Screening,Purification And Immunological Activity Of Exopolysaccharides From Lactic Acid Bacteria

Lactic acid bacteria exopolysaccharides(LAB EPS)have many biological activitie.In this study,the EPS which can enhance cellular immunity were selected from LAB stored in the laboratory and then the separation,purification and structure identification were carried out.And on this basis we investigated the effects of EPS on the immunologic activity and the OVA-specific immune responses.The results were as follows:1.10 lactic acid bacteria which could obtain higher yield of EPS have been screen out from the 110 lactic acid bacteria stored in the laboratory.The EPS produced by Lactobacillus harbinensis TCP086,Lactobacillus kefiri SXJ29 and Lactobacillus casei SXJ30 could significantly enhance the proliferation,phagocytosis and NO release of macrophage RAW264.7 and Ana-1.2.The crude EPS produced by Lactobacillus harbinensis TCP086,Lactobacillus kefiri SXJ29 and Lactobacillus casei SXJ30 were extracted and purified by DEAE-Sepharose Fast Flow and Sepharose CL-6B,then TCP086 EPS,SXJ29 EPS and SXJ30 EPS were obtained during the purification steps.The three polysaccharides fractions had the absence of protein and nucleic acid.TCP086 EPS,SXJ29 EPS and SXJ30 EPS were characterized by infrared spectrum and showed that they all had characteristic absorption peak of polysaccharides and ?-pyranose ring configurations.The average molecular weight of three EPS was 4.908 × 104 Da,3.423 × 104 Da and 3.737 × 104 Da,respectively.TCP086 EPS was mainly composed of glucosamine,D-galactosamine,mannose,glucuronic acid in an approximate molar ratio of 2.6:1.6:1.2:1.SXJ29 EPS was mainly composed of glucose,D-galactosamine,glucosamine in an approximate molar ratio of 3.1:1::1.SXJ30 EPS was mainly composed of glucose,glucosamine,mannose,in an approximate molar ratio of 1.4:1.1:1.3.To explor the effect of proliferation,phagocytosis,NO release,cytokine secretion and the expression of the cell surface markers CD40,CD80,CD86 and MHC-? on macrophage,the TCP086 EPS,SXJ29 EPS and SXJ30 EPS were used to stimulate RAW264.7 and Ana-1,respectively.We found that the immune activities of macrophage and the concentration of EPS were in a concentration-dependent way,and SXJ30 EPS had the strongest immune enhancement activity in macrophages.4.Mouse bone marrow dendritic cells(BMDCs)were successfully induced by IL-4 and GM-CSF in vitro.We observed the effects of SXJ30 EPS on cellular morphology with inverted microscope and found that these colonies were covered with many sheet-like processes typical of DCs.Flow cytometroy and ELISA were used to analyze the expression level of CD40,CD80,CD86,MHC-? and cytokine.We found that SXJ30 EPS could enhance the expression level of CD40,CD80,CD86,MHC-? and cytokine.5.Mice were subcutaneously immunized with OVA and SXJ30 EPS.Results showed that SXJ30 EPS could enhanced the expression level of CD40,CD80,CD86 and MHC-II in mouse spleen DCs.Compared with Alum,SXJ30 EPS significantly enhance the titers of serum OVA-specific IgQ IgGl,IgG2a and IgG2b.SXJ30 EPS also significantly enhanced the proliferation and cytokines secretion in T lymphocytes.Compared with Alum,SXJ30 EPS significantly enhanced the ratio of IL-4+ CD4+ T cells?IFN-?+ CD4+ T cells and IFN-y+ CD8+ T cells.The results demonstrated that SXJ30 EPS not only enhanced the immune activities of macrophage and BMDCs in vitro,but also enhanced OVA-specific immune response and both Thl and Th2 type immune responses in vivo,and mainly in the Th2 type immune response.This study provides reliable theoretical guidance and experimental data for the screening and application of immunopotentiator.