Preparation,Structural Characterization And Bioactivity Of Extracellular Polysaccharides Of Rufibacter Sediminis H-1

Microbial exopolysaccharides have various biological activities including antioxidant properties.Compared with traditional antioxidants butylated hydroxytoluene（BHT）and dilauryl thiodipropionate（DLTP）,microbial exopolysaccharides have the advantages of high yield,short production cycle and easy extraction.In this study,a novel bacterial strain Rufibacter sediminis H-1 was used to extrait the extracellular polysaccharide to analyze its primary structure,emulsification,moisture absorption,moisturizing properties,and further explore its protective effect on oxidative stress of cells.In addition,the yield of polysaccharides was improved by optimizing fermentation conditions.The main results were as follows:1.The extracellular polysaccharide（RSP）of strain H-1 was extracted by water extraction and alcohol precipitation;and further separated and purified by column chromatography.The monosaccharide composition,thermal stability,spatial structure,apparent morphology,molecular weight and functional group composition of RSP were analyzed.The results indicated that the RSP produced by strain H-1 was acidic polysaccharide,containing a variety of uronic acids and 0.46%protein.It was composed of two polysaccharides with different molecular weights of 2662 Da and 27945 Da,and the molar ratio was glucose:mannose:galactose:glucuronic acid:guluronic acid:galacturonic acid:glucosamine hydrochloride:N-acetyl D-glucosamine as 33.5:25.8:13:7.6:6.6:5.9:3.1:2.6.The RSP containsα-configuration pyranose andβ-configuration pyranose units.The surface of the RSP is loose and porous,which has a triple helix structure and good thermal stability.2.The moisture absorption and moisture retention rate of RSP were calculated by a constant temperature and humidity dryer.The emulsification and the in vitro antioxidant activity of RSP were detected by hydrophobic substrate and different free radicals,respectively.The results suggested that RSP had good properties of moisture,water absorption and emulsification.The RSP possessed dose-dependent activities on scavenging hydroxyl radical,1,1-diphenyl-2-trinitrophenylhydrazine（DPPH）radical,2,2’-azino-bis（3-ethylbenzothiaz-oline-6-sulfonic acid）（ABTS）radical and ferric iron.3.MTT method was used to analyze whether RSP was toxic to IPEC-1 cells,and an IPEC-1 cell oxidative stress injury model was constructed to detect intracellular lactate dehydrogenase（LDH）,superoxide dismutase（SOD）;and the activities of aldehyde（MDA）and catalase（CAT）,and the changes of Keap1,Nrf2 and HO-1 m RNA expression levels in cells were detected by RT-PCR.The results demonstrated that RSP had no toxic effects on cells.The level of LDH and MDA in H₂O₂-treated IPEC-1 cells dose-dependently decreased（P<0.05）from 539.27 U/L to 462.21 U/L protein and from 1.54 nmo L/mg to 1.03 nmo L/mg protein,respectively.The RSP greatly（P<0.05）increased the levels of SOD and CAT in H₂O₂-treated IPEC-1 cells from 4.57 to 6.18 U/mg protein and 6.56 to 9.58 U/mg protein in a dose-dependent manner,respectively.RT-PCR results showed that the m RNA expression of the Nrf2 inhibitory protein Keap1 in the H₂O₂ injury group was significantly increased（P<0.05）when compared with the control,while the m RNA expressions of Nrf2 and HO-1 were significantly decreased（P<0.05）.The enhancement of cellular antioxidant capacity by RSP may be through up-regulating Nrf2 signaling pathway,inhibiting or slowing down the oxidative damage caused by H₂O₂ to IPEC-1 cells.4.Response surface methodology was used to optimize the polysaccharide yield of H-1.The results illustrated that the optimal conditions for the production of exopolysaccharide by strain Rufibacter sediminis H-1 were trehalose 2.6 g/L,yeast extract 1.50 g/L,K₂HPO₄·3H₂O0.30 g/L,Mg SO₄·7H₂O 0.05 g/L,acetone sodium 0.30 g/L,p H 7.0,fermentation temperature27.5℃,and inoculum volume 8.5%.Under these conditions,the exopolysaccharide yield of H-1 was 140.49 mg/L after 54 h fermentation,which was 4.77 times higher than that before optimization.This study provides theoretical reference for the development of polysaccharide application of Rufibacter Sediminis H-1 bacterial species,and is conducive to expanding the understanding of microbial extracellular polysaccharide resources and structure.