Structural Characterization And Immunomodulatory Effects Of The Extracellular Polysaccharide Of Bacillus Amyloliquefaciens JM033

Extracellular polysaccharides(EPS)are biopolymers with structural diversity produced by microorganisms.Because of its structural diversity,EPS has various biological activities,such as antioxidant,antiviral,anticancer and immunomodulatory.Bacillus.amyloliquefaciens,a probiotic bacterium belonging to the genus Bacillus,has strong enzymatic and carbohydrate synthesis capabilities,and the EPS produced by it has various biological activities such as antiviral,antitumor,immunomodulatory,and antiox idant.More and more probiotic bacteria in the genus Bacillus are being discovered,but there are fewer studies related to these probiotics,so the study of extracellular polysaccharide production by probiotic bacteria in the genus Bacillus is of great importance.In this study,whole genome sequencing was performed to explore the EPS synthesis potential of B.amyloliquefaciens JM033,which has high EPS production capacity,at the genetic level.The fermentation substrate carbon source of B.amyloliquefacie ns JM033 was screened based on the functional gene annotation results combined with a single-factor test,and the fermentation conditions were optimized by Box-Behnken design for response surface to improve EPS yield.The EPS produced by B.amyloliquefaciens JM033 was extracted,isolated and purified,and the neutral polysaccharide fraction(BAP-1)was selected for structural analysis.Constructive analysis was performed based on the results of structural analysis,and the safety and immunomodulatory abilit y of BAP-1 were investigated by in vitro cellular assays.An immunosuppressive mouse model was established by Cyclophosphamide(CTX),and the effects of BAP-1 on gut microbiota and host immunity were investigated in vivo in terms of cellular immunity,humo ral immunity,monocyte-macrophage phagocytosis,NK cell activity and gut microbiota regulation.The following results were obtained from these studies:(1)The whole genome of B.amyloliquefaciens JM033 showed a complete EPS synthesis gene cluster and a strong EPS synthesis potential.Functional gene annotation yielded 220 and 363 genes related to carbohydrate metabolism in the eggNOG and KEGG databases,respectively;108 genes for enzymes related to EPS synthesis were annotated in the CAZy database.A complete EPS synthesis gene cluster from glycosyl transfer to polysaccharide polymerization and translocation was identified in the whole genome of B.amyloliquefaciens JM033.(2)B.amyloliquefaciens JM033 had a high EPS yield.The alternative fermentation carbon sources were screened based on the functional gene annotation results combined with single-factor tests,and the fermentation conditions were optimized by Box-Behnken design with response surface.A 6.12-fold increase in EPS yield from 0.75 mg/mL to 5.34 mg/mL was obtained with sucrose as the carbon source,a fermentation temperature of 37 ℃,an initial pH value of 7.0 and a fermentation time of 50 h.(3)BAP-1 is a novel hybrid fructan.two fractions(BAP-1 and BAP-2)were isolated by anionic column purification of B.amyloliquefaciens JM033 produced EPS,and the neutral polysaccharide fraction(BAP-1 with a molecular weight of 17.6 k Da)was selected for structural analysis.The results showed that BAP-1 consisted of two monosaccharides,with99.15% fructose and 0.85% glucose.Combined with methylation and NMR spectroscopy analysis,it was concluded that BAP-1 is a hybrid fructan with 13(2 → 1)-linked β-Dribofuranose in the main chain of the repeating unit and(2 →6)-linked β-D-ribofuranose in the branched chain(4)BAP-1 has immune-enhancing effects in vitro and in vivo.The in vitro cellular assay investigated that BAP-1 was not cytotoxic and promoted the secretion of NO,interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in RAW264.7 cells with neutral red phagocytosis.In vivo,the immune-enhancing effects of BAP-1 were investigated in an immunosuppressed mouse model using CTX,and it was found that BAP-1 intake significantly improved host immunity in terms of humoral immunity(increased antibody secretion),monocyte-macrophage phagocytosis and NK cell killing capacity.(5)BAP-1 intake alleviated gut microbiota disorders in immunosuppressed mice and shifted the flora composition to the normal group.BAP-1 intake significantly increased the flora abundance of beneficial bacteria such as Verrucomicrobia and decreased the flora abundance of potentially pathogenic ba cteria such as Actinobacteria and Proteobacteria at the phylum level.BAP-1 intake at the genus level significantly increased the abundance of Akkermansia,Lachnospiraceae and Oscillospiracea,the next generation of probiotic bacteria that produce short-chain fatty acids,in the gut microbiota of immunosuppressed mice.BAP-1has the potential to influence host immunity by regulating gut microbiota.