Technological aspects related to the use of bifidobacteria in dairy products Denis Roy

To cite this version:

Denis Roy. Technological aspects related to the use of bifidobacteria in dairy products. Le Lait, INRA Editions, 2005, 85 (1-2), pp.39-56. ￿hal-00895593￿

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait 85 (2005) 39–56 © INRA, EDP Sciences, 2005 39 DOI: 10.1051/lait:2004026 Review

Technological aspects related to the use of bifidobacteria in dairy products

Denis ROY*

Institute of Nutraceuticals and Functional Foods and Centre STELA, Laval University, Quebec City, Quebec, Canada

Abstract – Dairy-related bifidobacteria are already used in a wide variety of dairy products including , cheese, frozen yoghurt-like product and ice cream. The survival of bifidobacteria in fermented dairy products depends on varied factors such as the strain of used, fermentation conditions, storage temperature, and preservation methods. Growth of bifidobacteria in milk is often slow or limited compared with bacteria used in fermented dairy products, and this appears partially due to low proteolytic activities. When manufacturing cheese or yoghurt-like pro- duct, addition of probiotic cultures to the normal starters generally results in slower growth of the probiotic strains than if they were added alone in milk. The use of higher inocula of bifidobacteria and the addition of growth promoting factors as a nitrogen source should further enhance the growth and viability of the bifidobacteria. Although the most common processing stage at which to add the bifidobacteria to the milk would be in conjunction with the other starter cultures, other alternative processing protocols have been successful in incorporating the bifidobacteria into cheese and cultured dairy products. The success of the incorporation of bifidobacteria into cheeses is dependent on the bifidobacteria strains, the activity of used in the manufacture of the cheese, the composition of the cheese, and the conditions of processing and ripening. Changes in the chemical composition and the texture of the fermented products can occur in cheeses and fer- mented without affecting sensorial properties. / yoghurt-like product / cheese / viability / stability

Résumé – Les aspects technologiques de l’utilisation des bifidobactéries dans les produits lai- tiers. Les bifidobactéries d’intérêt laitier sont déjà employées dans une grande variété de produits laitiers probiotiques comme le lait, le fromage, le yaourt congelé et la crème glacée. La survie des bifidobactéries dans les produits laitiers fermentés dépend de facteurs divers tels que la souche des bactéries utilisées, les conditions de fermentation, la température d’entreposage et les conditions de conservation. La croissance des bifidobactéries dans le lait est souvent lente ou limitée comparée à celle des bactéries lactiques utilisées dans produits laitiers fermentés, et ceci semble partiellement dû à de faibles activités protéolytiques. En fabriquant le fromage ou le yaourt, l’addition des cultures probiotiques aux ferments a généralement comme conséquence une croissance plus lente des sou- ches probiotiques comparé à si elles étaient ajoutées au lait seules. L’utilisation de plus hauts niveaux d’inoculum de bifidobactéries et l’addition de facteurs de croissance comme la source d’azote devraient améliorer la croissance et la viabilité des bifidobactéries. Bien que la pratique la plus commune serait d’ajouter les bifidobactéries au lait en même temps que les autres ferments, d’autres protocoles de traitement alternatifs ont été proposés pour incorporer les bifidobactéries dans les fromages et les produits laitiers fermentés. Le succès de l’incorporation des bifidobactéries

* Corresponding author: [email protected] 40 D. Roy dans les fromages dépend des souches de bifidobactéries, de l’activité des bactéries lactiques utili- sées dans la fabrication du fromage, de la composition du fromage, et des conditions de fabrication et d’affinage. Les changements de la composition chimique et de la texture des produits fermentés peuvent se produire dans les fromages et les laits fermentés à la suite de l’incorporation des bifido- bactéries, sans affecter les propriétés sensorielles.

Bifidobacterium / yaourt / fromage / viabilité / stabilité

1. INTRODUCTION monly used for the production of fermented milks [53, 76]. Dairy products containing During the last 35 years, attempts have bifidobacteria are made with pure cultures, been made to improve the health status of alone or in combination with other lactic human by modulating the intestinal micro- acid bacteria such as Streptococcus thermo- flora using live microbial adjuncts called philus, delbrueckii subsp. bul- . Indeed, different products con- garicus, and L. acidophilus group or L. casei taining probiotic bacteria have gained in group [36]. Dairy-related bifidobacteria are popularity with consumers. Strains of Lac- already used in a wide variety of probiotic tobacillus acidophilus and Lactobacillus dairy products including milk, bifidus milk, casei complex are well represented in com- cheese, frozen yoghurt-like product and ice mercial probiotic products, followed by cream. Yoghurt-like products are made Bifidobacterium spp. (B. animalis subsp. using a single species of bifidobacteria in lactis, B. bifidum, B. breve, B. longum subsp. combination with lactic acid bacteria. Bifi- infantis and B. longum), some other lactic dobacteria can be incorporated to yoghurt- acid bacteria (lactococci, leuconostocs, like products before or after fermentation. enterococci) and non-lactic acid bacteria Fermented milks containing B. longum or (propionibacteria and yeasts) [29]. B. breve have obtained “Foods for specific health use” (FOSHU) approval in Japan The incorporation of bacteria of intesti- which are recognized to regulate intestinal nal origin into human diet corresponds to microflora and allow a healthier gastroin- the emergence of functional food, which testinal condition. uses the beneficial effects of these bacteria on intestinal metabolism [46]. Functional The aim of this review is to address the food can be defined as any food that may technological factors involved in the use of provide a health benefit beyond the tradi- bifidobacteria as probiotics: addition level, tional nutrients it contains. selection criteria, incorporation in fer- mented milks and cheese, stability during Bifidobacteria are emerging as possibly storage and change in sensorial properties. one of the most important group of intesti- This review is a complement to many excel- nal organisms with regard to human health. lent reviews that have been published on It is estimated that over 400 species of bac- development of functional foods with bifi- teria inhabit the human gastrointestinal dobacteria [8, 63, 68, 72]. tract and Bifidobacterium spp. belongs to the dominant anaerobic flora of the colon. The main species present in human colon 2. RECOMMENDED ADDITION are B. adolescentis, B. bifidum, B. longum LEVEL subsp. infantis, B. breve and B. longum [29]. According to Marteau and Shanahan Dairy products appear to be good vehi- [40], the best criterion for the expression of cles for the delivery of probiotics to humans. the results in pharmacokinetics studies is B. bifidum, B. longum subsp. infantis, B. lon- probably the concentration of probiotic at gum and B. animalis subsp. lactis are com- the target site. Probiotic products containing The use of bifidobacteria 41 bifidobacteria should be standardized on it would be impossible to control microbial the basis of viable count because it is pre- counts of probiotic products at the time of sumed that probiotic viability is a reasonable consumption. The manufacturers have the measure of probiotic activity. The number responsibility to undertake shelf-life tests at of cells present in products is a useful indi- the date of minimum durability; at the point cator to estimate the probiotic viability with of sale to the consumer; and at the time the assumption that this is the important when the product leaves the manufacturer. factor to consider in product functionality. Separate minimum counts should be To perform this functionality, probiotics developed for individual micro-organisms must be viable at the time of consumption such as probiotics bacteria. and maintain their viability throughout the . Recommendations for the minimum suggested level for probi- otics in product to attain this viability are 3. SELECTION CRITERIA quite variable. More critical than the con- OF BIFIDOBACTERIA centration of probiotics in the functional food, however, is the daily intake of the pro- Selection of probiotics can be based on biotics necessary to attain a therapeutic effect. general microbiological criteria that refer to The concentration of probiotics needed to safety, technology, performance and health obtain a clinical effect is often quoted as benefits [20]. From the safety’s point of ≥106 cfu·mL–1 in the small bowel and view, bifidobacteria are considered to be ≥108 cfu·g–1 in the colon although a thera- beneficial for the host by inhibiting the peutic minimum dose of 1 × 105 viable growth of potential harmful bacteria in the cells/g or mL product was proposed intestinal tract and by exhibiting beneficial [39,73]. The small bowel concentration effects for the host. The production of lactic limit has been proposed because it leads to acid and acetic acid contributes to the a clinical effects (diarrhoea) in subjects bifidobacteria’s defence against pathogens with bacterial colonization of the small through the toxic effect of the undissociated bowel; the concentration in the colon has acids on the microorganisms and stimulation been proposed as it represents the lower of intestinal peristalsis. Administration of limit of the dominant flora [11, 40]. Hence, probiotics has been proposed to increase the a daily intake of at least 108–109 viable cells number of bifidobacteria in the gastro- has been suggested as the minimum intake intestinal tract. The widespread use of to provide a therapeutic effect [21, 71]. bifidobacteria in fermented foods and dairy However, the dose required for a probiotic products has a long history, and most strains effect may be dependent on the food form are considered commensal microorganisms in which the probiotic is ingested and on the with no pathogenic potential. No case of local probiotic strain used. or systemic infections, including septicemia, In general, the food industry has targeted meningitis, and endocarditis occurred due populations of ≥106 cfu·g–1 at the time of to dairy-related bifidobacteria has been consumption of strain that has been added reported. to food. Initially, this standard appears to Strain selection based on technological have been adopted to provide bacterial con- aptitudes is a critical aspect of developing centrations that were technologically attain- a probiotic-based functional food. Probiotic able and cost-effective, rather than to achieve bacteria must exhibit resistance to techno- a specific health effect in humans [8, 57, logical processes used in preparing the 66]. As more data on bacterial population vehicle of probiotic delivery [43]. Unfortu- requirements are available, it becomes clear nately, not all strains of bifidobacteria can that numbers will vary as a function of the be easily produced industrially because of strain and the health effect desired. However, low yields in the growth media or poor survival 42 D. Roy to freezing or freeze-drying [67], and not all utes to the probiotic survival. Experiments promising strains can be marketed. The involving human intubation and sampling selection of a probiotic strain to be added in of B. animalis subsp. lactis from the cecum the product is a crucial step. showed that these probiotics, when given in High viable counts and survival rates fermented milk, survive to the extent of during stomach passage are necessary to 23.5% of the administered dose [51]. The allow live bifidobacteria from the fermented delivery of B. bifidum and L. acidophilus to ≈ milk products to play a biological role in the the cecum was 30% and 10% of the admin- human intestine. Many bifidobacteria added istered dose, respectively [41]. In comparison, to dairy products may not exert probiotic it has been observed that the concentration effects because they die after exposure to of viable yoghurt bacteria reaching the duo- acid during or after fermentation, oxygen denum after the ingestion of yoghurt con- taining 107 cfu·mL–1 was at its peak around during refrigerated distribution and storage, 5 –1 and/or acid in the human stomach [43]. 10 cfu·mL [50]. Intrinsic resistance to gastric acid and bile of L. delbrueckii subsp. Delivery of viable bifidobacteria to the bulgaricus and S. thermophilus is poor [10]. , where they would be able to Bifidobacteria survived passage through function, has been limited because of the the stomach and small intestine in humans extreme acidity found in the human stomach and reached the appropriate fecal concen- [2]. Thus, the success of food products trations to exert metabolic activities, but did containing bifidobacteria depends on the not colonize the human colon [6, 7]. In sum- viability of bifidobacteria in the product mary, many variables can determine the during its shelf life as well as on the degree to which probiotics survive passage resistance of the bacteria to the conditions through the upper gastrointestinal tract: the system existing in the upper gastrointestinal degree of stomach acidity, the length of tract [74]. The survival of bifidobacteria at exposure to acid, the concentration of and the pH values of fermented dairy products length of exposure to bile salts, the level of and gastric fluid varies. In one strain of bile salt hydrolase activity, and other as yet bifidobacteria, growth was inhibited by unspecified properties of the probiotics only 0.5 log units, whereas population themselves [3]. It has been noted that, levels declined by 4 log units in the other whereas the majority of bile salts-resistant strain [2]. B. bifidum was found to be bifidobacteria may be intrinsically sensitive tolerant to the acidity of a model gastro- to gastric transit, they are intrinsically intestinal tract system, with only a 20% resistant to small intestinal transit [10]. decrease in numbers as the pH decreased The mucosal adhesion of bifidobacteria from 5.0 to 1.8 over an 80 min period [41]. might be strain-specific and dependent on The viability of B. animalis subsp. lactis substrate availability such as mucins [27]. remained unchanged at a pH of 3 for The mucus covering the epithelial cells is 180 min, declined slowly at a pH of 2, and the initial surface that ingested micro- was zero after 60 min at a pH of 1 [51]. organisms confront in the human gut. Mucus B. animalis subsp. lactis exhibited much is continually subjected to degradation; greater tolerance to human gastric juice than conversely new mucins are constantly towards simulated gastric juice containing secreted. Mucins are high molecular mass milk protein and mucin. However, it is not glycoproteins that are synthesized and secreted clear why this occurred [10]. Viability of by the goblet cells. They form a gel-like bifidobacteria, as expected, depends on the layer on the mucosal surface, and provide pH, the length of the exposure to acid, and favourable conditions for the resident the species and strains used [3, 10]. by forming a specific micro-envi- In the small intestine, tolerance to bile ronment. Thus, bacteria able to adhere to salts is an important factor which contrib- mucus but unable to reach the epithelial The use of bifidobacteria 43 cells might be dislodged from the mucosal The performance aspects for selection of surface with the degraded mucin and washed strains should include the following crite- away with the luminal contents. This may ria: strains claimed to be present in a prod- partly explain the transient pattern of colo- uct should survive in relatively high viable nisation characteristic for most probiotic cell numbers, retain metabolic activity and bacteria [33, 49]. In addition, it is postulated provide desirable sensorial properties [28]. that milk proteins and mucin may function The survival of bifidobacteria for example as both buffering agents and inhibitors of in fermented dairy products depends on digestive protease activity in vivo, thereby varied factors such as the strain of bacteria protecting ingested bacterial strains during used, fermentation conditions, storage tem- upper gastrointestinal transit. perature, and preservation methods [68]. One strategy for enhancing bacterial tolerance to The viability of probiotic bacteria is stress such as temperature or pH is prior affected by inhibitory substances such as exposure to sub-lethal levels of the given lactic acid produced during production and stress. The stress response phenomenon, cold storage. During production of yoghurt- which occurs when this treatment results in like product, yoghurt bacteria and probiotic greater survival, has been widely reported bacteria produce sufficient hydrogen perox- for a variety of micro- and macro-organisms ide to be auto-inhibitory [61]. High num- [43, 64]. Rapid growth rate and rapid acid- bers of viable bifidobacteria in yoghurt-like ification of milk are important criteria for product may not always be maintained the selection of probiotic bacteria. These because of the acidity of yoghurt-like prod- characteristics of probiotics would reduce uct, the low acid tolerance of bifidobacteria costs by requiring short incubation times and the cell death due to oxygen toxicity [3, during the manufacture of fermented dairy 9]. Acid tolerance is an important property products. of probiotic bifidobacteria, enabling the The presence of bifidobacteria in the cells to survive gastric acidity and volatile human intestine is almost universally accepted fatty acids produced during fermentation in to be a contributing factor to a healthy well- the intestine. Bifidobacteria encounter pH being. There is accumulating evidence that values ranging from 2.0 to 8.0 in the stom- bifidobacteria are associated with benefi- ach, depending upon whether food is being cial health effects. It has been claimed that consumed and must resist to exposure to ingestion of specific bifidobacteria could pH 3 for 1.5 to 2 h. In addition, survival of contribute to reestablishment of a bifido- ingested bifidobacteria in the stomach bacterial flora in humans after antibiotic obviously is influenced by the buffering therapy; alleviation of constipation; pre- capacity of food components [10]. Mature vention against diarrhea and other gastro- Cheddar cheese compares very favourably intestinal infections and; alleviation of the with fresh yoghurt-like product as a deliv- symptoms of lactose intolerance [48]. The ery system for viable probiotic microorgan- biological effect of the probiotic culture is isms to the gastrointestinal tract [19]. On generally linked to the strain used, as well the other hand, survival in fermented milk as the form and the numbers consumed [3]. during long-term storage is a complex process because it is influenced by other stress factors including oxygen, light, nutrient exhaustion 4. TECHNOLOGICAL and metabolites other than organic acids PERSPECTIVES produced during fermentation [75]. Differ- ence in acid tolerance between yoghurt bac- From the manufacturer’s point of view, teria and bifidobacteria suggest that various the effects of the environment of the dairy responses of cytoplasmic pH product during processing and storage must system may occur [75]. also be considered to ensure that the 44 D. Roy concentration of bifidobacteria at the time tially be related to the production of bacte- of consumption provides a therapeutic dose riocins or other inhibitors such as lactic and to consumers. Strain of bifidobacteria must other organic acids and exhibit resistance to technological processes produced by the starter cultures [42, 78]. In used in preparing the vehicle of probiotic addition, starter cultures grow faster, acid- delivery. However, the survival of bifido- ification occurs rapidly, and fermentation bacteria in fermented milk is mainly limited times are much shorter in their presence by their sensitivity to the high acidity. Only which resulted in reduced availability of a few Bifidobacterium strains seem to be nutrients [68]; thus, probiotic cultures do suitable to obtain sufficiently large numbers not have time to grow extensively. of viable cells at the time of consumption The use of higher inocula of bifidobacteria of these products. In addition, the growth of and the addition of growth promoting bifidobacteria seems to be suppressed in the factors as a nitrogen source should further presence of yoghurt starter organisms. How- enhance the growth and viability of the ever, compared with yoghurt bacteria, bifi- bifidobacteria [25, 26]. High inoculum lev- dobacteria have a higher resistance to acid els must be used because a food product and bile present in the gastrointestinal tract. should contain between 1 and 100 million The ingestion of bifidus fermented milk led bifidobacteria per gram when placed on to an increase in total bifidobacteria which store shelves. Hence, it is recommended was related to the colonic transit of the that during the manufacture of dairy prod- exogenous bifidobacteria [7]. ucts with bifidobacteria, the milk should be inoculated with the final number of bifido- 4.1. Dairy products production bacteria required for the product. Growth of bifidobacteria in milk is often The addition of yeast extracts, casein slow or limited compared with other lactic hydrohysates or combination of amino acids, acid bacteria used in fermented dairy prod- minerals and ribonucleotides or casein hydro- ucts, and this appears partially due to low lysates, amino acids, peptides and other proteolytic activities [14, 25]. Probiotic micronutrients to supplement the growth of bacteria are added to fermented milks, in bifidobacteria in milk can be beneficial. which the consumer expects the presence of However, the redox conditions might be live bacteria. Starters such as lactococci, inappropriate for growth of the bifidobac- thermophilic lactobacilli or streptococci are teria. Redox conditions indeed appear to be always added for technological purposes linked to the growth-promoting properties (acidification, texture, flavour). Lactobacilli of various supplements [31]. In yoghurt- have also become important as adjunct like product, the addition of ascorbic acid cultures for the production of bifidobacteria- and cysteine decreased the redox potential, containing dairy products especially the providing an environment more favourable L. acidophilus group (L. acidophilus and to the growth of bifidobacteria [15, 16]. L. johnsonii) and L. casei group (L. casei Oxygen may affect bifidobacteria due its and L. rhamnosus). toxicity to cells and production of H2O2, When manufacturing cheese or yoghurt- presumably due to intracellular production like product, addition of probiotic cultures of H2O2 and by the H2O2 produced by other to the normal starters generally results in cultures in the environment [68]. A syner- slower growth of the probiotic strains than gistic inhibition of bifidobacteria by acid if they were added alone in milk [59, 65]. and hydrogen peroxide has been demon- These starters produce environments that strated [38]. Bifidobacteria are considered inhibit the growth of not only pathogens as highly susceptible to oxygen although and spoilage microorganisms, but also of the oxygen tolerance of these organisms is probiotic [79]. The phenomenon could par- species dependent [17, 69] because B. animalis The use of bifidobacteria 45 subsp. lactis, isolated from fermented milk modify the environment of bifidobacteria, was found to display good oxygen tolerance thus creating favourable conditions for [45]. With respect to susceptibility to oxy- them to proliferate. The production of H2O2 gen, a particularly important feature for that is detrimental for bifidobacteria under growth and stability of bifidobacteria in aerobic conditions is effectively suppressed milk and fermented milk, it has been shown in the presence of the bifidogenic growth that a correlation exists between the synthe- stimulator. ACNQ acts as a mediator of the sis of NAD-oxidase and NADH-peroxidase electron transfer from NAD(P)H to dioxy- and oxygen susceptibility of the bifidobac- gen (O2) and hydrogen peroxide (H2O2). teria [69]. High levels of these enzymes The generation of H2O2 by B. longum under were found in the most aerotolerant Bifido- aerobic conditions is effectively suppressed bacterium spp. It is believed that intracellular in the presence of ACNQ. These ACNQ- levels of H2O2 block fructose-6-phosphof- mediated reactions would play roles as ructoketolase, a key enzyme in the sugar NAD(P)(+)-regeneration processes [80]. metabolism of bifidobacteria and therefore The production of functional cheeses scavenging H2O2 becomes important for cell was recently proposed as a suitable and survival [17]. Bifidobacterium spp. is promising alternative as vehicle carrying devoid of catalase, a key enzyme for the probiotics as compared to fermented milks breakdown of H2O2 and has to rely on because the cheese could offer certain enzymes such as NADH oxidase and NADH advantages (Tab. I). The cheeses having peroxidase to scavenge environmental oxy- higher pH (pH range 4.8–5.6) than gen. The activities of NADH oxidases in fermented milks (pH 3.7– 4.3) can provide bifidobacteria give rise to H2O2, prompting a more stable medium to support the long- NADH peroxidase to scavenge H2O2 and term survival of probiotics. Moreover, the prevent cell death. cheese matrix and the high lipid content of Some species of probiotics are more cheeses can protect them during during pro- oxygen tolerant than the others [45]. Hence, cessing and digestion [18, 72]. In addition, creating an anaerobic environment during an anaerobic environment will be devel- yoghurt-like product manufacture should oped due to the metabolism of lactic flora promote the development of Bifidobacte- within a few weeks of ripening, favoring the rium spp. Klaver et al. [35] found this to be survival of bifidobacteria [77]. Bifido- the case, by influencing redox values with bacteria have been used as probiotic culture ascorbic acid. Yoghurt cultures that have to produce Cottage cheese, Crescenza, proteolytic or oxygen-scavenging proper- Cheddar, Fresco, fresh or white-brined ties have been shown to be beneficial to cheeses, Canestrato Pugliese cheese made bifidobacteria and could be considered in of ewe’s milk [5, 12, 13, 18, 23, 26, 54, 79, the selection of cultures compatible to pro- 81] and Swiss-type cheese (Roy D., biotic strains [52]. It seems possible to Mainville I., Bélanger G., unpublished). Roy affect H2O2 synthesis by bifidobacteria by et al. [58] studied the growth and acid taking advantage of the benefits of propionic production of bifidobacteria in milk under cultures. Propionibacterium freudenreichii conditions typical of cheese-making, and produced extracellularly growth stimulator(s) evaluated the survival of the selected strains for bifidobacteria, which appeared to be dif- during storage in the presence of starters ferent from propionic acid. A bifidogenic used for cheese-making. The bifidobacteria growth stimulator produced by P. freuden- species differed in their ability to grow in reichii was purified by Mori et al. [47]. The different media, including milk, rennetted chemical structure of the bifidogenic growth milk, and acidified milk in the presence of stimulator was 2-amino-3-carboxy-1,4- starter cultures and in their ability to naphthoquinone (ACNQ). It has been produce acetic and lactic acids. B. longum shown that this molecule plays a role to strains demonstrated high survival rates in 46 D. Roy [60] [12] [26] [79] [23] [18] [44] [13] [24] [4, 5] [4,

–1 cheese), cfu·g gradually –1 5 –1 cheese –1 –1 during storage cfu·g B. longum longum B. 8 cfu·g 7 at concentrations up up to at concentrations to 10 o log to 0.75 to 10 × 6 decreased one by log cycle lost up to 1 log during 70 d 70 during log 1 to up lost during first week, but stable week, but during first were reduced to 10 Lb. acidophilus Lb. were at log 8.05 and were at 8.05 log decreases in colony counts weredecreases in colony Bifidobacterium infantis Bifidobacterium , reaching a value , reachingof a value within 70 d of ripening –1 B. longum B. B. infantis viability drops between 0.5 and 3 log and 3 log between 0.5 drops viability cfu·g L. acidophilus acidophilus L. 7 B. longum at 56 d Lb. acidophilus Lb. increased from 10 for at least 90 d of ripening. ripening. at least d of 90 for –1 , respectively, whichnumbers were similarto respectively, , and and cells survived in cheeses cells survived –1 survived at high numbers (×10 numbers at high survived –1 –1 and decreased by one to tw decreased by . Lower viability of . Lower cfu·g B. bifidum B. 7 B. lactis B. bifidum bifidum B. 1. after 36 d of storage 12 °C storage at 4 or d of after 36 B. lactis showed a lower survival a lower showed log cfu·g 5.0 B. bifidum 6 log 6 log cfu·g lower than 1 log order for bifidobacteria and null for for and null bifidobacteria for 1 log order than lower L. casei while numbers of to 0.6 to 3 × 10 to 0.6 during 24 wks storage 24 wks storage during 2. cheese, following six months ripening of six months cheese, following log of 0.5 Loss 3. wks 11 the following during found at 1 d of ripening. ripening. d of at 1 found decreased to a final cell number of log 5.23 cfu·g 5.23 of log cell number to a final decreased Stability during storage Ref. 7.12 cfu·g 7.12 of storage storage of Bifidobacterium Bifidobacterium at 4 storage 14 d of during strain of a function as °C B. lactis 10 14 d d 168 d 184 84 d 57 d57 log between 5 and 6 dropped viable counts Bifidobacteria 70 d 90 d 60 d60 the storage, d of 60 After time sp.), and , B. infantis , L. acidophilus acidophilus L. sp. as starters 70 d B. bifidum B. Bifidobacterium Bifidobacterium B. infantis and 1% wt/wt) of bifidobac- of wt/wt) 1% , and , and B. bifidum B. B. lactis B. longum B. B. longum L. casei Bifidobacterium or or or fermented by B. bifidum , of milk from a freeze-dried powder of milk from a freeze-dried powder and and –1 added to salted, milled curds of milk from of milk from a freeze-dried powder –1 –1 incorporated individually or as multispecies as or incorporated individually grown in the cream dressing grown 14 d B. lactis B. breve B. breve , or both both species , or cfu·mL B. longum B. 8 cfu of fresh cells per gram of gram cells per of fresh cfu cfu·mL cfu·mL 6 7 8 B. bifidum bifidum B. B. longum longum B. mixtures and as free cells or as cells immobilized in alginategel culture of 3. Enriched cream culture of Starter concentrates of inoculum levels added at different 10 B. longum B. Frozen cultures (inoculum of teria ( L. acidophilus 2. 10 10 B. infantis Addition mode Storage L. acidophilus Semi-hard goat cheese Fresh cheese Argentinian Argentinian Fresco cheese Cheddar 1. Canestrato Pugliese Cottage Cheese Gouda Crescenza 10

Use and stability of bifidobacteria in cheeses. bifidobacteria of Use and stability L. casei L. L. acidophilus Bifidobacterium Probiotics of Type Bifidobacterium Bifidobacterium and Bifidobacterium, Bifidobacterium, L. acidophilus and Table I. The use of bifidobacteria 47 the presence of mesophilic starters would uation of the viability of bifidobacteria in be acceptable for use in cheese-making. several types of cheeses, B. bifidum and However, B. adolescentis strains were B. longum were among the strains shown to unable to grow in conditions typical of demonstrate good viability through the cheese-making. In cheese manufacturing, processing and storage of the cheese. How- Dinakar and Mistry [18] were concerned ever, B. longum subsp. infantis and B. ado- that the addition of B. bifidum with the lescentis demonstrated poor survivability starter might be detrimental to the culture and would be less preferable for incorpora- because of the aerobic conditions of cheese- tion into dairy products. making, the cooking conditions and the In cream cheeses produced with four cul- presence of the mesophilic starter. They tures of bifidobacteria (B. breve, B. longum thus opted for addition of dried bifidobac- subsp. infantis, B. longum and B. bifidum), teria to the milled curds. Stanton et al. [72] the growth of bifidobacteria during cheese- indeed found that the bifidobacteria did not making was not affected by the presence of grow during cheese-making, presumably Lc. lactis subsp. lactis and Lc. lactis subsp. because of the redox conditions (filling of cremoris in association or not with Lc. lactis the cheese vat with milk aerates the milk). subsp. lactis biovar diacetylactis and Leuco- Although in general, the results obtained nostoc mesenteroides. Addition of native for incorporation of bifidobacteria in phosphocaseinate to increase milk protein cheese are promising, the success of addi- content and hence buffering capacity did tion of probiotic bacteria depends on spe- not result in an increase of growth of bifi- cies used as well as interactions with lactic dobacteria in cream cheese [1]. Cream starters, conditions of fermentation, final dressing added to Cottage cheese was fer- pH of the product, presence of oxygen, and mented by adding B. longum subsp. infantis. storage temperature. Several factors which The buffering capacity of the cream was can influence the capacity of probiotic to increased by the use of ultrafiltered reten- survive in cheese and to remain active must tate of milk [4]. Higher level of lactic acid be considered. These factors include (1) production was observed whereas the addi- physiological state of probiotic cultures (if tion of a tryptic casein hydrolyzate did not the cells are in logarithmic or stationary increase the fermentation rate of cream [4]. phase of growth), (2) physical conditions of storage of the product (for example, tem- The temperature of the production of perature), (3) chemical composition of the fresh cheese was the principal factor which product to which probiotics are added (for has affected the growth of the bifidobacte- example, acidity, content of sugars availa- ria. The presence of bifidobacteria during ble, nitrogen sources, content, the fermentation of milk did not influence water activity and oxygen content), and (4) the growth of the mixed cultures of lactic possible interactions between probiotic cul- starters. However, the presence of bifido- tures and starters (for example, bacteriocin bacteria modified the final characteristics production, antagonism and synergism). of cream cheese. A slight modification of Four types different of interactions between the process such as a temperature of manu- probiotic and lactic starters are possible facture of 35 °C instead of 30 °C is thus (stimulation, delay and inhibition of the favourable to the growth of bifidobacteria growth and any effect). The interactions without affecting that of lactic leuconostocs between probiotic and the cheese matrix and other bacteria in cream cheeses [1]. The and starters can be much more intense when survival reduction survival by the temper- probiotic bacteria are also used in the same ature of cooking must also be taken into time that starters. Careful strain selection is account at the time of the manufacture and mandatory to warrant the survival of bifi- during the period of maturation of cheese dobacteria in the cheese matrix. In the eval- such as Cheddar cheeses [13]. 48 D. Roy

Daigle et al. [13] did not observe any temperatures remains a problem in most of growth of B. longum subsp. infantis during the fermented products, which are acidic in different steps of manufacture of Cheddar nature. Several factors have been claimed to cheese. Ten minutes after addition of affect the viability of probiotic cultures in cheese starters, bifidobacteria counts were . Acidity, pH and the same in the cheddar cheese-making hydrogen peroxide have been identified to milk as in the inoculum. After cooking, have an effect during manufacture and stor- there was no decrease of the viable counts age. Other factors, such as temperature of of bifidobacteria. There was an increase in storage, oxygen content, concentrations of the number of cells in the as expected. lactic acid and acetic acid also have been Bifidobacteria counts in curds were of presumed to affect the viability of bifido- 7.3 log cfu·g–1 in curd and of 5.7 log cfu·g–1 bacteria [15, 68]. in [13]. These results were in agree- In yoghurt-like product, pH, oxygen and ment with those of Gomes et al. [26] who starter production conditions have signifi- observed no growth of Bifidobacterium sp. cant effects. The main factors for loss of strain Bo that was used as starter for the viability of bifidobacteria have been attrib- manufacture of Gouda cheese [26]. uted to the decrease in the pH of the medium Although the most common processing and accumulation of organic acids as a stage at which to add the bifidobacteria to result of growth and fermentation [68]. the milk would be in conjunction with the Hence, the manufacture of these products other starter cultures, other alternative containing bifidobacteria requires the selection processing protocols have been successful of strain with low susceptibility to acid [35]. in incorporating the bifidobacteria into Previous studies showed that not all strains cheese and cultured dairy products. Many of bifidobacteria exhibited equivalent sta- of these alternative protocols have been bility during storage of dairy products [35, used to develop Cheddar cheese with bifi- 70] suggesting that performance of strains dobacteria. Dinakar and Mistry [18] added should be evaluated individually prior to commercial or immobilized freeze-dried commercial use in new dairy fermented strains of B. bifidum to the matrix of Ched- dar cheese, following cheddaring and salt- products. Viability of bifidobacteria and lactic acid bacteria in milk and yoghurt-like ing. In the production of Cheddar cheese, B. 6 longum subsp. infantis was initially cul- product remained above 10 cfu/ml or g tured in the cream prior to adding to the until the expiration date of the respective products sold in USA [70]. High bifidobac- skim milk and inoculating with the lacto- 6 –1 cocci starter cultures [13]. The production teria counts (>10 cfu·g ) were also of acetic and lactic acids in the cream prior observed in commercial yoghurt-like prod- to the initiation of cheese-making reduced ucts manufactured and sold in Germany and the time required for curd formation. At the France even in yoghurt-like products with beginning of storage of Cheddar cheese, a pH as low as 4.0, suggesting selection of Daigle et al. [13] observed that viable count more acid-tolerant strains by the industry in of bifidobacteria was 7.3 log cfu·g–1 which these countries. Indeed, most bifidobacteria was similar to the number of bifidobacteria strains isolated from Central European and estimated in curd at the draining. These French milk products were classified and results indicate that viability B. longum identified as B. animalis subsp. lactis which subsp. infantis did not decrease during showed identity to B. animalis ATCC cheese-making process. 27536 [59]. The survival of B. bifidum, B. breve, 4.2. Stability during storage B. longum subsp. infantis and B. longum was Viability of bifidobacteria in dairy prod- drastically affected during storage of yoghurt- ucts over a long shelf-life at refrigeration like product, except for yoghurt-like product The use of bifidobacteria 49 with B. animalis used because viable counts dobacteria may thus be reduced if the pH of remain >106 cfu·g–1 after 28 d of storage at the final product is high or the ratio of strep- 4 °C [37]. The level of bifidobacteria were tococci and lactobacilli is modified. This <106 cfu·g–1 after 7 d of storage in yoghurt- inhibitory effect of starters on probiotics is like products made with B. bifidum, B. breve, major and, in commercial products, low B. longum subsp. infantis and B. longum. levels of bifidobacteria are correlated the These results are in agreement with previ- addition of the starters used for technolog- ous studies because the viable cells of bifi- ical purposes. This has led to the develop- dobacteria in yoghurt-like product could ment of fermented milks where the probi- not be maintained in sufficient amounts otic cultures are the only starters added. A (>106 cfu·g–1) for more than 1 wk during few strategies have been developed to try to storage at 4 °C [37, 61]. Enhancement of reduce the strong inhibiting effect of the bifidobacterial cold- and/or acid-tolerance starter strains on the probiotic cultures: could increase the number of viable bifido- omission of a portion of the starter strains, bacteria ingested via refrigerated dairy and changes in the respective inoculation products [43]. It has been observed that the rates are the most common. It appears non-commercial species, B. longum, gener- important to select lactobacilli that have ally had less cold- and acid-tolerance than weak post-acidification properties, or to the commercially used B. animalis subsp. reduce and even exclude L. delbrueckii lactis. The two species also responded very subsp. bulgaricus from the starter. Exam- differently to cold-tolerance and acid-tolerance ples of such cultures are the so called ABT tests after exposure to multiple-adaptation cultures (ABT standing for L. acidophilus, and standard treatments. These differences Bifidobacterium and S. thermophilus) [60]. indicate that stress-responses can be species- dependent and that probiotic bifidobacteria Many studies suggest that consumption strains should be carefully chosen for use in of synbiotic products has higher beneficial various foods or dietary supplements. The effects on the human health than probiotic yoghurt fermentation process itself could or products [22, 55, 56]. Prebiotic be considered an acid adaptation treatment has been defined as “a nondigestible food (pH drops from 6.5 to 4.7 in about 5 h) [43]. ingredient that beneficially affects the host The yoghurt-like product environment was by selectively stimulating the growth and/ shown to effectively enhance subsequent or activity of one or a limited number of acid-tolerance of both untreated and treated bacteria in the colon” [20]. In this context, B. animalis subsp. lactis, suggesting that a prebiotic is a dietary ingredient that acid-tolerance of certain probiotic bifido- reaches the large intestine in an intact form bacteria would be enhanced by addition of and has a specific metabolism therein, one cells at the start of fermentation [43]. directed toward beneficial rather than The problem of sensitivity to acidity of harmful bacteria. are mixtures bifidobacteria in yoghurt-like product is of prebiotics and probiotics that would ben- increased by the fact that acidity may efit the host by improving survival and increase during storage. In yoghurt-like prod- implantation of the selected microbial sup- uct, acidification may continue during cold plements [83]. Indeed, the presence of probi- storage, a phenomenon called post-acidifi- otic and prebiotic in a single food improved cation, and the pH may drop to 3.6. Because survival of probiotic bacteria during the most strains of bifidobacteria are sensitive storage of the product and during the pas- to pH values below 4.6, in practical sage of the intestinal tract. Moreover, the applications, the pH value of the final product synbiotic product may allow an efficient must be maintained above 4.6, otherwise implantation of probiotic bacteria in colonic the bifidobacterial population will decline microbiota because prebiotic has a stimu- rapidly. Hence, the loss of viability of bifi- lating effect on the growth and/or activities 50 D. Roy of the exogenous and the endogenous bac- Viability of bifidobacteria in cottage cheese teria [55]. declined rapidly after 15 days of storage at Synbiotic dairy products are already 4 °C because the pH and titratable acidity marketed in Europe and Japan. In synbiotic of this product did not favor the bifidobac- fermented milks, the strains of Lactobacil- teria survival (Tab. I). In fact, strains of bifi- lus acidophilus, and dobacteria do not have the same stability Bifidobacterium subsp. (B. animalis, B. bifi- during maturation and storage of cheeses, dum, B. breve, B. longum subsp. infantis and suggesting that the survival of strains must B. longum) are widely used as probiotic be evaluated individually in the various whereas fructo-, galacto- types of cheese before their commercial oligosaccharides, lactulose and inulin-derived use. In Cottage cheese, the survival of products are widely used as prebiotic [34]. B. longum subsp. infantis was moderate [5]. The use of a mixture of probiotic and preb- In Cheddar cheese, B. longum subsp. infantis iotic results in an increasing of cost produc- and B. bifidum remained viable during 12 tion that can slow down the development of and 24 wks, respectively [13, 18]. Daigle new synbiotic products. The use of probi- et al. [13] did not observe any significant otic bacteria, which are able to synthesize difference for the viable count of bifidobacte- prebiotics might overcome this limitation. ria during storage at 4 °C. Through a 12-week Many authors reported the capacity of bifi- storage period, counts of bifidobacteria did dobacteria to synthesize galacto-oligosac- not change significantly and were main- charides [30, 37, 62]. tained at levels higher than 106 cfu·g–1 of High survival rates of bifidobacteria can cheese. The B. lactis Bb-12 strain survived at be obtained in none or low fermented prod- high number (>108 cfu·g–1 of cheese) as ucts such as fluid milk, ice cream, low-acid compared to B. longum BB536 (105 cfu·g–1) yoghurt-like product and cheese. It is rec- after six months of ripening of Cheddar ognized that Cheddar has a pH of 5.2, fresh [44]. B. bifidum and B. longum exhibited cheeses are at 4.6, and yoghurt-like product better survival in Crescenza cheese than has a pH of 4.2. During ripening, the pH of B. longum subsp. infantis during a period of cheeses such as Camembert rises some- 14 d of storage. At the first day after man- times over 6.0. Ice cream and milk have pH ufacture of white-brined cheese, while the values close to 6.5. Therefore there are sub- counts of B. bifidum BB-02 in cheeses inoc- stantial variations in the pH of dairy prod- ulated at levels of 2.5% and 5.0% were 7.0 × ucts, even within those that are fermented. 108 cfu·g–1 and 1.2 × 109 cfu·g–1, respec- The success of the incorporation of tively, these figures dropped to 4.0 × bifidobacteria into cheeses is dependent on 106 cfu·g–1 and 1.1 × 107 cfu·g–1 after 90 d the bifidobacteria strains, the activity of in the same order [81]. The traditional tech- lactic acid bacteria used in the manufacture nology of manufacture of Canestrato Pugliese of the cheese, the composition of the cheese made of ewe’s milk was slightly cheese, and the conditions of processing modified to support the survival of the pro- and ripening. Although numerous studies biotic bacteria inoculated at a rate of 7.0 log report great losses in viability of probiotic cfu·g–1. After 56 d of maturation, the sur- strains during the storage of yoghurt-like vival of B. bifidum and B. longum was 6.0 product, the data on cheese shows that bifi- and 5.0 log cfu·g–1, respectively [12]. The dobacteria incorporated can be stable dur- use of bifidobacteria and L. acidophilus as ing storage (Tab. I). Thus, the pH value probiotic culture or starter for the manufac- seems to be a critical factor in the stability ture of half-firm goat’s milk cheese and of probiotic strains during storage. There Gouda showed that these probiotic bacteria are significant differences between species had a satisfactory viability (106 cfu·g–1) and strains with respect to survival in an during at least 9 wks [24, 26]. Finally, the acid environment. survival of the bifidobacteria, L. acidophilus The use of bifidobacteria 51 and L. casei was also satisfactory in the Fresco dobacteria while a temperature of 16 °C has cheese. After 60 d of storage, the reductions in little effect on survival of those, in particu- viable counts were lower than one log cfu·g–1 lar for B. bifidum (Tab. II). The various lac- for bifidobacteria [78]. tic starter cocktails have few effects on the Blanchette et al. [5] observed that in the fate of bifidobacteria. However, it has been Cottage cheese that the probiotic culture of observed that the presence of B. bifidum B. longum subsp. infantis introduced at the during the period of storage led to a delay time of the addition of the cream to the of growth of propionibacteria (Tab. III). cheese curds could reach 7.0 log cfu·g–1 of Those developed more quickly at 22 °C cheese after 1 d of storage, but they although the final number is not different observed a fast decline of their viability between the two temperatures for the warm after 15 d of storage to 4 °C. The pH and room. titratable acidity of this product did not favour the survival of this strain of bifido- 4.3. Changes in sensory properties bacteria. However, these authors observed that this strain modified the content lactose Changes in the chemical composition significantly in the Cheese Cottage [5]. and the texture of the fermented products Roy et al. [60] used strains of B. breve can occur in cheeses and fermented milks. and B. longum to produce a fresh cheese in In yoghurt-like product, it is possible to combination with strains of Lactococcus observe that the level of metabolites lactis subsp. lactis and Lc. lactis subsp. cre- (mainly acetic acid) produced by the bacte- moris. The final pH of cheese was higher rial strain(s) can influence the organoleptic than that of the cheese Cottage (pH 4.6). assessment [76]. Stanton et al. [72] noted The cheeses were stored at two tempera- that no difference between cheeses pro- tures (4 and 12 °C). The populations of bifi- duced with or without B. infantis with dobacteria could be maintained longer at a regard to chemical and organoleptic prop- rate higher than 6 log cfu·g–1. The survival erties of cheeses was obtained. In Cheddar of B. breve and B. longum was higher at cheese, bifidobacteria remained metabolically 12 °C than at 4 °C because, the survival of active during the storage period, but did not lactococci was lower at 12 °C. These adversely affect the sensory characteristics authors also noted that the presence of bifi- and composition of the cheese without any dobacteria had an effect on the biochemical adverse impact on organoleptic properties changes of fresh cheese. The lactose con- [13]. The residual lactose in cream cheese centration decreased more strongly in containing B. breve, B. bifidum and cheeses containing bifidobacteria than in B. longum subsp. infantis was lower than control cheese [60]. that of cheese made without bifidobacteria The survival of various strains of bifido- and those containing B. longum. The cream bacteria in Swiss-type cheeses has been cheese without bifidobacteria contained studied (Roy D., Mainville I., Bélanger G., less acetaldehyde than those with bifidobac- unpublished). The process had to be modified teria. The concentration of diacetyl was in order to be able to add the bifidobacteria lower in the presence of bifidobacteria. The to cheeses. The modifications made to the concentration of ethanol increased when original process were the reduction of the bifidobacteria were employed with the temperature of cooking of 53.5 °C with mixed cultures of lactococci, aromatic lac- 41 °C and addition of a delactosing step in tococci and leuconostocs [1]. The higher order to create a shock, similar to that of the amount of acetaldehyde might be due to the temperature of cooking on starters. The use metabolism of bifidobacteria. Yuguchi et al. of a temperature of the warm room of 22 °C [82] mentioned that the strains B. longum, B. has a drastic effect on survival of the bifi- bifidum and B. breve produced acetaldehyde. 52 D. Roy

Table II. Fate of during manufacture of Swiss-type cheeses (Roy D., Mainville I., Bélanger G., unpublished).

Starter cultures Warm room Viable counts temperature (log cfu·g–1) Ripening period (days) 1 2236435071 TA 8.42 7.89 7.83 7.41 7.10 7.17 TA+MA 8.49 8.10 7.67 7.40 7.00 6.75 16 °C TA+LH 8.48 8.15 7.60 7.61 7.36 7.43 TA+MA+LH 8.66 8.15 7.94 7.52 7.12 6.60

TA 8.41 7.90 6.56 5.37 4.64 3.26 TA+MA 8.44 7.98 5.60 3.78 3.43 2.92 22 °C TA+LH 8.30 8.00 6.49 5.69 2.84 2.00 TA+MA+LH 8.24 8.24 5.88 4.98 4.39 3.63 Starters used: TA: Streptococcus salivarius subsp. thermophilus (TA 060); MA: Lactococcus lactis subsp. lactis and L. lactis subsp. lactis (MA 011); LH: Lactobacillus helveticus and L. delbrueckii subsp. lactis (LH 100), Propionibacterium freudenrei- chii subsp. shermanii, B. bifidum R071. Small scale experimental Swiss cheeses were made from pasteurized milk by a standard procedure des- cribed previously [9, 32]. Eight cheeses were produced and three repetitions were carried out. Samples were taken just after brining (day 1), at the end (day 22) of the time in the cold room (12 °C), twice (days 36–43) during the time in the warm room (16 °C or 22 °C), at the end (day 50) of the time in the warm room, and after 21 days at cold storage (day 71).

The production of acetaldehyde by bifido- are not identified. If the product can offer bacteria and the transformation of this prod- something measurable to the consumer in uct into ethanol by the leuconostocs and the long term, there is a greater chance of their ability to transform lactose into pyru- repeat buying [73]. Sensorial attributes in vate or ethanol can also explain the greater this respect are as important as health ethanol concentration in cream cheeses attributes. containing bifidobacteria as compared with those without bifidobacteria. Finally, the economical consequences 5. CONCLUSION such as price, market positioning, advertising, and understanding of market conditions and The functional cheese may be more consumer attitudes for the production of the effective than yoghurt-like product to functional cheeses are not well-known but deliver probiotic bacteria to the intestinal it is important to take in consideration that tract. It is possible to include bifidobacteria the interest in and acceptance of functional in the traditional production of Cheddar foods is gaining momentum. A strong health cheese and fresh cheeses. It is also possible claim or inference, although being an impor- to produce Swiss-type cheese after slight tant purchase motivator, does not guarantee modifications. Bifidobacteria do not dete- a product’s success. The customer must be riorate the product, enhance the develop- encouraged to repeat the purchase, which ment of flavours in fresh cheeses and give may not happen if the benefits of the product to the products a functional character. The use of bifidobacteria 53

Table III. Fate of Propionibacterium freudenreichii subsp. shermanii during manufacture of Swiss-type cheeses (Roy D., Mainville I., Bélanger G., unpublished).

Starter cultures Warm room Viable counts temperature (log cfu·g–1)

Ripening period (days) 1 2236435071 Control TA 6.23 8.81 8.78 8.90 8.90 8.89 TA+MA 6.28 7.32 8.68 8.97 9.08 9.00 TA+LH 6.26 7.54 8.79 9.00 8.90 8.97 TA+MA+LH 6.34 7.50 8.68 8.61 9.00 9.00 16 °C B. bifidum TA 6.29 7.94 8.40 8.70 8.72 8.90 TA+MA 6.27 7.30 8.73 8.67 8.90 8.95 TA+LH 6.33 6.78 8.30 8.58 8.68 8.81 TA+MA+LH 6.35 7.26 8.47 8.56 8.64 8.76

Control TA 6.26 8.52 9.22 9.23 9.14 9.05 TA+MA 6.40 7.87 9.26 9.33 9.12 8.98 TA+LH 6.18 7.74 9.19 9.04 8.96 8.93 TA+MA+LH 6.31 7.94 9.21 9.06 8.92 8.68 22 °C B. bifidum TA 6.36 7.98 9.22 9.22 9.06 8.96 TA+MA 6.33 8.08 9.27 9.23 8.96 8.93 TA+LH 6.27 7.32 9.02 8.93 8.88 8.88 TA+MA+LH 6.39 7.16 9.01 8.89 8.84 8.63 Starters used: TA: Streptococcus salivarius subsp. thermophilus (TA 060); MA: Lactococcus lactis subsp. lactis and L. lactis subsp. lactis (MA 011); LH: Lactobacillus helveticus and L. delbrueckii subsp. lactis (LH 100), Propionibacterium freudenrei- chii subsp. shermanii, B. bifidum R071. Small scale experimental Swiss cheeses were made from 10 pasteurized milk by a standard procedure described previously [9, 32]. Eight cheeses were produced and three repetitions were carried out. Sam- ples were taken just after brining (day 1), at the end (day 22) of the time in the cold room (12 °C), twice (days 36–43) during the time in the warm room (16 °C or 22 °C), at the end (day 50) of the time in the warm room, and after 21 days at cold storage (day 71).

Acknowledgements: I thank Sylvie Lortal and REFERENCES the organisers of the first Symposium on Propi- [1] Baron M., Roy D., Vuillemard J.C., Bio- onibacteria and Bifidobacteria who invited me chemical characteristics of fermented milk to present my work. I would like also thank Fran- produced by mixed-cultures of lactic starters cine Mondou for her scientific assistance and and bifidobacteria, Lait 80 (2000) 465–478. Isabelle Mainville and Gaétan Bélanger for their [2] Berrada N., Lemeland J.-F., Laroche G., technical assistance. Thouvenot P., Piaia M., Bifidobacterium 54 D. Roy

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