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 characterized 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 intraperitoneally transplanted Sarcoma 180 cells. In addition, heat-killed BL2928 administered by gavage significantly suppressed the growth of subcutaneously inoculated 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 of effectiveness varied accord-

Table 4. Groups of microorganisms classified according to their immunomodulating effects and characteristics of typical strains selected based on their mitogenic activity.

aMitogenic activity was measured by MTT assay . Bacteria were categorized by their stimulation index, which indicated degree

of spleen cell activation. bBALB/c female mice were immunized intraperitoneally with DNP -OVA (0.1 mg i.p.) and each bacterial preparation (0.1 mg:

washed, heat-killed and lyophilized). Ten days later, anti-DNP-OVA antibody titer was determined by ELISA.

BALB/c male mice were inoculated intraperitoneally with Meth A fibrosarcoma (1 •~ 105 cells per animalc i.p.) on day 0, and

these mice were treated with intraperitoneal injections of each bacteria (0.1 mg/treatment) on days 0, 2, 4, 6 and 8. 228 S.FUJIWARA

Fig. 2. Effect of heat-treated BL2928 administered by gavage Fig. 1. Effect of heat-treated BL2928 administered by gavage on delayed-type hypersensitivity in CDF1 female mice bear- on the growth of Sarcoma 180 cells transplanted subcutane- ing subcutaneously transplanted Sarcoma 180 cells . Sched- ously in CDF1 female mice. CDF1 female mice (6 weeks of ules for tumor inoculation were the same as those described age; 22/group) were inoculated subcutaneously with Sarcoma in the legend of Fig. 1. Foot pad swelling was measured 24 180 cells (5 •~ 105 cells/mouse) on day 0. Tumors were resected , 48 and 72 hr post-challenge with sheep red blood cells (I x 21 days after the inoculation. 108 cells) and expressed as population means and their 95%. confidence intervals. ing to the route of administration , BL2928 modified the host cell-mediated immune responses when admin- A B istered either parentcral or orally .

EFFECT OF BL2928 ON THE FUNCTIONS OF MACROPHAGES

To study the capability of BL2928 to activate macrophage functions in vitro, the levels of cytotoxicity , glucose consumption and IL-1 production of macrophages were examined by co-culturing murine perito- neal macrophages with BL2928 and other B. longum strains with different levels of mitogenic activity (36) . All B. longum strains tested induced a cytotoxic effect of macrophages against tumor cells. Glucose consumption by macrophages was also equally enhanced by all strains tested at concentrations up to 20 ,ƒÊg/ml. Fig. 3. Incidenceof hepatomaand numberof hepatomaper However, at higher concentrations of B . longum cells animalin positivecontrol (PEG) and test groups. A: rate of in the assay system, a concentration-dependent reduc- occurrenceof hepatoma,B: numberof hepatomaper animal tion of glucose consumption was observed when mac- in both groups.Statistical significance: p = 0.066(cumulative rophages were incubated with B. longum strains other X2test). than BL2928. IL-1 production was stimulated to vary- ing degrees by the strains tested. Notably, BL2928 stimulated IL-1 production even at low doses . It was EFFECTOF BL2928ON THE INCIDENCE also observed that the stimulatory activity of each strain OF SPONTANEOUSTUMORIGENESIS on IL-1 production corresponded well with its mito- To investigate the effect of BL2928 on the incidence genic activity. Thus, the effect of BL2928 on IL-I pro- of spontaneous liver tumorigenesis, gnotobiotic C3H/ duction might he closely related to its immunostimu- He male mice harboring Escherichia coli , Enterococ- lating effect in vivo. cus faecalis and Clostridium paraputrificum (Dr. Mizu- tani's model), which spontaneously develop liver tu- BL2928: A BIOLOGICAL RESPONSE MODIFIER 229 mors at a high rate, were employed (45). The effect of BL2928 established in the gastrointestinal tract of gnotobiotic mice on the rate of occurrence of liver tumors and intestinal bacterial metabolism was potentially related to the risk of carcinogenesis (23). I observed that the occurrence of liver tumors was significantly reduced by BL2928 established in the in- testines (Fig. 3). However, no significant changes in enzymatic activities of the intestinal microflora, which have been claimed to relate to carcinogenesis, nor in the amounts of tyrosine and tryptophan metabolites, which might be relevant to the promotion of liver tumorigenesis, were observed. These results suggest that the activation of cellular immunity by BL2928 colonization, but not a decrease of the fecal concentration of phenols, may be involved in the reduced occurrence of liver tumors. Fig. 4. Time-dependentchanges in the fecal populationsof Bifidobacteriumlongum SBT2928SR after oral administra- INTESTINALTRANSIT OF BL2928 tion.Each line correspondsto one individual. Manipulation of the gastrointestinal microflora in humans is currently being used as a means to introduce isolated from fecal samples. new microorganisms into the digestive tract that are In an experiment involving healthy volunteers, the beneficial to the host and able to produce beneficial long-term survival of BL2928SR in the human intes- changes regarding the equilibrium and metabolism of tine was confirmed. BL2928SR was detected in the fe- the intestinal microflora (7). Live microorganisms cal specimens of some of the volunteers 30 days after which beneficially affect the host by improving micro- completion of the administration (Fig. 4). However, the bial balance are referred to as 'probiotics' (26). An fate of ingested BL2928SR in the gastrointestinal tract important property of a probiotic is its ability to sur- of humans varies from individual to individual. It is vive in a 'sustained transient state' in the gastrointesti- necessary to clarify the mechanism(s) underlying the nal tract (19, 29, 57). In other words, the essential de- long-term survival of ingested BL2928SR in the hu- terminants for the selection of a probiotic is its ability man gastrointestinal tract. It was suggested that the to reach, survive and persist (or colonize) the environ- parent strain, BL2928, might behave in a similar man- ment which it is intended to affect. ner in the gastrointestinal tract of humans, because the It has been suggested that a probiotic cannot influ- only difference between BL2928 and BL2928SR is its ence the environment unless its population reaches a streptomycin and rifampicin resistance. certain minimum level of between 106 to 108 colony- The kinetics of the fecal clearance of the spores of B. forming units (cfu)/g of intestinal contents (41, 42, 55). subtilis (transit marker) and the selected BL2928SR The intrinsic resistance of Lactobacillus delbrueckii strain after cessation of oral administration were not subsp. bulgaricus and Streptococcus thermophilus to parallel. BL2928SR was retained for a longer period of gastric acid, pancreatic juice and bile is poor (11, 40). time in the intestinal tract than the transit marker. This On the other hand, bifidobacteria and L. acidophilus indicated that BL2928SR survived better during its have been reported to be more resistant, but large dif- passage through the gastrointestinal tract, and suggested ferences exist between strains (12, 40, 46, 56). that ingested BL2928SR bound to the gastrointestinal Consequently, the aim of the study described in this mucosal membrane of the volunteers. It was reported section was to accurately investigate the fate of an orally that the kinetics of the fecal clearance of a strain of administered BL2928 in the human gastrointestinal Bifidobacterium spp. (administered at about 1011cfu of tract. In order to distinguish ingested BL2928 and in- the strain/day) was similar to that of a transit marker digenous bifidobacteria, a streptomycin- and rifampi- (6, 58). This may reflect a wide variation in the gas- cin-resistant variant of BL2928 (BL2928SR) was se- trointestinal transit tolerance and/or ability to colonize lected. In addition, randomly amplified polymorphic the gastrointestinal tract among the strains and species DNA(RAPD)-PCR was used to identify BL2928SR of bifidobacteria. 230 S. FUJIWARA

Streptomycin and rifampicin resistance were used to intestinal microflora. This may explain why the kinet- isolate the BL2928SR strain from the fecal samples of ics of fecal clearance of the administered bacterial strain volunteers. Selectivity of the means employed for the seemed to depend on the individual. At present, the isolation of BL2928SR was not always satisfactory. A molecular mechanism(s) by which bifidobacteria ad- small population of streptomycin- and rifampicin-re- here to the intestinal mucosal surface remains unknown. sistant Eubacterium spp. was found simultaneously in some cases. To avoid the risk of mistaking colonies of EFFECT OF ORAL ADMINISTRATION OF Eubacterium spp. grown on BL-SR medium for those BL2928 ON INTESTINAL MICROFLORA of BL2928SR, RAPD-PCR was employed for identi- AND METABOLISM fication in this study. It remains debatable whether any of the LAB cur-

In a recent study (62), an excellent method for dis- rently used in cultured milk products can exert a major tinguishing ingested bacteria using specific monoclonal activity on the resident intestinal microflora of humans antibodies was reported. An ELISA technique was used after oral administration (27, 38). Since strains of Bifido- to specifically detect and quantify the ingested CBM588 bacterium species have been considered to be potent strain in rat fecal samples. This method should be fur- candidates as superior probiotics, these species consti- ther developed and applied more widely to fully evalu- tute a significant proportion of the cultures used in de- ate the fates of probiotics in the gastrointestinal tract. veloped countries (27). However, the influence of the

Intestinal colonization is defined as a bacterial popula- oral administration of B. longum yogurt on the compo- tion in the gastrointestinal tract that remains stable over sition of the human intestinal microflora and its meta- time without the need for periodic re-introduction of bolic activity is controversial (3, 52). The wide varia- the bacteria. BL2928SR did not fulfill this criterion com- tions in the gastrointestinal transit tolerance and/or abil- pletely, but long-term retention of the bacterium in the ity for colonization among these strains may account gastrointestinal tract of several volunteers was shown. for such unpredictable effects. The detection of BL2928SR in the feces up to one month The effect of the oral administration of BL2928SR after stopping its administration suggested that the strain on the composition and metabolism of the human in- showed affinity, at least to some extent, to the mucosal testinal microflora was next investigated. With regard surface of the human intestinal tract. It was suggested to its effects on the composition of the intestinal that the parent strain, BL2928, may behave in a similar microflora, orally administered BL2928SR was asso- manner. ciated with a decrease of the populations of Entero-

The reason(s) for the long-term survival of BL2928 bacteriaceae and clostridia, including lecithinase-posi- SR in the gastrointestinal tract need to be further inves- tive Clostridium spp. (Fig. 5). It was considered that tigated. Since survival seemed to depend on the indi- these changes were specific because fecal numbers of vidual, it is possible that the density of the binding no other bacterial species or genera changed. Further- receptor(s) for BL2928SR on the mucosal surface also more, the contents of organic acids in feces, which serve depends on the individual. The concept of colonisation as anti-infectious agents (33), did not change during resistance posed by endogenous anaerobic microflora this study. These data suggest that BL2928SR specif- has been well established (8, 13, 62, 63, 65). The in- ically competes with these bacteria in the gastrointesti- digenous intestinal microflora exerts resistance to colo- nal tract. nization by exogenous microorganisms (5). This bar- As for the impact on intestinal metabolism, ƒÀ-glucu- rier effect has been shown for several bacteria whose ronidase activity in feces, which has been repeatedly fecal excretion was compared with that of an inert reported to relate to carcinogenesis, was significantly marker administered at the same time, and it is consid- reduced. Microbial ƒÀ-glucuronidase is believed to be ered to be caused by bacteriostasis when fecal elimina- primarily responsible for the intestinal hydrolysis of tion of the two components shows the same kinetics , glucuronides. This reaction is potentially important in and bacteriolysis when the exogenous bacteria are elimi- the generation of toxic and carcinogenic substances nated more quickly than the passive marker (14). From since many compounds are detoxified by glucuronide this viewpoint, it may be considered that orally admin- formation in the liver, whereupon as bile, they enter istered BL2928SR somehow evades the colonization the bowel through enterohepatic circulation (28). The resistance posed by the indigenous anaerobic action of microbial ƒÀ-glucuronidase may delay the ex- microflora. The degree of such evasion of colonization cretion of many exogenous compounds (64). As BL resistance may be influenced by the composition of the 2928SR was found to repress fecal ƒÀ-glucuronidase BL2928: A BIOLOGICAL RESPONSE MODIFIER 231

Fig. 5. Changes in the composition of the intestinal microflora. Data are expressed as the mean ± SEM (n = 7). Detection limit of the bacterial count procedure is 10/g. activity, oral administration of the bacterial prepara- nisms underlying the physiological roles of the genera tion may contribute to reduce the risk of colon carcino- have been clarified. genesis. In addition, a questionnaire survey of changes Basic research on the anti-infectious mechanism(s) in the physical characteristics of feces showed that color of action of bifidobacteria against enterotoxigenic E. and odor were significantly modified during its admin- coli seems to be necessary. A better understanding of istration. the relationship between both types of bacteria may In conclusion, these results suggest that following explain the beneficial action of bifidobacteria on the its oral administration to humans, strain BL2928SR af- well-being of the host. fected the composition of the intestinal microflora as Recently, there has been renewed interest in the age- well as the intestinal glucuronide hydrolysis activity. old concept of using bacterial preparations with inhibi- Thus, BL2928SR and its parent strain, BL2928, are tory activity against enteric pathogens to protect do- good candidates for further evaluation in vivo of their mestic animals and humans. This concept is often re- probiotic effects in humans. ferred to as bacterial interference. Protection could be obtained because the lactose fermenters produce me- SPECIFIC COMPETITIVE EXCLUSIONOF tabolites antagonistic to the growth of pathogens. In ENTEROTOXIGENICESCHERICHIA addition, the concept of competitive exclusion of patho- COLI BY BL2928 genic microbes by lactic acid bacteria has been studied Infections of the gastrointestinal tract are still a ma- to understand their beneficial action (10). jor health problem for adults and children worldwide. For example, a relationship has been reported be- One of the major causes of postweaning diarrhea in tween the oral administration of bifidobacteria and the neonates is the presence of enterotoxigenic E. coli decrease in the number of fecal Enterobacteriaceae (23, strains, which express colonization factor antigens 45, 61). In this study, fecal numbers of both Entero- (CFAs) I and II (39). On the other hand, normal intes- bacteriaceae and Clostridium spp., including lecithi- tinal microbes and some probiotic bacteria are believed nase-positive Clostridium spp., decreased during to enhance the host's defense mechanisms against BL2928SR administration as mentioned above. It was pathogens. Since bifidobacteria were first isolated by considered that these changes were specific because Tissier (59) from a breast-fed infant, it has been widely fecal numbers of no other bacterial species or genera acknowledged that they are the most predominant com- changed. Furthermore, the contents of organic acids in ponent of the intestinal flora of infants (44, 47). Al- feces, which serve as anti-infectious agents (33), did though the physiological roles of bifidobacteria in host not change during the study. These data suggest that resistance to infection have been repeatedly suggested BL2928SR specifically competes with these bacteria from various observations (30, 50), only a few mecha- in the gastrointestinal tract. 232 S. FUJIWARA

Fig. 6. Inhibitory activity of neutralized cultured supernatants of several species of bifidobacteria on

the binding of ETEC (E. coli Pb176) to GA1 . Radiolabeled ETEC were suspended in each neutralized cultured supernatant and overlaid on TLC plates on which GA 1 (5 ƒÊg) had heen developed . Binding strength was measured densitometrically and compared with the binding strength of the

control (in 1% BSA-PBS) being 100 . Data are expressed as the mean relative binding strength •} SD

(n=5).

To clarify the specific antagonistic action of BL2928 factor(s) that prevents the binding of ETEC to the bind- against Enterobacteriaceae in gnotobiotic mice and ing receptor GA 1 We speculated that this protein con- healthy volunteers, we examined the competitive bind- tributes to the normal anti-infectious activities of the ing of several species of bifidobacteria and an intestine by preventing the binding of pathogenic strains enterotoxigenic E. coli strain [E. coli Pb176 express- of E. coli to the mucosal surface. ing colonization factor antigen (CFA) II, which con- Since adhesion by bacteria to host tissue is regarded sists of coli surface-associated antigens (CS) 1 and 3 as a prerequisite for infection and colonization (4, 17), (ETEC)] to asialomonosialoganglioside 1 (asialo GM 1; inhibition of bacterial adhesion to the intestinal surface GA 1), a common binding structure in vitro, and identi- may prevent the intestine from becoming a reservoir fied the factor(s) that inhibit the binding of ETEC to for E. coli pathogens. In this section, purification and GA 1 in the cultured supernatant of bifidobacteria . characterization of the ETEC-binding inhibitory factor While bifidobacteria inhibit the binding of ETEC to from the cultured supernatant of BL2928 are described. GA 1, ETEC do not substantially affect the adherence To identify the factor(s) produced by BL2928 against of bifidobacteria to GA 1. The competitive exclusion of ETEC in the gastrointestinal tract, purification of this ETEC from GA 1 by bifidobacteria is estimated to be anti-ETEC factor from the cultured supernatant of due to a non-specific hydrophobic interaction between BL2928 was attempted. A novel protein (BIF) that in- bifidobacteria and the ceramide moiety of GA 1. hibited the binding of ETEC to GA 1 was isolated from On the other hand, the neutralized cultured superna- the cultured supernatant of BL2928 at the stationary tant of bifidobacteria, including BL2928, also inhib- phase of culturing. The homogeneity of the final prepa- ited the binding of ETEC to GA 1 (Fig . 6). The results ration was demonstrated by SDS-PAGE, polyacryl- of the analyses performed suggested that the inhibitory amide gel electrofocusing and N-terminal amino acid factor(s) produced by BL2928 was a proteinaceous sequencing. BIF was characterized as (i) a protein with factor(s) with a molecular weight around or over 100 a molecular weight of approximately 104 kDa when kDa and a neutral isoelectric point. The proteinaceous chromatographed on a gel filtration column and 52 kDa factor(s) produced by BL2928 and other bifidobacteria when separated on SDS-PAGE, and (ii) having an iso- is estimated to contribute to the anti-infectious activi- electric point of 5.9. No change in size was produced ties and play a role in the exclusion of the E. coli popu- by thiol reduction. These results suggest that BIF is a lation from the human intestinal microflora. homodimer consisting of identical 52 kDa monomers (Fig. 7). The purified BIF at the concentration of 25 jig IDENTIFICATIONAND CHARACTERIZATION of protein/ml caused a 50% reduction in the binding of OF THE ETEC-BINDING INHIBITORY FACTOR OF BL2928 the ETEC strain to GA 1 (Fig. 8). These findings show that BL2928 produces a pro- We clarified that BL2928 produced a proteinaceous teinaceous factor, BIF, that prevents the binding of the BL2928: A BIOLOGICAL RESPONSE MODIFIER 233

a) b) c)

Fig. 7. SDS-PAGE analyses of BIF under reducing and non- reducing conditions. M, 5 ƒÊg of low molecular weight marker Fig. 8. Inhibitory activity of purified BIF on the binding of ETEC to GA 1. (A) TLC overlay: a: binding of ETEC to GA 1 (Bio-Rad): R, BIF (0.5 ƒÊg) was processed in the presence of 2-mercaptoethanol; NR, BIF (0.5 ƒÊg) was processed in the without BIF, b: binding of ETEC with BIF 25 ƒÊg/ml, c: bind- absence of 2-mercaptoethanol. The samples were loaded on a ing of ETEC with BIF 50 ƒÊg/ml. Lanes 1 and 2 contained 5 10% acrylamide gel and run at 18 mA constant current for and 2.5 ƒÊg of GA 1 , respectively. (B) Dose-dependency of the 100 min. Staining was carried out with Copper staining (Bio- inhibitory activity of BIF. The 50% inhibitory concentration Rad). (IC5()) of binding of BIF was estimated to be 25 ƒÊg/ml.

ETEC to GA1 in vitro. This is the first time a specific than blocking of a bacterial adhesin has also been shown

ETEC inhibitory product of a Bifidobacterium strain by Blomberg et al. (5) in the case of a proteinaceous has been isolated. The ETEC adhesion was reduced by factor produced by Lactobacillus spp. of porcine ori-

BIF in a dose-dependent manner. The N-terminal amino gin. acid sequence of the 52 kDa peptide of BIF is similar There are several other pathogens that show affinity to that of thymidine phosphorylase of Lactobacillus for GA 1. For example, Jagannatha et al. (35) reported

asei (1), but the molecular weights of cboth proteins enteropathogenic E. coli strains of localized adherent are quite different. Separate experiments (not presented) phenotypes also bound to GA 1. They claimed the mini- were carried out to show that purified BIF bound to a mum common sequence of the GA 1 molecule as a bind- column of GA1-coated latex beads and was eluted as ing epitope is Ga1NAcƒÀ1-4Gal. Yu et al. (68) reported the major protein. that Candida albicans bound to the same binding While there have been many studies demonstrating epitope. Willoughby et al. (66) reported that Rotaviruses that strains of bifidobacteria show anti-infectious prop- also bound to GA1 A strain of P. aeruginosa (SBT erties against enteropathogenic bacteria (31, 32, 67), 3092) exhibited a decrease in binding to GA1 in vitro few have shown their interference with the adhesion of after treatment of GA 1 with a protein fraction contain- the pathogen to the receptor molecule, and none has ing BIF (20, 21). However, it is possible that some varia- reported a mechanism for the inhibition observed. tion exists with respect to the specific binding sequence

Pretreatment of a TLC plate, on which GA 1 had been on GA 1 from one pathogen to another (37). developed with BIF, significantly decreased the bind- The precise binding site on GA1 for BIF remains to

ing of ETEC to GA1. However, pretreatment of the be determined. Some of the possibilities that have not ETEC cells with BIF showed little effect. These find- been investigated as yet include the lectin-like carbo-

ings suggest that BIF works by blocking the binding hydrate interaction of BIF with GA 1 , a glycosidase-

site on the GA1 molecule. BIF apparently does not block like activity in which the carbohydrate chain of the GA 1 ETEC adhesin directly, which is assumed to be coloni- molecule is hydrolysed or has less specific (e.g. hydro-

zation factor CFA-II. Blocking of receptor sites rather phobic or electrostatic) interaction. Fontaine et al. (18) 234 S. FUJIWARA

reported that a strain of B. bifidum showed hemaggluti-

nation activity, suggesting that a lectin-like substance(s)

is located on the cell surface of the bacteria .

BL2928, as a lactose fermenter , may supplement the natural anti-infectious properties of milk . Ashkenazi and Mirelman (2) found that a glycoprotein present in human milk could inhibit CFA I- and II-mediated adhe-

sion to the small intestine of the guinea pig upon pre-

treatment of the bacteria. Recently, Ouwehand et al . (53) showed that bovine milk protein like ƒÀ-lactoglo-

bulin inhibited the binding of S-fimbriated E . coli to human ileostomy glycoproteins . These results support the concept that milk has a beneficial effect in prevent-

ing gastrointestinal infection. It is interesting, therefore , to speculate that BL2928 could reinforce the anti-in- Fig. 9. Adherence of E. coli Pb 176 to HCT-8 cells pre-incubated with different concentrations of the binding inhibitory fectious function of milk by adding its metabolic prod- protein fraction P100V for 30 min at 37•Ž. After washing, E. ucts to fermented milk. coli Pb 176 at a density of 109 cells per 1 ml of HEPES-Hanks'

were incubated with HCT-8 cells for 60 min at 37•Ž . The EFFECT OF THE BIF-CONTAINING FRACTION number of bound bacteria was determined by scintillation ON THE BINDING OF ETEC TO HCT-8 CELLS counting.

An essential stage in the pathogenesis of intestinal infections, especially diarrhea of bacterial origin, in- In addition, I provide evidence suggesting the impor- volves the adhesion of the microorganisms concerned tance of the GA 1 molecule on the binding of the ETEC to the intestinal epithelial cells . In principle, bacterial strain to the cell line and of the importance of the bind- diarrhea could be treated by preventing adhesion of ing of BIF to GA 1 present on HCT-8 cells in the bind- enteropathogenic bacteria. Three main methods have ing competition with the ETEC strain (22) . been described: vaccination against bacterial adhesion P100V fraction containing BIF produced by BL2928 factors (16), administration of antibiotics that inhibit also inhibited the binding of ETEC to the human intes- the expression of adhesion factors (9), and oral admin- tinal epithelial cell line HCT-8 (ATCC CCL 244) in a istration of substances containing structures similar to dose-dependent manner (Fig. 9). ETEC-binding experi- those of the adhesion factors of pathogens (49) or struc- ments using crude colonization factor antigen (CFA)- tures that mimic receptors of the intestinal mucosa (48) . II prepared from ETEC, rabbit anti-GA 1 antiserum , Orally administered strains of Bifidobacterium and medium containing GA 1 and media containing lectins , Lactobacillus have also been found to inhibit the im- as the binding inhibitors, suggested that the interaction plantation of various pathogens in the digestive tract between the CFA-II antigen present on the cell surface (34, 60), and have been shown to exert beneficial ef- of ETEC and GA 1 expressed on HCT-8 cells played a fects on diarrhea in humans and animals (32 , 54). Es- significant role in the binding of ETEC to GA 1 . It is pecially, Bifidobacterium has been repeatedly suggested strongly suggested that the P100V fraction works as a to contribute to host resistance to infection (31); but to blocking agent for the ETEC receptor GA 1 on HCT-8 date little is known about the mechanism(s) by which cells. this might occur in the human gastrointestinal tract . I clearly showed here that the P 100V fraction pre- In the previous section, I mentioned the inhibitory pared from the spent broth of BL2928 inhibited the ad- activity of BIF produced by BL2928 on the binding of herence of ETEC to HCT-8 cells, a human intestinal an ETEC strain (E. coli Pb 176) to the glycolipid bind- carcinoma cell line. Furthermore , it was considered, ing receptor GA 1 (11, 21); however, the activity of BIF because of the facts described below, that BIF affected on the binding of the ETEC strain to intestinal epithe- the interaction of the GA 1 molecule present on the sur- lial cells has not been examined as yet. To gain further face of HCT-8 cells and the ETEC strain. information, I used here the enterocyte-like HCT-8 cell First, pretreatment of HCT-8 cells with crude CFA- line to investigate the adherence of the ETEC strain to II antigen isolated from the ETEC strain inhibited the the cell line in the presence of the BIF-enriched extra- binding of ETEC to the cells in a dose-dependent man- cellular protein fraction (P100V) produced by BL2928 . ner. This showed that the binding of the ETEC strain to BL2928: A BIOLOGICAL RESPONSE MODIFIER 235

Speculated mechanism #1: binding competition

Speculated mechanism #2: blocking of colonization factor

Speculated mechanism #3: blocking of binding receptor

Fig. 10. Binding competition between BL2928 and ETEC on the intestinal mucosal surface. Mecha- nism #3 is likely the answer for the binding competition.

HCT-8 cells depends on the function of CFA-II anti- binding competition between CFA-II present on ETEC gen. as reported by Evans and Evans (15). and BIF. Because the binding affinity of the ETEC Second. OrƒÓ et al. (51) reported that the ETEC strain strains for GA 1 is greater than that for ganglio-tetra- bound specifically to the neutral sphingoglycolipid osylceramide (GA2) (20). However, further studies are GA 1. In addition, we found that BIF significantly in- needed to confirm this hypothesis (Fig. 10). hibited the binding of ETEC to GA1 as described in These findings also suggested that BIF might inter- the preceding section (21). I also found that pretreat- fere in vivo with the binding of pathogens which show ment of HCT-8 cells with rabbit anti-GA1 serum affinity for GA 1 on the intestinal (or other) epithelium. significantly inhibited the binding of ETEC to HCT-8 This hypothesis needs to be confirmed in vivo not only cells. Furthermore, pretreatment of the ETEC strain with in gnotobiotic animals but also in conventional animals. GA1 reduced binding in a dose-dependent manner. Further research is necessary to examine the possible Moreover, pretreatment of HCT-8 cells with the P100V in vivo significance of the GA 1-binding phenomenon fraction containing BIF also inhibited the binding of as the in vitro system differs from the intestinal envi- ETEC. These results suggest that the GA1 molecule ronment. In this regard, human intestinal microflora- present on the surface of HCT-8 cells plays a critical bearing mice (23) will be an appropriate model to simu- role in the interaction between HCT-8 cells and the late the conditions of the human gastrointestinal tract. ETEC strain. In addition, it is important to carry out a survey on On the other hand, pre-incubation of HCT-8 cells with the resistance of BIF against the activities of digestive three different types of lectins independently suggested enzymes in the gastrointestinal tract of humans. Fur- that the galactose molecule was important for the in- ther studies are required to fully clarify the physiologi- teraction. The sequence of the carbohydrate moiety of cal role of BIF or the BIF-enriched protein fraction GA1 [Gal(ƒÀ1-3)GaINAc(ƒÀ1-4)Gal(ƒÀ1-4)Glc] sug- P100V in human intestinal tract. But on the basis of gested that BIF might interfere with the interaction be- my studies, I can emphasize that BIF possibly works tween CFA-II antigens on the ETEC strain and GA1 as a physiological component in the intestinal tract of on the surface of HCT-8 cells by its binding to the ga- infants and/or adults to protect them against enteric in- lactose moiety of the GA1 molecule. Especially, the fections. It will be important to determine whether all terminal galactose moiety in the oligosaccharide se- strains or all species of bifidobacteria produce BIF or a quence of GA1 would play an important role on the similar protein(s) that prevents the binding of patho- 236 S. FUJIWARA gens showing affinity for GA1 to this glycolipid recep- (8) Charteris WP, Kelly PM, Morelli L, Collins JK. 1998. De- tor on the surface of the intestinal (or other) epithe- velopment and application of an in vitro methodology to lium. If the theory is developed on the basis of this as- determine the transit tolerance of potentially probiotic Lac- sumption, BIF or a similar protein(s) might work as a tobacillus and Bifidobacterium species in the upper human colonization resistant factor(s) of indigenous anaero- gastrointestinal tract. J Appl Microbiol 84: 759-768. (9) Chopra I, Hacker K. 1986. Inhibition of K88 mediated ad- bic microflora (62) against invasive pathogens. hesion of Escherichia coli to mammalian receptors by antibiotics that affect bacterial protein synthesis.J Antimicrob CONCLUSION Chemother 18: 441-451. These results suggest that, following its oral admin- (10) Conway PL. 1989. Lactobacilli: fact and fiction. In The istration, BL2928 showed persistence in the gastrointes- Regulatory and Protective Role of the Normal Microflora, Grubbe R, Midtvedt T, Norin E (eds), MacMillan Press, tinal tract, significantly affected the composition of in- New York, p. 263-281. digenous microflora as well as intestinal glucuronide (11) Conway PL, Gorbach SL, Goldin BR. 1987. Survival of hydrolysis activity, modified host cellular immunity, lactic acid bacteria in the human stomach and adhesion to and possibly suppressed the binding of ETEC to mu- intestinal cells. J Dairy Sci 70: 1-12. cosal epithelial cells by producing a novel anti-infec- (12) Dreher RM, Gerhard J, Schonborn W, Lauer E. 1991. De- tious protein, BIF. tection of Bifidobacterium longum in fecal samples after Thus, it is suggested that BL2928 is a good probiotic orogastric intubation using immunological test systems. Microb Ecol Health Dis 4: 271-273. for humans and animals. (13) Dubos R. 1965. The composition of the indigenous microbiota. In Man Adapting, Dubos R (ed), Yale Univer- Acknowledgments. I wish to thank Dr. Tomotari sity Press, New Haven, p. 110-128. Mitsuoka (Professor Emeritus, The University of To- (14) Ducluzeau R. 1989. Role of experimental microbial ecol- kyo) and Dr. Shuu-ichi Kaminogawa (Professor, The ogy in gastroenterology. In Microbial Ecology in Intesti- University of Tokyo) for the opportunity to publish this nal Infection, Bergogne-Berezin E (ed), Springer Verlag, Paris, p. 7-26. review. (15) Evans DG, Evans DJ Jr. 1978. New surface associated heat- labile colonization factor antigen (CFA/II) produced by REFERENCES enterotoxigenic Escherichia coli of serogroups 06 and 08. (1) Abraham Y, Grosswicz N, Yashphe J. 1990. Purification Infect Immun 21: 638. and characterization of uridine and thymidine phosphory- (16) Evans DG, Graham DY, Evans DJ. 1984. Administration lase from Lactobacillus casei. Biochim Biophys Acta 1040: of purified colonization factor antigens (CFA/I, CFA/II) of 287-293. enterotoxigenic Escherichia coli to volunteers. Gastro- (2) Ashkenazi S, Mirelman D. 1987. Nonimmunoglobulinfrac- enterology 87: 934-940. tion of human milk inhibits the adherence of certain (17) Finlay BB, Falkow S. 1990. Common themes in microbial enterotoxigenic Escherichia coli strains to guinea pig in- pathogenicity. Microbiol Rev 53: 210-230. testinal tract. Pediatr Res 22: 130-134. (18) Fontaine IF, Aissi EA, Bouguelet SJ-L. 1994.In vitro bind- (3) Bartram H-P, Scheppach W, Gerlach S, Ruckdeschel G, ing of Bifidobacteriumbifidum DSM 20082 to mucosal gly- Kelber E, Kasper H. 1994. Does yogurt enriched with coproteins and hemagglutinating activity. Curr Microbiol Bifidobacterium longum affect colonic microbiology and 28: 325-330. fecal metabolites in healthy subjects? Am J Clin Nutr 59: (19) Freter R. 1992. Factors affecting the microecology of the 428-432. gut. In Probiotics: The Scientific Basis, Fuller R (ed), (4) Beachey EH. 1981. Bacterial adherence: adhesin-receptor Chapman and Hall, London, p. 111-144. interactions mediating the attachment of bacteria to mu- (20) Fujiwara S, Hashiba H, Hirota T, Forstner JF. 1997. Pro- cosal surfaces. J Infect Dis 143: 325-345. teinaceous factor(s) in culture supernatant fluids of (5) Blomberg L, Henriksson A, Conway PL. 1993. Inhibition bifidobacteriawhich prevents the binding of enterotoxigenic of adhesion of Escherichia coli K88 to piglet ileal mucus Escherichia coli to gangliotetraosylceramide. Appl Environ by Lactobacillus spp. Appl Environ Microbiol 59: 34-39 . Microbiol 63: 506-512. (6) Bouhnik Y, Pochart P, Marteau P, Arlet G, Goderel I, (21) Fujiwara S, Hashiba H, Hirota T, Forstner JF. 1999. Rambaud JC. 1992. Fecal recovery in humans of viable Purificationand characterizationof a novel protein produced Bifidobacterium sp ingested in fermented milk. Gastroen- by Bifidobacteriumlongum SBT2928 that inhibits the bind- terology 102: 875-878. ing of enterotoxigenic Escherichia coli Pb 176 (CFA/II) to (7) Charteris WP, Kelly PM, Morelli L, Collins JK. 1997. Se- gangliotetraosylceramide. J Appl Microbiol 86: 615-621. lective detection, enumeration and identification of poten- (22) Fujiwara S, Hashiba H, Hirota T, Forstner JF. 2001. Inhi- tially probiotic Lactobacillus and Bifidobacterium species bition of the binding of enterotoxigenic Escherichia coli in mixed bacterial populations. Int J Food Microbiol 35: Pb 176 to human intestinal epithelial cell line HCT-8 by an 1-17. extracellular protein fraction containing BIF of Bifidobac- BL2928: A BIOLOGICAL RESPONSE MODIFIER 237

terium longum SBT2928: suggestive evidence of blocking uctscontaining bifidobacteria. In TherapeuticProperties of of the binding receptor gangliotetraosylceramideon the cell FermentedMilk, Elsevier AppliedFood ScienceSeries , surface. Int J Food Microbiol 67: 97-106. Elsevier,London, p. 117-157. (23) Fujiwara S, Hirota T, Nakazato H, Mizutani T, Mitsuoka (39) Levine MM. 1987.Escherichia coli that cause diarrhea: T. 1991. Effects of Bifidobacterium longum SBT 2928 enterotoxigenic,enteropathogenic, enteroinvasive, entero- (BL2928) on spontaneous liver tumorigenesis and intesti- hemorrhagic,and enteroadherent.J Infect Dis 155: 377- nal metabolism in gnotobiotic C3H/He male mice. J Jpn 389. Soc Nutr Food Sci 44: 261-266 (in Japanese with English (40) LindwallS, Fonden R. 1984.Passage and survivalof L. summary). acidophilusin the humangastrointestinal tract. IDF Bull (24) Fujiwara S, Kado-oka Y, Hirota T, Nakazato H. 1990. 179: 21. Screening for mitogenic activity of food microorganisms (41) MarteauP, PochartP, BounikY, RambaudJ-C. 1993.The and their skim milk culture supernatants. J Jpn Soc Nutr fate and effectsof transiting,non-pathogenic microorgan- Food Sci 43: 203-208 (in Japanese with English summary). isms in the humanintestine. In WorldReview of Nutrition (25) Fujiwara S, Kado-oka Y, Hirota T, Nakazato H. 1990. and Dietetics,Vol 74, IntestinalFlora, Immunity, Nutri- Immunopotentiating effects of Bifidobacterium longum tion,and Health,Simopoulos AP, Corring T, ReratA (eds), SBT2928 (BL2928) showing mitogenic activity in vitro. J Krager,London, p. 1-21. Jpn Soc Nutr Food Sci 43: 327-333 (in Japanese with (42) MarteauP, RambaudJ-C. 1993.Potential of using lactic English summary). acid bacteriafor therapy and immunomodulationin man. (26) Fuller R. 1989. Probiotics in man and animals. J Appl FEMSMicrobiol Rev 12: 207-220. Microbiol 66: 365-376. (43) Metchnikoff E. 1908. The Prolongation of Life, C.P. (27) Fuller R. 1992. Problems and prospects. In Probiotics: The Putnam'sSons, NewYork. Scientific Basis, Fuller R (ed), Chapman and Hall, Lon- (44) MitsuokaT, HayakawaK, KitamuraN. 1974.Die Faekal- don, p. 377-386. flora bei Menschen.II. Mitteilung:die Zuzammensetzung (28) Goldin BR, Dwyer J, Gorbach SL, Gordon W, Swenson L. der Bifidobakterien-florader vershiedenen Altersgruppen. 1978. Influence of diet and age on fecal bacterial enzymes. ZentralblBakteriol Parasitenk I Abt OrigA 226: 469-478. Am J Clin Nutr 31 (Suppl 10): 136-140. (45) MizutaniT, MitsuokaT. 1979.Effect of intestinalbacteria (29) Havenaar R, Brink BT, Huis in't Veld JHJ. 1992. Selec- on incidenceof liver tumors in bnotobioticC3H/He male tion of strains for probiotics use. In Probiotics.The Scientific mice. J NatlCancer Inst 63: 1365-1370. Basis, Fuller R (ed), Chapman and Hall, London, p. 209- (46) ModlerHW, McKellar RC, YagushiM. 1990.Bifidobac- 224. teria and bifidogenicfactors. Can Inst Food Sci Tech 23: (30) Honma N. 1974. Intestinal flora of infants and resistance to 29-41. infection. Jpn J Pediatr 27: 1267-1275 (in Japanese with (47) MoreauMC, Thomasson M, DucluzeauR, RaibaudP. 1986. English summary). Cinetiqued'etablissement de la microfloredigestive chez (31) Honma N. 1988. Bifidobacteria as a resistance factor in le nouveau-nehuman en fonctionde la naturedu lait.Replod human beings. Bifidobacteria Microflora 7: 35-43. Nutr Develop26: 745-753. (32) Hotta M, Sato Y, Iwata S, Yamashita N, Sunakawa K, (48) Mouricot M, Petit JM, Julien R. 1986. Mode d'action Oikawa T, Tanaka R, Watanabe K, Takayama H, Yajima d'agents inhibitures de 1'adhesionde Escherichiq coli M, Sekiguchi S, Arai S, Sakurai T, Mutai M. 1987. Clini- enterotoxinogenes (ECET) aux glycoproteines de cal effects of Bifidobacterium preparations on pediatric in- l'quaueuse intestinqlebovine. Rev Inst PasteurLyon 19: tractable diarrhea. Keio J Med 36: 298-314. 161-168. (33) Ibrahim SA, Bezkorovany A. 1993. Inhibition of Escheri- (49) NeeserJR, ChambazA, HoangKY, Link-AmsterH. 1988. chia coli by bifidobacteria. J Food Protect 56: 713-715. Screeningfor complexcarbohydrates inhibiting hemaglu- (34) Itoh K, Freter R. 1989. Control of Escherichia coli popula- tinationsby CFA/I-and CFA/II-expressing enterotoxigenic tions by a combination of indigenous clostridia and lacto- Escherichiacoli strains. FEMSMicrobiol Lett 49: 301- bacilli in gnotobiotic mice and continuous-flow cultures. 307. Infect Immun 57: 559-565. (50) OkamuraN, Nakaya R, Yokota H, Yanai N, Kawashima (35) Jagannatha HM, Sharma UK, Ramaseshan T, Surolia A, T. 1986.Interaction of Shigellawith bifidobacteria. Balganesh TS. 1991. Identification of carbohydrate struc- bacteriaMicroflora 5 : 51-55. tures as receptors for localised adherent enteropathogenic (51) Oro HS, Kolsto A-B, Wenneras C, SvennerholmA-M. Escherichia coli. Microb Pathog 11: 259-268. 1990.Identification of asialo GM 1as a binding structure (36) Kado-oka Y, Fujiwara S, Hirota T. 1991. Effects of bifido- for Escherichiacoli colonizationfactor antigens. FEMS bacteria cells on mitogenic response of splenocytes and MicrobiolLett 72: 289-292. several functions of phagocytes. Milchwissenschaft 46: (52) OrrhageK, LidbeckA, NordCE. 1991.Effect of Bifidobac- 626-630. teriumlongum supplements on thehuman faecal microflora. (37) Karlsson K-A. 1989. Animal glycolsphingolipids as mem- MicrobEcol HealthDis 4: 265-270. brane attachment sites for bacteria. Ann Rev Biochem 58: (53) OuwehandAC, Conway PL, SalminenSJ. 1995.Inhibi- 309-350. tion of S-fimbria-mediatedadhesion to humanileostomy (38) Kurman JA, Rasic JL. 1991. The health potential of prod- glycoproteinsby a proteinisolated from bovine colostrum. 238 S. FUJIWARA

Infect Immun 63: 4917-4920. role in microbial colonization of mice. Bifidobacteria (54) Perdigon G, Nader de Mecias ME, Alvarez S, Oliver G, Microflora 8: 13-22. Pesce de Ruiz-Horgado AA. 1990. Prevention of gas- (62) Van der Waaij D, Berghuis JM, Lekkerkerk-van der Wees trointestinal infection using immunological methods with JEC. 1971. Colonization resistance of the digestive tract in milk fermented with Lactobacillus casei and Lactobacil- conventional and antibiotic-treated mice. J Hyg 69: 405- lus acidophilus. J Dairy Res 57: 255-264. 411. (55) Rambaud J-C, Bouhnik Y, Marteau P, Pochart P. 1993. (63) Vollaard EJ, Clasener HAL. 1994. Colonization resistance. Manipulation of the human gut microflora. Proc Nutr Soc Antimicrob Agents Chemother 38: 409-414. 52: 357-366. (64) Weisburger JH. 1971. Colon carcinogens,their metabolisms (56) Sakai K, Mishima T, Tachiki T, Kumagai H, Tochikura T. and mode of action. Cancer 28: 60-70. 1987. Mortality of bifidobacteria in boiled yogurt. J Fer- (65) Wells CL, Maddaus LA, Jechorek RP, Simmons RL. 1988. ment Tech 65: 215-220. Role of intestinal anaerobic bacteria in colonization resis- (57) Salminen S, Deighton M, Gorbach S. 1993. Lactic acid tance. Eur J Clin Microbiol Infect Dis 7: 107-113. bacteria in health and disease. In Lactic Acid Bacteria, (66) Willoughby RE, Yolken RH, Schnaar RL. 1990. Rota vi- Salminen S, Von Wright A (eds), Marcel Dekker, New ruses specificallybind to the neutral glycolipid asialo-GM1. York, p. 199-235. J Virol 64: 4830-4835. (58) Sato R, Tanaka M. 1996. Multiplication of orally adminis- (67) Yamazaki S, KamimuraH, Momose H, KawashimaT, Ueda tered Clostridium butyricum in rats. Microb Ecol Health K. 1982.Protective effect of Bifidobacterium-monoassocia- Dis 9: 115-122. tion against lethal activity of Escherichia coli. Bifidobac- (59) Tissier H. 1923. La putrefaction intestinale. Bull Inst Pas- teria Microflora 1: 55-59. teur 21: 361, 409, 577, 625. (68) Yu L, Lee KK, Sheth HB, Lane-Bell P, Srivastava G, (60) Tojyo M, Oikawa T, Morikawa Y, Yamashita S, Iwata Y, Hindsgaul O, Paranchych W, Hodges RS, Irvin RT. 1994. Satoh J, Hanada J, Tanaka R. 1987. The effects of Bifido- Fimbria-mediated adherence of Candida albicans to bacterium breve administration on Campylobacter enteri- glycosphingolipid receptors on human buccal epithelial tis. Acta Pediatr Jpn 29: 160-167. cells. Infect Immun 62: 2843-2848. (61) Umesaki Y. 1989. Intestinal glycolipids and their possible