Challenges in the Addition of Probiotic Cultures to Foods

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CLAUDE P. CHAMPAGNE and NANCY J. GARDNER. Food Research and Development Center, Agriculture and Agri-Food Canada, St-Hyacinthe, Quebec, ...
Critical Reviews in Food Science and Nutrition, 45:61–84 (2005) C Taylor and Francis Inc. Copyright  ISSN: 1040-8398 DOI: 10.1080/10408690590900144

Challenges in the Addition of Probiotic Cultures to Foods CLAUDE P. CHAMPAGNE and NANCY J. GARDNER Food Research and Development Center, Agriculture and Agri-Food Canada, St-Hyacinthe, Quebec, Canada

DENIS ROY INAF, Universit´e Laval, Quebec, Canada

Probiotic cultures are increasingly being added to foods in order to develop products with health-promoting properties. Although the literature is abundant on the beneficial effects of bifidobacteria and Lactobacillus acidophilus on health, little information is available on the challenges industry faces in adding these probiotic cultures to food products. The aim of this article is to examine seven issues that should be addressed when developing functional foods: 1) type or form of probiotic that should be used; 2) addition level required to have a beneficial effect; 3) toxicity; 4) effect of the processing steps on viability; 5) determination, in the product, of the cell populations added; 6) stability during storage; 7) changes in sensory properties of the foods. Keywords

bifidobacteria, flavor, lactobacilli, processing, stability, storage, technology

PROBIOTICS AND FOODS

intestinal metabolism. Recent advances in research in intestinal flora are the background for the appearance of functional foods.10 Functional foods can be defined as any food that may provide a health benefit beyond the traditional nutrients they contain. Such foods may contain one or a combination of components that have desirable cellular or physiological effects on the body.11 Functional foods can include probiotics, prebiotics, and synbiotics. The dairy industry, in particular, has found probiotic cultures to be a tool for the development of new products. It is estimated that there are 80 probiotic-containing products in the world.12 Examples of commercial products that have been marketed with probiotics are found in Table 1, and other lists are found in the reviews of Gomes and Malcata,12 as well as Mital and Garg.13 Markets shares of probiotic products are increasing. In 1997, the market share of probiotic yogurts was between 5% and 20% of that of total yogurts in Europe,14 but it has now reached 25% in France, Germany and Sweden,12 Today in the U.S., approximately 60% of refrigerated yogurts contain the probiotic cultures, Lactobacillus acidophilus and/or Bifidobacterium spp.15 Reported microbial contents of commercial products are presented in Table 1, but this is only aimed at serving as examples, since data differ as to their exact species contents.16 Dairy products appear to be good vehicles for the delivery of probiotics to humans. The dairy industry in Japan, South Korea, European, North African, North, Central, and South American countries uses bifidobacteria to produce fermented milks that are less sour than traditional yogurt. Fermented milks containing

The lactic acid bacteria (LAB) have long been used for the fermentation of milk (cheese, yogurt), meats (dry sausages), cereals (sourdough), vegetables (sauerkraut), and fruits (wine). The original aim of fermentation by LAB was to preserve foods by acidifying them, or to develop specific flavors or textures. However, there is increasing evidence that the consumption of LAB may affect the composition of indigenous microflora and have several beneficial effects on the human health. Many studies have examined the potential role of LAB on the improvement of lactose assimilation, food digestibility, hypercholesterolaemia, and immune response, or on the prevention of intestinal infections, vaginal infections, cancer, food allergies, and constipation.1–6 However, it is important to stress that any postulated benefits from consumption of probiotics should be accepted only after extensive testing in human clinical studies.7 Food cultures that have such beneficial effects on human health have been termed “probiotic.” Although definitions vary,8 probiotics can be defined as “live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance.”9 The incorporation of bacteria of the intestinal origin into human diet corresponds to the emergence of a new generation of food products that use the beneficial effects of these bacteria on Address correspondence to C.P. Champagne, Food Research and Development Center, Agriculture and Agri-Food Canada, 3600 Casavabt, Saint-Hyacinthe, QC J2S 8E3, Canada.

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62 Table 1

C. P. CHAMPAGNE ET AL. Examples of dairy foods that contain probiotic cultures (adapted from Tamime and Robinson176 ) Mainly as starter thermophilus1

Examples of commercial products

S.

Lb.

Lunebest, Mil-Mil Olifus Biogarde, Aktifit Progurt BA, Biobest Biokys Gaio, Praghurt Bioghurt Bifighurt, Yoke AB, Cultura, LA7, Miru-Miru, Bifilact, BRA, Symbalance Bifilact, Bifilus, Onaka, Procult, BBA Pro Viva, Yakult, Prima Liv, LCI, Fysig

  







  



bulgaricus2

Mainly as a probiotic adjunct culture Lc.  

lactis3

Lactobacillus

Bifidobacterium

   

     

   

Enterococcus



Pediococcus



  

1 Streptococcus

thermophilus delbrueckii ssp bulgaricus 3 Lactococcus lactis 2 Lactobacillus

B. longum or B. breve have obtained ‘foods for specific health use (FOSHU)’ approval in Japan. FOSHU foods are defined by the Japanese Ministry of Health and Welfare as “processed foods containing ingredients that aid specific bodily functions, as well as being nutritious.” Lactic acid bacteria, including B. longum and B. breve, are one of the twelve categories of healthenhancing ingredients and are recognized to regulate intestinal microflora and allow a healthier gastrointestinal condition. The high buffering capacity of milk has been helpful in protecting the cells against the acid secretions of the stomach.4 The fat in cheese could also provide some protection.17 After the dairy products, soya-based products seem to be the most appropriate carrier for probiotic cultures. Soymilk is a popular drink in East Asian countries,13 and its consumption is on the rise in North America. Some probiotic cultures, particularly the bifidobacteria, assimilate raffinose, stachyose, and sugars that may cause flatulence. Therefore, soymilk fermentation with probiotic strains can have the triple benefits of preserving the product, reducing flatulence-causing sugars, and improving health. Other fermented foods that have been examined with respect to their potential in carrying probiotic cultures include kimchi,18 mayonnaise,19 meats,20,21 baby foods,22 confectionary,22 edible spreads,23 plant seed extracts, such as cowpea, sorghum, and peanut,24 cucumber juice,25 catfish fillets,26 and fish sausages.27 Novel foods with probiotics are particularly numerous in Japan. They are malted milk, milk powder, sweets, cakes, bavaroise, chewing gums, various fibre preparations, and even beer.28 The improvement of foods for specific health purposes is not limited to supplementation by probiotic cultures. There is much activity in the development of functional foods via the addition of active compounds, often referred to as “nutraceuticals.” Calcium29 or antioxidants30 are examples of nutraceuticals used for the development new functional foods. Many excellent reviews have been published on the health benefits of probiotics or nutraceuticals, but only a few have addressed the technological challenge associated with the devel-

opment of functional foods with probiotics.1,13,22,31–33 Whether one wishes to supplement a beverage with antioxidants or probiotic cultures, one has to face the same challenges. At least seven questions must be addressed in either case: 1. 2. 3. 4. 5.

What type or form of ingredient/probiotic should be selected? How much must be added to have a beneficial effect? Are there toxicity issues? Are they destroyed during processing? Can the concentration of the active compounds (strains or cell populations in the case of probiotics) be determined? 6. Are the nutraceuticals/probiotics stable during storage? 7. Does supplementation bring changes in sensory properties? The aim of this article is to address these issues.

PROBIOTIC CULTURES USED IN FOODS Many lactobacilli are used in functional foods (Table 2). However, bifidobacteria are emerging as possibly one of the most important groups of intestinal organisms regarding human health. It is estimated that over 400 species of bacteria inhabit the human gastrointestinal tract, and the Bifidobacterium species belongs to the dominant anaerobic flora of the colon. Twenty-nine species of bifidobacteria are now included in this genus, and nine of these species have been found in the intestine of man and/or as human clinical isolates. The remaining 20 species have been isolated from fermented milk, the intestinal tracts of various animals and honeybees, and also found is sewage and anaerobic digesters. Bifidobacteria constitute 5% to 10% of the total colonies flora of healthy children and adults. In the days following birth, infants have intestinal flora dominated by bifidobacteria. With age and changes in dietary habits, bifidobacteria tend to be suppressed by other microorganisms, and their population even decreases in humans late in life.10 The main species present

PROBIOTICS IN FOODS Table 2 Potentially probiotic cultures used as nutraceuticals and/or in fermented milks (adapted from Tamime and Robinson176 ; Shah177 and Mercenier et al.52 ) Genera

Species

Lactobacillus

acidophilus delbrueckii subsp. bulgaricus casei crispatus johnsonii lactis paracasei fermentum plantarum rhamnosus reuteri salivarius adolescentis bifidum breve essensis infantis lactis longum faecalis faecium acidilactici freudenreichii boulardii thermophilus

Bifidobacterium

Enterococcus Pediococcus Propioniobacterium Saccharomyces Streptococcus

in humans are Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum in the colon. Their presence in the human intestine is almost universally accepted to be a contributing factor to a healthy well-being. Lactobacilli have also become important as adjunct cultures for the production of probiotic foods.34 Lactobacillus species are essential to the dairy industry in cheeses, yogurt, and other fluid products.35 Species of this genus can also be found in the gastrointestinal tract of human and animal.36 However, DNA homology data revealed that some strains belonging to Lactobacillus acidophilus had been misclassified.34,37 Thus, it seems important that probiotic products should contain well characterized strains in an effort to understand the factors that dictate probiotic functionality and host benefits. Lactobacillus acidophilus, which is a natural inhabitant of mammalian gastrointestinal systems, is of considerable industrial and medical interest, because this species is believed to play an important role in human health and nutrition by its influence on the intestinal flora. However, the systematics of this species, which exhibits heterogeneity as reflected by phenotypic characterization and DNADNA hybridization, remained confusing for a long time.38 The Lb.acidophilus complex strains were divided into six groups at the species level. Presently, Lb. acidophilus, Lb. crispatus, Lb. amylovorus, Lb. gallinarum, Lb. gasseri, and Lb. johnsonii have been proposed as valid species in the L. acidophilus group. Strains of the Lactobacillus acidophilus complex are widely used as starter or probiotic cultures for the production of fer-

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mented milks, but only Lb. gasseri, Lb. johnsonii, and, in part, Lb. acidophilus, and Lb. crispatus are used as probiotics.36 With respect to Lb. casei–related species, strains were divided into three species (Lb. casei, Lb. paracasei, and Lb. rhamnosus), according to DNA-DNA relatedness by Collins et al.39 The Lb. casei group might also comprise the recently revived Lb. zeae species.36,40 It must be emphasized that the strain itself is an important element in the selection of a probiotic culture. The investigation of probiotics has focused primarily on lactic acid bacteria and bifidobacteria. Propionibacteria do not belong to the group of lactic acid bacteria, but are increasingly being considered as potential probiotics.41 They are generally divided into dairy and cutaneous groups. The dairy propionibacteria are used to produce typical flavor and eyes in swiss-type cheese. The dairy propionibacteria include the species P. freudenreichii, P. jensenii, P. thoenii, and P. acidipropionici. These species are not typical of human microflora.42 The production of vitamin B12 , CO2 , bacteriocins, and organic acids are associated with probiotic effects of propionibacteria. Three bacteriocins were isolated from propionibacteria, and their mineral content was higher than the ones from lactic acid bacteria or bifidobacteria, contributing to their probiotic characteristics.41 There is considerable interest in the use of propionibacteria as probiotics ingredients in animal feeds or as dietary adjuncts to human foods. Zarate et al.43 showed that P. acidipropionici would be a good source of betagalactosidase in the intestine. In P. freudenreichii, a transient exposure to pH 5 increased the resistance toward acid stress,44 which shows that adaptation to acid stress is possible with this group of bacteria. Propionibacteria were shown to influence the metabolic activities of the intestinal microflora of mice.45 So far, the active use of yeasts as probiotics has been limited, despite the presence of yeasts as part of the microflora of many dairy products. Surplus yeast from breweries has been used widely in the animal feed industry for cattle, pigs, and poultry for improved performance and product quality46 S. boulardii has been used since the 1950s as a preventive and therapeutic agent for the treatment of a variety of diarrheal disease.47 Many antogonistic interactions have been reported between culture yeasts, especially the genus Saccharomyces, and enteric pathogens. Moreover, it was demonstrated that Saccharomyces can survive the passage through the intestinal tract.48

FACTORS THAT SHOULD BE CONSIDERED IN THE DEVELOPMENT OF PROBIOTIC-CONTAINING FOODS Strain Selection Strain selection is a critical aspect of developing a probioticbased functional food,22 and a list of potential criteria appears in Table 3. Unfortunately, not all strains can be easily produced industrially because of low yields in the growth media or poor survival to freezing or freeze-drying,33 and not all promising strains can be marketed. This explains why the producers of probiotics

64 Table 3

C. P. CHAMPAGNE ET AL. Some technological criteria for the selection of probiotic strains for supplementation to dairy foods

Industry

Criteria

Product

Reference

Suppliers of cultures

Inexpensive cultivation Ease of concentration to high cell densities Stability in dried or frozen from Growth in milk Can be produced at a large scale (ex. starter tanks) Compatibility with other lactic cultures

Cultures for all products Cultures for all products Cultures for all products Cheese, Acidophilus milk, yoghurt Products made in large manufacturing plants, such as cheese All fermented products

Stability during storage in acid conditions

Acidophilus milk, yoghurt, cheese

Stability during storage in unfermented milk Bacteriophage resistance Survival to cream aging and freezing Tolerance to preservatives Tolerance to high shear conditions Stability during storage at −20◦ C

Sweet acidophilus milk All fermented products Ice cream Non-sterilized products Edible spreads Ice cream, frozen products

Flavour and sensory properties Oxygen tolerance for growth Low activity below 15◦ C Assimilation of pentanal and n-hexanal

All products All fermented products All products Soya-based products

Fermentation of raffinose and stachyose

Soya-based products

Charteris et al.35 Charteris et al.55 Koch and Carnio53 Koch and Carnio53 ; Gomes and Malcata12 Gomes and Malcata12 Samona and Robinson91 Nighswonger et al.178 Micanel et al.88 Gobbetti et al.179 Brashears and Gilliland134 Richardson2 Christiansen et al.153 Charteris et al.55 Charteris et al.55 Modler et al.155 ; Christiansen et al.153 Koch and Carnio53 Gomes and Malcata12 Gomes and Malcata12 Scalabrini et al.180 ; Murti et al.96 Scalabrini et al.180

Users of cultures in foods and beverages

have their own requirements for probiotics (Table 3). Nevertheless, a wide range of species are available as probiotic cultures (Table 2). In addition to the species listed in Table 2, various other LAB are used in the production of fermented dairy products (Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc mesenteroides), but they do not have the reputation of growing in the gastro-intestinal (GI) tract and, thus, have lower probiotic potential. It should be remembered, however, that they reach high numbers in fermented milks, and that even if they do not grow in the GI tract, they may produce health-promoting metabolites, such as exopolysaccharides or peptides. The selection of a probiotic strain to be added in the product is a crucial step. Selection of probiotics can be based on general microbiological criteria that refer to safety, technology, performance, and health benefits49 (Table 3). Biological Properties Obviously, the first point is the desired biological effect. High viable counts and survival rates during the passage through the stomach are necessary to allow live bifidobacteria from the fermented milk products to play a biological role in the human intestine. Suppliers of probiotic strains have traditionally relied on extensive in-vitro testing of their cultures to select the most adapted strains. Survival to the acid conditions of the stomach and to bile salts are, thus, of prime concern. Many more properties have been examined in vitro, such as sensitivity to oxygen, stability during storage, resistance to proteases of the digestive system, sensitivity to lysozyme or phenolic compounds resulting from amino acid metabolism, antioxidative ability, or adhesion to animal cells.2,50,51 In the past, such testing was the basis for the selection of cultures by suppliers.

Many culture suppliers have now gone a step further and have carried out clinical studies of their most promising strains with animals and humans.2,4 These suppliers are, thus, in a position to provide solid evidence of the purported health claims. In a “probiotic” mind frame, the health claim is probably the first to be looked at, this applies for all products. At this point, no single strain has demonstrated all the health properties that have been ascertained for LAB.4 Some strains are effective in demonstrating immunomodulation, while others can reduce problems associated with the assimilation of lactose. Therefore, strain selection must be made in light of the health claims that will be made or for the consumer group that is targeted. One strategy is to add many strains with different clinical effects. Even if the various legislation do not always permit health allegations on the labels, it still appears critical to use strains having demonstrated clinical effects. It is important to emphasize this point. If a specific biological effect is to be hoped for, one should expect data to this effect from the supplier. Many strains are now characterized in this respect.52 An example of an extensive selection process is one carried out by Nestle. From their collection of 3,500 strains, they conducted a rigorous selection program that included the following criteria: technologically exploitable, human origin, adherence to intestinal epithelial cells, and stimulation of the immune response.2 This example shows the importance of the health properties of the strains, but it also shows that technological considerations must be examined. Selection on a Technological Basis From a culture supplier’s perspective, technological qualities the strains should possess are biomass yield at large

PROBIOTICS IN FOODS

scale and ease of concentration and survival to freezing and drying.53 However, from the manufacturer’s point of view, other technological qualities are demanded, and many examples are given in Table 3. As strain related variations in viability losses during processing or storage are provided in the sections to follow, it will become clear that strain selection is critical in the development of a probiotic-carrying functional food. With respect to sensitivity to oxygen, a particularly important feature for growth and stability of bifidobacteria in foods, it has been shown that a correlation exists between the synthesis of NAD-oxidase and NADH-peroxidase and oxygen sensitivity of the bifidobacteria.54 Molecular biology techniques are now used to characterize the genes associated with various properties of the probiotic strains,34 and this approach should expand in the future. It must also be emphasized that, in the strain selection process, it is not only the strain per se that is important, but also the method in which it is prepared is critical. Aspects of fermentation technology, drying technology, and microecapsulation will significantly influence the functionality of probiotics.22 Some data on the effects on encapsulation are available, and this aspect will be examined later. However, most of the data on the effects of fermentation and drying technologies are proprietary. In the selection of probiotics, it is, thus, necessary to establish a good working relationship with the suppliers of the cultures.

Addition Level It is not clear how many cells are required in the GI tract to significantly affect the environment of the gut, but it is believed to be between 106 and 108 CFU/g of intestinal contents.2 Charateris et al.55 suggest the addition of 109 to 1010 CFU/100 g of product. In general, the food industry has targeted populations over 106 bifidobacteria/g at the time of consumption of strain that has been added to food.56 Initially, this standard appears to have been adopted to provide bacterial concentrations that were technologically attainable and cost effective, rather than to achieve a specific health effect in humans.57 As more data on bacterial population requirements are available, it becomes clear that numbers will vary as a function of the strain and the health effect desired. The number of cells is not the only aspect to consider. It is believed that the food carrying the cultures also influences the level of activity.4 An example of how food influences the delivery of probiotics is found in the data of Stanton et al.17 In this study using piglets, a Cheddar cheese sample enabled the ingestion of 109 CFU, while 1011 were ingested with yogurt; the intestinal contents were at 105 CFU/mL with the cheese, but only 7 × 104 CFU/mL with yogurt. Thus, Cheddar appeared to enable a higher efficiency in delivering viable bacteia to the intestine than did yogurt, presumably because cheese has a higher buffering capacity.

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Consequently, it is difficult to come up with a specific number of cells required to have a biological effect. The best approach is to work with suppliers. Ideally, one would base his/her decision on scientific data obtained for a given food, with a given number of cells of a specific strain. This is not always possible, and the decision must then be based on legislation or recommendations from organizations that have credibility. An example is the International Dairy Federation (IDF), which recommends that dairy products, such as Acidophilus milk, contain at least 107 . colony forming units (CFU) per mL of the probiotic culture.58 However, when bifidobacteria are the adjunct cultures, such as is the case for yogurt, the IDF suggests that the product contains at least 107 CFU of LAB per mL, of which at least 106 CFU/mL are bifidobacteria. Thus, high inoculum levels should be used, because a food product must contain between 1 and 100 million bifidobacteria per g at the term of the storage period on the shelves. Hence, it is recommended that during the manufacture of yogurt-related products with bifidobacteria, the milk should be inoculated with the final number of bifidobacteria required for the product.

Toxicity Vitamin A, when taken excessively, can negatively affect bone formation. Vitamin D and minerals, such as Fe, Cu, and Se, are necessary for body development, but can also become toxic if very large quantities are consumed. Therefore, when considering fortification of foods, toxicity is an issue. Many authors believe that probiotic cultures should preferably be of human origin. At first glance, this is logical. However, LAB are quite ubiquitous in nature, and strains isolated from plant material, for example, could prove to be excellent probiotics. Instead of looking at the source of the strain, another approach, suggested by Richardson,2 would be to demand that the strain have a long history of occurrence in foods for human consumption. This is the case with yeasts. Traditionally and from an economic point of view, yeasts are the most important microorganisms exploited by man. Infections arising from the pathogenic yeasts, such as Candida albicans, are not transmitted through food. Therefore, the health risk associated with the use of yeast in foods is minimal.59 There are occurrences of endocarditis as well as bloodstream, chest, and urinary tract infections associated with LAB.60 However, it is considered that LAB are only opportunistic bacteria producing local or bloodstream infections in debilitated patients suffering from a breakdown of host defences. Gasser60 states that LAB have a long history of widespread consumption in the general population. There is currently insufficient evidence to suggest that their use in food fermentation poses any danger. This being said, Collins et al.61 recommend that the strain selection process take into account various safety features in addition to the technological properties (Table 3), which include: GRAS status, possession of a desirable antibiogram

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profile, nonpathogenic (even to immunocompromised hosts), noninflammation-causing microorganisms. In this spirit, Hammes and Hertel62 indicated that some authors question the use of enterococci as probiotics. Many probiotics are intestinal isolates and, at first glance, bringing them back to their place of origin should not have harmful effects. However, these intestinal bacteria may exhibit properties in the food that are not revealed or of concern in the intestinal tract.62 An example is the potential to from biogenic amines in food. The presence of probiotic cultures can enhance the safety of foods.63 Feeding a consortium of 29 live bacteria to chickens has helped exclude some pathogenic species in the chick intestines. Such an approach could help reduce the Salmonella contamintion of poultry.64 With respect to chicken breasts, it is expected that the presence of probiotic cultures on the meat is of limited effect to the consumer, since cooking of the meat will kill the organisms. However, many foods are eaten fresh and uncooked (fruits, vegetables, fish/sushi), and the presence of probiotics at the surface of the products could potentially reduce the development of pathogenic bacteria on the food itself and even, subsequently, in the gastro-intestinal tract. It is known that some probiotic strains do indeed help prevent bacterial65 or viral infections in the intestinal system.66 For instance, yeast cells are known to bind to enterotoxins produced by enterobacteria through a mannose-specific reaction. Some of them secrete killer factors, which are proteins that inhibit many microorganisms. Finally, yeasts produce metabolites with known toxic effects against undesirable microorganisms in the intestinal tract.67 The addition of the probiotics to fresh foods could be carried out at the grocery store or at restaurants having salad or sushi bars. No data is available to support this hypothesis, however, and research is warranted on these potential food applications. Adaptation of Processing Steps Numerous technological operations are used in the processing of foods. Many have an effect on how the probiotics grow and survive in the food product. Data are presented in Table 4. It is deemed that the substrate, competing lactic cultures, and heating warranted special attention. Type of Substrate Mattila-Sandholm et al.22 state that the food matrix formulation is a major technological factor that influences the functionality of probiotics. Several studies have shown that nonviable probiotics can have beneficial effects in the host. Thus, for certain probiotic strains, it might not be necessary to maintain high viability during storage, and good growth during the manufacturing process could be sufficient to obtain a health benefit. This section aims to examine how the composi-

tion of the food matrix affects growth during processing at the plant. Data suggests that probiotic cultures can grown in many types of milk. Although most studies were performed on cow’s milk, data is also available on camel,68,69 buffalo,70 and goat milk.71 Although one study reports unsatisfactory growth of Lb. acidophilus in goat milk,71 this might be a strain-related occurrence. With respect to B. longum, much higher populations were obtained in fermented sheep milk compared to those in fermented cow milk; populations in fermented camel milk were also higher than those in cow milk.72 At this point, no clear conclusion can be reached as to which milk is better suited for the growth of probiotic cultures. Most studies have been conducted on cow milk, and, unless otherwise stated, in this article, the use of the term “milk” will refer to cow milk. Many probiotic cultures cannot multiply well in pure milk, but variations occur between strains of the same species in this respect,33,73 (Table 4). Growth in milk is often slow or limited74 and this appears to be partially due to low proteolytic activities.75 Probiotic cultures, such as Lb. acidophilus or Bifidobacterium, generally grow faster on synthetic media than on milk,76,77 but nevertheless appear to be better suited for the milk substrate than are cultures of Lb. rhamnosus.78 The bifidobacteria are generally not highly proteolytic, and their growth in milk is also dependent on their β-galactosidase activities.79 In some instances, mixing nonproteolytic probiotic strains with a highly proteolytic LAB will be helpful, but if the LAB grows too fast, the probiotic strains are overwhelmed.74 Therefore, many groups have supplemented milk with various compounds (Table 5). Supplementation of milk with yeast extracts (YE) often solves this problem,33 and YE can be replaced by a combination of amino acids, minerals, and ribonucleotides73 or casein hydrolysates (Table 5), but there are instances where the strategy is ineffective, particularly with bifidobacteria.80 Presumably, in this last instance, factors other than amino acids were lacking in the supplements, or the redox conditions were simply inappropriate for the growth of the bifidobacteria. Redox conditions, indeed, appear to be linked to the growth-promoting properties of various supplements. Thus, in a study where different peptones, extracts, or proteins were tested for their stimulatory effect on bifidobacteria, all materials lost growth-promoting activity when their disulfide bonds were reduced.81 The addition of milk hydrolysate may, however, negatively affect the flavor of the product, as was the case for Gouda cheese.80 The potential of probiotic yeasts in dairy products is linked with their ability to grow at low pH values, low water activities, low temperatures, and high salt concentrations.59 Traditionally, yeasts have been used as starter culture in the production of kefir and soft cheese.67 It is now admitted that many yeasts have the capability to become established in a dairy and act as a spontaneous or as a controlled starter culture. Although many yeasts are unable to grow in milk, it has been shown that S. boulardii is capable of using the yogurt constituents (organic acids, galactose, and glucose) as growth substrates.32 In fruit yogurt, the presence of sugars from the added fruit are bound

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PROBIOTICS IN FOODS Table 4

Effects of processing steps on growth or viability of probiotic cultures

Product

Process step

Effect on viability

References

Frozen yogurt

Freezing

1. Laroia and Martin181 2. Modler and VillaGarcia182

Yogurt

1. Sugar addition (up to 16%) at the beginning of fermentation. 2. Addition of starter 3. Presence of oxygen in milk Carbonation

1. No survival of B. bifidum if pH is 3.9-4.6. Lb. acidophilus has better resistance. 2. B. longum: 1 log drop in low acid yogurt (pH 5.85) but 2 log drop in viable counts when pH is 4.47 1. Decrease in Bifidobacterium and Lb. acidophilus during fermentation as sugar concentration increases 2. Less growth of the probiotics

No effect overall on probiotic populations during manufacture. CO2 stimulated growth of Lb. acidophilus but production time was shorter 6 to 150 fold reduction of viable populations after drying. Bifidobacterium more sensitive than Lb. acidophilus 1. Less than 1 log drop in viability of Lb. acidophilus and B. bifidum 2. 90% drop in viability 3. 10% drop in viability of Bifidobacterium population 4. 40% drop in viability of Bifidobacterium 5. 50% drop in Lb. acidophilus viability High salt levels causes higher losses during ripening Addition of 1.8% salt causes a 0.5 log drop in B. infantis viability during fermentation. Lb. acidophilus does not grow well on goat milk

Vinderola et al.107

Carbonated fermented milk Dried yogurt

Freeze-drying

Ice cream

Aging prior to freezing and freezing itself

Gouda Cottage cheese

Salting Salting the cream dressing

Semi-hard goat cheese Pasteurized milk

Fermentation Heating

Kimchi

Salt content

Fermented soya milk

1. Protein content (soya:water ratios) 2. Soaking soybeans in alkali 3. Heating of soymilk

D values at 62.8◦ C were 4.6–7.2 min for Lb. acidophilus and Z values of 7.3–10.8◦ C. Increase in salt from 2 to 3.5% increases losses of bifidobacteria during storage 1. Higher protein content increases buffering capacity and growth 2. Resulting soymilk has too high a pH for the lactic bacteria 3. 60◦ C/15 min and over 100◦ C provides good growth; media heated 80◦ C/5-60 min was unsuitable.

to be fermented and to cause gas and ethanol production by the probiotic yeasts. In many instances, the strategy for improving the growth of probiotic cultures by the addition of novel ingredients has led to the development of new dairy products. Such novel dairy blends include tomato juice,82 peanut milk,70 soy milk,70 buffafo whey/soya milk,83 and rice84 (Table 5). These data suggest that some plant-based supplements are valuable in promoting the growth of probiotics, and there is increasing number of reports on the value of plant-based substrates for the growth of probiotics. Lactobacillus acidophilus is reported to grow much better on soymilk than on cows’ milk.13,85,86 This explains why supplementation of cow milk with soya extracts has been proposed (Table 5), and why many groups have examined the production of yogurt-type products based on soymilk.83,85–87 The bifidobacteria, on the other hand, seem to prefer cow milk over soy milk for growth.83 Lactic cultures also grow well in vegetable juices from cabbage and carrot.89 Unfortunately, little data is available for meats. One study suggests that Lb. gasseri is well suited for pork-based fermented sausage manufacture.20 In addition to the food matrix as such, the effects of ingredients or additives have been examined. Some components of milk

1. Shah and Ravula183 2. Roy et al.184

Rybka and Kailasapathy185 1. Hekmat and McMahon186 2. Christiansen et al.153 3. Modler et al.155 4. Kebary et al.154 5. Duthie et al.156 Gomes et al.80,93 Blanchette et al.187 Gomes and Malcata71 Collins and Hartlein188 Lee et al.18 1. Chang and Stone189 2. Mital and Steinkraus190 3. Mital and Steinkraus190

do not seem to influence the growth of the probiotics, for example, fat content of yogurt mix.88 However, some ingredients added to foods, such as salts, sweteners, aroma compounds, and some preservatives (nisin, natamycin or lysozyme), may influence growth of the probiotic bacteria. Sensitivities vary between strains, but fruit juices, strawberry flavorings, vanilla flavors, and nisin, at concentrations used in dairy products, can inhibit growth of probiotic bacteria.90 It must be said that the starter (technological) cultures can also be affected by the ingredients,90 and that it is, therefore, difficult to predict how the addition of supplements will affect the ratios of starter to probiotic cells. Competing Lactic Cultures Probiotics are typically added to fermented products, in which the consumer expects the presence of live bacteria. Starters are always added for technological purposes (acidification, texture, flavor). When manufacturing cheese or yogurt, the addition of probiotic cultures to the normal starters generally results in slower growth of the probiotic strains than if they were added alone in milk91,92 Such is the case with the manufacture of acidophilus milk93 and Cheddar cheese.94 The phenomenon could partially be related to the production of bacteriocins or

68

C. P. CHAMPAGNE ET AL. Table 5

Adapting processing steps to improve growth or survival of probiotic cultures

Product

Means

References

Yogurt

1. Use only S. thermophilus with the probiotics (and thus omit Lb. delbrueckii ssp. bulgaricus) 2. Reduce the S. thermophilus inoculation level 3. Use pre-refrigerated milk (107 CFU/mL psychrotrophic count) 4. Pack in glass rather than plastic so as to lower oxygen content 5. Add cysteine 6. Add ascorbic acid; effective on Lb. acidophilus 7. Inoculate with ruptured cells of yogurt starter 8. Add buffers, such as glycerophosphate 9. Use S. therhophilus strains having oxygenscavenging properties. 10. Inoculate with probiotics, and delay the inoculation of the yogurt culture. 11. Decrease incubation temperature to 37◦ C 12. Add yeast extracts 13. Add Ginseng extracts 14. De-aeration of milk 1. Add glucose/fructose or yeast extracts/casitone to favour growth 2. Grow the cultures separately and mix the various fermented milks in appropriate ratios just before packaging 3. Add soy products to favour growth 4. Add tomato juice to milk 5. Have a portion of milk be pepsin-digested 6. Add peanut milk 7. Add amino acids or protein hydrolysates 8. Apply stress conditions to starters (pressures . . .) 9. Increase fermentation temperature from 30 to 35◦ C 10. Add fruit juices to milk 11. Use high inoculation rates of probiotics (up to 10%) 12. Add cereals 13. Add β-galactosidase 14. Sterilize milk (more extensive growth in sterilized milk rather than steamed milk) 15. Higher B. longum populations obtained if UF retentates are used 16. Add honey to milk 1. Increasing inoculation rate from 1 to 5% increases final B. infantis population in cream dressing from 1 to 8 × 108 CFU/mL 2. Growing the bifidobacteria in the cream dressing 1. Add 0.3% milk hydrolysate 2. Increase cooking temperature by 2◦ C, to 40◦ C 3. Inoculating only with the probiotic cultures 1. Add milk hydrolysate. Effective with Lb. acidophilus but not with Bifidobacterium 2. Increase inoculation rate of probiotic (3.5%) 3. Mesophilic starter not added 4. Increase processing temperatures: stirring at 38 rather than 36◦ C, holding at 25-40◦ C rather than 20◦ C 1. Adding bifidobacteria to the milled and salted curds 2. Using a Lb. casei stran that can grow in the cheese during ripening Addition of yeast extracts, soy extracts or β-galactosidase

1. Rybka and Kailasapathy106 2. Khedkar et al.191 3. Srivinas et al.157 4. Dave and Shah125 5. Dave and Shah126 ; Dave and Shah 1998192 6. Dave and Shah193 ; Roy et al.194 7. Shah et al.195 8. Roy et al.194 9. Ishibashi and Shimamura196 10. Reddy197 11. Kneifel et al.111 12. Kim et al.160 13. Goh et al.198 14. Tamime et al., 199556

Fermented milk, starters, bifidus or acidophilus milk

Cottage cheese

Semi-hard goat cheese Gouda

Cheddar

Kariesh cheese

1. Yoshiharu et al.199 ; Badran and Reichart200 ; Saxelin et al.33 ; Saxena et al.201 ; Dave and Shah192 2. Mital and Garg13 3. Yajima et al.202 4. Babu et al.203 ; Prajapati et al.204 Badran and Reichard200 5. Misra and Kuila76 6. Murad et al.70 7. Murad et al.70 ; Elli et al.73 , Oliveira et al.205 8. Knorr31 9. Baron et al.206 10. Young Tae and Mi Hwa207 11. Kisla and Unluturk208 ; Khattab et al.209 12. Kyung-Hee and Young-Tae210 13. Khattab et al.209 14. Misra and Kuila101 15. Al-Saleh72 16. Ustunol and Gandhi211

1. Blanchette et al.187 2. Blanchette et al.212

1. to 3. Gomes and Malcata71

1. to 4. Gomes et al.80

1. Dinakar and Mistry103 2. Stanton et al.17

Murad et al.213 (Continued on next page)

69

PROBIOTICS IN FOODS Table 5

Adapting processing steps to improve growth or survival of probiotic cultures (Continued)

Product

Means

References

Milk powders

Pre-adaptation of cells prior to spray-drying by sublethal heat or salt stress treatments, improves survival by up to 18 times. 1. Immobilization in gel beads improves survival by up to 80% 2. Addition of glycerol and mannitol to gel beads improves survival by 20% For bifidobacteria: addition of cysteine

Stanton et al.100

Ice milk

Fermented soya milk

other inhibitors produced by the starter cultures,22,95 but the fact that the typical starter cultures grow faster, acidification occurs rapidly, and fermentation times are much shorter in their presence, is probably the dominant factor in the limitation of the growth of probiotics during manufacture; thus, probiotic cultures do not have time to grow extensively. It is noteworthy that interactions between strains can be affected by the medium. Vinderola et al.95 noted that cell free supernatants (CFS) of Lb. casei were more inhibitory to Lb. acidophilus when obtained from MRS broth than from milk, but the opposite was observed for CSF of Bifidobacterium strains. This inhibitory effect of starters on probiotics is major and, in commercial products, low levels of bifidobacteria are correlated in the addition of the starters used for technological purposes. This has led to the development of cheeses80 or fermented milks (Table 1) where the probiotic cultures are the only starters added. Yogurt cultures that have proteolytic or oxygen-scavenging properties have been shown to be beneficial to bifidobacteria (Table 5) and could be considered in the selection of cultures compatible to probiotic strains. A few strategies have been developed to try to reduce the strong inhibition of the starter strains on the probiotic cultures. The most common strategies are the omission of a portion of the starter strains and changes in the respective inoculation rates (Table 5). One must be careful in decreasing the starter inoculation rate too much, because probiotics can produce inhibitory compounds against starters,95 which could slow down the acidification process. Probiotic bacteria can even generate inhibitors between themselves, and Lb. acidophilus seem to be feared in this respect.95 The data of Murti et al.96 shows that Lb. casei and Lb. acidophilus grow much better in soya extracts than Lb. bulgaricus or Lb. helveticus. Therefore, soya-based substrates appear well suited for the production of fermented foods that contain the probiotic lactobacilli, since, in contrast to cow’s milk, these lactobacilli are able to compete with the “technological” lactobacilli typically used in the manufacture of yogurt and cheese. The propionibacteria are even slower growing cultures than the bifidobacteria, and promoting the development of Propionibacterium in a fermented food is a challenge. Since the propi-

1. Kebary et al.154 ; Sheu and Marshall214 2. Kebary et al.154 ; Sheu et al.139 Kamaly88

onibacteria can use lactate as a carbon source, one option is to provide conditions that will enable their growth following the lactic fermentation. Swiss cheese is an example of a process based on this sequenced growth, and it remains to be seen if other products will be manufactured with this approach. In sour milk, selected strains of P. jensenii were able to grow with B. longum. The propionibacteria survived well, as their viable counts remained above the recommended level after a period of cold storage. As for the bifidobacteria, their level was clearly higher in milks inoculated with the P. jensenii strains.42 There are, thus, examples of associations between probiotic cultures, such approaches warrant further study. Little data is available with respect to interactions between probiotic yeasts and starters. Yeasts in dairy products may interact in different ways with the starter. They may inhibit the LAB or the mold starter cultures of cheese with their by products, such as alcohol, or by the killer factor with its possible broad antimicrobial spectrum. But they may also contribute positively to the fermentation or maturation by supporting the function of the starter culture.67 Heating In food processing, heating serves two purposes. High temperatures over 65◦ C are applied to destroy microorganisms. Lower temperatures, such as a cheddarization step at 39◦ C, are introduced for technological purposes. Temperatures under 45◦ C are generally not detrimental to probiotics. Obviously the heating of products over 45◦ C will destroy at least a fraction of the population, depending on the temperature97 and the strain. Lactobacillus acidophilus cultures are rather sensitive to heating (Table 4), and would experience approximately a 4 log destruction level during a typical pasteurization process. Food processes that include a heating step above 65◦ C are highly detrimental to probiotic cultures. Therefore an option is to add the cultures after pasteurization and when the product is packaged immediately after heating, proceed with aseptic packaging. This was proposed for mayonnaise.19 Microencapsulation of the dried probiotic culture with lipids may be the best way of protecting the cells against a heat treatment. Siuta-Cruce and Goulet98 have developed an encapsulation process that offers good protection against short exposure to

70

C. P. CHAMPAGNE ET AL.

high temperatures. Although no data is provided for this claim, such a culture could find wide application in pasteurized foods, as well as those that incorporate a heating step as part of the process, such as bread. Lemay et al.99 have used a similar approach for the introduction of protective cultures into sausages. It is to be expected that such novel technologies will increasingly be used for the protection of cultures against detrimental food processes. The production of spray dried milk powder harboring high levels of probiotic cultures has been carried out. The high temperatures involved in spray drying are often very detrimental to probiotic cultures. It was possible to improve survival levels by pre-adapting the cells with sub-lethal stresses with NaCl, bile salts, heating, and hydrogen peroxide.100 This approach of pre-adapting cells in increasingly used and can be applied to improve resistance to other conditions, such as the acid environment. In the previous lines, the effect of heating was considered in the perspective of food that already contained the probiotics. The effect of heating as a technological step must also be examined from a standpoint of its effect on the raw material’s subsequent ability to support the growth of the cultures. Cheese milk is generally pasteurized (73◦ C for 15 s), but milk formulation destined for yogurt manufacture are typically heated between 85◦ C and 95◦ C. Therefore, a question to be asked is: “Is there an optimal set of heating parameters for milk that favors the growth of the probiotics?” Data by Misra and Kuila (1992)101 suggest that sterilized milk (121◦ C–15 min) enables more extensive growth of bifidobacteria than does steamed milk. Data seems to show that milks heated at 85◦ C to 95◦ C are appropriate for the subsequent growth of probiotic cultures.

Other Processing Steps Apart from the supplementation of milk with various growth supplements, many changes in processing steps have been proposed to promote the growth of the probiotic cultures: changes in incubation temperature, addition of enzymes, adding compounds that affect the redox conditions of the medium and encapsulation are the most notable (Table 5). Two practices are observed in plants carrying out food fermentation with respect to inoculation: direct-to-the-vat inoculations (also known as DVS for “direct vat set”), or preparation of cultures in starter tanks under controlled conditions. To our knowledge, probiotic bacteria are inoculated with DVS cultures because of the difficulties involved in propagating probiotic microorganisms at the production site.22 This increase costs of the products, and the potential of growing the probiotics in combination with the main starters can be raised. Theoretically, probiotic strains could be combined with thermophilic cultures, such as those used in yogurt or swiss cheeses, and grown in mixed cultures. However, controlling the ratios of the different strains can be complicated, and the trend is towards preparing pures. One attempt has been made to grow a mixed culture of mesophilic strains destined for cheese manufacture with a Lb. rhamnosus

strain.102 Cheese starters are typically propagated between 19◦ C and 28◦ C, which is not appropriate for extensive development of probiotics. As can be expected, better development of the lactobacilli was noted when the mixed culture was propagated at 32◦ C rather than at 22◦ C.102 At this point, no highly successful low-cost strategy of preparing probiotic-containing mixed cultures at the plant site, using dairy-based commercial media, has been published. In Cheddar cheese manufacturing, Dinakar and Mistry103 were concerned that the addition of B. bifidum with the starter might be detrimental to the culture because of the aerobic conditions of cheesemaking, the cooking conditions, and the presence of the mesophilic starter. They, thus, opted for the addition of dried bifidobacteria to the milled curds (Table 5). Stanton et al.,17 indeed, found that the bifidobacteria did not grow during cheese making, presumably because of the redox conditions (filling of the cheese vat with milk aerates the milk). Hence, creating an anaerobic environment during cheese or yogurt manufacture should promote the development of Bifidobacterium. Klaver et al.74 found this to be the case, by influencing redox values with ascorbic acid. Rennetting of milk, an important cheese processing step, does not have a significant effect on the growth of probiotic bacteria.92 Bacteriophage are a big concern in cheesemaking. In 1995, there were still no reports of bacteriophage attacks against Bifidobacterium on plant sites.56 Nevertheless, it is to be feared that some problems could also occur with probiotics if a given strain is used extensively during manufacture. It has, thus, been suggested that phage resistance properties of probiotic cultures would be desirable.222 If bacteriophages do occur, two strategies would be available. The first is strain rotations, as is done with the technological cultures. The second is the addition of the probiotic at the very end of the manufacturing process. In the first option, the disadvantage lies in the fact that a different strain would not necessarily have an identical biological effect on health. In the second option, a disadvantage would be increased costs. Indeed, since no growth of the probiotic cultures during manufacture would be allowed, higher innoculation rated would be required to obtain the desired population of probiotics in the finished product. An interesting product for the delivery of probiotics is ice cream. Survival levels vary as a function of strains and pH of the dairy blend, and many examples are found in Table 4. The freezing process does not seem to affect the cells’ sensitivity to bile salts.56 However, the question of the effect of freezing on the subsequent sensitivity to stomach acid remains to be determined.

Identification and Enumeration of the Viable Populations The determination of the viable population in functional foods is an important feature in assuring the consumer that they are purchasing a product that answers to certain norms. In foods having only the probiotic cultures, enumeration is rather easy.

PROBIOTICS IN FOODS

However, in yogurts, for example, it is not only necessary to distinguish between the bifidobacteria and Lb. acidophilus, but also between the various lactobacilli. The difficulty is compounded by the fact that there are now novel species of Lactobacillus that have probiotic effects: Lb. paracasei, Lb. rhamnosus, Lb. reuteri, and Lb. plantarum. The need exists for rapid, reliable methods for enumeration of mixed cultures of probiotics. Such methods are needed to routinely determine the initial inoculum, and to estimate the storage time period, these different organisms remain viable. Several methods could be applied for the detection of probiotics in food products, like plate count methods, molecular genetic methods, or enzymatic methods. Plate count methods are still preferable for quality control measurements in food products. It is, therefore, necessary to have a medium that selectively promotes the growth of the different probiotic bacteria, whereas other bacteria are suppressed. The selection of an adequate culture medium for probiotics should be based on the following parameters: supply of nutritive and growth substances, low oxidation-reduction potential, maintenance of the pH value during growth, and the buffering capacity and final pH of the prepared medium. Anaerobic conditions are also an important factor in detecting and enumerating bifidobacteria. Several methods and media have been developed,75,104–107 and good reviews are those of the Expert Group on Lactic Acid Bacteria and Starters of the International Dairy Federation,108 as well as of Shah109 or Tamime et al.56 Independent evaluations of commercial yogurts gave surprising results, some having less than 103 colony-forming units (CFU) per mL of the claimed probiotic culture.88,110 This was also found with commercial starter cultures.111 The populations of Lactobacillus acidophilus, Bifidobacterium spp., and Lactobacillus casei were estimated in 26 in commercial fermented milk products represented by 14 companies in Australia.110 The population of bifidobacteria dropped below 106 cfu/g in 94% of the products that claimed to contain them. These data point to a problem with viability during storage, and this aspect will be examined in the next section. It has been our experience, however, that certain strains react atypically on the various selective or differential media. It is important to assess individual strains in pure cultures before mixed cultures are evaluated. The characterization of probiotic bacteria is important for food industries, as the manufacture of some products requires a particular strain. Again, it must be stressed that the health effects are sometimes related to given strains. Therefore, it is not only necessary to determine if the population of a given species is adequate, but also to ascertain if the product contains the strain it claims to have. The identification of these strains can be difficult when conventional methods are used. Some information exists on the DNA analysis of bifidobacteria, such as DNA-DNA hybridization, pulsed field gel electrophoresis (PFGE), and the randomly amplified polymorphic DNA profiles (RAPD).36,112–114 The 16S rRNA gene was also used for the systematic identification of bifidobacteria.40 The develop-

71

ment of new approaches and techniques of molecular biology would make it possible to clarify in which species strains of probiotics belong. Amplified ribosomal DNA restriction analysis (ARDRA) has an excellent potential for characterization at the species level. This technique is based on the amplification of the DNA sequence in the 16S rDNA region, followed by the digestion of PCR products with restriction enzymes.115 A few authors used conserved sequences other than the 16S rRNA gene for the characterization of species, such as the conserved gene encoding for the L-lactate dehydrogenase (ldh) studied in B. longum or the gene recA in humans.116 Like the 16S rRNA, the recA gene is considered to be universally present in bacteria. A phylogenetic analysis of the short sequence recA products was used to accurately classify strains of bifidobacteria.116 Fructose-1,6-biphosphate(FDB)-dependent L-lactate dehydrogenase is a key enzyme in lactic acid fermentation by most lactic acid bacteria. Many publications indicated that it is not possible to characterize bacterial species only by one method. With the information from the 16S rRNA sequences and the conserved genes, it is possible to combine these techniques and to develop a new approach for the characterization of bifidobacteria.

Stability During Storage Consumers obviously demand that the product they purchase contain the probiotic cultures at the time of consumption. Thus, companies provide the required viable population at the time the product is marketed, but are also concerned with the evolution of the population throughout storage. Typically, the “best before” date is given in order to provide a period guaranteeing the desired population. The ability to survive during processing and storage are not linked.82 Therefore, it is necessary to specifically examine factors that affect survival during storage. The biological effect of the probiotic culture is generally linked to the strain used, as well as the form and the numbers consumed. Little attention has been given to the effect of technology or storage on their specific probiotic attributes. Gilliland and colleagues have paid attention to these aspects and have followed bile salt resistance or cholesterol-assimilation abilities. With respect to cholesterol-assimilating ability, freezing had no effect on the culture’s properties, but they observed that this character could be reduced in Lb. acidophilus following storage in unfermented milk at 5◦ C for 21 days.117 However, this observation was strain dependant, which raises a concern. Studies on the effect of technology on probiotics have typically examined CFU levels, but rarely the expression of the probiotic character of the remaining cells. Numerous studies report on the stability of probiotics during storage (Table 6). The fat content of yogurt does not affect stability during storage,88 but pH, oxygen, and starter production conditions have significant effects, and deserve a more detailed analysis.

72

C. P. CHAMPAGNE ET AL. Table 6

Stability of probiotic cultures during storage

Product

Stability during storage

References

Yogurt

1. Bifidobacterium: 0 to 99% loss during 5 weeks, depending on strain; Lb. acidophilus: 0 to 80% loss in 6 weeks, depending on strain 2. Bifidobacterium: 20 to 90% loss in viable population over 62 d at 4◦ C; Lb. acidophilus: 25% drop in viability 3. Bifidobacterium: 1 log loss in viable population during first 14 d, much greater thereafter; same phenomenon with Lb. acidophilus, but lower losses 4. Addition of fruit or extracts may decrease viability by up to 2 logs 5. Drop of up to 6 logs of viable bifidobacteria at 4◦ C at pH 4.2 6. Drop of 0.2 to 2 log in viability depending on species, yoghurt composition and overincubation Drop of 85% in viability of Bifidobacteium over 24 d at 7◦ C. pH were between 4.57 and 3.71. Lb. acidophilus drop in viable population between 0.2 and 1.3 log in 28 days depending on strain; Lb. casei remains stable 1. B. bifidum: increase from 106 to 107 CFU/g during 24 week storage 2. Lb. casei grows to 3 × 108 CFU/g cheese during first 100 d of ripening; stable up to 150 d 3. Loss of 0.5 log B. infantis during first week, but stable during the following 11 weeks. During 9 week ripening storage at 13◦ C, Bifidobacterium looses one log while Lb. acidophilus loses 2 log cycles B. lactis and Lb. acidophilus lose up to 1 log during 70 d storage at 6◦ C. Lactobacilli more resistant B. longum and B. bifidum more stable than B. infantis, which loses 2 log in viability during 14 d storage Bifidobacterium viability drops between 0.5 and 3 log during 14 storage at 4◦ C as a function of strain Bifidobacteria viable counts drop between 5 and 6 log after 36 d storage at 4 or 12◦ C 1. No loss of viability for 2 of 3 strains of Lb. acidophilus during 28 d at 7◦ C 2. 1.5 log drop in Lb. acidophilus viable counts over 21d 3. Survival of Lb. acidophilus between 14 and 58% over 23 days at 4◦ C 1. No loss of activity of Bifidobacterium or Lb. acidophilus cultures for 15 d at 4◦ C 2. Depending on strain of Bifidobacterium, 0 to 3 log drop in viability over 14 d at 4◦ C 3. Viability drop between 0.2 and 1.4 log of Lb. acidophilus cultures over 21 d at 5◦ C Lower viability of Lb. acidophilus during storage

1. Micanel et al.88 2. Rybka and Kailasapathy106 3. Shah et al.110 4. Koch and Carnio53 5. Roy et al.184 6. Dave and Shah 199675

Loss of 1 log of bifidobacteria over 2 months storage

Tamime et al.56

Viability of Lb. acidophilus and Lb. rhamnosus drops by 2 logs over 28 days at 4◦ C; pH of products between 4.3 and 4.8

Vinderola et al.90

1. Small drop of B. bifidum (less than 40%) over 6 weeks at −29◦ C. 2. Loss of 3 log in Lb. acidophilus viability over 12 weeks at −18◦ C 3. B. longum: stable during 11 weeks at −30◦ C in low acid yoghurt (pH 5.85), but loss of 2.5 log in acid (pH 4.47) yoghurt. 1. Up to one log decrease after 16 weeks at −20◦ C for B. bifidum and Lb. acidophilus 2. Approximately 0.5 log decline over 70 d at −17◦ C 3. Loss of 1 log after 17 weeks at −29◦ C for B. bifidum, but loss of 2 logs for Lb. acidophilus

1. Laroia and Martin181 2. Shah and Ravula218 3. Modler and Villa-Garcia182

Spanish fermented milk Cultured buttermilk Cheddar

Gouda Semi-hard goat cheese Crescenza chese Cottage cheese Fresh cheese Sweet acidophilus milk

Unfermented milk

Carbonated fermented milk Baby feeds-powder Fermented whey-milk beverage Frozen yoghurt

Ice cream

Medina and Jordano215 Nighswonger et al.178 1. Dinakar and Mistry103 2. Stanton et al.17 3. Daigle et al.94

Gomes et al.80 Gomes and Malcata71 Gobbetti et al.179 O’Riordan and Fitzgerald161 Roy et al.104 1. Brashears and Gilliland134 2. Mitchell and Gilliland132 3. Young and Nelson216 1. Hughes and Hoover77 Sanders et al.57 2. Dechter and Hoover217 3. Piston and Gilliland117

Vinderola et al.107

1. Christiansen et al.153 2. Modler et al.155 3. Hekmat and McMahon186 (Continued on next page)

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PROBIOTICS IN FOODS Table 6

Stability of probiotic cultures during storage (Continued)

Product

Stability during storage

References

Edible spread

Four adaptations required: split-stream parteurization, reduction of a tube-chiller agitation rate, processing under oxygen-free nitrogen, near neutral pH Loss of 6 log in two weeks for free cells but only of 4 logs in 16 weeks if entrapped in alginate Bifidobacteria loss of 1 to 4 log over 10 days at 4◦ C Between 1 and 7 log reduction in viable populations depending on species

Charteris et al.23

Mayonnaise Kimchi Cucumber juice

pH Most fermented dairy products are acidic in nature. It is recognized that Cheddar has a pH of 5.2, fresh cheeses are at 4.6, and yogurt has a pH of 4.2. During ripening, the pH of cheeses, such as Camembert, rises sometimes over 6.0. Ice cream and milk have pH values close to 6.5. Therefore, there are substantial variations in the pH of dairy products, even within those that are fermented. Although numerous studies report great losses in the viability of probiotic strains during the storage of yogurt, the data on Cheddar shows that probiotics incorporated into this cheese can be stable during storage (Table 6). Thus, the pH value seems to be a critical factor in the stability of probiotic strains during storage. There are significant differences between strains with respect to survival in an acid environment (Table 6). In general, Lb. acidophilus cultures are more resistant to acid environments than bifidobacteria 1,71,77,88 and Lb. casei seems more acido-tolerant that Lb. acidophilus or Lb. fermentum 118,119 . On the other hand, the presence of acetic acid under undissociated form may have an antagonistic effect on the survival of bifidobacteria. For example, at pH 6.0, 8% of the acetic acid produced by bifidobacteria can be in undissociated form. We do not know if there is a correlation between the ability to survive short-term exposures to high acidic environments (such as a few hours in the stomach) and its ability to survive long-term storage in fermented dairy products. High bifidobacteria counts (>106 CFU/g) were observed in commerical yogurts manufactured and sold in Germany and France, even in yogurts with a pH as low as 4.0, suggesting a selection of more acid-tolerant strains by the industry in these countries. Indeed, most bifidobacteria strains isolated from central European and French milk products were identified as B. animalis,36,120 which showed identity to B. animalis ATCC 27536.121 Therefore, it is recommended to ask culture suppliers for data specifically related to strain stability in acidic foods. It is probably wise for a manufacturer to test a few promising strains in their own products. The problem of sensitivity to acidity of the probiotic cultures is compounded by the fact that acidity may increase during storage. In yogurt, acidification may continue during cold storage, a phenomenon called “over-acidification” and the pH may drop to 3.6.1 To prevent this, it appears important to select lactobacilli that have weak over-acidification properties, or to reduce and even exclude Lb. delbrueckii ssp. bulgaricus from the starter (Table 7).

Khalil and Mansour19 Lee et al.18 Chavasit et al.25

In many cheeses, particularly the surface ripened varieties, such as Camembert or Oka, the pH increases during ripening. From a pH perspective, these cheeses would be better carriers for probiotic cultures than yogurt, but no data is available to verify this assumption. Although Lb. casei strains appear less sensitive than those of Lb. acidophilus during storage, they have the disadvantage of being more active at low temperatures. In certain instances, this results in continued acidification during storage at 4◦ C,118 which could be undesirable. There are instances where numbers of probiotic cells increase during storage, but this could be related to the splitting of the bacteria from the chains into single cells.33 This is an important observation. Most LAB grow in chains, and not enough attention is given to chain length in relationship to CFU counts. The moment the culture is added to yogurt may affect its survival during storage. Thus, Hull et al. 122 found that greater viability losses of Lb. acidophilus occurred during storage of yogurt at 5◦ C when the probiotic strain was added to the yogurt prior to storage rather than with the starter at the beginning of fermentation. They also showed that the addition of catalase partially prevented this extensive drop in viability, which brings us to the effect of oxygen. Of the various methods used to enhance the survival of cells to acidic environments, encapsulation seems promising. Microentrapment (ME) in gel beads has shown to be very effective against freezing, and some data shows that this approach could also be used to increase stability during storage in yogurt.123 However, there is evidence that the benefit of ME to stability in yogurt is due to protection from oxygen rather than protection against the acid in the environment.124 Oxygen Oxygen affects the probiotic cultures in two ways. The first is a direct toxicity to cells. Certain probiotic cultures are very sensitive to oxygen and die in its presence,125,126 presumably due to the intracellular production of hydrogen peroxide. The second way oxygen effects the probiotic cultures is indirect. When oxygen is in the medium, certain cultures, particularly Lb. delbrueckii,125,127 excrete peroxide in the medium. A synergistic inhibition of bifidobacteria by acid and hydrogen peroxide has been demonstrated.128 This suggests that the probiotic strains can, therefore, be affected by the H2 O2 produced by other cultures in the environment. This partially explains why the removal of Lb. delbrueckii ssp. bulgaricus from fermented milk

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C. P. CHAMPAGNE ET AL. Table 7

Improving the viability of probiotics during storage

Product

Means

References

Chocolate bars Milk powders Semi-hard goat cheese

Microencapsulation of the probiotic cultures Microencapsulation of the probiotic cultures 1. Adding 0.3% milk hydrolysate to the manufacturing milk increases the subsequent stability of B. lactis and Lb. acidophilus in cheese 2. Lower salt content seems to improve stability Immobilization in alginate beads Immobilization in gel beads with addition of glycerol and mannitol improves survival of B. bifidum to 10 weeks by 30%. 1. Addition of soy protein after fermentation 2. Increase inoculation level of the probiotic 3. Add Lb. reuteri with starter rather than at the end of fermentation 4. Addition of honey to the milk medium Microentrapment in alginate beads increases stability of bifidobacteria at 4◦ C. 1. Add Lb. acidophilus with starter rather than at the end of fermentation 2. Addition of catalase 3. Select compatible S. thermophilus and Lb. delbrueckii ssp. bulgaricus culture 4. Lower milk solids, but higher whey solids 5. Add cysteine, but only when S. thermophilus is used as starter 6. Add ascorbic acid. Effective with Lb. acidophilus but not with Bifidobacterium 7. Packaging under nitrogen atmosphere 8. Fruit in separate chambers 9. Preparation of dietary products. Some artificial sweeteners are protective in presence of fruit 10. Reduce or eliminate Lb. delbrueckii ssp. bulgaricus from the starter to prevent over-acidification. 11. Inactivate yoghurt cultures by treating yoghurt (heat or pressure), thus preventing over-acidification. 12. Stop incubation at a higher pH. 13. Add catechins or tocopherol 14. Microencapsulate the culture in carrageenan gel beads

Siuta-Cruce and Goulet98 Siuta-Cruce and Goulet98 Gomes and Malcata71

Frozen yogurt Ice milk Fermented milk, buttermilk

Unfermented milk Yogurt

starter cultures has had success in improving the survival of Lb. acidophilus in fermented milks (Table 5). Many studies have focused on the way to prevent the detrimental effects of oxygen on the probiotic cultures (Table 5), the most common being the addition of anti-oxidants, such as ascorbic acid, and the elimination of peroxide producing strains. Some strains of probiotics are more oxygen tolerant than the others.129 It seems possible to affect H2 O2 synthesis by bifidobacteria by taking advantage of the benefits of propionic cultures. Propionibacterium freudenreichii produced extracellularly growth stimulator(s) for bifidobacteria, which appeared to be different from propionic acid. A bifidogenic growth stimulator produced by P. freudenreichii was purified by Mori et al.130 The chemical structure of the bifidogenic growth stimulator was 2-amino-3carboxy-1,4-naphthoquinoe (ACNQ). It has been shown that this molecule plays a role to modify the environment of bifidobacteria, thus, creating favorable conditions for them to proliferate. The production of peroxyde that is detrimental for bifidobacteria under aerobic conditions is effectively suppressed in the

Shah and Ravula218 Kebary et al.154 1. Yajima et al.202 2. Rodas et al.151 3. Rodas et al.151 4. Ustunol and Gandhi211 Truelstrup Hansen et al.143 1. and 2. Hull et al.122 3. Nighswonger et al.178 4. Gardini et al.152 5. Dave and Shah126 6. Dave and Shah193 7–9. Koch and Carnio53 10–12. Kailasapathy and Rybka1 13. Akahoshi and Takahashi162 14. Adhikari et al.144

presence of the bifidogenic growth stimulator. ACNQ acts as a mediator of the electron transfer from NAD(P)H to dioxygen (O2 ) and hydrogen peroxide (H2 O2 ). The generation of H2 O2 by B. longum under aerobic conditions is effectively suppressed in the presence of ACNQ. These ACNQ-mediated reactions would play roles as NAD(P)(+)-regeneration processes.131 Probiotic Production Procedure Although the selection of the appropriate strain in itself critical to a successful introduction of probiotics, the way the strain was prepared can also have a significant effect. The growth medium used to prepare Lb. acidophilus cultures influences their ability to survive subsequent storage in Sweet Acidophilus milk. Thus, when cells were grown in 5% pepsinized whey solids, a 2.8 log drop in viability occurred during 21 d storage at 5◦ C, while it was of only 1.5 log when cells were grown in 2.5% pepsinized whey medium.132 The pH at which the cultures are produced affects their subsequent stability to storage in milk. There are variations between

PROBIOTICS IN FOODS

strains, but B. longum cells grown at pH 6.0 and 6.5 are generally more stable than those grown at pH 5.5 or 7.0.133 Some Lb. acidophilus cultures collected during the stationary growth phase show better stability during their subsequent storage at 7◦ C in Sweet Acidophilus milk, than do cells that were recovered at the end of the logarithmic phase.134 This is unfortunately something manufacturers of dairy products have no control over. Testing the commercial strains in one’s product seems to be the only solution. In addition to the reports of benefits of microentrapment or encapsulation in protecting probiotics against the stressful conditions of the gastro-intestinal tract,135,136 there is increasing evidence that they are helpful in protecting the probiotic cultures destined to be added to foods.137,138 Entrapment of the cultures in alginate beads significantly increases the survival of probiotic cultures to freezing in the production of ice cream type products (Table 5). The success of this approach is often related to the addition of glycerol or mannitol in the gel bead, which creates a protective micro-environment. The positive effect of encapsulation is limited to the freezing step, and no benefit seems to occur during storage. The effectiveness of the method also improves when beads have high alginate contents.139 Adding 2% glycerol to the ice cream mix itself did not improve the survival of probiotic cultures to freezing,140 which suggest that the micro-environment created by the entrapment procedure is, indeed, one of the keys to the success. Other examples of the benefits of microencapsulation are listed in Table 6. In the same line as microentrapment, microencapsulation seems to be the most promising technology to protect cells from the environment. A proprietary technology based on lipid coating of freeze-dried culture particles was effective in increasing survival rated of lactobacilli and pediococci at storage temperatures between 30◦ C and 40◦ C.98 Encapsulation has been widely used in the pharmaceutical industry for the controlled release of many medicinal compounds. Microencapsulation is also widely used in the food industry,141 but is mainly applied to flavors and various other food additives. Probiotic cultures with enteric coatings are currently available, and coatings for technological purposes are now being developed. This would enable the preparation of many novel functional foods stable at room temperature, such as yogurt-covered raisins, nutrient bars, chocolate bars, and tablets.98 With respect to polysaccharide gels, alginate does not seem to be as effective as carragheenan to protect cells against acid environments.142–144 Furthermore, the question the level of cell release in the intestinal environment of alginate or carrageenan-entrapped cultures is not clear. Some specialty starches also show promise for the encapsulation of the cells.22 In addition to their protective effect during storage, some starch products only enable the release of the cells in the lower intestine.22 Hull et al.122 suggested that Lb. acidophilus cells adapt to H2 O2 during yogurt fermentation, which lead to increased stability during storage. The LAB are known to synthesize stress proteins in response to acidification, osmotic stress, or high temperatures.145 Data is increasingly available on the improvement of survival of LAB to stressful conditions through adapted

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starter production techniques. These strategies are worth examining in a perspective of survival to food processing steps and to storage conditions.

Starter Culture When mixing probiotics with yogurt cultures, severe viability losses can result during storage; this depends on the composition of the starter culture (Table 6). It would be too long to address the many strategies that help prevent the loss of the probiotic strain during storage; the reader is referred to Table 7 for a list. The results of an extensive European FAIR project33 has shown that for every probiotic culture it is important to select an optimal support culture to obtain good survival of the strain in fresh fermented products. Therefore, in probiotic strain selection, relationships between the probiotic culture and that of the traditional starter must be examined not only from the perspective of competitive growth during the manufacture of the product, but also from the perspective of stability during storage.

Effects on Other Flora Most studies carried out on the stability of probiotic cultures during storage have data limited to the evolution of the multiple probiotic populations themselves. It is interesting to note that the addition of probiotics may contribute in reducing the growth of unwanted microorganisms. This has been the case for mayonnaise,19 acidophilus-yeast milk,146 and unfermented milks.77 Dairy propionibacteria, beside producing propionic acid that may serve as a food preservative, also produce several bacteriocins that inhibit a variety of bacteria as well as various yeasts and molds. The addition of the organisms themselves to food or feed and allowing acid production provides natural preservative action. For instance, the addition of propionibacteria to lactic acid bacteria as starter cultures for silages fermentations increases the storage stability of the silage.147

Cultures that Grow During Storage In cheeses, most probiotic cultures lose their viability during storage. But there are examples of species that grow in this environment. This is the case of propionibacteria in swiss-type cheese, where their number increases up 109 CFU per g during the 4–8 wk maturation period.148 However, the growth of propionibacteria in cheese varies, depending on the seasonal variations of milk composition, the contaminating milk flora, and the manufacturing procedure.149 Lactobacillus casei can also grow during the ripening of Cheddar.17 In a study of 33 lactobacilli as potential adjunct cultures in semi-hard cheeses, Antonsson et al.150 found that most strains of Lb. casei, Lb. rhamnosus, and Lb. plantarum were reisolated from the cheeses after 13 wk storage, which adds to the list of potential species that

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could at least survive in ripened cheeses. Lactobacillus reuteri populations increased by 1 log during the first 2 d of storage of cultured buttermilk151 suggests that this species should be further examined with respect to use in fermented milks. It would also be worthwhile to examine the various ripening cultures that are used in cheese manufacture and to determine if they have probiotic potential. Although the cultures would not have the “isolated from humans” label, their safety profile would nevertheless by supported by years of safe use in foods. Another approach would be to examine probiotic strains as adjunct cultures and to determine their effects on sensory properties, as was done by Antonsson et al.150

Changes in Sensory Properties The concern that the addition of probiotics to foods will strongly affect their sensory properties should not be disregarded. A successful market introduction of the functional food will be linked to good flavor and texture of the food. Thankfully, data suggests that this is not the main problem facing manufacturers who wish to develop products containing probiotic cultures. In many instances, based on sensory evaluation, no clear differences can be observed between the tastes of products that contained the probiotic strain and the control made only with the support culture.33,152 However, when the typical starters used in a technological process are replaced by the probiotic strains, the resulting products can be very different (Table 8). There are often changes in the chemical composition and the texture of the fermented products (Table 8), but these changes do not necessarily have a noticeable effect of flavor. Obviously, this will depend on the extent of growth of the probiotics in the product. When probiotic cells represent less than 10% of the total population in the fermented product, their effect on the sensory properties is often minimal. Ice cream and ice milk appear to be good products for the delivery of probiotic bacteria. When the cream blend is prepared by adding a fermented milk, the resulting product’s flavor can be effected.153 However, when small quantities of concentrated cultures are introduced, the sensory properties are not effected.154,155 Counts attaining 2 × 108 CFU/g have been achieved with this method.139 Strain or species does seem to be important, since ice creams manufactured with 10% of a Lb. reuteri culture were “more sour” than those made from corresponding cultures of Lb. acidophilus, Lb. rhamnosus, or B. bifidum.140 It was not determined if pH neutralization before mixing the probiotic cultures with the ice cream mix could prevent this effect. Another product that does not seem to be effected by the addition of probiotics is Sweet Acidophilous milk. The lactobacilli are added to milk that is then kept refrigerated. If maintained under 4◦ C, the milk’s flavor is not effected by the lactobacilli after 11 d of storage.156 There appears to be a limited time during which the addition of probiotics does not effect the sensory properties of unfermented milk. Abu-Tarboush et al.69 found a

significant increase in the products of proteolysis in camel milk after 9 d of storage of milk inoculated with 2 × 106 CFU/mL of bifidobacteria at 4◦ C. This was not the case for cow’s milk, however. Consequently, manufacturers will want to test various cultures in their specific manufacturing and storage conditions, and the duration of storage may be a limiting factor. An unusual method for improving the flavor of probiotic yogurt is the use of prerefrigerated milk, aged up to 4 days, with psychrotrophic populations between 107 and 108 CFU/mL.157 Normally, such milk would be undesirable for the manufacture of most dairy products. The prerefrigerated milks generated products having much higher bacterial populations, which might explain the improvement in flavor. The products were tasted less than one day after production, and it remains to be seen if the improvement on flavor is maintained during storage of the yogurts. Prabha and Shankar158 also found increased development of the starter cultures and improved sensory attributes when milk was preincubated with psychrotrophic bacteria, but flavor was negatively effected if the milk was pre-incubated for more than 6 d. The shelf life of fermented milks was prolonged by initial levels of 2 × 107 cells of both Lb. rhamnosus and P. freudenreichi. Their cell number did not increase during storage of fermented milks at 6◦ C for 4 w. With the same culture in quark, the sensory quality was superior to the control product due to the production of diatecyl by the protective culture.159 There are instances where the addition of probiotic cultures can extend the shelf life of products. In catfish fillets,160 adding probiotic cultures inhibited the development of undesirable psychrotrophic flora. The inhibition of psychrotrophs by the probiotics has also been demonstrated in milk-based products.57,161 Although this would presumably delay the appearance of the spoilage flavors associated with the psychrotrophic bacteria, it remains to be determined if flavors specifically produced by the probiotic cultures would appear instead. Some manufacturers add neutraceutical compounds to yogurt, in addition to the probiotic cultures, in order to further enhance the health properties of the product. If fish oil is added, it is possible to suppress the offensive odor by including cathesins or tocopherols in the formulation.162 The cathesin/tocopherol addition also improves the stability of the probiotic cultures during storage. In soya-based products, fermentation by probiotic bacteria can actually improve the flavor. Pentanal and n-hexanal contribute to the beany flavor of soymilk, and this flavor is generally considered undesirable. A strain of Bifidobacterium breve was reported to metabolize these compounds, thus, generating products having low levels of alkylic aldehydes. Some strains of Lb. acidophilus also reduce n-hexanal levels in fermented soya products.96

SYNBIOTICS Many studies suggest that the consumption of synbiotic products12 has higher beneficial effects on the human health

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Table 8

Effect of the addition of probiotic cultures to dairy products on their sensory properties

Product

Addition mode

Effect

Reference

Cheddar

1. 106 CFU/g B. bifidum added to salted, milled curds. One treatment with ICT1

1. No effect on moisture, total protein, soluble protein or fat content, but higher ash content when cells immobilized in K-carrageenan were added. No effect on flavor, body or texture of free or ICT culture. 2. With one B. lactis strain, acidification was more rapid and the cheese had a higher moisture level. Flavour development was faster during ripening, but texture was negatively affected. 1. If content in cultured milk is greater than 50%, noticeable physical changes occur. Over 25%, different overrun and melting properties are registered 2. Beads < 30 µm do not affect texture B. lactis and Lb. acidophilus both affect proteolysis but not lipolysis B. longum reduces acetaldehyde and diacetyl levels in yoghurt formulation enriched with whey solids. Flavour is affected. S. boulardii ferments the fruit carbohydrates and produce gaz and alcohol. 1. Lb. casei and Lb acidophilus give unpleasant fermentation flavor if population >3 × 108 CFU/mL 2. Different viscosity obtained if S. thermophilus is added when compared to products obtained if Lb. rhamnosus or Lb. acidophilus are used alone 3. Lower flavour scores if casitone is added to milk in order to improve growth Addition of B. bifidum increases soluble N and acetic acid levels, and has a slightly detrimental effect on flavour after 14 d Off-flavours after 16 d storage at 4◦ C of microentrapped bifidobacteria, but not free cells The sensory quality was superior to the control product due to the production of diatecyl. Lower acceptability by consumers when cream was fermented to pH 4.5 Addition of probiotics affects flavor; increased acetic acid level; no openings

1. Dinakar and Mistry103 2. McBrearty et al.219

2. 108 CFU/mL of milk from a freeze-dried powder

Ice cream or ice milk

1. Mixing cultured milks to cream mix

Semi-hard goat cheese

2. ICT (alginate) cultures added to cream mix As a starter

Yogurt

With the starter

Fruit yogurt

With the starter

Fermented milk

As a starter

Crescenza cheese

As a starter

Unfermented milk

Microentrapment in alginate beads.

Quarg cheese

Propionibacteria as a starter

Cottage cheese

Bifidobacteria grown in the cream dressing

Gouda cheese

As a starter

1 ICT:

Immobilized cell technology, mostly cells immobilized in k-carrageenan or alginate.

1. Christiansen et al.153 2. Sheu et al.139

Gomes and Malcata71 Baig and Velore220

Lourens-Hattingh and Viljoen32 1. Yoshiharu et al.199 2. Oliveira et al.78 3. Saxena et al.201

Gobbetti et al.179

Truelstrup Hansen et al.143

Suomalainen and Mayra-Makinen159 Blanchette et al.212

Gomes et al.80

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than probiotic or prebiotic products.11,163–165 Indeed, the presence of probiotic and prebiotic in a single food improved the survival of probiotic bacteria during the storage of the product and during the passage of the intestinal tract. Moreover, the synbiotic product may allow an efficient implantation of probiotic bacteria in colonic microbiota, because prebiotic has a stimulating effect on the growth and/or activities of the exogenous and the endogenous bacteria.11 Synbiotic dairy products are already marketed in Europe and Japan, including Symbalance yogurt, manufactured by Tonilait (contains three probiotic strains), the branded prebiotic Raftiline from Orafti, fermented milks Probiotic Plus Oligofructose (which contains two probiotic strains), and the prebiotic Raftilose by Bauer. In synbiotic fermented milks, the strains of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium sp. (B. animalis, B. bifidum, B. breve, B. infantis, and B. longum) are used as probiotic, whereas fructo-oligosacharides, galactooligosacharides, lactulose, and inulin-derived products are used as prebiotic.166,167 Specialty starches that are only hydrolyzed once reaching the colon could also be used for that purpose.22 The use of a mixture of probiotic and prebiotic results in an increasing of cost production that can slow down the development of new synbiotic products. The use of probiotic bacteria, which are able to synthesize prebiotics, might overcome this limitation. Many authors reported the capacity of bifidobacteria to synthesize galacto-oligosaccharides.168–173 However, the production of galactooligosacharides (GOS) by bifidobacteria during manufacture of fermented milks is not well-known in the literature.

CONCLUSION Foods that contain probiotic cultures seem destined to a bright future. As was shown in this article, the addition of probiotic cultures to foods is challenging, but means are available to achieve this goal. Most foods containing probiotics are currently dairybased. There is a need to develop technologies for nondairy or novel probiotic applications.22 The ability to ensure the consumer that they are receiving the claimed quantity of probiotic cells is an important step in providing credibility to the probiotic food sector. However, the question of specific health claims that will be allowed will be a strategic feature in their marketing. The data of Stanton et al. 17 shows that the carrier food influences the efficiency of delivery of viable probiotic cells in the intenstine. Thus, for example, can data on health properties obtained from the consumption of a given strain in yogurt extend to fermented vegetables or meats? With respect to meats, Hammes and Hertel21 contend that proof of a beneficial effect should be based on sound studies performed with the probiotics in the meat matrix as it is consumed. After all the efforts put into adding probiotic strains into a given food result in a marketable product, it would, thus, appear the task is not yet complete. A question must be raised as to whether each novel probiotic-containing food needs to be tested in order for health

claims to be allowed? Obviously, this will depend on the specific allegation. Animal trials that are required for some claims are very expensive and are prohibitive to small and medium size industries. Means of generating in-vitro data that provide accurate predictions of animal reactions need to be further developed. As an example, the SHIME system174,175 was created to simulate the gastro-intestinal tract, with respect to its microbial flora, and good correlations have been achieved between SHIME and invivo data. Tools such as these must continue to be developed. This review has shown that the single most important element in successfully developing a functional food containing probiotics is the selection of the strain with respect to how it will react to the specific constraints of the targeted food. But, sometimes, one wishes to add a specific strain because of a particular demonstrated health benefit and not go through a selection process. Then, two aspects need to be examine: How the probiotic culture is prepared, and how it is used at the plant. In the first instance, such considerations address the manufacturers of probiotic cultures. Applying specific stresses to the cultures in order to prepare them for those they will encounter in the foods is one way of intervening. A second way a culture producer can intervene is by protecting the cells, and microencapsulation/microentrapment technologies are the most promising approaches. As for how the cultures are used at the plant, this review shows that numerous options are available. Creativity is necessary in order to adapt a process for the probiotics. An increasing number of people have reduced immunological response because of aging, AIDS, cancer treatments or, paradoxically, improved sanitation in our society that exposes us less to pathogens. Adding probiotics to foods is a response to this problem. Although the addition of probiotics to foods is challenging, the benefits to our society are certainly worth the effort.

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