Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect via Bacillus subtilis natto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types of B. subtilis natto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant from B. subtilis strain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted by B. subtilis natto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC.

The effects of a microbial inoculant (Thervelics ® : a mixture of cells of Bacillus subtilis C-3102 and carrier materials) on rice (Oryza sativa cv. Milkyprincess) and barley (Hordeum vulgare cv. Sachiho Golden) were evaluated in four pot experiments. In the first and second experiments, the dry matter production of rice and barley increased significantly by 10-20% with the inoculation of the mixture at a rate of 10 7 cfu ⋅ g -1 soil compared with the non-inoculated control. In the third experiment, the growth promoting effects of the mixture, the autoclaved mixture and the carrier materials were compared. The dry mater production of rice grains was the highest in the mixture, and it was significantly higher in the three treatments than in the control, suggesting that the carrier materials may also have a plant growth promoting effect and the living cells might have an additional stimulatory effect. To confirm the efficacy of the living cells in the mixture, only B. subtilis C-3102 cells were used in the fourth experiment. In addition, to estimate the mechanisms in growth promotion by B. subtilis C-3102, three B. subtilis strains with similar or different properties in the production of indole-3-acetic acid (IAA), protease and siderophore and phosphatesolubilizing ability were used as reference strains. Only B. subtilis C-3102 significantly increased the dry matter production of rice grains and the soil protease activity was consistently higher in the soil inoculated with B. subtilis C-3102 throughout the growing period. These results indicate that the microbial inoculant including live B. subtilis C-3102 may have growth promoting effects on rice and barley.

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