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Background Products from the fermentation process of soybeans by Bacillus subtilis ( natto) have been shown to possess anti-tumour and immunomodulatory activities. However, the formulations previously examined were not chemically pure, and this is a major limitation for elucidation of the molecular mechanisms for their activities. Objective In order to determine which components in soybean mucilage exert immunostimulatory activities, we examined the activities of their purified forms in vitro and in vivo in mice. Methods B. subtilis ( natto) and fractions including levan and poly-γ-glutamic acid (γ-PGA) from fermented soybean mucilage were prepared. Levels of cytokine production by mouse macrophage cells after treatment with the fractions were measured by means of ELISA. In vivo effect of levan delivered intragastrically on ovalbumin (OVA)-specific T-helper type 2 (Th2) response with IgE production was examined in BALB/c mice that had been immunized intraperitoneally with OVA. Results Levan but neither γ-PGA nor killed B. subtilis ( natto) was found to exert strong activity to induce production of IL-12 p40 and TNF-α by macrophage cell lines in vitro. Results of experiments using Toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of levan. Oral administration of levan in vivo significantly reduced the serum levels of OVA-specific IgE and Th2 response to OVA in mice immunized with OVA. Conclusion Levan is an immunostimulatory moiety in products from the fermentation process of B. subtilis ( natto) and may be useful for prevention of allergic disorders with IgE production. [ABSTRACT FROM AUTHOR]