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 USA Key words: Lactobacillus, Bifidobacterium, milk, probiotics, storage stability, nutritional benefits, health benefits Introduction 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 longum L. ca sei adole scentis L. paracasei anim a lis L. rh a m n o su s

b ifid u m breve 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

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is provided by the milk. Milk proteins also provide important protection to the probiotic bacteria during passage through the stomach (Charteris et al 1998). Compared to cells suspended in buffered saline all cultures tested survived exposure to simulated gastric juice much better when milk protein was present. While traditional starter cultures used in the dairy industry are selected for their ability to rapidly produce desirable organoleptic qualities of cultured dairy products, the probiotic bacteria should be selected for the potential to provide specific health or nutritional benefits following consumption. Unfortunately, in many cases the probiotic bacteria included in the cultured dairy products have not been selected for specific functions, but rather are added just so the manufacturer can say the organism is there. Considerable variation occurs among strains of these bacteria with regard to their potential for producing health and nutritional benefits. Thus, proper selection of the strain to be included as a probiotic becomes important. A number of dairy products are marketed as containing probiotic bacteria. However, the most widely encountered one is yogurt. It is not uncommon today to read on the label of yogurt products that the product was made with a culture including L. acidophilus or bifidobacteria species. These two cultures are not ones that have traditionally been used to manufacture yogurt.

The traditional yogurt starter cultures of course include

Streptococcus salivarius ssp thermophilus and Lactobacillus delbrueckii ssp bulgaricus. In the United States, we also have what is referred to as nonfermented acidophilus milk in which cells of L. acidophilus are added to freshly pasteurized milk. The milk is then stored under refrigeration so the probiotic bacteria does not grow in the milk, but rather the milk serves as the carrier for the organism. There are many other fermented products in the world with a dairy base, which contain probiotic bacteria. An example is Yakult, which is made with a selected culture of

Lactobacillus casei. Other that health food store and pharmaceutical products few if any dried milk products containing probiotics are available.

Potential benefits

There are a number of potential benefits that might be derived from consuming dairy products containing probiotic bacteria (Table 2). In this presentation, 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.

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Table 2. 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. 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. Unfortunately, many of the early studies on the use of lactobacilli to control intestinal infections were not well designed. In most cases, the lactobacilli were used as a therapeutic rather than a preventative agent and in many cases adequate controls were not included in the studies. Furthermore, in many of these studies, there was very little information concerning the particular culture of lactobacilli involved, especially with regard to its origin or its potential for producing the desired effect. It may be more reasonable to consider probiotics as a prophylactic rather than a therapeutic treatment for intestinal infections.

One of the studies which confirms the advantage of considering L. acidophilus as a

prophylactic treatment for controlling intestinal infections was done by Watkins and Miller (1983). In their studies they used germ-free chickens as the animal model. In the prophylactic experiment, the chickens were fed a culture of L. acidophilus initially. Following two days, the chicks were divided into three groups. One group served as a control. The other two groups were challenged with either Salmonella typhimurium or Staphylococcus aureus. One group for each pathogen received no further treatment, and the third group was additionally fed L. acidophilus at two-day intervals for ten days.

In the therapeutic experiment, germ-free chickens were fed the pathogenic

organisms initially, then divided into three groups. One group served as control; one group was fed L. acidophilus on day two; and the third group received L. acidophilus at two-day intervals for a total of five treatments following inoculation with the pathogen. In the therapeutic experiments, L. acidophilus had minimal affect. Results indicated that the animals fed L. acidophilus initially (i.e. prior to the challenge with the pathogen) survived much better than did those that were challenged with the pathogen first. Furthermore, the continued feeding of L. acidophilus following the challenge of the chickens with the pathogens was the best form of treatment. These results suggest it

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is important to have L. acidophilus initially and to provide the organism on a continuing basis in the diet for best control of these 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. Thus, 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. Furthermore, 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 organisms. Feeding trials are difficult to conduct and very expensive with regard to showing the influence of probiotic organisms on intestinal pathogens. Thus, some sort of laboratory screening of the cultures prior to their use as probiotics seems desirable. As an example, we have conducted studies recently comparing six strains of L.

acidophilus of bovine origin for the ability to inhibit Escherichia coli 0157:H7 in associative cultures in laboratory media. For these experiments, two strains of the pathogenic E. coli were included. Broths were inoculated with each at approximately 3x104 colony forming units (CFU)/ml. The inoculated broths were divided into seven parts. One for each strain of E. coli served as control and each of the other portions was inoculated with different strains of

L. acidophilus (1x105/ml). All tubes were incubated six hours at 37°C after which the numbers of E. coli were enumerated on Violet Red Bile Agar. The results revealed variation among the strains of L. acidophilus with regard to the percentage inhibition of the pathogen. Four of the strains were significantly more inhibitory than the other two. Thus, if we were to select a strain of L. acidophilus as a probiotic for helping control E. coli 0157:H7 in cattle, we would want to select one of these which exhibited the greatest amount of inhibition in the laboratory tests. Similar variations should be expected when selecting strains of a probiotic to control intestinal pathogens in humans. Various probiotic bacteria have been shown in scientific studies to aid in control of intestinal pathogens in humans. Examples of these are listed in Table 3. The studies listed in this table focus on control of diarrhea in children through the use of probiotics.

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Table 3. Examples of published studies reporting use of probiotic bacteria to exert control of intestinal infections in humans. Benefit Reference Significant reduction in duration ot diarrhea in Gozalez et al. 1995 children in Argentina. Milk formulated with L. casei & L. acidophilus Beneficial effect in control of rotavirus diarrhea in children by L. reuteri .

Shornekova et al. 1996

Reduced severity of diarrhea in children by L. casei.

Pedone 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. These include the production of antimicrobial agents, competitive exclusion, competition for nutrients, and stimulation of the immune system. Of these possible mechanisms, the one which seems to be receiving the most attention today is stimulation or modulation of the immune system (Alvarez et al, 1998, Perdigon & Alvarez, 1992, Perdigon et al, 1995). In this group’s studies, the consumption of L. casei caused the body to secrete antimicrobial substances into the intestine. Bifidobacteria fed to mice also caused similar results in another study (Fukushima et al, 1999). 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. While most of the work on modulation of the immune system has involved animal studies, there have been studies focusing on the effect of feeding cells of L. acidophilus on the immune system of humans. In one study (Link-Amster et al, 1994) an increase in the secretion into the intestine was observed of substances which were considered inhibitory for certain intestinal pathogens.

Currently the final answer regarding the most likely mechanism for controlling

intestinal pathogens by probiotic bacteria is not clear. Certainly additional research is needed. Ideally this will involve challenge studies using human subjects, but it is difficult to get people to participate in such a study and to get approval by university research review committees. Modulation of the immune system through the use of probiotics also has been shown to suppress some tumors in animal studies. Oral administration of L. casei for instance, was effective in suppressing chemically induced tumors in mice (Matsuzaki, 1998) and the recurrence of superficial bladder cancer in humans (Aso et al, 1995)

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Consumption of milk containing cells of L. acidophilus can improve lactose indigestion in humans classified as “lactose maldigestors”(Kim & Gilliland, 1983). As part of this study, lactose maldigestors were subjected to breath hydrogen test (BHT) at seven day intervals using control milk or milk containing cells of L.

acidophilus as the test dose (5 ml/kg body weight) following 12 hour fasts. 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. They were instructed not to consume milk products between the test days. The results revealed significantly lower levels of breath hydrogen when milk containing the lactobacilli was the test dose. The benefit was due to the presence of ß-galactosidase in the L.

acidophilus. 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. 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, Kolars et al, 1984). The primary factor required to provide this benefit is adequate levels of ß-galactosidase in the bacterial cells. 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. 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, we tested the influence on weight gain of feeding a culture of L. acidophilus selected for its ability to hydrolyze starch. The culture was grown in a broth using starch as carbohydrate source. The cells were harvested and resuspended in nonfat milk for the feeding trial. Two levels of L. acidophilus L23 tested produced significant increases in daily weight gain during a five-week feeding period compared to the control group. The results likely were due to the amylase activity of the culture. 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. Because of the risk of coronary heart disease and fatal heart attacks occurring in hypercholesterolemic individuals, there has for a number of years been interest in means whereby serum cholesterol levels could be reduced in these people. The risk of coronary heart disease can be significantly reduced by lowering serum cholesterol levels (Lipid Research Clinics Program, 1984). Serum cholesterol levels can be influenced by the intestinal microflora. For example, 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, 1973). The conventional animals excrete more cholesterol in the feces than do the

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germ-free animals. This suggests the possibility that microorganisms in the intestinal tract interfere with cholesterol absorption from the intestines. In the 1970s, 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 first of these involved feeding fermented milk to a group of 25 men (Mann & Spoerry, 1974). The men were fed milk that had been fermented with what was described as a “wild” strain of lactobacillus. The fermented milk was consumed for a period of six days (4-5 liters per day per man). Because of the large intakes of the milk, all men on the trial gained weight and were expected to exhibit increases in levels of serum cholesterol. While it was not the objective of the study to find a beneficial effect of the fermented milk, much to the surprise to the investigators, the serum cholesterol levels decreased in all men. 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. Unfortunately, the main organism involved in the fermentation of this milk was not isolated and identified. The other study involved the addition of cells of 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, 1975). 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.

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. Because of these studies, considerable research has focused on the potential of L. acidophilus and related bacteria to help control serum cholesterol levels. In our laboratories, we have shown that during anaerobic growth

L. acidophilus will remove cholesterol from the laboratory media supplemented with cholesterol and bile salts (Gilliland et al, 1985). The ability to assimilate cholesterol in this manner varied among strains. In order to test the theory that L. acidophilus might exert hypocholesterolemic activity in vivo, we used pigs as an animal model. 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. Our investigation revealed that a few strains of L. acidophilus isolated from pigs assimilated little or no cholesterol during growth in the laboratory media while others assimilated considerable amounts. Because of this wide variation among strains, our feeding trial was conducted using two strains of the lactobacilli. Lactobacillus acidophilus RP32, which assimilated the greatest

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amount of cholesterol, and L. acidophilus P47, which assimilated little or none in the laboratory tests, were selected for use in the trial. Pigs were fed twice daily a diet high in cholesterol. Additionally each pig in group one (control) was given 50 ml of milk per day. Pigs in group two were given 50 ml of milk containing 5x1010 L. acidophilus P47 per day and those in group three were given 50 ml of milk containing 5x1010 cells of L. acidophilus RP32 per day. 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.

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. The group receiving L. acidophilus P47, which did not assimilate cholesterol in the laboratory medium, had levels of serum cholesterol comparable to those in the control group on both days. However, those in the group receiving L.

acidophilus RP32 had significantly lower serum cholesterol than observed in the control group on day 10. These data suggest that a strain which assimilates cholesterol during growth in a laboratory medium has the potential of influencing serum cholesterol level, whereas those which do not assimilate cholesterol during growth in a laboratory medium do not. Thus, 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. There have been a number of other research studies that have shown similar potential for L. acidophilus or related lactobacilli in controlling serum cholesterol levels (Akalin et al, 1997; Danielson et al, 1989; Grunewald, 1982; Jin et al, 1998). Most of these have been done using animal models. In another of our studies involving pigs (deRodas et al, 1996), 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. 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, suggesting that the lactobacilli reduced the absorption of bile acid from the intestinal tract. These findings suggested the possibility that L. acidophilus interfered with the enterohepatic circulation of bile acids which in itself could be an important factor in reducing serum cholesterol levels.

Lactobacillus acidophilus can deconjugate bile acids. Thus this activity likely resulted in the lower concentrations of bile acids in the serum of the pigs that received L. acidophilus. Others (DeSmet et al, 1998) have reported the involvement of bile acid deconjugation in the cholesterol lowering effect of similar probiotic bacteria. Free or deconjugated bile acids would not be readily absorbed from the small intestine into the blood (Chickai et al, 1987).

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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, we have tested a number of commercially available strains of L. acidophilus for the ability to assimilate cholesterol during growth in laboratory media (Gilliland and Walker, 1990). All these strains of L. acidophilus were commercially available in the United States. A total of seven commercially available cultures were compared to L. acidophilus ATCC 43121 (the one used in our pig trial). None of them were as active as the latter in assimilating cholesterol from a laboratory medium. Because of this we undertook a project to isolate new strains of human origin. In this study, isolates were made from sixteen human volunteers (Buck and Gilliland, 1994). A total of 123 isolates of L. acidophilus were obtained and were compared for the ability to assimilate cholesterol. Of the isolates compared in this study, eight assimilated more (although not significantly) cholesterol than did L. acidophilus ATCC 43121 most of the rest assimilated significantly less. This again shows variation among strains of L. acidophilus with regard to the ability to assimilate cholesterol during growth in a laboratory medium and thus, variation with regard to the potential for providing a benefit in controlling serum cholesterol levels. One of these strains, L. acidophilus L 1, has been used in a human feeding trial involving hypercholesterolemic humans (Anderson & Gilliland, 1999). For this trial, the L. acidophilus was used along with S. thermophilus to produce a yogurt type product. Consumption of the yogurt caused a significant (P