Isolation,Purification,structural Characterization And Cholesterol-Lowering Effect Of Exopolysaccharide From Levilactobacillus Brevis M-10

In the previous study,a strain of lactic acid bacteria with significant cholesterol-lowering effect was isolated from the naturally fermented broomcorn millet sour porridge in Northwest of Shanxi province of China,and it was identified and named as Levilactobacillus brevis M-10.In this paper,the exopolysaccharide produced by Levilactobacillus brevis M-10 was isolated and purified by DEAE-Sepharose Fast Flow and Sepharose CL-6B chromatography.The cholesterol-lowering activities of main components were determined by the method of binding cholate in vitro,and EPS1(exopolysaccharide 1)with the highest cholesterol-lowering activity was obtained.Furthermore,the structure of EPS1 was characterized.The mice fed by high-cholesterol diet were given EPS1 by gavage,the cholesterol-lowering effect of EPS1 was evaluated by measuring the cholesterol-related biochemical indexes of mice.Real-time quantitative PCR was used to detect the effect of EPS1 on the expression of genes associated with cholesterol metabolism.The beneficial effect of EPS1 on intestinal health was explored by measuring the content of short-chain fatty acids in intestine.The ability of EPS1 to regulate gut microbiota was evaluated using high-throughput sequencing techniques.The results showed that EPS1 was consisted of mannose and glucose with a molar ratio of 9.78︰1.It did not have the triple helix structure and the molecular weight was approximately 1.591×10~6Da.Its main chain was composed of→3)-α-D-Manp-(1→and 2)-α-D-Manp-(1→.The branch chain was composed of4)-α-D-Glcp-(1→that was connected with the O-3 position of→3)-α-D-Manp-(1→,andα-D-Manp-(1→that was linked to the O-4 position of→2,4)-α-D-Manp-(1→on the main chain.In mice fed with high-cholesterol diet,the intervention of EPS1significantly reduced the levels of total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT)in serum,and liver TC compared with the high-cholesterol diet(HC)group.The total bile acid(TBA)levels in feces were significantly increased.The gene expression levels of sterol regulatory element binding protein 2(SREBP-2),3-hydroxy-3-methyl-glutaryl coenzyme A reductase(HMGCR),and Niemann-Pick-C1-Like 1(NPC1L1)in small intestine were down-regulated.Histopathological analysis showed that liver fat drops decreased significantly,and liver cell structure tended to be normal after intervention of EPS1.In addition,EPS1 could significantly increase the contents of short-chain fatty of acid(acetic acid,propionic acid and butyric acid)in intestine,adjust the composition of gut microbiota in mice,increase the relative abundance of intestinal beneficial bacteria(unclassified＿Atopobiaceae,Parasutterella,Blautia),reduce the relative abundance of intestinal harmful bacteria(Lachnospiraceae＿NK4A136＿group).These indicated that EPS1 could improve the destruction of gut microbial structure by high-cholesterol diet to a certain extent.In correlation analysis,Parasutterella was negatively correlated with the level of ALT,and positively correlated with the level of TBA in feces and the content of acetic acid and propionic acid in intestine.Blautia was positively correlated with the level of TBA and the content of acetic acid in intestine.Unclassified Atopobiaceae was positively correlated with the level of TBA in feces and the content of propionic acid in intestine.Lachnospiraceae＿NK4A136＿group was positively correlated with the levels of TC,LDL-C and aspartate aminotransferase(AST)in serum,and negatively correlated with the content of acetic acid and n-butyric acid in intestine.This research indicated that EPS1 could be used as a exopolysaccharide in the development of functional food to prevent hypercholesterolemia.