The Study On Structure Of Cell-bound Polysaccharide From Lactobacillus Fermentum S1 And Life Span And Tolerance Of Caenorhabditis Elegans

In recent years,exopolysaccharides(EPSs)metabolized by lactic acid bacteria have been widely used in food industry and pharmaceutical industry because of its unique physicochemical properties,high safety and strong physiological activity.The lactic acid bacteria EPSs generally exist in two forms,one is release exopolysaccharides(r-EPS)which released outside of cell,and the other one is cell-bound exopolysaccharides(c-EPS)which closed integration to cell wall.At present,among the studies on lactic acid bacteria EPSs,the r-EPS had been studied widely,while the research of c-EPS not only progressed slowly,but also had the problems such as various extraction method,low extraction efficiency,less precise construction parse and less research on the mechanism about the corresponding activity enhancement.In this study,three ways of ultrasonic,phenol and EDTA were used to extract c-EPS from L.fermentum S1 which separated independently from Fuyuan kimchi,the crude c-EPSs were purified,and the structure and antioxidant activity of purified c-EPSs were studied.The model organism Caenorhabditis elegans was used to test the vivo antioxidant activity of c-EPS,and combined the antioxidant activities in vitro to comprehensively evaluate the antioxidant capacity of c-EPS from three extraction methods.At the same time,the effects of the life span and tolerance ability of C.elegans of c-EPS with better antioxidant capacity were studied,and the effects mechanism of improving life span was analyzed at the gene level.The results of research are lists as follows:1.The yields of c-EPS extracted from L.fermentum S1 by ultrasonic,phenol and EDTA were 57.71 mg/L,25.20 mg/L and 33.74 mg/L,respectively.The c-EPS samples purified by DEAE-52 ion exchange column and G-100 gel chromatography column were named CFU,CFP and CFE,respectively.The MW of CFU,CFP and CFE were 7.19×105 Da,2.89×105 Da and 2.44×105 Da,respectively.The composition analysis showed that the neutral sugar content of CFU,CFP and CFE was 93.80%,89.36% and 84.04%,the content of protein was 0.32%,0.73% and 1.52%,and the content of sulfate group was 1.05%,1.32% and 2.46%,respectively,and the uronic acid did not be detected in all samples.2.Monosaccharide analysis demonstrated that CFU,CFP and CFE were composed by mannose,rhamnose,glucose and galactose in a molar ratios of 1.00:3.81:7.96:5.23,1.00:3.70:5.54:6.40 and 1.00:1.90:3.55:2.40,respectively.FTIR spectrum showed that all typical polysaccharides characteristic absorption peaks of all samples were detected.Methylation reaction and GC-MS and NMR analysis demonstrated that the main chain of three polysaccharide samples were composed by ?-D-Galp-(1?3),?-L-Rhap-(1?2),?-D-Glcp-(1?3),?-D-Galp-(1?3),?-D-Glcp-(1?2)and ?-L-Rhap-(1?3,4),and a tail by?-D-Manp-(1?.Congo red experiment showed that both CFP and CFE had three helical structure,while CFU did not have three helical structure.FE-SEM analysis indicated that the surface of CFU had porous structure,CFP had reticular structure and CFE had a cupped block structure.Thermodynamic analysis indicated that the thermal degradation temperatures of CFU,CFP and CFE were 288.0?,253.6?and 310.6?,and melting 1 g of CFU?CFP and CFE required about 273.1 J,301.9 J,and 315.3 J quantity of heat,respectively.3.In vitro antioxidant activity analysis showed that CFU,CFP and CFE had strong scavenging activities of DPPH,hydroxyl,ABTS+ free radical and medium reduction potential of FRAP ferrous ion.In vivo antioxidant experiments showed that CFU,CFP and CFE all had strong total antioxidant capabilities and total superoxide dismutase activities.4.C.elegans life and tolerance experiment analysis showed that CFU at 50 ?g/m L could effectively extend the life and tolerance ability of C.elegans,at the same time,CFU at this concentration could also improve the spawning ability of C.elegans.Compared with C.elegans control group,CFU at 50 ?g/m L could reduce the expression of Daf-2 gene in C.elegans,improve the expression of Daf-16 gene to improve the life span of C.elegans,CFU also significantly increased the expression of Sir-2.1,Hsf-1 and Hsp-16.2 genes to improve the life span of C.elegans.The above results showed that there were some differences in structure and physiological of c-EPS extracted from Lactobacillus fermentum S1 by three methods,and the c-EPS had good antioxidant and anti-aging activities,so it could be used as an ideal natural antioxidant and anti-aging drug in functional food and medicine field.