Isolation,Purification And Antioxidant Activity Of Exopolysaccharides From A Bacterial Strain

Objective:In recent years,researchers have discovered a variety of biological activities of microbial polysaccharides.Its antioxidant activity has been used in food,cosmetics and other fields.In the field of medicine,antioxidants are mainly derived from polysaccharides extracted from animals and plants.Due to the long production cycle and low yield,they can no longer meet the demand.Compared with plant polysaccharides and animal polysaccharides,Microbial polysaccharides have the advantages of higher yield,easy extraction,short production cycle and ability to obtain a large amount of products in a short time.Therefore,as a source of natural antioxidants,it has great development and application potential.In this study,a strain of BY bacteria preserved in the Applied Microbiology Laboratory of Northwestern University was used as the research object.The extracellular polysaccharide of the bacteria was extracted and its antioxidant activity was studied at the chemical and cellular levels.It hopes to provide a reference as a potential antioxidant drug for adjuvant treatment of diseases.Method:The strain was identified by 16Sr RNA sequence analysis.The extracellular polysaccharides(EPS)of this strain were extracted by water extraction and alcohol precipitation method.We conduct qualitative and quantitative analysis of extracellular polysaccharide EPS and determine free radical scavenging ability.The EPS was further separated and purified by column chromatography,and the monosaccharide composition,molecular weight and functional group composition of EPS2 were analyzed.We used ELISA method to detect the effect of EPS2 on the content of MDA,LDH,and SOD in cardiomyocytes H9C2 and human umbilical vein endothelial cells HUVEC.Using H₂O₂as the oxidative stress induction model and H9C2 cardiomyocytes as the oxidative stress injury model,the effects of EPS2 on H9C2apoptosis,ROS levels and mitochondrial membrane potential were detected by fluorescent staining,and the expression of apoptosis-related proteins was detected by WB.Result:1.The BY strain is G^-bacterium,Paenibacillus sp.The Molish reaction,the iodine-starch reaction and the micro-ultraviolet spectroscopy analysis results of EPS show that it is a polysaccharide substance with a total sugar content of 63.4%,a protein content of 1.5%,a uronic acid content of 1.9%and no sulfate ions.When the concentration is 0-10 mg/ml,EPS has scavenging ability to ABTS⁺free radicals,superoxide anion(O^2-)free radicals,can chelate ferrous ions and has reducing ability.And the scavenging ability of free radicals is concentration-dependent.2.DEAE cellulose column chromatography shows two peaks,one is the absorption peak of neutral sugar(EPS1),and the other is the absorption peak of acid sugar(EPS2).The weight average molecular weight of EPS2 is 398.4 KDa and the monosaccharide composition is Fuc(0.466%),Rha(0.110%),Ara1(1.019%),Gal(21.755%),Glc(33.815%),Xyl(0.983%),Man(25.557%),Gal UA(12.925),Glc UA(3.368).The results of Fourier infrared spectroscopy analysis of EPS2 showed two characteristic peaks of polysaccharides.3.Compared with H₂O₂ can cause the increase of MDA,LDH content and the decrease of SOD content in H9C2 cardiomyocytes and human umbilical vein endothelial cells HUVEC,EPS2 can reduce the content of MDA,LDH and increase the level of SOD.4.Compared with the H₂O₂ induction group,the nucleus fluorescence intensity of the EPS2polysaccharide group was weakened,ROS levels decreased,mitochondrial membrane potential increased,Bax protein and cleaved caspase-3 protein expression decreased and Bcl-2 protein expression increased.Conclusion:These results indicated that the extracellular polysaccharides produced BY strain had antioxidant activity.The acidic sugar component EPS2 can protect H9C2 cardiomyocytes from oxidative stress injury by inhibiting the expression of related apoptotic proteins.This research provides an idea for microbial extracellular polysaccharide as a natural antioxidant in the adjunctive treatment of diseases.