Food allergy is thought to be caused mainly by the reaction of IgE antibody to food antigens. Immunoglobulin class switch to IgE is induced by the transcription of germ line transcript from I region and splicing of S region present in upstream of the gene of IgE constant region. These reactions are triggered by the stimuli to IL-4 receptors and CD 40 molecules on B cells and suppressed by the action of interferon-γ. Therefore, balance between Th 2 cells producing IL-4 and Th 1 cells producing interferon-γ is important for IgE production and the factors providing Th 2 dominancy are related to the occurrence of allergy. The mucosal immune system is somehow independent from general immune system and there present many T cells producing TGF-β or IL-10 in the gut associated lymphoid tissues. TGF-β induces class switch to IgA and therefore provides the condition to produce secretory IgA antibodies. In contrast, IL-10 suppresses Th 1 cells and therefore provides the condition to produce IgE. Unmethylated CpG motif of bacterial DNA causes IL-12 production from macrophages and IL-12 induce Th 1 cells. Thus activated Th 1 cell would suppress IgE production. The development of food allergy seems to be controlled by the two opposing conditions namely the presence of antigens or other substances which induce Th 2 reaction and by the presence of the substances such as bacterial DNA which induce Th 1 reaction.

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Recently, a great deal of attention has focused on mucosal immunization. Oral or mucosal immunization wiil be important as a means of inducing secretory IgA antibodies directed against specific pathogens of mucosal surfaces. Despite great interest in mucosal immunization, the success of mucosal immunizations has been very limited. Recently, component vaccine strategies have become popular for the development of new vaccines, and there have been reports of several excellent antigens delivery systems suitable for mucosal administration. One such system is a live attenuated cell invasible bacterium that expresses protective antigens of unrelated microorganisms, and the second is chemically synthesized microparticles, which are conjugated with antigenic epitopes. Live attenuated vectors have to be converted into nonpathogenic systems that are still capable of inducing protective immunity. However, it is difficult to evaluate the safety of these vectors. From this point of view, the chemically synthesized microparticles are more useful. This system requires the purification of antigens prior to particle formation, which may be time consuming and expensive. Our research has been directed toward developing carrier systems using nonpathogenic bacteria. Lactic acid bacteria have a long history of use in food fermentation, and therefore are classified: “generally regarded as safe.” They are able to survive or grow in the gastrointestinal tract of humans and animals. Lactococcus lactis, for which various genetic modification systems are available, is thought to be potentially useful for the production of heterologous peptides and proteins in foods and medicines, but for use in vaccination, this bacterium needs some modifications, because it does not invade nor colonise the mucosal surfaces, nor does it evoke strong immune responses to itself. In this report, I will summarize the present studies about developing mucosal vaccines against enteric pathogens, and discuss the potential for development of the recombinant vaccines with lactic acid bacteria as antigens delivery vehicles for mucosal immunization.

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Bifidobacteria are Gram-positive, anaerobic, GC-rich bacteria constituting a major part of the normal microflora in the large intestine of human and animals. These intestinal organisms have been believed to have health-promoting properties for their host, involving enhancement of immune response, inhibitory effect for carcinogenesis, protection of the host against virus infection and possible production of antibacterial substances. Some Bifidobacterium species are widely used for the preparation of fermented milk products in the world. Here, we report reproducible and stable transformation of B. longum by plasmid vectors, and transcription mode of a gene encoding histone-like protein of B. longum. Highefficiency transformation of B. longum by electroporation was achieved with novel shuttle vectors pBLES 100 and pMASK 23, which were constructed by cloning B. longum plasmid and a gene encoding spectinomycin AAD (9) from Enterococcus faecalis into Escherichia coli vector pBR 322. Transformation efficiency of 2×10⁴/μg DNA could be obtained under optimal condition of 10.0kV/cm, 200 Ω and 25 mF, using cells harvested at late log phase of growth. Bifidobacterium longum 105-A transformed with these shuttle vectors showed normal cell growth rate, and the plasmids were maintained stably in the transformants without rearrangement of the molecules. Histone-like protein HU is a structural component of bacterial nucleoids, which has been well conserved during evolution of prokaryotes, and is expressed highly and constitutively. We cloned hup gene (gene encoding HU protein) from B. longum ATCC 15707, and found that the ORF is identical to HU family protein HB1, a DNA binding protein of B. longum reported previously. We identified promoter, transcription start point, ribosome binding region and transcription termination site in this gene. The hup gene is expressed constitutively through cell cycle in ATCC 15707, and is transcribed efficiently when it is integrated in pBLES 100 and transferred into B. longum 105-A.

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To establish safety is most important when genetically-modified microorganisms like lactic acid bacteria are planned to apply for food production. For this reason, it would be necessary to use exclusively safe genetic materials which are suitable for food production, and also to guarantee the genetic stability and the safety of the genetically-improved microorganisms. The authors have tried to establish the fundamental techniques to construct safe recombinant strains of yogurt starters: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. So far two techniques have been established including a double cross-over, which ensures exclusive introduction of the objective gene in the host chromosome, and a host-vector system composed of only safe genetic materials. These results would be useful to establish safety of the future recombinant lactic acid bacteria to be used for food production.

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Effect of yogurt (LKM 512 yogurt) prepared Bifidobacterium lactis LKM 512, Lactobacillus delbrueckii subsp. bulgaricus LKM 1759 and, Streptococcus thermophilus LKM 1742 on fecal microflora and properties in 30 volunteers (5 males and 25 females, average age 30.1 yr, defecation frequency<4times/week) given this yogurt 100g/day for 2 weeks were analyzed. During the consumption of LKM 512 yogurt, defecation frequency was significantly increased (p<0.05) and fecal water content tended to increase. LKM 512 yogurt contained 4.7×10⁸cfu/g of L. delburueckii subsp. bulgaricus and S. thermophilus and 5.2×10⁷ cfu/ g of B. lactis. At week 2 of the consumption, the number of bifidobacteria in the fecal bacteria increased significantly (p<0.05) and the proportion in the total fecal bacteria also increased significantly (p<0.05) from 6.6% to 17.0%. In contrast, the difference between before the yogurt consumption and during the placebo yogurt (contained 5.0×10⁸cfu/g of L. delburueckii subsp. bulgaricus and S. thermophilus) consumption was not significant, although the similar tendencies to LKM 512 yogurt were recognized in both the fecal microflora and defecation during the placebo yogurt consumption. The results in this study demonstrated that the consumption of LKM 512 yogurt was effective to increase the number of fecal bifidobacteria and to improve the fecal properties.

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A many kind of antimicrobial agents were used for therapy. But the same time, the intestinal flora was disturbed by these therapies using antimicrobial agents. The MICs of nine antimicrobial agents were determined about Collinsella species and related bacteria, to determine what kind of antimicrobial agent effect to flora. All Collinsella strains were susceptible to some antimicrobial agents tested. A few agents of cephem and monobactum, however, have some effects to these strains. For the therapy, these agents are useful better than other agents are. Furthermore, it is clear that the susceptibility patterns are can be differential characters of three Collinsella species.

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