Preparation, Antioxidant Activity, And Mechanism Of Sulfated Polysaccharides Produced By Enterobacter Cloacae Z0206

In this research, sulfated derivatives of exopolysaccharide (EPS) produced by Enterobacter cloacae Z0206 were prepared by chlorosulfonic acid-pyridine (CSA-Pyr) method and their chemical structures were analyzed based on fermentation optimization and kinetic modeling. The antioxidant activities, of EPS and its sulfated derivatives were evaluated in vitro, by scavenging abilities on superoxide radical, hydroxyl radical and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical. And the protective effects of sulfated derivatives against H2O2-induced RAW264.7 murine macrophages oxidative damage and the possible mechanism were studied using cell culture and proteomics technology. Results are as bellow:1. Technology optimization and kinetic characteristics of EPS production by fermentation of E. cloacae Z0206The medium composition and cultivation conditions of fermentation for EPS produced by E. cloacae Z0206 were optimized according to one-factor-at-a-time method and the orthogonal test. Then response surface methodology based on a three level Box-Behnken factorial design of experiments was employed to optimize the level of three factors significantly affecting the yield of EPS, i.e., concentration of maltose, tryptone and beef extract in the medium. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods. By solving the regression equation and analyzing the response surface contour plots, the optimal medium was determined. The optimal fermentation conditions were:initial pH 7.5, fermentation temperature 32℃, inoculation amount 4%, K2HPO4·3H2O 0.3%, MgSO4·7H2O 0.15%, KN030.05%, maltose 3.72%, tryptone 0.51% and beef extract 0.56%. Under the optimal conditions, EPS production was 12.89 g/L of fermentation liquor, which was also verified by validation experiments. Based on Logistic equation, the kinetic model describing cell growth was established. Meanwhile, the change of monosaccharide composition of EPS during the fermentation was analyzed by Precolumn Derivatization High Performance Liquid Chromatography. Galactose and fucose were determined as the characteristic monosaccharides during the fermentation.2. Sulfated modification and structural analysis of EPSEPS was prepared through fermentation, deproteinization, decolorization and dialysis. The nine sulfated derivatives of EPS, with various degrees of substitution (DS), were prepared by CSA-Pyr method which nine modification conditions were designed according to orthogonal test focusing on three affecting factors such as the ratio of CSAto Pyr, reaction temperature and reaction time. The results indicated that the extent of the impact of variables on DS followed the order:molar ratio of CSAto Pyr> reaction time> reaction temperature. With the increase of the molar ratio of CSAto Pyr and reaction temperature from 50℃to 70℃, DS of product increased very rapidly. However, the DS decreased when the temperature up to 90℃, The increased reaction time from 1 h to 3 h caused the decrease of DS. The chemical structure analysis of EPS and its sulfated derivatives SEPS indicated that sulfation of EPS were successful obtained, the SO3-group was partially sulfated at C-6 of the sugar unit. And the average molecular weight of EPS, SEPS-2 and SEPS-4 was 24.046×104 Da,1.954×104 Da and 18.221×104 Da. The average size of EPS, SEPS-2 and SEPS-4 was 163.50 nm,62.69 nm and 94.47 nm respectively.3. Study on the antioxidant activity of EPS and its sulfated derivativesThe antioxidant activities of EPS and its nine sulfated derivatives were evaluated in vitro, by scavenging abilities on superoxide radical, hydroxyl radical and DPPH radical. The results indicated that sulfated derivatives of EPS showed noticeable effects on scavenging superoxide radical and hydroxyl radical compared with native one. Then the protective effect of sulfated derivatives of EPS against H2O2-induced RAW264.7 murine macrophages oxidative damage and the possible mechanism were studied. The results showed that H2O2 treatment (0.658 mM) reduced the viability of cells by MTT analysis compared with control (p< 0.05). Addition of EPS, SEPS-2 and SEPS-4 (0.5 and 2.0μg/mL) increased the viability of oxidative damage cells significantly (p< 0.05). SEPS-4 (0.5μg/mL) significantly increased (p< 0.05) the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and significantly reduced (p< 0.05) the activation of Caspase-3 compared with H2O2-treated cells only. SEPS-4 (2.0μg/mL) treatment significantly increased (p< 0.05) the mitochondrial membrane potential, and reduced the DNA fragmentation and structure damage of cells induced by H2O2 obviously. Moreover, the antioxidant activity of sulfated derivative SEPS-4 (DS=0.17) was better than EPS and SEPS-2 (DS=0.60). The results indicated that sulfate modification could be considered as the effective approach to enhance the antioxidant activities of EPS, and a moderate DS of the sulfated derivatives was necessary for high biological activities. The sulfated polysaccharides could protect oxidative damage and apoptosis of RAW264.7 cells induced by H2O2 through protection of cell structure, improvements of antioxidant enzymes'activities and mitochondrial membrane potential, inhabitations of Caspase-3 activation and DNA fragmentation.4. Proteomic analysis of antioxidant effect of sulfated polysaccharide SEPS-4 on RAW264.7 cells induced by H2O2 A proteomic approach was used to indentify proteins involved in protective effect of sulfated polysaccharide SEPS-4 against H2O2-induced RAW264.7 murine macrophages oxidative damage. The cells were treated with 0.658 mM H2O2 to induce oxidative damage or pretreated with 2.0μg/mL SEPS-4 before H2O2 treatment. Two-dimensional gel electrophoresis (2-DGE) in combination of MALDI-TOF/TOF MAS was used to identify proteins whose expression level increased or decreased. The results showed that the isoelectric point and molecular weight of proteins distributed broadly, and 132 protein spots were differently expressed. Among the 132 differential protein spots,30 spots were analyzed by MALDI-TOF/TOF MS, and their peptide mass fingerprintings (PMF) were obtained. The PMF maps were searched in Mascot database, and 26 proteins were preliminarily identified. The results showed that SEPS-4 pretreatment downregulated the expression of Peroxiredoxin-2, Eef1D, Eef1G, Phosphatidylethanolamine-binding protein etc., and upregulated the expression of Heat-shock protein hsp84, Rho GDP-dissociation inhibitor 2, T complex protein 1, Alpha-tubulin isotype M-alpha-2, Enol etc.. Those proteins are involved in cell immune and stress, structure and skeleton, protein metabolism and modification, energy metabolism, signal transduction, and they may play important role in protective effect of sulfated polysaccharide SEPS-4 against H2O2-induced RAW264.7 oxidative damage.