdles which directly influence the maintenanceof the functional activities of probiotic bacteria in cheese:addition of the probiotic inoculum, salting, packaging,ripening and storage conditions.

Addition of the probiotic inoculumThere are two options for the addition of probiotic bac-

teria during cheese processing which can directly affect thesurvival rate of these microorganisms: probiotic bacteriacan be added before the fermentation (together with thestarter 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 bechecked according to the process. If the second option ischosen, immediate cooling must be performed (below8 �C, preferentially), as metabolic activities of startersand 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

olutions

inary tests to choose the most suitable probiotic and starternation;f strains from the same supplier;different moments of addition of the probiotic inoculum (observingpact on the final cost of the product and probiotic survival).

encapsulation;le strain selection (information from the strain supplier).

e suitable packaging system: film plastic with low permeability ton, vacuum packaging or active packaging;cubation under sub-lethal conditions to develop salt resistance;le strain selection (information with the strain supplier).

encapsulation;ize ripening conditions through preliminary tests.

control of storage temperature.

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exactly controlled and the adverse effects of the high scald-ing temperature are avoided. It is also important to considertheir physiological state in order to have an idea of theirsurvival throughout ripening and/or storage (Heller et al.,2003). In terms of the growth curve, microbial cellsbetween the late exponential and the stationary phase arethe favourite option, and, sometimes, the preparation ofa previous substrate to inoculate the strain might bebeneficial.

Daigle, Roy, Belanger, and Vuillemard (1999) useda cream fermented by B. infantis for the manufacture of pro-biotic cheese (Cheddar like-cheese). Viable counts of thismicroorganism were above 3.0� 106 CFU/g through 84days of storage at 4 �C and there was no metabolic activitywhich had impact on the sensory properties. This seemedto 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-hardcheese: a freeze-dried powder dispersed in milk and a sub-strate containing milk and milk fat. The second trial didnot improve the survival of probiotic bacteria during theripening period, although it increased the probiotic popula-tion in the initial inoculum by one log cycle. This can bea more economical option and will have an impact on thefinal cost of the probiotic product.

Several other techniques have been adapted to enhancethe viability of probiotic bacteria to harsh conditions, typi-cal to many cultured dairy products, and may also be usedin the production of probiotic cheeses, including the selec-tion of oxygen-tolerant, acid-tolerant and bile-resistantstrains, 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 stresssuch as temperature, pH or bile salts is prior exposure tosub-lethal levels of the given stress. Stress responses maybe used to enhance the survival of probiotic bacteria instressful conditions and to improve their technologicalproperties (Roy, 2005; Saarela et al., 2004). The bacterialresponses can be adaptive (when an acid treatment at pH3e4 protects the bacteria against pH 2.5 or a treatment at47 �C protects against 55 �C) or cross-protective (whenheat protects against low pH or bile) (Saarela et al.,2004). The exposure of early stationary phase bacterialcells to combinations of reduced temperature, reduced pHand starvation could enhance the probiotic cold- and/oracid-tolerance and increase the number of viable probioticsingested via refrigerated dairy products (Maus & Ingham,2003), which could be useful in the production and storageof fresh cheeses, for e.g.

Moreover, an alternative for protecting bifidobacteria tooxygen stress is the use of selected strains of S. thermophiluswith high oxygen consumption as starter for the productionof 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

steps involved in the traditional cheese processing, espe-cially the scalding temperature normally used by producers,which can have detrimental effect on the survival duringprocessing. It is also important to consider the interactionsbetween probiotic bacteria and starters cultures, which mayact in a negative manner on the processing and stability ofthe product.

Searching for the interaction between probiotic andstarter strains, Vinderola, Mocchiutti, and Reinheimer(2002) observed that probiotic bacteria proved to be moreinhibitory towards lactic acid bacteria than vice versa, sincethe latter did not exert any effect on the growth of the for-mer, except for cell-free supernatants of some strains ofLactobacillus delbrueckii subsp. bulgaricus that weakly in-hibited the growth of certain L. acidophilus strains in well-diffusion agar assays. In addition, Bifidobacterium sp. andL. casei strains did not show any effect on the growth ofL. delbrueckii subsp. bulgaricus strains. Moreover, strainsof S. thermophilus and Lactococcus lactis showed variableresults, depending on the strain considered. To avoid thecompetition between the strains, it is advisable to usestarters 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 forthe production of organic acids, may also produce other an-timicrobial compounds, including hydrogen peroxide, alco-holic compounds, diacetyl, and bacteriocins. Thisinhibitory activity creates a hostile environment for patho-gens and spoilage organisms. Lactobacilli species, in par-ticular those from the L. casei group, are frequentlyresponsible 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).

SaltingApart from Domiati cheese, all cheeses are salted after

rennet coagulation and curd formation. Three methods areavailable: dry salting, surface dry salting and brine saltingor brine immersion (Guinee, 2004). Salt exerts an effecton the improvement of sensorial attributes of cheeses, aswell as on the biochemical reactions during the storage ofthe product. It is well established that the survival of micro-bial strains is restricted by the level of salt (Yilmaztekin,Ozer, & Atasoy, 2004). Gobbetti, Corsetti, Smacchi, Zoc-chetti, and de Angelis (1998) reported that the viability ofprobiotic bacteria is drastically reduced in cheese whensalt concentration is above 4%. Therefore, cheeses whichnaturally 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 probioticbacteria from the hostile environment. One alternative ismicroencapsulation or cell incubation under sub-lethal

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conditions. The former is a promising alternative used inthe processing of probiotic cheese, being responsible forimproving the survival of probiotic bacteria, without nega-tive effects on texture, aroma and acceptance by the con-sumers (Dinakar & Mistry, 1994; Ozer, Kirmaci, Sxenel,Atamer, & Hayaloglu, 2009; Ozer, Uzun, & Kirmaci,2008; Yilmaztekin et al., 2004). Sometimes, however, thetechnique cannot offer the adequate protection. Kailasapa-thy and Masondole (2005) reported that the use of microen-capsulation of probiotic bacteria (L. acidophilus DD910and Bifidobacterium lactis DD920) in calcium-induced al-ginate-starch capsules did not improve their viability ina Feta cheese matrix during storage in brine solution; lossesof 2e3 log cycles were observed in free and immobilizedcells during storage. Possible reasons given by the authorswere the open texture of the cheese and the high salt con-centration, 7.3e8.4%, causing death of the cells.

PackagingThe packaging system ought to be considered as an im-

portant stage of the processing of probiotic dairy foods andshould be taken into consideration in order to improve thestability of probiotic bacteria in foods. In general, probioticdairy foods, like cheese, are packaged in plastics filmswhich 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 tooxygen, due to anaerobic metabolism (Robertson, 2006).

Several factors affect the survival of L. acidophilus andBifidobacterium spp. when they are added to a food matrix.These include strains of probiotic cultures, pH, hydrogenperoxide, storage atmosphere, concentration of metabolitessuch as lactic acid and acetic acids, dissolved oxygen andbuffers such as whey proteins. Among these factors, expo-sure to dissolved oxygen during manufacture and storage isconsidered highly significant. Both L. acidophilus and Bifi-dobacterium spp. are human gut-derived microorganismsand, as mentioned previously, they are microaerophilicand anaerobic, respectively. Unlike aerobic bacteria, whichcompletely reduce oxygen to water, oxygen-scavengingsystem in these probiotic bacteria is either reduced or com-pletely absent. Consequently, accumulation of toxic oxygenmetabolites e superoxide anion (O2

- ), 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 sugarmetabolism of bifidobacteria (Shah, 1997). A correlationbetween the levels of two enzymes e NAD-oxidase andNAD-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 andprevent cell death (Roy, 2005).

Therefore, plastic films with low permeability to oxygenshould be chosen to pack these functional products;

alternatively, the practice of adopting other alternatives,such as the use of vacuum packaging should be followed.Kasımoglu, Goncuoglu, and Akgun (2004) investigatedthe effect of a packaging system using vacuum and brineon Turkish white cheese ripening. The authors observedthat vacuum packaged cheese presented best performancein the sensorial evaluation (good flavour and texture) be-sides a high level of proteolysis. Indeed, packaging systemsought to be taken into consideration when developingnew probiotic food products. A review on this topicwas published elsewhere (Cruz, Faria, & Van Dender,2007).

RipeningThe ripening process of cheese is very complex and in-

volves microbiological and biochemical changes in thecurd, resulting in the flavour and texture characteristic ofa particular variety. The biochemical changes occurringduring 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 probioticculture, as its survival through this period cannot be pre-dicted with accuracy. Biochemical changes occurring insidethe cheese environment, as water activity decreases, some-times together with a decrease in pH, create a hostile andstressful environment for the adjunct cultures.

An additional problem is the proliferation of a populationof non-pathogenic adventitious bacteria, usually lactobacilliand pediococci, which often become the dominant micro-biota in cheese. They are also called non-starter lactic acidbacteria (NSLAB) (Hayes et al., 2006). This microbial groupcompetes for nutrients and sometimes creates a problem forthe quantitative determination of probiotic viability. Incorpo-ration of probiotic bacteria in cheese does not generallyaffect primary proteolysis, performed by a coagulant agentand, to a less extent, by plasmin, residual coagulant andenzymes from the starter microbiota (Sousa, Ardo, &McSweeney, 2001). However, probiotic bacteria enzymesact in the secondary proteolysis, increasing the total freeaminoacid content, which contributes decisively to cheeseflavour (sweet, bitter or malty) and can be precursors forthe synthesis of other flavours or volatile aroma, resultingin off-flavours (Ardo, 2006).

Lipolysis is not influenced by probiotic bacteria, as theirenzymes have a lower lipolytic activity, when compared tostarters and NSLAB (Grattepanche et al., 2008). Despitethis, several ripened probiotic cheeses have been devel-oped, without or with minimum changes in the proteolyticand lipolytic profile, exerting a positive effect on the overallquality of the cheese (Fernandez, Delgado, Boris, Rodrı-guez, & Barbes, 2005; Kalavrouzioti, Hatzikamari, Lito-poulou-Tzanetaki, & Tzanetakis, 2005; Kourkoutas et al.,2006; Ong, Henriksson, & Shah, 2006; Ong, Henriksson,

351A. 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 conditionsConsumers obviously demand that the product they pur-

chase contain the probiotic cultures at the time of consump-tion. Thus, companies provide the required viablepopulation at the time the product is marketed, but arealso concerned with the evolution of the populationthroughout storage. Typically, the ‘‘best before’’ date isgiven in order to provide a period guaranteeing the desiredpopulation (Champagne & Gardner, 2005).

The supplementation of cheeses with probiotic and/orstarters cultures can help to decrease growth of contami-nant, since these cultures are able to produce natural anti-microbial substances in order to inhibit undesirablemicroorganisms in foods. Moreover, NaCl is widely usedin the food industry as a preservative agent (Vinderola,Costa, Regenhardt, & Reinheimer, 2002). However, hightemperature found in most of sales points has a negativeimpact on the survival of probiotic populations, and causesundesirable 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 veryimportant, as failure may endanger the functional statusof the product and lead to the growth of contaminant andpathogenic microorganisms.

Sensorial aspects of probiotic cheeseProbiotic cultures do not tend to strongly modify the

sensorial properties of the products to which they areadded (Champagne & Gardner, 2005). The main concernis ripened cheeses containing bifidobacteria, since theyproduce a high amount of acetic acid, as well as lacticacid in a molar ratio of 2:3 from lactose fermentationvia the fructose-6-phosphate shunt pathway. In smallamounts, acetic acid exerts a positive influence on thearoma of probiotic cheeses. However, excessive concentra-tions are undesirable, causing off-flavours (Grattepancheet 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 toobtain more precise conclusions on this attribute of theproducts. If possible, the sensorial profile should be inves-tigated using appropriated sensorial techniques likeDescriptive Quantitative Analysis. A review on this topicwas published (Drake, 2007), and these principles canbe applied to development of probiotic cheeses.

Other challenges related to development of probioticcheeses

Nowadays, the current consumers’ interest towardsproducts that contribute to decreased risks of chronic-degenerative diseases encourages the development of probi-otic cheese with reduced contents of fat or sodium.

However, the reduction of these components may affectthe sensorial features of the final product. The propercombination of probiotic strains used as adjunct culturescan influence favourably the flavour and texture, particu-larly for low-fat cheeses.

Compared to yogurt, the problem for cheese e especiallysemi-hard and hard cheese e acting as carrier for probioticsresults from the high fat and salt content and the relativelylow recommended daily intake. It follows that the concentra-tion of probiotics in cheese should be about four to five timeshigher than in yogurt. However, this does not apply to freshcheese, such as Cottage cheese, which can easily be adjustedto low fat and salt contents, and for which recommendeddaily intake is rather high. Low-fat fresh cheese may thusserve as a food with a high potential to be applied as a carrierfor probiotics (Heller et al., 2003).

Ryhanen, Pihlanto-Leppala, and Pahkala (2001) studiedthe behavior of L. acidophilus and Bifidobacterium sp.added to Festivo cheese with reduced-fat content. The fatcontent was reduced to approximately 11% and no adverseeffect on sensory quality was observed. Also, L. acidophi-lus and Bifidobacterium sp. remained viable for up to sevenmonths 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, reaching1� 108 CFU/g during the whole storage period.

Because reduced and low-fat cheeses contain moremoisture and are generally produced using lower cookingtemperatures, lactic acid bacteria are able to grow to highpopulations in these cheeses (Drake & Swanson, 1995).So, low-fat probiotic cheese manufacture with suitable pro-biotic populations is possible, so as to obtain cheeses withadded health benefits.

There are a limited number of studies available concern-ing the probiotic response to salt concentration. Kasımogluet al. (2004) studied the viability of L. acidophilus added toTurkish white cheeses manufactured with and without theaddition of salt, and observed populations above1� 107 CFU/g and above 1� 106 CFU/g, respectively.

It is, therefore, convenient to establish the effect of salton the growth and survival of probiotic bacteria, in order topredict their potential utilization in a cheese containing dif-ferent salt concentrations.

PerspectivesThe use of cheese as probiotic food carrier presents po-

tential advantages and it is a valuable alternative for thedairy industry. However, development on an industrial scalerequires knowledge of all technological steps involved inthe traditional process, and adaptations of the existing pro-tocol are usually necessary.

As there are lots of cheese varieties available on themarket, 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 thecheese environment for traditional and low-fat processes.Such tests must be completed with laboratorial analysistesting parameters which are decisive for the marketingof the product, like organic acid profile, and typical aromacompounds of the product. Additionally, the use of senso-rial techniques can help to determine the important attri-butes which may influence consumers.

AcknowledgementsThe authors would like to thank to Fundac~ao de Amparo

a Pesquisa do Estado de S~ao Paulo (FAPESP), Coordenac~aode Aperfeicoamento de Pessoal de Nıvel Superior(CAPES), and Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) for financial support andscholarships.

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