Extraction,Isolation,Structural Characterization And Immune Activity Of Cell Wall Peptide Polysaccharide From Bacillus Natto

Bacillus natto is a probiotic isolated from Japanese natto and belongs to a subspecies of Bacillus subtilis.The live bacterial preparation and the produced metabolite have important physiological effects and have been widely concerned.Peptidoglycan is a special component of prokaryotic cell walls such as bacteria.Eukaryotic cells do not contain peptidoglycan,so peptidoglycan is an ideal target for eukaryotic immune system recognition.Peptidoglycan recognition molecules can induce the release of immunomodulatory substances,thereby playing an immunomodulatory role.In order to explore the immunomodulatory function of Bacillus natto cell wall peptidoglycan,this paper studied the extraction and isolation conditions of Bacillus natto cell wall peptidoglycan,analyzed the physicochemical properties and structural characteristics of mice,and studied its effect on immune function of mice.,Below are key research findings:1.Optimization of extraction conditions of Bacillus natto cell wallThe laboratory-preserved Bacillus natto was used as the research material.After the value-added culture,the optimal process parameters for ultrasonic-assisted extraction were determined by Box-Behnken test design and response surface analysis: ultrasonic power was 314 W,processing time was 27 min,OD600 was 0.66,the maximum extraction rate of cell wall under this condition was 44.51%.Preparation and chemical structure analysis of cell wall of Bacillus natto2.Preparation and chemical structure analysis of cell wall of Bacillus nattoThe obtained cell wall of Bacillus natto was used as a raw material,and the teic acid,lipid substance and protein in the cell wall were removed by the trichloroacetic acid method,and the peptidoglycan with higher purity was obtained after dialysis.The peptidoglycan constituents were analyzed by lysozyme assay,Bradford method,aldehyde method,phenol-sulfuric acid method.The structure of Bacillus natto peptidoglycan was studied by infrared spectroscopy and SDS-PAGE.The analysis results showed that the total amount of purified sugar was 39.77%,protein accounted for 54.47%,and total lipid content was 4.5%.After lysozyme treatment,the soluble peptidoglycan has a molecular weight of about 60 ku.By HPLC profiling,it was determined that the peak time of the lysate was consistent with the peak time of the standard peptidoglycan lysate.Its sugar chain structure is formed by linking N-acetylglucosamine and N-acetylmuramic acid through ?-1,4-glycosidic bonds.3.Effect of Bacillus natto peptidoglycan on immune function in mice96 Kunming mice with healthy body weight were selected and weighed 18-22 g.They were randomly divided into blank control group and low,medium and high dose groups ofpeptidoglycan solution,24 in each group.The intragastric cycle was 7d every 24 h,and the changes of immune index and cytokine secretion in different dose groups were detected.According to the results of SPSS analysis,Bacillus natto peptidoglycan can significantly increase the immune organ index of mice;peptidoglycan can significantly increase serum hemolysin content,mouse carbon clearance index and delayed type hypersensitivity reaction,and small The proliferation of murine spleen lymphocytes and the phagocytic ability of peritoneal macrophages were enhanced.Peptidoglycans increased the metabolic activity of macrophages and significantly increased IL-2,IL-10,IFN-r and TNF.-The amount of alpha secretion increases the expression levels of NO and iNOS.