The Isolation,purification And Immunomodulatory Activity Of Polysaccharide From Bacillus Amyloliquefaciens

Since there are few studies on the structure and function of polysaccharide from bacillus amyloliquefaciens(BAP)and polysaccharides,this study used bacillus amyloliquefaciens as a sugar-producing strain to prepare polysaccharides by fermentation,extraction,separation and purification to study its physicochemical properties,chemical structure and immune activity.Firstly,bacillus amyloliquefaciens was used as a fermentation strain,and the purified BAP was obtained through culture,fermentation,centrifugation,alcohol precipitation,dialysis,chromatography,etc..The components of BAP were determined by high performance liquid chromatography.The results showed that BAP was composed of Two components.Determined by the polysaccharide standards,the molecular weight of the two components was determined to be approximately 6.65 kDa and 683.68 kDa,respectively.The structure of BAP was identified by MALDI-TOF,UV,IR,and gas phase.The molecular weight of BAP were 5.31 kDa and 687.77 kDa.The content of BAP was 99.2%,and the protein content was 0.6%.The BAP was neutral polysaccharides withα-glucosidic bonds and pyranose ring structures consisted of rhamnose,arabinose,fucose,mannose,glucose,and sorbose(the measured molar ratio of each monosaccharide is 0.5:1.2:1.7:2.1:3.7:2.3)consisting of no uronic acid,no nucleic acids and proteins.Secondly,the antioxidant capacity of ABTS,DPPH free radical scavenging capacity and total reducing capacity(FRAP)was determined.The result showed that the Trolox and FeSO4·7H2O equivalent values of the samples without BAP were at a low level,and the Trolox equivalent values of the samples added with BAP showed an increasing trend with the increase of BAP concentrations.When the concentration of BAP samples reached 160μg,the equivalent values of ABTS,DPPH free radical scavenging capacity are as high as 753.5,110.5 μM and total reducing capacity is 1301.5 μM,respectively.It can be seen that there is a certain concentration dependence between the Trolox and FeSO4·7H2O equivalent values and the BAP sample concentration within the determined BAP concentration range.Then,BAP was used as a test sample with 6 corresponding concentration gradients in the MTT and Neutral Red experiments and co-cultivated with RAW264.7 cells.It revealed that BAP significantly enhanced the cell viability and phagocytic activity compared to the control group,and showed the maximum of viability and phagocytic activity were 138±1.4%and 0.27 ± 0.004%in the dose of 80 μL/mL BAP group,respectively.Finally,by measuring the effect of BAP on the secretion of immune factors in RAW264.7 cells,it was demonstrated that BAP can induce and activate RAW264.7 cells to exert autoimmune regulation.The results showed that in 80 μM BAP experimental group,the cell genes relative expression levels of tumor necrosis factor-a(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-12(IL-12),interferon-gamma(IFN-y)peaked at 5500,3500,3600,2500,and 7000 pg/mL,respectively,and the cell genes relative expression levels of inducible nitric oxide synthase(iNOS)cell genes was no clear correlation with the BAP concentration.In summary,BAP can significantly increase the expression level of immune-related genes and enhance the immune function of phagocytic cells.