Probiotics have been defined as the living organisms that give advantageous effects to host through improving the factors related to the balance of intestinal bacterial flora. Lactobacilli and bifidobacteria are recognized as the representative probiotics. The present report summarizes recent progress in basic studies concerning the probiotic activity, especially immunoregulatory function of L. casei and B. breve according to the following manners. A) Immunoregulatory functions of L. casei. 1) Prevention of methylcholanthrene-induced carcinogenesis in mice and improvement of downregulated immune responses of lymphocytes. 2) Prevention of autoimmune diseases such as diabetes and chronic rheumatoid arthritis in experimental animal models. 3) Prevention of the allergic responses induced by immunization of mice with ovalbumin. B) Immunoregulatory functions of B. breve. 1) Enhancement of antigen-specific immunoglobulin production by B. breve. 2) Antiinfectious activity of B. breve against experimental viral infections in mice. The importance of antigen presenting cells such as macrophages and dendritic cells in the mechanisms of the immunoregulatory functions of probiotics are also discussed.

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Probiotic bacteria must remain viable in stomach content containing HC1 and in small bowel content containing conjugated bile acids (CBA) at toxic concentrations. Lactobacillus strains vary in their capacity to survive in HCl at concentrations approximating those in the stomach. More study is needed, however, of the mechanisms in lactobacilli and bifidobacteria of intrinsic resistance to hydrogen ion. Likewise, more study is needed of the mechanisms by which such bacteria survive in small bowel content containing CBA. One mechanism may be a capacity to express conjugated bile salt hydrolase (BSH) which catalyzes hydrolysis of the conjugating amino acid from the steroid moiety of the CBA. The steroid product of such deconjugation has physical properties rendering them less toxic to bacteria than the conjugated form. We are developing methods for testing the hypothesis that BSH activity enhances resistance in lactobacilli to the toxicity of CBA. Two peptides with BSH activity have been purified from the intracellular content of Lactobacillus johnsonii 100-100. The peptides form in the organism four trimeric isozymes. The activity of the isozymes is enhanced by an extracellular factor called BSH-EF induced in the bacterium by CBA. The gene encoding one BSH peptide and two adjacent genes encoding CBA transporters have been cloned into Escherichia coli. The transport activity of those genes is enhanced by BSH-EF. Sequences of the cloned BSH gene have been used as primers and probes in PCR and Southern blotting, respectively, for detecting BSH genes in various Lactobacillus species. Also assessed was the capacity of those strains to express BSH activity and to resist the toxicity of CBA. High BSH activity is a predictor of resistance to CBA toxicity. However, BSH activity appears to be coded by several genes of unrelated sequence. Our goal in characterizing these genes is to develop methods for enhancing resistance of lactobacilli to CBA toxicity.

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The human intestines harbor 100 trillion viable bacteria, reaching 100 kinds, which consist of so-called intestinal flora. These bacteria grow on the food components ingested and bio-components secreted into the alimentary tract as nutrients. The composition of the intestinal flora can be altered by many factors such as aging, physiological state, drugs, various diseases, diet, and stresses, and has an influence on intestinal metabolisms, which have a great influence on nutrition, drug efficacy, physiological function, aging, carcinogenesis, immunity, infection and others in the host. Thus, it was revealed that the intestinal flora have a close relationship to both the health and diseases of the host. Furthermore, it was found that it is important to increase beneficial bacteria such as bifidobacteria and to decrease harmful bacteria such as clostridia among the intestinal flora for prophylaxis against geriatic diseases including cancers. It is now well established that foods and food components contribute to physiologic and biologic well-being. Optimal intestinal flora and intestinal environment are possibly achieved by a nutritionally well-balanced diet and the active intake of functional foods such as oligosaccharides, dietary fiber and fermented milks, which promote useful bacteria or suppress harmful bacteria. Recent advances of research in intestinal flora are the background for the appearance of functional foods. The functional foods are classified into 3 groups based on their mechanisms of action: “probiotics, ” “prebiotics” and “biogenics.” Probiotics are viable microorganisms, such as lactobacilli and bifidobacteria, which beneficially affect the host by improving the intestinal bacterial balance. Prebiotics are non-digestible food ingredients, such as oligosaccharides and dietary fiber, which beneficially affect the host by selectively stimulating the growth or activities of beneficial intestinal bacteria in the colon, and thus improve the health of the host. Biogenics include biologically active peptides, including immunopotentiator (biological response modifier: BRM), plant flavonoids, etc. They act directly or indirectly, through the modulation of intestinal flora, on health of the host. Probiotics and prebiotics act on the intestinal flora and improve the balance of the flora by enhancing the growth of beneficial intestinal bacteria and/or inhibiting the growth of harmful ones, resulting in scavenging in the intestinal environment. They often induce the production of biogenics such as antibacterial substances, immunopotentiators, etc. by the proliferation of beneficial intestinal bacteria. Thus, the functional foods enhance bio-regulation such as stresses, appetite and absorption, bio-defence such as immunity and anti-allergy, prevent diseases including diarrhea, constipation, cancer, cholesterolemia and diabetes, and suppress aging through immunostimulation as well as the suppression of mutagenesis, carcinogenesis, oxidation processes, intestinal putrefaction and cholesterolemia.

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Bifidobacteria (Lac-B; a Bifidobacterium preparation for oral use) were orally administered in serial doses to germ-free mice (ICR, IQI) with Escherichia coli O157: H7 NK2 intestinal infections. The effects of the oral administration of bifidobacteria on E. coli O157: H7 cell counts in the feces of the infected mice and on the in vivo production of verotoxins were then investigated. Bifidobacteria and norfloxacin (NFLX) were administered in serial doses according to three different schedules. The localization of bifidobacteria in the intestine was markedly disturbed when the serial administrations of bifidobacteria and NFLX were initiated on the same day. However, no disturbance in bacterial colonization was found when the administration of bifidobacteria was initiated prior to the administration of NFLX. No mice died when bifidobacteria (2.6 ∼ 10⁷ CFU/kg ∼ 2/day) were given orally to 4 mice infected with E. coli O157: H7 for 18 days, beginning 5 days before the start of the NFLX administration regime (3 mg/kg x 2/day for 6 days). However, when the oral administration of bifidobacteria and NFLX was initiated on the same day, 3 of the 4 infected mice died. The same results were obtained in a group of mice that did not receive bifidobacteria. When mice infected with E. coli O 157: H7 were given oral doses of NFLX for 6 days, the production of verotoxins in the intestine was markedly enhanced, and large amounts of toxins were excreted in the feces of the mice and then several of the mice died. However, the NFLX-induced production of toxins in the infected mice was inhibited when bifidobacteria were administered in serial doses prior to the administration of NFLX. No toxins were found in the feces of the mice who received this treatment regime. In the present study, serial doses of bifidobacteria, a major component of the normal intestinal flora in infants, effectively demonstrated the inhibitory effects of bifidobacteria on the in vivo production of verotoxins by E. coli 0157: H7 in germ-free mice, providing protection against the fatal action of verotoxins.

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The populations of Lactobacillus acidophilus, Bifidobacterium spp. and Lactobacillus casei were examined in 26 commercial fermented milk products represented by 14 companies. Six products contained L. acidophilus only, 12 products contained L. acidophilus and bifidobacteria, 6 products claimed L. acidophilus, bifidobacteria, and L. casei, and 2 products contained L. casei only as the probiotic organism. The initial bacterial count was enumerated immediately after purchase and the final count at the expiry date. L. acidophilus was enumerated on MRS-salicin agar and MRSsorbitol agar, bifidobacteria on MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride, paromomycin sulfate) agar, and L. casei on LC agar. The counts of L. acidophilus decreased below 10⁶ cfu/g in all the 6 products that contained L. acidophilus only. The counts of L. acidophilus decreased below 10⁶ cfu/g in 75 % of the products that contained L. acidophilus and Bifidobacterium spp. at the expiry date. The population of bifidobacteria dropped below 106 cfu/g in 94 % of the products that claimed L. acidophilus, Bifidobacterium spp. or L. acidophilus, Bifidobacterium spp. and L. casei. Similarly, 50% of the products that contained L. acidophilus, Bifidobacterium spp. and L. casei showed L. casei count of < 10⁶ cfu/g. The counts of L. casei in one of the two products that contained L. casei only dropped below 10² at the expiry date. The pH in all the products decreased during storage.

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A pilot clinical trial was carried out in Egypt to investigate the effect of probiotic preparation containing both Lactobacillus rhamnosus strain LC-705 (L. rhamnosus strain LC-705) and Propionibacterium freudenreichii spp. shermanii JS (P. freudenreichii spp. shermanii JS) on the levels of aflatoxin in human faecal samples. Faecal sampling was the only practical sampling method for this pilot study and faecal levels of aflatoxin are thought to reflect the exposure. For this study, 20 normal healthy volunteers were selected and randomized into two groups of 10. The study was divided into three stages: baseline period (1 week), supplementation period (2 weeks), follow-up period (1 week). During the baseline period the subjects consumed their normal diets and gave two faecal samples (in the beginning and at the end). During supplementation period two faecal samples (after the first and second supplementation week) were collected. During the follow-up a faecal sample was collected at the end of the week. The faecal samples of 11 of the recruited 20 volunteers were positive for aflatoxin B₁ (AFB₁) with levels ranging between 1.8 and 6 μg AFB₁/kg faeces. For volunteers who were administered the probiotic preparation, there was a significant reduction in the level of AFB₁ after the second week of the trial, a reduction which continued during the follow up period. There was no difference in the consumption of foods known to be sources of exposure to AFB₁ between the group receiving the treatment and the control group. These results suggest that the strains of probiotics used in this trial have the ability to influence the faecal content of AFB₁.

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We evaluated the direct effectiveness of pediocin, produced by Pediococcus acidilactici P420. Pediocin has strong antibacterial activity against all serotypes of Listeria monocytogenes. When fermented sausage artificially contaminated with Listeria were experimentally manufactured, Listeria cells were not detected after 14 days of incubation. This antilisterial activity was caused by pediocin rather than lactic acid. In hamburger patty preserved containing pediocin powder and Listeria cells in an ordinary refrigerator, the bacterial cell number decreased markedly within 3 days. We conclude that pediocin reduces and eliminates bacterial cells; therefore, we recommend its use as a good food additive to remove Listeria from dairy products.

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Thirteen dietary substances were examined by a modified in vitro fecal incubation method, with an additional amounT of L-methionine (Met) present, to assess their suppressive effect on the generation of volatile methanethiol (MeSH) in feces. Three kinds of carbohydrates (glucose, inulin, and fructooligosaccharides (FOS)) were found to suppress McSH generation to a level below half that of the control after 3-hr incubation, and it was suggested that the effect wasn't simply derived from the decrease of pH during incubation. In a human in vivo study, FOS-tofu intake for 2 weeks was found to reduce both the frequency of MeSH detection (5/11→3/11) and the average concentrations (3.57 ± 4.81→. 1.02 ± 1.92 ppm) of fecal MeSH as compared to no intake. A comparison of the fecal bacterial counts between MeSH. undetectable (MeSH^-) and MeSH-detectable (MeSH⁺) groups showed interesting features that the counts of ^{Bifidobacterium} indicated a significant increase (p = 0.002) only between the MeSH^- groups in the FOS-tofu intake and non-intake periods, and the frequencies of Bacillus detection showed a high number in the MeSH⁺ group of the nonintake periods.

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