Bifidobacterium Longum SBT2928 and Its Biological Significance

Review Bioscience Microflora Vol. 21 (4), 225-238, 2002 Bifidobacterium longum SBT2928 and Its Biological Significance Shigeru FUJIWARA* Technologyand Research Institute, Snow Brand Milk Products Co., Ltd., 1-1-2, Minamidai,Kawagoe, Saitama 350-1165, Japan Presented at the 6th Annual Meeting of Intestinal Bacteriology,held in Tokyo, May 30-31, 2002. Received for publication, May 31, 2002 Probiotics are initially defined as food supplements containing live microorganisms which beneficially affect the host by improving intestinal microbial balance. Although the definition has been expanded by other researchers from time to time, in this view, lactic acid bacteria (LAB) are unalterably considered to be the main components of probiotics as they fulfill, in the best course, the requirements targeted by the basic idea of these expanded definitions. It has been believed that periodical supplementation of special cultures of probiotic LAB is helpful to keep the person who consumes it in good health. Recently, the ingestion of LAB as a probiotic has drawn much interest all over the world because of growing health consciousness and concern. A typical example is seen in Japanese society. Well-being is one of the most important matters of concern for Japanese because of the rapid aging of the society. The beneficial image of probiotics has led to an increase in the consumption of fermented milk products in Japan as well as in other countries. Bifidobacteria are often used as dietary supplements or as starter cultures in the production of fermented milk products used as health foods. I have developed the probiotic strain Bifidobacterium longum SBT2928 (BL2928) over a decade for this purpose. This review summarizes the research done to demonstrate the biological functions of BL2928, especially regarding its effect on the host's immune system, on intestinal transit as well as the intestinal microflora composition and metabolism, and specific competitive exclusion of enterotoxigenic Escherichia coli by a novel anti-infectious factor (BIF). Key words: BL2928; competitive exclusion of ETEC; immune response modifier; intestinal transit; probiotics strains of microorganisms that work as modifiers of the INTRODUCTION host immune response. At the same time, there have Lactic acid bacteria (LAB) are regarded as safe mi- been few screening studies on immunomodulatory croorganisms (GRAS: generally regarded as safe), and strain(s) of GRAS microorganisms. In particular, re- some of them have been claimed to contribute to the ports regarding microorganisms that exhibit excellent health and fitness of the individual who consumes them. immunomodulating activity have been scarce. Lately, The idea goes back to the cradle of bacteriology as it is concomitant to an increased fat intake, the rates of death understood today (43). It should be noted that fermented due to colonic and liver cancer have been increasing in milk was known to be beneficial for health even before Japan. In this aspect, edible microorganisms, which are it became understood that the fermentation of milk in- considered to enhance cellular immunity of the host by volved the participation of bacteria that is now known oral administration, may play a crucial role to prevent as LAB. The favorable image and biological effects of cancer of the digestive system. (some kinds of) LAB led to the concept of probiotics. In the following several sections, I will present the However, little is known about the underlying mecha- results of screening of GRAS microorganisms done to nism(s) behind the beneficial effects of LAB. select those strains that enhance cell-mediated immu- The main reported effects of probiotics are related to nity. In addition, the characteristics of the BL2928 the improvement of disturbances of the indigenous strain, especially regarding its transit and colonization microflora, amelioration of the development of micro- of the human gastrointestinal tract, amelioration of hu- flora, health enhancement through an inhibition of car- man intestinal microflora, improvement of human in- cinogenesis and non-specific activation of the host im- testinal metabolism, competitive exclusion of Escheri- mune system. However, some of these assertions have chia coli-including the enterotoxigenic Escherichia not been scientifically confirmed. coli Pb 176 (ETEC) and the mechanism of the com- In recent years, researchers have reported many petition between BL2928 and ETEC are summarized. These studies were done to determine whether the *Corresponding author . Mailing address: Technology and Research Insti- BL2928 strain would fulfill the criteria of probiotics tute, Snow Brand Milk Products Co., Ltd., 1-1-2, Minamidai, Kawagoe, Saitama 350-1165, Japan. Phone: +81-49-242-8161. Fax. +81-49-242- and to obtain scientific evidence of its beneficial prop- 8969. E-mail: [email protected] erties. The results were as follows. 225 226 S. FUJIWARA Of the 112 strains of lactobacilli, nine (four strains of SCREENINGOF POSSIBLEIMMUNOMODU- L. acidophilus, three of L. casei and two of L. helveticus) LATINGGRAS FOOD MICROORGANISMS showed mitogenic activity; however, none of the L. Whole heat-killed cells and metabolites of GRAS mi- delbrueckii strains assayed showed mitogenic activity. croorganisms, including bifidobacteria and lactobacilli Two strains of B. subtilis exhibited detectable mito- isolated from the human intestinal flora, lactobacilli genic activity, while the 33 strains of Streptococci as- isolated from fermented milk products, some other lactic sayed did not show any activity (Table 1). acid bacteria, fungi isolated from commercialized Koji At the same time, the cultured supernatants from skim and B. subtilis strains, were screened for their mito- milk cultures of 83 strains of lactobacilli and other bac- genic and immunostimulatory activities by the MTT teria were assayed and those of only three strains of L. method as the mitogen assay using murine spleen cells helveticus exhibited mitogenic activity (Table 2). In as the target cells. addition, of the 56 strains of fungi examined, the cul- First, 170 strains of heat-killed whole cells were as- tured supernatant of only one of them (Aspergillus sayed. Of the 23 strains of Bifidobacterium assayed, oryzae EF-08) showed obvious mitogenic activity only one strain, BL2928, showed mitogenic activity. (Table 3). The mitogenic activity exhibited by the skim Table 1. Spectrum of mitogenic activity in the heat-killed cells of food microorganisms. aData are expressed as stimulation indices (SI) and summarized in a contingency table . Classification: -, SI < 1.00; •}, SI 1.00-1.19; +, SI 1.20-1.39; ++, SI 1.40-1.59; +++, SI > 1.60. Table 2. Spectrum of mitogenic activity in the skimmed milk culture supernatants of food microorganisms. aData are classified by stimulation index (SI) and summarized in a contingency table . Classification: -, SI < 1.00; •}, SI 1.00-1.19; +, SI 1.20-1.39; ++, SI 1.40-1.59; +++, SI > 1.60. BL2928: A BIOLOGICAL RESPONSE MODIFIER 227 milk cultured supernatant reached a maximum at 48 hr of culturing, when 75% of the milk proteins had under- CHARACTERIZATION OF THE IMMUNO- MODULATING ACTIVITIES OF gone proteolysis. The mitogenic activity increased in SCREENED STRAINS proportion to the degree of proteolysis. These results indicate that the mitogenic activity of food microor- The in vivo immunomodulating effects of GRAS mi- ganisms is considered to depend on the characteristics croorganisms with mitogenic activity were investigated of each strain. It is also expected that a novel mito- using two animal models which allow the evaluation genic factor(s), including peptides, is produced in the of cell-mediated and humoral immune responses (25). skim milk cultured supernatants of L. helveticus and A. Three different categories of immunomodulating GRAS oryzae EF-08 (24). microorganisms with mitogenic activity were found. Microorganisms of group I, including BL2928, mark- edly stimulate cell-mediated immunity; those of group II, including L. helveticus SBT2192A, are more likely Table 3. Spectrum of mitogenic activity of the skimmed to enhance humoral immune responses, and those of milk cultured supernatants of food fungi. group III, including L. acidophilus SBT2080, moder- ately stimulate both functions (Table 4). The immunostimulatory activity of BL2928, as char- acterized by its superior antitumor activity, was pre- eminent over the other strains selected by their mitogenic activity. When heat-killed BL2928 was administered parenteral, it exhibited marked anti-tumor activity in both allogeneic and syngeneic tumor models. In contrast, BL2928 did not stimulate primary antibody production in mice. In addition, administration of car- rageenan, an anti-macrophage agent, inhibited the antitumor activity of BL2928. Cultured milk of BL2928 slightly, but significantly, prolonged the life span of ICR mice bearing intraperi- toneally transplanted Sarcoma 180 cells. In addition, heat-killed BL2928 administered by gavage significantly suppressed the growth of subcutaneously inocu- lated Sarcoma 180 cells (Fig. 1), and simultaneously aData are classified by stimulation indices (SI) and summa- enhanced delayed-type hypersensitivity in CDF1 mice rized in a contingency table. (Fig. 2). These findings suggest that BL2928 adminis- Classification: -, SI < 1.00; •}, SI 1.00-1.19; +, SI 1.20- tered parenteral potentiates macrophage and Th 1 cell 1.3 9; ++, SI 1.40-1.59; +++, SI > 1.60. functions but not those of Th2 cells. Although the degree

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