You are on page 1of 15

Technological & Commercial Applications of Lactic Acid Bacteria;

Health & Nutritional Benefits in Dairy Products
Stanley E. Gilliland, PhD
Department of Animal Science
Food & Agricultural Products Center
Oklahoma State University
Stillwater, OK 74078
Key words: Lactobacillus, Bifidobacterium, milk, probiotics, storage stability, nutritional benefits, health benefits
Probiotics have been defined as selected viable microorganisms used as dietary supplements having
potential for improving health or nutrition of man or animal following ingestion. The primary probiotic bacteria
associated with dairy products have been Lactobacillus acidophilus, Lactobacillus casei and bifidobacteria. More
recent taxonomic studies involving sophisticated techniques have resulted in reclassification within some of the
species of Lactobacillus listed as probiotics to the Lactobacillus acidophilus group and the L. casei group (Table 1).

T a ble 1 . Prob iotic ba cte ria th a t m ay b e associated w ith m ilk p rod ucts
L a c to b a c illu s a c id o p h ilu s group
L a ctobacillus reuteri
L. a cid op h ilu s
L. am ylovorus
L a ctobacillus plan ta ru m
L. crispatus
B ifidobacterium sp ecies:
L. g a sseri
L. jo h n so n n i
la c tis
L a ctobac illu s c a sei group
L. ca sei
adole scentis
L. paracasei
anim a lis
L. rh a m n o su s

b ifid u m
infa ntis

Other lactobacilli include L. reuteri and L. plantarum. Bifidobacterium species include longum, adolescentis,

animalis, bifidum, breve infantis, and lactis (Holzapfel et al 1998, Klein et al 1998, Reid 1999). Some of them or
closely related organisms have been used for centuries in the manufacture of cultured dairy products. For this
reason, they are generally regarded as safe. Which means there is minimal concern to their being used as dietary
adjuncts in dairy products.
Milk or milk products provide an excellent carrier for these probiotic organisms. Most of them can readily
utilize lactose as an energy source for growth. Thus, an important requirement for the growth in the intestinal tract


However. proper selection of the strain to be included as a probiotic becomes important. but rather the milk serves as the carrier for the organism. These two cultures are not ones that have traditionally been used to manufacture yogurt. Potential benefits There are a number of potential benefits that might be derived from consuming dairy products containing probiotic bacteria (Table 2). Considerable variation occurs among strains of these bacteria with regard to their potential for producing health and nutritional benefits. A number of dairy products are marketed as containing probiotic bacteria. Other that health food store and pharmaceutical products few if any dried milk products containing probiotics are available. the probiotic bacteria should be selected for the potential to provide specific health or nutritional benefits following consumption. acidophilus or bifidobacteria species. While traditional starter cultures used in the dairy industry are selected for their ability to rapidly produce desirable organoleptic qualities of cultured dairy products. 2 . Unfortunately. Compared to cells suspended in buffered saline all cultures tested survived exposure to simulated gastric juice much better when milk protein was provided by the milk. Milk proteins also provide important protection to the probiotic bacteria during passage through the stomach (Charteris et al 1998). which contain probiotic bacteria. In the United States. The traditional yogurt starter cultures of course include Streptococcus salivarius ssp thermophilus and Lactobacillus delbrueckii ssp bulgaricus. In this presentation. which is made with a selected culture of Lactobacillus casei. There are many other fermented products in the world with a dairy base. acidophilus are added to freshly pasteurized milk. the most widely encountered one is yogurt. An example is Yakult. The milk is then stored under refrigeration so the probiotic bacteria does not grow in the milk. Thus. but rather are added just so the manufacturer can say the organism is there. I will not attempt to discuss in detail all of the potential benefits that might be derived from the consumption of dairy products containing probiotic bacteria. we also have what is referred to as nonfermented acidophilus milk in which cells of L. It is not uncommon today to read on the label of yogurt products that the product was made with a culture including L. in many cases the probiotic bacteria included in the cultured dairy products have not been selected for specific functions.

acidophilus at two-day intervals for a total of five treatments following inoculation with the pathogen. One of the studies which confirms the advantage of considering L. Following two days. In the therapeutic experiments. and the third group received L. One group for each pathogen received no further treatment. then divided into three groups. the lactobacilli were used as a therapeutic rather than a preventative agent and in many cases adequate controls were not included in the studies. germ-free chickens were fed the pathogenic organisms initially. In the prophylactic experiment. one group was fed L. These results suggest it 3 . the chickens were fed a culture of L. In their studies they used germ-free chickens as the animal model. Potential benefits from probiotics * Control of intestinal infections * Stimulation or modulation of the immune system * Improve lactose (or other nutirent) utilization * Control of some cancer * Control of serum cholesterol levels Since the time of Eli Metchnikoff (1904) much has been learned about microbiology and many reports have been published concerning the benefits of lactobacilli in relation to the intestinal tract. Furthermore. acidophilus at two-day intervals for ten days. The other two groups were challenged with either Salmonella typhimurium or Staphylococcus aureus. In the therapeutic experiment. acidophilus following the challenge of the chickens with the pathogens was the best form of treatment. acidophilus initially. the chicks were divided into three groups. acidophilus initially (i. many of the early studies on the use of lactobacilli to control intestinal infections were not well designed. The interest in the role of lactobacilli and other lactic acid bacteria in the microecology of the intestinal tract was regenerated in the last twenty years. the continued feeding of L. Furthermore. L. acidophilus had minimal affect. especially with regard to its origin or its potential for producing the desired effect. there was very little information concerning the particular culture of lactobacilli involved. One group served as control. acidophilus on day two. and the third group was additionally fed L. One group served as a control. Results indicated that the animals fed L.Table 2. It may be more reasonable to consider probiotics as a prophylactic rather than a therapeutic treatment for intestinal infections. Unfortunately. prior to the challenge with the pathogen) survived much better than did those that were challenged with the pathogen first.e. acidophilus as a prophylactic treatment for controlling intestinal infections was done by Watkins and Miller (1983). in many of these studies. In most cases.

coli 0157:H7 in cattle. we would want to select one of these which exhibited the greatest amount of inhibition in the laboratory tests. The studies listed in this table focus on control of diarrhea in children through the use of probiotics. acidophilus as a probiotic for helping control E. some sort of laboratory screening of the cultures prior to their use as probiotics seems desirable. Feeding trials are difficult to conduct and very expensive with regard to showing the influence of probiotic organisms on intestinal pathogens. In the dairy industry. we have known for many years that there was variation among strains and species of starter culture bacteria with regard to their ability to produce the desired changes in the milk being fermented. As an example. Examples of these are listed in Table 3. Similar variations should be expected when selecting strains of a probiotic to control intestinal pathogens in humans. acidophilus with regard to the percentage inhibition of the pathogen. it should not be surprising that there would be variation among strains and species of probiotic microorganisms with regard to their ability to produce inhibitory action toward pathogenic microorganisms. coli were included. One for each strain of E. we should not expect one strain or species of probiotic microorganisms to provide all of the potential benefits that might be possible from consumption of these important to have L. if we were to select a strain of L. The inoculated broths were divided into seven parts. Various probiotic bacteria have been shown in scientific studies to aid in control of intestinal pathogens in humans. acidophilus initially and to provide the organism on a continuing basis in the diet for best control of these pathogens. acidophilus of bovine origin for the ability to inhibit Escherichia coli 0157:H7 in associative cultures in laboratory media. Furthermore. For these experiments. Thus. two strains of the pathogenic E. we have conducted studies recently comparing six strains of L. 4 . Broths were inoculated with each at approximately 3x104 colony forming units (CFU)/ml. coli served as control and each of the other portions was inoculated with different strains of L. Thus. Four of the strains were significantly more inhibitory than the other two. The results revealed variation among the strains of L. All tubes were incubated six hours at 37°C after which the numbers of E. acidophilus (1x105/ml). coli were enumerated on Violet Red Bile Agar. Thus.

Of these possible mechanisms. Bifidobacteria fed to mice also caused similar results in another study (Fukushima et al. Certainly additional research is needed. Perdigon et al. was effective in suppressing chemically induced tumors in mice (Matsuzaki. casei. competitive exclusion. casei & L. This mechanism appears to have a lot of potential as far as answering the questions as to how these bacteria might exert inhibitory action toward intestinal pathogens. Pedone et al. Perdigon & Alvarez. 1995). In one study (Link-Amster et al. and stimulation of the immune system. reuteri . 1995 children in Argentina. These include the production of antimicrobial agents. 1999). acidophilus Beneficial effect in control of rotavirus diarrhea in children by L. competition for nutrients. 1998) and the recurrence of superficial bladder cancer in humans (Aso et al. Benefit Reference Significant reduction in duration ot diarrhea in Gozalez et al. 1999 Control of viral diarrhea in children through use of Lactobacillus & Bifidobacterium McNaught & MacFie. 2001 There have been several possible mechanisms proposed to explain how probiotic bacteria can inhibit pathogenic microorganisms in the intestinal tract. the one which seems to be receiving the most attention today is stimulation or modulation of the immune system (Alvarez et al. Ideally this will involve challenge studies using human subjects. 1992. Shornekova et al. Modulation of the immune system through the use of probiotics also has been shown to suppress some tumors in animal studies.Table 3. Examples of published studies reporting use of probiotic bacteria to exert control of intestinal infections in humans. the consumption of L. there have been studies focusing on the effect of feeding cells of L. Oral administration of L. but it is difficult to get people to participate in such a study and to get approval by university research review committees. casei caused the body to secrete antimicrobial substances into the intestine. 1995) 5 . casei for instance. While most of the work on modulation of the immune system has involved animal studies. 1996 Reduced severity of diarrhea in children by L. 1994) an increase in the secretion into the intestine was observed of substances which were considered inhibitory for certain intestinal pathogens. Milk formulated with L. acidophilus on the immune system of humans. 1998. Currently the final answer regarding the most likely mechanism for controlling intestinal pathogens by probiotic bacteria is not clear. In this group’s studies.

Kolars et al. In a related study in our laboratories we have studied the potential of amylase positive cultures to improve starch digestion following their consumption. Using weaning aged pigs as an animal model. lactose maldigestors were subjected to breath hydrogen test (BHT) at seven day intervals using control milk or milk containing cells of L.Consumption of milk containing cells of L. acidophilus. 1984). Because of the risk of coronary heart disease and fatal heart attacks occurring in hypercholesterolemic individuals. For example. 1984). This presents another potential for a nutritional impact for using a selected probiotic for young children in developing countries where starchy foods are a staple. The conventional animals excrete more cholesterol in the feces than do the 6 . As part of this study. Yogurt containing viable starter cultures has been shown to have similar effects due to ß-galactosidase content of the traditional starter cultures (Kim and Gilliland 1984. germ-free animals on an elevated cholesterol diet accumulate approximately twice as much cholesterol in blood as do conventional animals on a similar diet (Eyssen. The above findings show that probiotic bacteria can be used to provide an enzyme(s) that may be useful in the digestive processes in the intestine. To realize the benefit from such a product it is essential that the cells for preparing the product be grown in a medium containing lactose as the sugar source since ß-galactosidase is inducible in this organism. acidophilus as the test dose (5 ml/kg body weight) following 12 hour fasts. we tested the influence on weight gain of feeding a culture of L. acidophilus can improve lactose indigestion in humans classified as “lactose maldigestors”(Kim & Gilliland. Serum cholesterol levels can be influenced by the intestinal microflora. there has for a number of years been interest in means whereby serum cholesterol levels could be reduced in these people. The culture was grown in a broth using starch as carbohydrate source. The results revealed significantly lower levels of breath hydrogen when milk containing the lactobacilli was the test dose. The primary factor required to provide this benefit is adequate levels of ß-galactosidase in the bacterial cells. The benefit was due to the presence of ß-galactosidase in the L. 1973). The cells were harvested and resuspended in nonfat milk for the feeding trial. The results likely were due to the amylase activity of the culture. 1983). On day 0 and 7 the test dose was control milk and on days 14 and 21 the milk containing lactobacilli was the test dose. The risk of coronary heart disease can be significantly reduced by lowering serum cholesterol levels (Lipid Research Clinics Program. acidophilus selected for its ability to hydrolyze starch. acidophilus L23 tested produced significant increases in daily weight gain during a five-week feeding period compared to the control group. They were instructed not to consume milk products between the test days. Two levels of L.

The decreases were associated with increased numbers of lactobacilli in the stools of the infants suggesting that the lactobacilli in the intestinal tract influenced serum cholesterol levels. 1974). acidophilus and related bacteria to help control serum cholesterol levels. Unfortunately. Lactobacillus acidophilus RP32. Our investigation revealed that a few strains of L. 1985). The ability to assimilate cholesterol in this manner varied among strains. our feeding trial was conducted using two strains of the lactobacilli. While it was not the objective of the study to find a beneficial effect of the fermented milk. all men on the trial gained weight and were expected to exhibit increases in levels of serum cholesterol. much to the surprise to the investigators. In order to test the theory that L. acidophilus to infant formula to determine the effect of the organism on serum cholesterol levels and on the intestinal flora of the infants (Harrison and Peat. the main organism involved in the fermentation of this milk was not isolated and identified. The serum cholesterol levels in the infants which received formula containing the lactobacilli decreased significantly during experimental period while those in the control group showed a slight increase.germ-free animals. we have shown that during anaerobic growth L. considerable research has focused on the potential of L. the serum cholesterol levels decreased in all men. we used pigs as an animal model. 1975). two studies were published which indicated the potential for organisms such as L. acidophilus to aid in control of serum cholesterol levels in humans. The men were fed milk that had been fermented with what was described as a “wild” strain of lactobacillus. which assimilated the greatest 7 . In our laboratories. For this study we felt it necessary to use a strain which originated from the intestinal tract of a pig since there is evidence in the literature to indicate that the organism exhibits host specificity. Because of this wide variation among strains. Because of these studies. acidophilus will remove cholesterol from the laboratory media supplemented with cholesterol and bile salts (Gilliland et al. In the 1970s. Because of the large intakes of the milk. acidophilus might exert hypocholesterolemic activity in vivo. The other study involved the addition of cells of L. The fermented milk was consumed for a period of six days (4-5 liters per day per man). The first of these involved feeding fermented milk to a group of 25 men (Mann & Spoerry. The decrease was greatest in those who gained the most weight suggesting that those consuming the largest portions of the fermented milk exhibited the greatest decline. acidophilus isolated from pigs assimilated little or no cholesterol during growth in the laboratory media while others assimilated considerable amounts. This suggests the possibility that microorganisms in the intestinal tract interfere with cholesterol absorption from the intestines.

Pigs were fed twice daily a diet high in cholesterol. acidophilus RP32 had significantly lower serum cholesterol than observed in the control group on day 10. The milk in these studies was not fermented thus any effect observed would not be due to something produced in the milk during fermentation prior to consumption. 1997. These findings suggested the possibility that L. were selected for use in the trial. Thus. acidophilus P47. In another of our studies involving pigs (deRodas et al. 1987). Jin et al. Thus this activity likely resulted in the lower concentrations of bile acids in the serum of the pigs that received L. which assimilated little or none in the laboratory tests. The animals receiving the milk supplemented with lactobacilli also exhibited lower levels of bile acid in the serum on all days than did those receiving the milk without lactobacilli. 1982. acidophilus. whereas those which do not assimilate cholesterol during growth in a laboratory medium do not. with diet induced hypercholesterolemia we observed that for animals receiving the milk supplemented with lactobacilli the serum cholesterol level declined sooner and reached the significantly lower level than was observed in the control group. There have been a number of other research studies that have shown similar potential for L. had levels of serum cholesterol comparable to those in the control group on both days. Additionally each pig in group one (control) was given 50 ml of milk per day. acidophilus P47 per day and those in group three were given 50 ml of milk containing 5x1010 cells of L. Most of these have been done using animal models. screening the cultures in a laboratory test would be very useful in selecting a strain for use as a probiotic to aid in control of serum cholesterol. 1998) have reported the involvement of bile acid deconjugation in the cholesterol lowering effect of similar probiotic bacteria. Pigs in group two were given 50 ml of milk containing 5x1010 L. which did not assimilate cholesterol in the laboratory medium. acidophilus RP32 per day. Free or deconjugated bile acids would not be readily absorbed from the small intestine into the blood (Chickai et al. suggesting that the lactobacilli reduced the absorption of bile acid from the intestinal tract. Analysis of blood samples taken on days 0 and 10 of the experimental period revealed that the serum cholesterol levels in the control group increased from day 0 to 10. Lactobacillus acidophilus can deconjugate bile acids. and L. 1996). The group receiving L. 1998). those in the group receiving L. These data suggest that a strain which assimilates cholesterol during growth in a laboratory medium has the potential of influencing serum cholesterol level. Grunewald. Danielson et al. acidophilus or related lactobacilli in controlling serum cholesterol levels (Akalin et al. Others (DeSmet et al. acidophilus P47. acidophilus interfered with the enterohepatic circulation of bile acids which in itself could be an important factor in reducing serum cholesterol levels. 1989. However.amount of cholesterol. 8 .

1990). We feel that better hypocholesterolemic results could be obtained if we could develop the product which contains higher numbers of cells of the L. variation with regard to the potential for providing a benefit in controlling serum cholesterol levels. None of them were as active as the latter in assimilating cholesterol from a laboratory medium.01) serum cholesterol level reduction of 3. 9 . The numbers of L. Conclusions as to the mechanisms whereby L. the L. 1994).2% in the first phase. A total of seven commercially available cultures were compared to L. eight assimilated more (although not significantly) cholesterol than did L. A total of 123 isolates of L. we were pleased with the results obtained in the feeding trial. acidophilus with regard to the ability to assimilate cholesterol during growth in a laboratory medium and thus. acidophilus for the ability to assimilate cholesterol during growth in laboratory media (Gilliland and Walker. Consumption of the yogurt caused a significant (P<. All these strains of L. we have tested a number of commercially available strains of L. thermophilus to produce a yogurt type product. acidophilus was used along with S. acidophilus were commercially available in the United States. In this study. acidophilus L 1. A 3% reduction of serum cholesterol level translates to approximately a twelve-fold reduction in the risk of developing coronary heart disease. L. 1999).Since our main interest in all of this research concerning control of serum cholesterol levels is related to reducing serum cholesterol levels in hypercholesterolemic humans. Of the isolates compared in this study. Thus. were not particularly high because this particular strain does not grow well in milk. acidophilus. acidophilus were obtained and were compared for the ability to assimilate cholesterol. acidophilus ATCC 43121 (the one used in our pig trial). isolates were made from sixteen human volunteers (Buck and Gilliland. acidophilus may exert a hypercholesterolemic effect include both the ability of the organism to assimilate cholesterol during growth and bile salt deconjugation activity. acidophilus ATCC 43121 most of the rest assimilated significantly less. One of these strains. Because of this we undertook a project to isolate new strains of human origin. For this trial. The latter is most likely due to the potential for increased bile acid excretion from the body due to deconjugation and to reduced efficiency of absorption of cholesterol from the gastrointestinal tract rather than coprecipitation of cholesterol with free bile acids as has been suggested by some researchers. which developed in the yogurt product during the fermentation period. This again shows variation among strains of L. acidophilus. has been used in a human feeding trial involving hypercholesterolemic humans (Anderson & Gilliland.

certain requirements are needed. The most important characteristic of course is that the culture will produce the desired effect. harvested by centrifugation and concentrated in the small volume of milk then frozen in liquid nitrogen.Importance of selection of culture for use as probiotic To help ensure that the probiotic culture being used has a positive effect. we have shown that the pH at which the cells of L. This is important because it makes getting regulatory agency approval much easier. However. Thus. Additionally. if it is to be used as a probiotic for humans. acidophilus had been grown influenced their stability and survival in the milk during refrigerated storage (Gilliland and Rich. One strain should not be expected to produce all potential benefits. We have shown. Thus. The culture being used must be stable during production and storage of the food containing the probiotic organism. for instance. it is highly desirable that the probiotic bacteria originated in human intestines. that a strain which assimilates cholesterol during growth in the laboratory medium had much better effect compared to one that assimilated little or no cholesterol from the broth media. it is desirable to use a selected strain of the bacteria that originated in the host species for which the product is to be used. which are added to milk as a dietary adjunct. Such a production and freezing process had no detrimental effect on the cultures. they must have a certain degree of bile tolerance. growth of two strains of L. There is evidence in the literature indicating that many of these bacteria exhibit host specificity. acidophilus at pH 5 resulted in cultures which were more stable during refrigerated storage in milk than were the same cultures which had been grown at higher pH levels. can have a major impact on survival of the culture during storage. I suggest use of the most bile resistant strain that will provide the desired beneficial effect. Stability of probiotic bacteria in milk In situations where the probiotic bacteria in the form of a concentrated culture are added to milk. based on relative ability to grow in the presence of bile acids. The bile resistance. However. In other words. it is desirable that bacterial species have a history of use for producing fermented dairy foods. some sort of laboratory screening test for the desired effect would be helpful. in the case of using a probiotic bacteria for control of serum cholesterol levels. these microorganisms should be a normal inhabitant of the intestinal tract. 10 . Thus. varies greatly among strains of each species of probiotic bacteria. 1990). the method of growing the culture of probiotic bacteria. Generally speaking. In these experiments the culture was grown in a broth medium. If they are to grow and function in the intestinal tract. At present time we do not know the minimum level of bile tolerance required.

Only one strain of L. Thus.5 or 7. there was significant variation among strains with respect to the maintenance of viability during 28 days of storage at 7°C (Nighswonger et al. These data indicate variability among the strains with respect to storage stability. 6. of the four strains tested. growth of the culture in broth also had no detrimental effect on the culture during frozen storage (Reilly and Gilliland. 1996). but there appeared to be difference between the two yogurt cultures with respect to their influence on stability of L. Best survival was obtained in the 25% solids milk using an outlet temperature of 75º C. selection of the yogurt culture may also be important in helping assure that adequate viable numbers of the probiotic bacteria are maintained during storage of the product. Espina and Packard (1979) compared the influence of spray drying at three outlet temperatures (75º C.8 x 107 /g in the final product.In the case of B. 11 . Based on variability among strains for other characteristics we should expect variation among strains with respect to survival during spray drying and subsequent storage of dried products containing probiotic bacteria.0 did not exhibit any decrease in viability during subsequent storage of the milk at 5°C for 21 days. However when added to milk.34 mg Ca++ and 41. When cells of L. 6. The experiment was replicated in yogurt made using two different yogurt starter cultures. to provide an initial population in the range of 1 to 7x107 per gram. The other three strains were less stable during refrigerated storage. Preparation of dried milk products containing probiotic bacteria offers a great challenge. longum. King and Lin (1995) reported 91-96% survival during freeze drying of cells of L. acidophilus can be very detrimental to viability of the organism. 1999). The composition of the medium and/or conditions in which the cultures are grown prior to drying likely would influence their survival. No storage stability data was reported. strain L1 exhibited best survival. acidophilus was included in each of these studies. Spray drying of milk containing added cells of L. Under these conditions. No storage stability tests were done. Lactobacillus acidophilus L1 exhibited no loss in survival during storage in yogurt made with one of the starter cultures for 28 days while it did exhibit a significant decline after 28 days in the other. greater than one log cycle of viability was lost yielding a population of 9. acidophilus suspended in 10% reconstituted nonfat milk supplemented with 5. acidophilus.0. Out of four strains tested. one (S9) which had been grown at either pH 5.5 mg glycerol per ml. Not only was there variation among the strains of L. acidophilus with regard to stability in yogurt. 80º C and 85º C) milk adjusted to 25% and 40% nonfat solids containing added cells of L.5. acidophilus during refrigerated storage. acidophilus from a frozen concentrated culture were added to cultured yogurt.

12 . The culture(s) should be selected on the basis of being able to provide the desired result. 2. To achieve this may require providing more than one strain and/or species of probiotic bacteria in the product. 3. This likely will involve the importance of packaging material as well as size of package. Evaluation of stability of the selected probiotic organisms during drying and storage in powdered milk. Recommendations If powered milk products containing probiotic bacteria are to be supplied for developing countries the potential benefits of controlling intestinal infections and improving nutrient (primarily lactose) utilization should be considered. This should involve laboratory screening test(s) as well as clinical trials. Research needs 1.Even though not published. The best approach to producing a dried milk containing probiotic bacteria probably will involve blending the freeze dried probiotic culture with milk powder. It also must be stable during drying and storage in the milk powder. Determine best way to deliver the powdered milk containing probiotic. The biggest challenge will be ensure stability during storage of the dried product. This might be accomplished using existing cultures or may involve the need to isolate and develop new strains. One strain of one species should not necessarily be expected to provide more than on benefit. Selection of probiotic to produce desired effect. the industry that produces starter cultures possess the knowledge and technology to stabilize cultures to maximize survival during drying and storage.

& D. Kawata. Dairy Sci. J. De Smet. Cholesterol lowering in pigs through enhanced bacterial bile salt hydrolase activity. Environ. W.D. T. Bru. I. E. 1998 Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus & Bifidobacterium species in the upper human gastrointestinal tract.E. B.. Stability during frozen and subsequent refrigerated storage of Lactobacillus acidophilus grown at different pH. 1996. Espina.Z. 1979. A.K. Anderson. Whalen & M. Nakao & K. H.D. Jr. DeBoever & W. 10:79-87. 1994. Danielson. R. Charteris. Alvarez. Düzel. Shahani.N. 1985. Collin.REFERENCES Akalin. Dairy Sci. & G. J. 32:59-63. E. J. College Nutr.. Intl. Oliver. Specific immunity induction at the mucosal level by viable Lactobacillus casei : A perspective for oral vaccine development. Hypocholesterolemic action of Lactobacillus acidophilus and calcium in diet induced hypercholesterolemic swine. Food Protect.M. Appl. 1998. S.. 79:185-194. Uchida. 46: 193-197. E. 73:1187-1192. Gonzalez. Kurisaki & T.E. Walker. S. Mitsuoka. S. K. Dairy Sci. Kotake. J. F. S. Verstraete.C. de Rodas. T. A.J. 1995. J. Effect of bifidobacteria feeding on fecal flora & production of immunoglobulins in lactating milk.Y. Apella & G. Brit. Amer.V.P. Y. and S. Nutr.. 80:2721-2725. Microbiol. L. Am. Anticholesteremic property of Lactobacillus acidophilus yogurt fed to mature boars. H. Lipids 22:669-672. 1997.E. J.. Gilliland. C. Nelson & C. & C. 8: 129-134. Kelly. Urol. Naito & the BLP Study Group. Proc. Maxwell. Effect of fermented milk (yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans. Tsukamoto. Gönc.E.. Rich. Buck. Food Microbiol. 1973. J. S. Imai. W. Biotherapy. Morelli & J. Role of the gut microflora in metabolism of lipis and sterols. 77:2925-2933. Gilliland.R. Dairy Sci. Influence of yogurt and acidophilus yogurt on serum cholesterol levels in mice. and S. J. Fkushima. J. 1999. Mizumachi. 1990. Microbiol. & VC. 84: 759-768. S. 1989.R Holagdo & G. Nutr. Sci.M.A.. Immunol. Appl. Factors to consider when selecting a culture of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans. Y. Biotherapeutic role of fermented milk. 1995. 1990. N. L. Soc. A. M.. 67:966-974. S. S. Eur. 13 . 1998. Dairy Sci. Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats. Gilliland & C. Akaza.. E.P. Chickai. Maxwell.N. H. Packard. K. J. P. Gilliland. 79:2121-2128. 42: 149-152. Peo. Survival of Lactobacillus acidophilus in a spray-drying process. Lewis. Gobbato. T. 73:905-911.J. Aso. Anim. 1987. 27:104-109. Preventative effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer in a double-blind trial. Assimilation of cholesterol by Lactobacillus acidophilus.R. Cardozo.. Eyssen. J. J. Perdigon. Gilliland. M. K. Comparisons of freshly isolated strains of Lactobacillus acidophilus of human intestinal origin for ability to assimilate cholesterol during growth. 18:43-50. Food & Agri. P. 1999.K. P. J. Gilliland. 49:377-381. A.

J. 1975. N. O. Dairy Sci. Poult.D. Sci. Aouji & D. G. Jin. S. and A. V. 21: 343-353. Eng.H. J. Nutr. Kim. 14 . Gilliland. Food. 1994. A. Dairy Sci. Pack. J.Grunewald. 1. The lipid research clinics coronary primary prevention trial results. 2001. 310: 1-3. 1995. G. Jalaludin.M. Am.F. H. The effect of supplementation with milk fermented by Lactobacillus casei (strain DN-114 001) on acute diarrhea in children attending day care centers. J. 77:1259-1265. 41: 103-125. Food Agric. Taxonomy and physiology of probiotic lactic acid bacteria. Ho. Intl. 1982. Pract. Clin. Huis in’t Veld. Food Sci. Bernabeu. T. 1983. Harrison. Bonaparte & G.S. 1908.. C. Lactobacillus acidophilus as a dietary adjunct for milk to aid lactose digestion in humans. & J. J. Med. J. Mann. Lee. Pedone. Studies of a surfactant and cholesteremia in the Maasai. Saviano. Microbiol. & G. H. Metchnikoff. Med.R. Haberer. Klein. 79:212-219. Intl. The prolongation of life. & H. Mignot & J. Brashears & S.. New York. Immunomodulation by treatment with Lactobacillus casei strain Shirota. H. 1996. M. Holzapfel. and serum cholesterol of broilers fed diets containing Lactobacillus cultures.. Sci. Microbiol. Food. FEMS Immunol. Kim. Rochat. Gilliland. Am. K. Yougurt – an autodigesting source of lactose. 1974. J. V. Am. 1984. 1998. Probiotics in clinical practice: a critical review of the evidence. J. Postaire. J. 41: 133-140. Levitt. Effect of viable starter culture bacteria in yogurt on lactose utilization in humans. Schillinger & J. C. M. Bouley & P. 1998. 2001. Amylolytic cultures of Lactobacillus acidophilus: Potential probiotics to improve dietary starch utilization. H.K. 68: 191-196. Kolars. Abdullah & S. King. Modulation of a specific humoral immune response and changes in intestinal flora mediated through fermented milk intake. Growth performance. E. Med. Reduction in incidence of coronary heart disease. Putnam Suns.J. Food. Y.Z.. L.M. O. 1998.H.S. 47:2078-2079. P. J. Clin.W. Reinert. McNaught. 1999. Matsuzaki. Gilliland & S.J. Carter. C. MacFie.E. 1st ed. J.A. Intl. Sci.A. E. 67: 1-6. Aeschlimann. Link-Amster. Studies on the effect of protectants on Lactobacillus acidophilus strain dehydrated under controlled low-temperature vacuum dehydration and freeze-drying by using response surface methodology. F.. Serum cholesterol levels in rats fed skim milk fermented with Lactobacillus acidophilus. Reuter. 28:13511355. Gilliland. N. 1984. Snel.D. NY. Nutr.. Peat. Nutr. J. intestinal microbial populations. Lipid Research Clinics Program.E. K. 53: 179-184. Intl.E. 10:55-64. & S.E. J. M. Nighswonger. 1998. J. and S.C. 27:464-469.. Microbiol. Dairy Sci. A. C. Serum cholesterol and bowel flora in the new born. Res. J. 66: 338-344. Overview of gut flora & probiotics. Lin. C. Clin. Saudan. U. Spoerry. Assoc. 251:351-363.. B. 41: 85-101. Food Microbiol.A. J. 66: 959-966. W.E. Viability of Lactobacillus acidophilus and Lactobacillus casei in fermented milk products during refrigerated storage. Y. V.S. 1984.

G. I. & S. E. Poult. J. Rachid. Probiotics and the immune state. 15 . G. Dairy Sci. Food Sci. London. 1999. Ltd. S. Immune system stimulation by probiotics. Aguero & N. Competitive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks. 1992. Environ. E. Appl. Bifidobacterium longum survival during frozen and refrigerated storage as related to pH during growth. & B. G.V. B. In Fuller. 78: 1597-1606. & S..F. R (ed) Probiotics The Scientific Basis. Miller. Ped. G. 1995. J. Sci. Gabbato. 64:714-718. 1997. Isolauri..Perdigon. Gastro. The scientific basis for probiotic strains of Lactobacillus. Chapman and Hall Sci. Microbiol. Gilliland. J. 65: 37633766.A. Reid. M. Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children. Perdigon. 24: 399-404. Alvarez. S.A. Reilly. Watkins. 1999. Shornikova.. S. 1983. & Nutr. Casas. 62:1772-1779. Vesikari. H. Alvarez. Mykkanen & T. A.