Probiotic Bacteria: Selective Enumeration and Survival in Dairy Foods

Probiotic Bacteria: Selective Enumeration and Survival in Dairy Foods

SYMPOSIUM: PROBIOTIC BACTERIA Probiotic Bacteria: Selective Enumeration and Survival in Dairy Foods N. P. Shah School of Life Sciences and Technology ...

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SYMPOSIUM: PROBIOTIC BACTERIA Probiotic Bacteria: Selective Enumeration and Survival in Dairy Foods N. P. Shah School of Life Sciences and Technology Victoria University of Technology PO Box 14428, Melbourne City Mail Centre Victoria 8001 Australia

ABSTRACT A number of health benefits have been claimed for probiotic bacteria such as Lactobacillus acidophilus, Bifidobacterium spp., and Lactobacillus casei. Because of the potential health benefits, these organisms are increasingly incorporated into dairy foods. However, studies have shown low viability of probiotics in market preparations. In order to assess viability of probiotic bacteria, it is important to have a working method for selective enumeration of these probiotic bacteria. Viability of probiotic bacteria is important in order to provide health benefits. Viability of probiotic bacteria can be improved by appropriate selection of acid and bile resistant strains, use of oxygen impermeable containers, two-step fermentation, micro-encapsulation, stress adaptation, incorporation of micronutrients such as peptides and amino acids and by sonication of yogurt bacteria. This review will cover selective enumeration and survival of probiotic bacteria in dairy foods. (Key words: Lactobacillus acidophilus, bifidobacteria, Lactobacillus casei, selective enumeration, survival) Abbreviation key: ABT = Lactobacillus acidophilus, Bifidobacterium spp., Streptococcus thermophilus, ACH = acid casein hydrolysate, β-gal = β-galactosidase, LAB = lactic acid bacteria, LC = Lactobacillus casei agar, MRS = deMann Rogosa and Sharpe agar, NNLP = nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate; RCA = reinforced clostridial agar; ST = Streptococcus thermophilus agar, WP = whey powder, WPC = whey protein concentrate. INTRODUCTION The health benefits derived by the consumption of foods containing acidophilus and bifidus (called AB products) products are well documented and more than 90 probiotic products are available worldwide. Probiotic food can be defined as “food containing live microorgan-

Received June 2, 1999. Accepted October 7, 1999. e-mail: [email protected] 2000 J Dairy Sci 83:894–907

isms believed to actively enhance health by improving the balance of microflora in the gut” (28, 29). To provide health benefits, the suggested concentration for probiotic bacteria is 106 cfu/g of a product (78). However, studies have shown low viability of probiotics in market preparations (4, 45, 88). The need to monitor survival of Lactobacillus acidophilus and bifidobacteria in fermented products has often been neglected, with the result that a number of products reach the market containing a few viable bacteria (4, 88). To assess viability and survival of probiotic bacteria, it is important to have a working method for selective enumeration of these probiotic bacteria. Several media for selective enumeration of L. acidophilus and Bifidobacterium spp. have previously been proposed. However, most of these methods are based on pure cultures of these organisms. Similarly, there are only few reports that have described selective enumeration of Lactobacillus casei in the presence of other probiotic bacteria and yogurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) (9, 76). An important parameter in monitoring viable organisms in assessing product quality is the ability to count L. acidophilus, Bifidobacterium spp., and L. casei differentially. Selective enumeration of Lactobacillus reuteri, Lactobacillus plantarum, and Lactobacillus rhamnosus has not been studied extensively. A number of factors have been claimed to affect the viability of probiotic bacteria in yogurt, including acid and hydrogen peroxide produced by yogurt bacteria, oxygen content in the product, and oxygen permeation through the package (41, 44, 55). Although L. acidophilus and bifidobacteria tolerate acid, a rapid decline in their numbers in yogurt has been observed (16, 38, 84). Bifidobacteria are not as acid tolerant as L. acidophilus; the growth of the latter organisms ceases below 4.0, while the growth of the Bifidobacterium spp. is retarded below pH 5.0 (83). L. acidophilus and Bifidobacterium spp. grow slowly in milk during product manufacture. Therefore, the usual production practice is to incorporate yogurt cultures along with probiotic cultures. However, L. delbrueckii ssp. bulgaricus produces lactic acid during fermentation and refrigerated storage. The latter process is known in the industry as ‘post-acidification.” Post-

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acidification is found to cause loss of viability of probiotic bacteria (88). It is important that the cells remain viable throughout the projected shelf life of a product so that when consumed the product contains sufficient viable cells. This paper will focus on two aspects: 1) development of selective enumeration techniques for enumeration of L. acidophilus, Bifidobacterium spp., and L. casei; and 2) viability and survival of L. acidophilus and Bifidobacterium spp. in dairy foods. DEVELOPMENT OF SELECTIVE ENUMERATION TECHNIQUES Yogurts are made from the symbiotic growth of the two bacteria: S. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. These yogurt bacteria do not survive the gastric passage or colonize the gut. Hence, the recent trend is to add L. acidophilus and Bifidobacterium spp. to yogurt. Although Lactobacillus johnsonii and Lactobacillus crispatus have been used in probiotic products, Australian manufacturers use L. acidophilus only. However, because of viability problems associated with L. acidophilus and Bifidobacterium spp. during storage (4, 19, 88), the more recent trend is to add L. casei to AB cultures. Several media have been developed for differential enumeration of yogurt culture organisms (L. delbrueckii ssp. bulgaricus and S. thermophilus), including lactic acid bacteria agar (25), Lee’s agar (62), and reinforced clostridial agar (RCA) adjusted to pH 5.5 (46). Jordano et al. (47) examined M17 media for the recovery of S. thermophilus. Hamann and Marth (36) evaluated four differential and two general purpose media for enumerating yogurt culture organisms. Several media have been suggested for the enumeration of L. acidophilus, including bile medium (14), Rogosa agar, deMan Rogosa Sharpe (MRS) medium containing maltose, raffinose or melibiose in place of dextrose (40), cellobiose-esculin agar (42), and agar medium based on X-Glu (52). Similarly, several selective media have been developed for enumeration of pure cultures of Bifidobacterium spp. including (NNLP) nalidixic acid- neomycin sulfate- lithium chloride- paromomycin sulfate agar (5, 8, 61, 65, 67, 80, 90, 91, 92). However, these media may not be suitable for selective enumeration of Bifidobacterium spp. in the presence of L. acidophilus and yogurt culture organisms. Further, differences exist among the strains of the same species regarding to sugar fermentation characteristics and tolerance of low pH and bile. Concern is growing that some media that contain bile or antibiotics might also restrict the growth of L. acidophilus or bifidobacteria and that counts obtained are

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not representative of the actual number of viable cells present in the product. Selective enumeration of L. casei in yogurt-type fermented milks containing probiotic bacteria based on 15°C incubation temperature and 14-d incubation time was studied by Champagne et al. (9). However, an incubation time of 14 d may not be practical for the dairy industry if the results are required in a short time. Lankaputhra et al. (60) proposed the use of MRSmaltose agar for selective enumeration of L. acidophilus in the presence of yogurt organisms in a product, which does not contain Bifidobacterium spp. Lankaputhra and Shah (57) developed a simple method for selective enumeration of L. acidophilus in the presence of yogurt bacteria and Bifidobacterium spp. based on sugar fermentation patterns. Table 1 summarizes the sugar utilization patterns of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and Bifidobacterium spp. Based on sugar fermentation patterns, salicin was found to be suitable for selective enumeration of L. acidophilus because this organism can utilize salicin. A salicin concentration of 0.5% was appropriate for producing proper size colonies. This work has been limited to L. acidophilus because yogurt manufacturers in Australia do not use other related species, including L. reuteri, L. plantarum, and L. rhamnosus. In another study, Dave and Shah (18) evaluated 15 media to determine their suitability for selective enumeration of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria with five to six strains of each of the four groups of organisms. Streptococcus thermophilus (ST) agar was found to be suitable for selective enumeration of S. thermophilus under aerobic incubation at 37°C for 24 h. The MRS agar at pH 5.2 or RCA at pH 5.3 could be used for the enumeration of L. delbrueckii ssp. bulgaricus when the incubation is carried out at 45°C for 72 h. MRS-maltose agar could be used to enumerate total counts of L. acidophilus and bifidobacteria. For selective enumeration of L. acidophilus, MRS-salicin agar or MRS-sorbitol agar could be used. For selective enumeration of bifidobacteria, MRSNNLP agar has been found to be suitable, however, for determining bifidobacteria by differential counts between L. acidophilus enumerated on MRS-salicin agar or MRS-sorbitol agar and the total counts of L. acidophilus and bifidobacteria obtained from MRS-maltose agar can also be done. However, counterchecking of pure strains of bifidobacteria in these media is recommended before adopting them for enumeration purposes in the presence of yogurt cultures and L. acidophilus. The recommended methods for selective enumeration of these four groups of organisms are shown in Table 2. Journal of Dairy Science Vol. 83, No. 4, 2000

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SHAH Table 1. Sugar utilization patterns by Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium spp.1 Sugar2 Strains S. thermopilus 2000 2002 2008 2010 2013 2014 L. delbrueckii ssp. bulgaricus 2501 2505 2515 2517 2519 L. acidophilus 2400 2401 2404 2405 2409 2415 Bifidobacterium spp. B. bifidum 1900 B. bifidum 1901 B. infantis 1912 B. adolescentis 1920 B. breve 1930 B. longum 1941 B. longum 20097 B. pseudolongum 20099 B. thermpilum 20210

Sal

Cel

Fru

Man

Sor

Glu

− − − − − −

− − − − − −

++ ++ ++ ++ ++ ++

− − − − − −

− − − − − −

++ ++ ++ ++ ++ ++

− − − − −

− − − − −

++ ++ ++ ++ ++

− − − − −

− − − − −

++ ++ ++ ++ ++

+++ +++ +++ +++ +++ +++

++ ++ ++ +++ +++ +++

++ ++ ++ ++ ++ ++

+ + + + + +

+ + + + + +

++ ++ ++ ++ ++ ++

− − − − − − − − −

+ − − − − + − ++ −

++ ++ ++ ++ ++ ++ ++ ++ ++

− − − − − ± − + −

− − − − − ± − + −

++ ++ ++ ++ ++ ++ ++ ++ ++

1 −, no growth; ±, pin point colonies; +, colony size 0.1 to 0.5 mm; ++, colony size 0.6 to 1.5 mm, +++, colony size > 1.5 mm. Source: Lankaputhra and Shah (57). 2 Sal = salicin, Cel = cellobiose, Fru = fructose, Man = mannitol, Sor = sorbitol, Glu = glucose.

The efficacy of the proposed methods was validated by Dave and Shah (18), who analyzed six commercial products with the different selective bacteriological meTable 2. Media recommended for selective enumeration of Streptococcus thermophilus, Lactococcus delbrueckii ssp. bulgaricus, Lactococcus acidophilus, and Bifidobacterium spp. in the presence of all the four groups of organisms (18). Agar

Bacteria Incubation conditions

S. thermophilus agar

S. thermophilus

Aerobic incubation 37°C for 24 h

MRS1 agar, pH 5.2 or L. delbrueckii ssp. RCA2 agar, pH 5.3 bulgaricus

Anaerobic incubation 45‡gC for ≥72 h

MRS-salicin agar or MRS-sorbitol agar MRS-NNLP3

Anaerobic incubation 37°C for 72 h Anaerobic incubation 37°C for 72 h

L. acidophilus Bifidobacteria

MRS = deMan Rogosa and Sharpe agar. RCA = Reinforced clostridial agar. 3 NNLP = Nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate. 1

2]

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dia proposed in Table 2. The samples that were analyzed included probiotic capsules (Blackmores Ltd., Balgowlah, NSW, Australia) claimed to contain L. acidophilus and bifidobacteria; a commercial yogurt sample (Yoplait, Echuca, Victoria, Australia); yogurt prepared in our laboratory with a commercial starter culture (Chr. Hansen, Bayswater, Victoria, Australia) containing all four groups of organisms; and three Bulla brand Fruit’N yogurt sticks (Regal Cream Products Pty Ltd., North Melbourne, Victoria, Australia) claimed to contain L. acidophilus and Bifidobacterium spp. The results are presented in Table 3. The proposed media appear to be selective for the four groups of organisms. However, there is little information on selective enumeration of L. casei in yogurt and fermented milk drinks, which may contain yogurt bacteria and probiotic bacteria. A selective medium L. casei (LC) agar has been developed by Ravula and Shah (76) for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain strains of yogurt bacteria (S. thermophilus and L. del-

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ENUMERATION AND SURVIVAL OF PROBIOTIC BACTERIA Table 3. Recovery of yogurt and probiotic bacteria from commercial products (18). Organisms

Agar

BDC

Yop

ChH

BCS

BS

BB

Streptococcus thermophilus

ST1 M17

<2.00 <2.00

9.28 9.24

7.86 8.65

8.90 7.73

8.26 8.77

8.26 8.20

Lactobacillus delbrueckii ssp. bulgaricus Lactobacillus acidophilus

MRS (pH 5.2)

<3.00

<3.00

7.18

7.13

7.18

7.45

MRS-salicin MRS-sorbitol MRS-maltose

8.40 8.38 7.96

7.48 7.33 7.08

7.26 7.21 6.91

4.72 4.69 4.46

4.95 4.84 4.62

5.20 5.15 4.83

Bifidobacteria

MRS-NNLP2 Substraction Method

7.90 8.18

6.23 6.88

7.21 7.59

2.08 <4.00

2.48 <4.00

3.04 <4.00

Log10 cfu/g

BDC = Blackmores capsule (Blackmore Ltd., Balgowlah, NSW, Australia), Yop = commercial yogurt sample (Yoplait, Echuca, Victoria, Australia), ChH = yogurt from culture (Chr. Hansen, Bayswater, Victoria, Australia), BCS = Bulla (Regal Cream Products Pty Ltd., North Melbourne, Victoria, Australia) chocolatecoated strawberry stick, BS = Bulla strawberry stick, and BB = Bulla banana stick. 1 Streptococcus thermophilus agar. 2 Nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate.

brueckii ssp. bulgaricus), Lactobacillus acidophilus, Bifidobacterium spp., and L. casei. The composition of LC agar is shown in Table 4. Table 5 shows the viable counts of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria, and L. casei on various bacteriological media (76). ST agar and M17 agar were selective for S. thermophilus, and MRS-pH modified agar was selective for L. delbrueckii ssp. bulgaricus. L. acidophilus and L. casei formed colonies on MRS-salicin, MRS-sorbitol agar, MRS-ribose agar, and MRS-gluconate agar. Thus, these media cannot be used for selective enumeration of L. casei. Hence, MRS-salicin medium proposed earlier cannot be used for selective enumeration of L. casei in products containing L. acidophilus and L. casei. LC agar inhibited the growth of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria; thus the medium was selective for L. casei. The efficacy of the medium was validated with nine commercial yogurts and fermented milk drinks claimed as containing single or mixed strains of S. thermophilus,

Table 4. Composition of Lactobacillus casei (LC) agar (76). Ingredients

Amount (g/L)

Bacteriological peptone Yeast extract Lab Lemco KH2PO4 Sodium acetate (trihydrate) Tri-ammonium citrate Magnesium sulphate (hepta hydrate) Magnesium sulphate (tetra hydrate) Acid casein hydrolysate Tween 80 Bacteriological agar

10.00 1.00 4.00 2.00 3.00 1.00 0.20 0.05 1.00 1.00 12.00

L. delbrueckii ssp. bulgaricus, L. acidophilus, Bifidobacterium spp. and L. casei. Table 6 shows the counts of L. casei in various commercial products. Products 4, 5, and 8 did not contain L. casei and this organism was not detected in these products. Products 1, 2, 3, and 9 were claimed to contain yogurt bacteria; L. acidophilus, bifidobacteria, and L. casei and L. casei respectively, was detected in these products. The populations of L. casei in products 2 and 3 were small. Products 6 and 7 claimed to contain strains of L. casei, and this organism was detected in these products. Thus, it appears that LC agar is selective for L. casei. In summary, MRS-salicin or MRS-sorbitol agar can be used for selective enumeration of L. acidophilus provided L. casei is not added into the product. However, if L. casei is added to the product, then MRS-sorbitol agar or MRS-salicin agar can be used to obtain counts of L. acidophilus and L. casei, and LC agar can be used to obtain a total count of L. casei. The counts of L. casei can be subtracted from the total population of L. acidophilus and L. casei enumerated with MRS-salicin or MRS-sorbitol agar. However, frequently used strains in commercial products, from culture suppliers and even from culture sources such as ATCC or CSIRO are not properly speciated by genetic methods. VIABILITY AND SURVIVAL OF L. acidophilus AND Bifidobacterium SPP. Several preparations employing L. acidophilus are well established in the market. In Japan, bifidobacteria containing products are very popular and these products account for more than one-third of the total yogurt sales. In France, products containing bifidobacteria and L. acidophilus have increased by ∼300% to capture 4% Journal of Dairy Science Vol. 83, No. 4, 2000

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SHAH Table 5. Counts (log10 cfu/g) of S. thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, bifidobacteria, and Lactobacillus casei on various bacteriological media (76).

Strains

ST Agar

M17 Agar

MRSbasal agar

MRSsalicin agar

MRSsorbitol agar

MRSribose agar

MRSgluconate agar

MRS-pHmodified agar

MRSNNLP1 agar

LC agar

ST2 2008 ST WJ7 LB3 2501 LB WJ7 LA4 2415 LA MJLA BB5 20099 BB BDB LC6

8.56 8.68 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00

8.72 8.59 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00

<3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00

<3.00 <3.00 <3.00 <3.00 8.62 8.68 <3.00 <3.00 8.89

<3.00 <3.00 <3.00 <3.00 8.41 8.46 <3.00 <3.00 8.68

<3.00 <3.00 <3.00 <3.00 8.68 8.55 <3.00 <3.00 8.73

<3.00 <3.00 <3.00 <3.00 8.85 8.55 <3.00 <3.00 8.50

<3.00 <3.00 7.66 7.91 <3.00 <3.00 <3.00 <3.00 <3.00

<3.00 <3.00 <3.00 <3.00 <3.00 <3.00 8.85 8.93 <3.00

<3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 <3.00 9.08

NNLP = Nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate. ST = Streptococcus thermophilus. 3 LB = Lactobacillus delbrueckii ssp. bulgaricus. 4 LA = Lactobacillus acidophilus. 5 BB = Bifidobacteria. 6 LC = Lactobacillus casei. 1 2

of total fresh milk sales (39). Presently, 11% of all yogurt sold in France contains added bifidobacteria. In Germany, one of the first bifidus products to be marketed, known as Biogarde, sold more than 400 million units in 1976 and the product is well established in the market. Biogarde is produced by 45 dairy companies in Germany. In Denmark, a product called Cultura was promoted as a completely safe and easily digestible food. Bifidus products are also produced in Canada, Italy,

Poland, the United Kingdom, Czechoslovakia, and Brazil. In the United States and Australia, bifidus products have been introduced in recent years (39). In Europe and Australia, yogurt containing L. acidophilus and Bifidobacterium spp. is referred to as ‘AB’ yogurt. The trend is to incorporate L. casei in addition to L. acidophilus and bifidobacteria and such products are known as ‘ABC’ yogurt. Traditionally, yogurt is manufactured using S. thermophilus and L. delbrueckii

Table 6. Counts (log10 cfu/g) of Lactobacillus casei from different commercial products enumerated on LC agar (76). Products Product 1—yogurt

Product 2—skim milk yogurt

Product 3—mild continental yogurt

Product 4—yogurt Product 5—yogurt Product 6—fermented milk drink Product 7—fermented milk drink Product 8—drinking yogurt

Product 9—dairy fruit drink

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Organisms claimed to be present in the products Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, bifidobacteria, and L. casei S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria, and L. casei S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria, and L. casei S. thermophilus, L. acidophilus and bifidobacteria S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus L. casei L. casei S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, and bifidobacteria S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and L. casei

L. casei counts 7.49

3.41

3.72

<3.00 <3.00 8.22 6.98 <3.00

8.08

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ssp. bulgaricus as starter cultures. These yogurt organisms are claimed to offer some health benefits; however, they are not natural inhabitants of the intestine. Therefore, for yogurt to be considered as a probiotic product, L. acidophilus and bifidobacteria (and L. casei or both) are incorporated as dietary adjuncts. Products such as Yakult contain L. acidophilus Shirota strain. Thus, fermented milk with only L. acidophilus or L. acidophilus and Bifidobacterium spp. could be manufactured; however, the longer incubation period and product quality are the two main factors that are sacrificed when fermenting milk with only ‘AB’ or ‘ABC’ bacteria. Thus, the normal practice is to make products with both yogurt and probiotic bacteria. To obtain the desired therapeutic effects, the yogurt and probiotic bacteria must be available in sufficient numbers. It has been suggested that these organisms should be present in a food to a minimum level of 106 cfu/g (78) or daily intake should be about 108 cfu/g (4). Such high numbers might have been suggested to compensate for the possible reduction in the numbers of the probiotic organisms during passage through the stomach and the intestine. According to the Australian Food Standards Code (Standard H8), yogurt must have a pH of ≤4.5 and must be prepared with S. thermophilus and L. delbrueckii ssp. bulgaricus or other suitable lactic acid bacteria. The Australian Food Standards Code does not specify any requirements regarding the numbers of yogurt or probiotic bacteria in the fermented products. However, in other countries standards have been developed regarding the requirement of the numbers of the probiotic bacteria in fermented products. In Japan, a standard has been developed by the Fermented Milks and Lactic Acid Bacteria Beverages Association, which requires a minimum of 107 viable probiotic bacteria cells per milliliter to be present in fresh dairy products (78). Therefore, efforts to select the right type of strain and improvement in viability are commercially significant. Beneficial and proven strains could be obtained through a reputed starter culture supplier; however, the viability of organisms during manufacture and storage is the sole responsibility of the manufacturers. Several reports have shown that the viability of these organisms is often low in yogurt (31, 41, 82). A number of brands of commercial yogurts have been analyzed in Australia (4, 88) and in Europe (45) for the presence of L. acidophilus and bifidobacteria. Most of the products contained very low numbers of these organisms, especially bifidobacteria. The viability of probiotic bacteria is affected by inhibitory substances such as lactic acid produced during production and cold storage. During production of yogurt, yogurt bacteria and probiotic bacteria produce

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organic acids. The pH of the product needs to be 4.5 or lower to meet legal requirements and to produce good quality yogurt. Also, depending on the extent of growth of bifidobacteria, concentrations of acetic acid could vary in the product. Several factors have been claimed to affect the viability of yogurt and probiotic cultures in fermented milk products. The viability of probiotic bacteria in yogurt depends on the strains used, interaction between species present, culture conditions, production of hydrogen peroxide due to bacterial metabolism, final acidity of the product, and the concentrations of lactic and acetic acids. The viability also depends on the availability of nutrients, growth promoters and inhibitors, concentration of sugars (osmotic pressure), dissolved oxygen and oxygen permeation through package (especially for Bifidobacterium spp.), inoculation level, incubation temperature, fermentation time and storage temperature (6, 17, 33, 93). However, the main factors for loss of viability of probiotic organisms have been attributed to the decrease in the pH of the medium and accumulation of organic acids as a result of growth and fermentation (38, 84). Bifidobacteria are anaerobic in nature; therefore, higher oxygen content may affect their growth and viability. The availability of growth factors is also reported to affect the growth and viability of yogurt and probiotic bacteria. Antagonism among the bacteria used in the starter cultures caused by the production of antimicrobial substances such as bacteriocins may decrease the numbers of any sensitive organisms that may be present in a product or starter cultures. Viability of Probiotic Bacteria as Affected by Antagonism between Various Groups of Organisms The precise mechanism by which lactic acid bacteria (LAB) cause inhibition of microorganisms seems to be rather complex and has not been fully understood. The inhibitory activity of LAB can be attributed to creation of a hostile environment for foodborne pathogens and spoilage organisms in foods. Mechanisms proposed for such effects include production of lactic and other organic acids, hydrogen peroxide, competition and nutrient depletion, altered oxidation-reduction potential, and production of bacteriocins or antibiotics (73). However, it is generally believed that the inhibitory activity is a composite effect of several factors. It is now known that hydrogen peroxide accumulates in cultures of lactococci, lactobacilli, leuconostocs, and pediococci. Anders et al. (3) observed that lactococci produced sufficient hydrogen peroxide to be auto-inhibitory. Accumulation of hydrogen peroxide in growth media can occur because lactobacilli do not possess catalase enzyme (49). Hydrogen peroxide produced by L. Journal of Dairy Science Vol. 83, No. 4, 2000

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acidophilus has been found to cause inhibition of S. aureus, E. coli, Sal. typhimurium, and Cl. perfringens (32). Lactococcus delbrueckii ssp. bulgaricus produces sufficient hydrogen peroxide to show inhibition of psychrotrophs (1). However, the concentration of hydrogen peroxide produced by starters may not be sufficient to directly affect the cells in the products. Hydrogen peroxide can react with other components to form inhibitory substances. Joseph et al. (48) studied antagonism between yogurt and probiotic bacteria isolated from commercial starter cultures and commercial yogurts using modified spot on lawn and agar well diffusion assays. Zones of inhibition for two Bifidobacterium isolates were observed with all L. acidophilus strains. The isolates of L. acidophilus were resistant and did not show inhibition by any of the four groups of microorganisms. Inhibitory activity of L. acidophilus was lost when the MRS broth was treated with the proteolytic enzymes, chymotrypsin, and papain. This confirmed that an active protein moiety was involved in the inhibition. Dave and Shah (23) and Shah and Ly (87) also observed antagonistic relationships between yogurt and probiotic bacteria. One strain of S. thermophilus was found to produce heat stable bacteriocin against 2 strains of bifidobacteria (87). Growth Requirements of Bifidobacteria Following early observations that the growth of bifidobacteria is stimulated by human milk numerous nutritional studies have been designed to elucidate the properties of bifidus factor/s present in human milk or to find substitutes (81). The essential factor in human milk, which was lacking in cow’s milk (bifidus factor) was identified as N-acetyl-D-glucosamine-containing saccharides (35, 66). Bifidus factor was reported to stimulate the growth of Bifidobacterium spp. (54, 74). Bifidus factor of human milk casein was further characterized by a number of workers (7, 68, 70). Lactulose (4O-β-D-galactopyranosyl-D-fructose) also has a growth promoting effect on bifidobacteria (64, 69). Unfortunately, milk is considered to be a less than optimal medium for the growth of bifidobacteria (10, 30). Therefore, many studies have tried to improve the growth of bifidobacteria in milk. Kosikowski (53) suggested the use of sterile milk supplemented with 0.5% Bacto-liver, 0.05% MgSO4, and 0.001% cysteine for growth of bifidobacteria in milk. Marshall et al. (63) fortified milk with whey protein and threonine to provide the bifidobacteria with nutritious medium and lower redox potential. Anand et al. (2) reported good growth of B. bifidum in sterile skim milk supplemented with 1% dextrose and 0.1% yeast extract. Journal of Dairy Science Vol. 83, No. 4, 2000

The slow growth of bifidobacteria in milk may be improved by the addition of growth promoting substances e.g., yeast extract, or pepsin-digested milk (72). Oxygen Because bifidobacteria are anaerobic microorganisms (81), oxygen toxicity is an important and critical problem. During yogurt production, oxygen can easily invade and dissolve in the milk. To exclude oxygen during the production of bifidus milk products, special equipment is required to provide an anaerobic environment. Oxygen can also enter the product through packaging materials during storage. Cheng and Sandine (11) found satisfactory growth of a variety of Bifidobacterium spp. without anaerobic conditions in whey-based medium containing L-cysteine (0.05%) and yeast extract (0.3%). APPROACHES TO IMPROVE THE VIABILITY OF PROBIOTIC BACTERIA Selection of Acid and Bile Resistant Strains One of the most important characteristics of probiotic microorganisms is their ability to survive through the acid in the human stomach and bile in the intestine. Several investigators have studied the survival of L. acidophilus and Bifidobacterium spp. in the presence of acid and bile salts (16, 34, 37, 43). Because it survives better, B. animalis is often used in fermented products. Iwana et al. (45) isolated B. animalis from several products available in Europe. Clark et al. (13) studied the survival of B. infantis, B. adolescentis, B. longum, and B. bifidum in acidic conditions and reported that B. longum survived the best. Clark and Martin (12) reported that B. longum tolerated bile concentrations of as high as 4.0%, whereas Ibrahim and Bezkorovainy (43) found B. longum to be the least resistant to bile. Many strains of L. acidophilus and Bifidobacterium spp. intrinsically lack the ability to survive harsh conditions in the gut and may not be suitable for use as dietary adjuncts in fermented foods. Lankaputhra and Shah (56) have shown that among six CSIRO strains of lactobacilli, three L. acidophilus strains survived best under acidic conditions. Two strains of L. acidophilus showed the best tolerance to bile. Among the nine strains of Bifidobacterium spp., B. longum and B. pseudolongum survived best under acidic conditions. Bifidobacterium longum, B. pseudolongum, and B. infantis showed the best tolerance to bile. However, B. infantis survived poorly in acidic conditions and may not be suitable for inclusion as dietary adjuncts. Thus, selection of appropriate strains on the basis of acid and bile

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tolerance would help improve viability of these probiotic bacterial strains. Glass Containers versus Plastic Containers Lactobacillus acidophilus is microerophilic and bifidobacteria are anaerobic. Because bifidobacteria are anaerobic, oxygen toxicity is an important consideration. Oxygen can easily dissolve in the milk. Dave and Shah (19) studied the survival of yogurt and probiotic bacteria in yogurt made in plastic containers and glass bottles. The increase in numbers and survival of L. acidophilus during storage were directly affected by the dissolved oxygen content which was shown to be higher in yogurts made in plastic containers than glass. For the samples stored in glass bottles, the counts remained higher than those stored in plastic cups. Bifidobacteria multiplied better in glass bottles than in plastic cups. The initial counts of the bifidobacteria population were 1.6-fold higher in yogurt prepared in glass bottles than in plastic cups. Although, the acid contents were similar in products stored in glass bottles and plastic cups at 4°C, the survival rate was 30 to 70% higher in products fermented and stored in glass bottles than in plastic cups. Better survival and viability of bifidobacteria in deaerated milk has been observed by Klaver et al. (51). Thus, it may be important to store the products in glass containers or to increase the thickness of the packaging materials used for AB or ABC products. Viability as Affected by Level of Inoculum Yogurt manufacturers rely mainly on starter culture suppliers for starter cultures. Accordingly the manufacturers may feel that the supplier is responsible for poor viability, especially of probiotic bacteria. Conversely, starter culture suppliers may suspect that manufacturers do not follow their instructions, particularly those for temperature of incubation and level of starter addition, which may lead to poor final counts. Dave and Shah (20) studied the effect of concentration of starter addition on the viability of yogurt and probiotic bacteria in yogurt made from four commercial starter cultures. The conditions used for incubation, pH, and storage temperature closely followed the recommendations of the starter culture supplier. Four commercial starter cultures, C1, C2, C3, and C4, were studied. Cultures C1 and C2 contained S. thermophilus and L. delbrueckii ssp. bulgaricus, L. acidophilus, and bifidobacteria as constitutive microflora, whereas cultures C3 and C4 contained L. acidophilus, bifidobacteria, and S. thermophilus (ABT culture) only. In all the commercial culture combinations studied, the strains of probiotic organisms (L. acidophilus and bifidobacteria)

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were kept the same, whereas strains of yogurt bacteria varied. The starter cultures in the freeze-dried concentrated Direct to Vat Set form were added at 0.5, 1.0, 1.5, or 2.0 g/10 L in separate containers. Streptococcus thermophilus multiplied in yogurt many more times with the lower concentration of inoculum than with the higher concentration. However, the final counts of the organism remained slightly higher at the higher concentration of inoculum, which could primarily be due to high initial numbers. Streptococcus thermophilus might be more active in samples prepared with less inoculum. Larger drops in pH of samples prepared with a low rate of inoculum were observed. The decrease in the count was highest for L. delbrueckii ssp. bulgaricus and the numbers declined to <105 after 15 to 20 d of storage. Lactobacillus delbrueckii ssp. bulgaricus remained viable for longer times in yogurt prepared with less inoculum. For C3 and C4, the viability was satisfactory (106 cfu/g) for 35 d in products prepared with higher rates of inoculum. For the other inoculum densities for starter cultures C3 and C4 and at all inoculum densities for C1 and C2, the viability was poor. The pH was the most crucial factor for the survival of L. acidophilus culture. If the pH in yogurt dropped below 4.4 at the time of fermentation, it resulted in a 3 to 4 log cycle decrease in L. acidophilus numbers. In yogurt prepared with C1 starter culture, the number of bifidobacteria remained ≥106 cfu/g for up to 35 d, whereas for starter cultures C2 and C3, the counts dropped to <106 cfu/g in yogurt prepared with the lower concentration of inoculum. In C1 and C2, the multiplication of bifidobacteria was higher possibly because proteolytic activity of L. delbrueckii ssp. bulgaricus resulted in availability of free amino acids, which have been reported to be essential growth factors for bifidobacteria. For bifidobacteria, the counts dropped to <106 cfu/g in yogurt with lower concentration of inoculum. Two-Step Fermentation Inhibitory substances such as acid and hydrogen peroxide produced by yogurt bacteria are claimed to be responsible for poor survival of L. acidophilus and bifidobacteria. Although yogurt bacteria produce inhibitory substances against probiotic bacteria, the former are essential in yogurt manufacture to provide the typical yogurt flavour. Generally, yogurt bacteria grow faster than probiotic bacteria during fermentation, and produce acids, which could reduce the viability of probiotic bacteria. Lankaputhra and Shah (58) studied the effect of twostage fermentation on viability of probiotic bacteria. Fermentation with probiotic bacteria followed by ferJournal of Dairy Science Vol. 83, No. 4, 2000

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mentation with yogurt bacteria may be helpful in improving the viability of these probiotic bacteria. Initial fermentation with probiotic bacteria was carried out for 2 h, followed by fermentation by yogurt bacteria. This allowed the probiotic bacteria to be in their final stage of lag phase or early stage of log phase and thus could dominate the flora, resulting in higher counts. The initial counts of probiotic bacteria have been found to increase by 4 to 5 times in the product made by the two step fermentation process. The final counts after 6 wk of storage were >107cfu/g of the product. The final counts of L. acidophilus 2409 and B. longum 1941 after 6 wk of storage were 6.85 and 7.93 log cfu/g and 7.60 and 8.84 cfu/g for yogurt prepared by single and twostep processes, respectively. Microencapsulation The numbers of culture bacteria in frozen fermented dairy desserts or frozen yogurt are reduced significantly by acid, freezing injury, sugar concentration, and oxygen toxicity. Microencapsulation is a process in which the cells are retained within the encapsulating membrane to reduce the cell injury or cell loss and may have applications in these products. Ravula and Shah (75) studied viability of probiotic bacteria in fermented frozen dairy desserts and found that the probiotic organisms do not survive very well in such products. About 16% sugar is added to frozen fermented dairy desserts; addition of sugar has been found to affect the growth of probiotic bacteria (R. R. Ravula and N. P. Shah, 1999 unpublished data). Several studies (71, 77) have reported on microencapsulation by using gelatin or vegetable gum to provide protection to acid sensitive bifidobacteria. Entrapment of living microbial cells in calcium alginate is simple and low cost. Furthermore alginate is nontoxic so that it may be safely used in foods. Alginate gels can be solubilized by sequestering calcium ions thus releasing entrapped cells (71). Kim et al. (50), Rao et al. (71), and Sheu and Marshall (89) have studied methods for microencapsulating lactic acid bacteria and their survival in acidic conditions. Ravula and Shah (77) have developed a microencapsulation technique with sodium alginate. Encapsulated organisms were incorporated in fermented frozen dairy desserts and viability of probiotic bacteria monitored. The counts of L. acidophilus and bifidobacteria decreased to <103 cfu/g in the control batch, whereas the counts were >105 cfu/g in the products made using encapsulated organisms (Figure 1). Stress Adaptation A strategy to improve the viability of L. acidophilus in yogurt is to adapt the organisms to harsh environments Journal of Dairy Science Vol. 83, No. 4, 2000

Figure 1. Survival of encapsulated and non-encapsulated cells of Lactobacillus acidophilus LA2400 in frozen fermented dairy desserts during storage for 12 wk at −28°C (77).

which normally cause loss in viability by first culturing cells in an environment. This then allows them to prepare for normally lethal conditions. This adaptation increases the survival rate of organisms in harsh conditions more than those that had been shifted directly to lethal acidic conditions. This mechanism is valid in other organisms including Salmonella typhimurium and Escherichia coli. Mechanisms contributing to the adaptive acid tolerance response include the induction of a new pH homeostasis system and the synthesis of a new set of proteins known as stress proteins (26, 79). Stress proteins can be transiently or constitutively produced and have been found to contribute to the adaptive acid tolerance response (26). Proteins induced after heat shock, including the chaperonin class proteins DnaK and GroEL, have shown homology with proteins produced during acid shock (26). These proteins are capable of refolding heat-denatured proteins into their native state and protection of proteins from denaturation during cellular stress. Transiently produced proteins confer only short-term protection to the organism, while constitutive production of proteins is associated with extended survival periods. The stress responses to acid have also been found to fully or partially protect the cell against other stress responses with the overlap of production of proteins (27). In a study by McKechnie and Shah (unpublished data), strains of L. acidophilus were cultured under optimal conditions to mid-log phase before being exposed to moderate acid conditions (acid adaptation). Following acid adaptation, cells were placed in either normally lethal acidic conditions or conditions that the organisms encounter in a product such as yogurt. Results have shown that exposure of cells during midlog phase to moderate acid conditions increased the survival rate of these organisms in normally lethal

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Conditions for adapting cells appear to depend on the pH that cells are being adapted for and the strain being tested. For example, strains A and B responded to acid stress and acid shock by maintaining better viability under lethal conditions, but failed to respond during long-term storage after acid adaptation. This suggests that responses to acid adaptation were transient and protected these strains for the short term only. In contrast, strain C responded under short term and longterm storage, suggesting that a different response system is induced to maintain survival under these conditions. Micronutrients

Figure 2. Survival of Lactobacillus acidophilus strains A, B, and C following acid adaptation and inoculation into lethal pH conditions. NA = non-adapted cells; ST = acid stressed cells; SH = acid shocked cells; SA = stepwise adapted cells.

acidic conditions as compared with the control that had been shifted directly from optimal into lethal acidic conditions. Strains of L. acidophilus A, B, and C underwent acid stress, acid shock, or stepwise adaptation and the viability was monitored hourly for five hours in lethal pH conditions (Figure 2). All strains improved in viability in lethal conditions compared with the control following acid stress and acid shock, and improvement following stepwise adaptation was strain dependent. Strains of L. acidophilus showed higher survival rates than the control (1.25%) following acid stress (43%) and acid shock (32%) (Figure 2).

During yogurt making, S. thermophilus dominates the early stage of yogurt fermentation. As redox potential of milk medium is reduced and the pH lowered from 6.5 to 5.5, growth of L. delbrueckii ssp. bulgaricus is stimulated. S. thermophilus dominates the early stage of yogurt fermentation; below pH 5.0 L. delbrueckii ssp. bulgaricus dominates yogurt fermentation and produces acetaldehyde and lactic acid, yielding the characteristic yogurt green apple flavor. Continued acid production lowers yogurt pH to near 4.6, which induces clotting. Fermentation is terminated at pH 4.5. Probiotic bacteria grow slowly in milk because of a lack of proteolytic activity (A. Shihata and N. P. Shah, 1999, unpublished data); the usual practice is to add yogurt bacteria to reduce the fermentation time. Lactobacillus delbrueckii ssp. bulgaricus produces essential amino acids because of its proteolytic nature, and the symbiotic relationship of L. delbrueckii ssp. bulgaricus and S. thermophilus is well established; the latter organism also produces growth factors for the former organism. However, L. delbrueckii ssp. bulgaricus also produces lactic acid during refrigerated storage. This process is known in the industry as post-acidification. Acid produced during refrigerated storage (i.e., postacidification) is found to cause loss of viability of probiotic bacteria. To overcome the loss of viability of probiotic bacteria due to post-acidification, the present trend is use starter cultures with no L. delbrueckii ssp. bulgaricus, such as ABT (L. acidophilus, bifidobacteria and S. thermophilus). Such starter cultures may require the incorporation of micronutrients (peptides and amino acids) through casein hydrolysate for reducing the fermentation time and for improving the viability of probiotic bacteria. Streptococcus thermophilus, which is less proteolytic than L. delbrueckii ssp. bulgaricus, is the main organism responsible for fermentation in ABT cultures. ABT starter cultures increase fermentation time significantly (up to 11 h) because there is no symbiosis Journal of Dairy Science Vol. 83, No. 4, 2000

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without L. delbrueckii ssp. bulgaricus, and the fermentation is carried out primarily by S. thermophilus. Longer incubation times are undesirable, given the rigid schedule in modern yogurt manufacturing. Dave and Shah (24), studied the effects of whey powder (WP), whey protein concentrate (WPC), and acid casein hydrolysate (ACH) on the viability of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria in yogurt made from 4 commercial starter cultures (C1, C2, C3, and C4). The incubation time to reach the pH of 4.5 was considerably affected by the added ingredients, as some ingredients provided peptides and amino acids for the bacterial growth. The addition of WPC, and ACH improved the viability of bifidobacteria to variable extents, but WP failed to improve their viability. Sodium dodecyl sulfate-PAGE and amino acid analyses suggested that a nitrogen source in the form of peptides and amino acids improved the viability of bifidobacteria. The number of bands and their relative area were different in each product. The degradation of casein by L. delbrueckii ssp. bulgaricus might have liberated additional peptides during fermentation and might have resulted in a greater number of bands. The rate of acid production was slower, and the viability of bifidobacteria was lower than in yogurt supplemented with WPC. After the SDS-PAGE and viability results were analyzed, it was presumed that intermediate peptides (15 to 30 kDa) might have affected the growth of starter bacteria in yogurts during fermentation. Proteins and peptides also seemed to be responsible for the time taken to reach pH 4.5 and for the differences in viability of starter bacteria in yogurts supplemented with various ingredients. WP and WPC also serve as sources of peptides and amino acids when heat treated in a yogurt mix. Whey proteins are rich in sulfur-containing amino acids, which are liberated during heat treatment, and these sulfur-containing amino acids lower the redox potential. Ascorbic Acid as an Oxygen Scavenger Oxygen content and redox potential have been shown to be important factors for the viability of bifidobacteria during storage. Ascorbic acid (vitamin C) can act as an oxygen scavenger and it is permitted in fruit juices and other products as a food additive. Furthermore, milk and milk products supply only 10 to 15% of the daily requirements of vitamin C (73). As a result, fortification of yogurt with ascorbic acid would increase its nutritive value. In a study by Dave and Shah (21) viability of yogurt and probiotic bacteria was assessed during manufacture and storage with four concentrations of ascorbic Journal of Dairy Science Vol. 83, No. 4, 2000

acid (0, 50, 150, or 250 mg/kg) using four commercial starter cultures (C1, C2, C3, and C4). Cultures C1 and C2 contained S. thermophilus, and L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria as constitutive microflora, whereas cultures C3 and C4 contained S. thermophilus, L. acidophilus, and bifidobacteria (ABT culture) only. The differences in counts of S. thermophilus were minimal during storage of yogurt with the different concentrations of ascorbic acid. The oxygen content and redox potential gradually increased during storage in plastic cups, but was lower with the higher levels of ascorbic acid. The counts of S. thermophilus were lower, whereas those of L. delbrueckii ssp. bulgaricus were higher, with increasing concentration of ascorbic acid. Use of ascorbic acid may lower redox potential by scavenging oxygen, thus affecting the growth of S. thermophilus. Addition of ascorbic acid helped improve the survival and viability of L. acidophilus, whereas the counts of bifidobacteria remained unchanged and addition of ascorbic acid did not improve the viability of bifidobacteria. The oxygen scavenging effect may not have been sufficient enough to improve the viability of anaerobic bifidobacteria. Addition of Cysteine Dave and Shah (19) reported improved viability of probiotic bacteria in products made in glass bottles, in which oxygen permeation was minimal. The viability of L. acidophilus was also improved in yogurts supplemented with ascorbic acid as an oxygen scavenging agent (21). Media used for enumeration of bifidobacteria often incorporate L-cysteine (0.5 to 0.1%) to improve recovery of bifidobacteria (61, 91). Cysteine, a sulfurcontaining amino acid could provide amino nitrogen as a growth factor while reducing the redox potential, both of which might favor the growth of anaerobic bifidobacteria species. Collins and Hall (15) reported improved viability of some bifidobacterial species in reconstituted milk containing 0.05% cysteine. Dave and Shah (22) studied the effect of L-cysteine on the growth and viability of yogurt and probiotic bacteria in yogurt made from four commercial starter cultures (C1, C2, C3, and C4) with four levels of L-cysteine (0, 50, 250, or 500 mg/L). Cysteine at 50 mg/L promoted the growth of S. thermophilus and decreased incubation time to reach pH of 4.5. Cysteine affects redox potential. A slight decrease in redox potential is beneficial for survival; however, when the concentration of cysteine is increased beyond 50 mg/L, the depression in redox potential will affect the growth. Higher levels of cysteine (250 or 500 mg/L) affected the growth of S. ther-

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mophilus. The cell morphology of S. thermophilus was adversely affected with increased concentration of cysteine (500 mg/L) as observed by an electron microscope, possibly as a result of reduced redox potential. The growth of L. delbrueckii ssp. bulgaricus was promoted with increased concentration of cysteine but was suppressed at 250 or 500 mg/L of cysteine. Counts of L. acidophilus for all four commercial starter cultures were higher in yogurt made with 250 or 500 mg/L of cysteine than those with no added cysteine or with 50 mg of cysteine. Lactobacillus acidophilus is microerophilic to anaerobic; at 50 mg of cysteine/L there was no marked effect on growth or multiplication of L. acidophilus cells. The decrease in redox potential was not sufficient to support the growth of L. acidophilus. Viability of bifidobacteria was improved by incorporating cysteine into yogurt made with C3 starter culture. This study supported an earlier observation (19) and confirms that redox potential plays an important role for viability of L. acidophilus. Fifty milligrams per liter of cysteine seemed to be optimal for improving viability. Use of Sonication to Release β-Galactosidase Enzyme from Yogurt Organisms Among the LAB, yogurt bacteria contain the highest lactase activity (84, 85). Lactase or β-D-galactosidase is an intracellular enzyme and whole microbial cells exhibit very little extracellular lactase activity (85). Activity of the β-galactosidase can be increased several times by cell lysis of yogurt bacteria induced by sonication. β-Galactosidase released after sonication could hydrolyze a portion of lactose in milk and the products of lactose hydrolysis, glucose, and galactose, could be used by slow growing organisms such as L. acidophilus and Bifidobacterium spp. Rupturing of yogurt bacteria could also reduce the viable count of yogurt bacteria. Shah and Lankaputhra (86) have studied the effect of rupturing yogurt bacterial cells to release their intracellular β-galactosidase (β-gal) and reduce their viable count, because yogurt bacteria seem to be responsible for the death of probiotic bacterial cells. The viable counts of probiotic bacteria were 2 log cycles higher in yogurt made with ruptured yogurt bacteria and whole cells of L. acidophilus and Bifidobacterium spp. The viability after 6 wk of storage remained above the recommended 106 cfu/g, possibly because more β-gal was released as a result of rupture of yogurt bacterial cells. Yogurt made with ruptured cells of yogurt bacteria contained less hydrogen peroxide during fermentation. Ruptured and whole cells of yogurt bacteria produced similar amounts of acetaldehyde.

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In summary, the viability of L. acidophilus and Bifidobacterium spp. could be improved by one of several approaches outlined. Fortification with acid casein hydrolysate as s source of peptides and amino acids may help improve the viability of probiotic bacteria especially in ABT starter cultures. CONCLUSIONS Among the various media developed for selective enumeration of yogurt and probiotic bacteria, ST agar could be used for selective enumeration of S. thermophilus, MRS agar (pH 5.2) is suitable for L. delbrueckii ssp. bulgaricus, MRS-salicin agar or MRS-sorbitol agar for L. acidophilus, and MRS-NNLP agar could be used for selective enumeration of bifidobacteria. However, MRSsalicin agar or MRS-sorbitol agar could not be used for selective enumeration of L. casei and L. acidophilus in products containing both. If L. casei is added, then MRS-salicin or MRS-sorbitol agar could be used to obtain a total population of L. casei and L. acidophilus and the population of L. casei could be subtracted from the total populations of L. acidophilus and L. casei enumerated using MRS-salicin agar or MRS-sorbitol agar. Viability of probiotic bacteria in yogurt or other fermented dairy foods made with commercial starter cultures could be improved by providing micronutrients in the form of amino acids and peptides, in particular in starter cultures that do not contain L. delbrueckii ssp. bulgaricus. At concentrations up to 50mg/L, cysteine could also be used to improve the viability of anaerobic bifidobacteria and microaerophilic L. acidophilus. Other methods to improve viability include stress adaptation, microencapsulation, two-step fermentation, use of sonication to release β-galactosidase enzyme from yogurt starter cultures, and selection of acid and bile resistant strains and appropriate containers that are impermeable to oxygen. REFERENCES 1 Abdel-Bar, N. M., and N. D. Harris. 1984. Inhibitory effect of Lactobacillus bulgaricus on psychrotrophic bacteria in associative cultures and in refrigerated foods. J. Food Prot. 47:61–64. 2 Anand, S. K., R. A. Srinivasan, and L. K. Rao. 1985. Antibacterial activity associated with Bifidobacterium bifidum—II. Cult. Dairy Prod. J. 20:21–23. 3 Anders, R. F., D. M. Hogg, and G. R. Jago. 1970. Formation of hydrogen peroxide by group N streptococci and its effect on their growth and metabolism. Appl. Microbiol. 19:608–612. 4 Anonymous. 1992. Yoghurt and probiotics. Choice 11:32–35. 5 Arroyo, L., L. N. Cotton, and J. H. Martin. 1994. Evaluation of media for enumeration of Bifidobacterium adolescentis, B. infantis and B. longum from pure culture. Cult. Dairy Prod. J. 29:2–24. 6 Bertoni, J., L. Calamary, M. G. Maiamti, and A. Azzoni. 1994. Factors modifying the acidification rate of milk. Lait 17:941–943. 7 Bezkorovainy, A., D. Grohlich, and J. H. Nichols. 1979. Isolation of a glycopeptide fraction with Lactobacillus bifidus var. pennsylJournal of Dairy Science Vol. 83, No. 4, 2000

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