Structural Characterization and Immunomodulatory Activity of a Mannose-Rich Polysaccharide Isolated from Bifidobacterium breve H4-2

In this study, a novel homogeneous mannose-rich polysaccharide named EPS-1 from the fermentation broth of Bifidobacterium breve H4-2 was isolated and purified by anion exchange column chromatography and gel column chromatography. The primary structure of EPS-1 was analyzed by high-performance liquid chromatography, Fourier-transform infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance. The results indicated that EPS-1 had typical functional groups of polysaccharides. EPS-1 with an average molecular weight of 3.99 x 10(4) Da was mainly composed of mannose (89.65%) and glucose (5.84%). The backbone of EPS-1 was -> 2,6)-alpha-d-Manp-(1 -> 2)-alpha-d-Manp-(1 -> 2,6)-alpha-d-Manp-(1 -> 2)-alpha-d-Manp-(1 -> 2,6)-alpha-d-Manp-(1 -> 6)-alpha-d-Glcp-(1 -> simultaneously containing two kinds of branched chains (alpha-d-Manp-(1 -> 3)-alpha-d-Manp-(1 -> and alpha-d-Manp-(1 ->). Besides, EPS-1 had a triple-helical conformation and exhibited excellent thermal stability. Moreover, the immunomodulatory activity of EPS-1 was evaluated by RAW 264.7 cells. Results indicated that EPS-1 significantly enhanced the viability of RAW 264.7 cells. EPS-1 could also be recognized by toll-like receptor 4, thereby activating the nuclear factors-kappa B (NF-kappa B) signaling pathway, promoting phosphorylation of related nuclear transcription factors, improving cell phagocytic activity, and promoting the secretion of NO, IL-6, IL-1 beta, and TNF-alpha. Thus, EPS-1 could activate the TLR4-NF-kappa B signaling pathway to emerge immunomodulatory activity on macrophages. The above results indicate that EPS-1 can serve as a potential immune-stimulating polysaccharide.