Preparation, Structural Analysis And Biological Function Research Of Selenium-enriched Exopolysaccharide Produced By Bacterium Enterobacter Cloacae Z0206

The Selenium-tolerant strain Z0206 Rosenth producing a large quantity of viscous exopolysaccharide isolated from Daphniphyllum oldhami (Hemsl.) belong to E.cloacae by phylogenetic analysis. We prepared crude Se-enriched exopolysaccharide through fermentation with E.cloacae Z0206 in enriched-selenium fermentation culture. Se-enriched exopolysaccharide was isolated and purified from crude Se-enriched exopolysaccharide; we further analyzed the structure of Se-enriched exopolysaccharide; On base of those, we investigated its immunomodulatory function on immunosuppressed mice and the protective effect against H2O2-induced Raw 264.7 murine macrophages oxidative damage and the possible mechanism of action. Results are as follows:1. Screening and Classification for Se-tolerant Strain Producing ExopolysaccharidesThe strain Z0206 producing exopolysaccharides isolated from Daphniphyllum oldhami (Hemsl.) Rosenth was identified as Selenium-tolerant strain through screening and domesticated by increasing the Se-concentration. It belongs to E.cloacae by identification with traditional physiological and biochemical methods, together with the 16S rDNA sequence homology and phylogenetic analysis. We obtained the accession number (EU647232) of E.cloacae Z0206 by submitting its 16S rDNA sequence to GenBank.2. Preparation of Enriched-Selenium ExpolysaccharidesThe experiment was aimed to prepared enriched-selenium exopolysaccharides. We first optimized culture medium and culture conditions for selenium expolysaccharide production. And the optimal culture medium was obtained, which contains tomato extract 200mL/L, glucose 20g/L, yeast extract 2g/L. Meanwhile the optimized culture conditions are:culture volume 100ml/500mL flash, inoculation amount 2%, initial pH 7.0, shake culture 48h,200rpm,30℃. Se-added time 6th h, Se-concentration 20μg/ml. The crude enriched-Selenium exopolysaccharides was prepared through fermentation with E.cloacae Z0206 in enriched-selenium fermentation culture medium. Yield of crude exopolysaccharide was 26.7 g/L, Se-enriched amount was 65.7 mg/kg. The enriched-selenium exopolysaccharide was isolated and purified by Sevag and enzymes method, frozen thaw method and with activated carbon, yield of purified exopolysaccharide was 17.5 g/L, Se-enriched amount was 35.7 mg/kg.3. Structural Analysis of Enriched-Selenium ExpolysaccharideThe obtained purified exopolysaccharide was loaded on a column of anion-exchange (DEAE-52) and gel-permeation chromatography (Sephadex G-100), concentrated, dialyzed and lyophilized. Finally obtained compound was named SPS-1. The structural characteristics was analysed by HMR, FTIR and HPLC methods. The results showed that SPS-1 was homogeneity and made up of Glucose, galactose and mannose with the molarity rate of 8.530:0.061:0.706. Its molecular weight is about 2.39kD. SPS-1 is a neutral heteropolysaccharide, and SPS-1 containedα,β-type glycosidic linkages, and may contain a-D glucopyranose, mammopyranoside. The molecule of SPS-1 had long dypass chain and more branch chains. The results of X-ray and DSC indicated that the state structure of the exopolysaccharides was noncrystal Elements. The particle size and zeta potential were 47.49nm and 11.9mV respectively. The dehydration temperature and gasification temperature were 85.98℃and 256.57℃respectively.4. Study on the Immune Function of Enriched-Selenium ExpolysaccharidesThe experiment was to investigate the immunomodulatory activity of selenium exopolysaccharide produced by E.cloacae Z0206 in enriched-selenium fermentation culture by establish of immunosuppressed mice model. Results indicated that CP showed suppressive effects on immune organs weight and cellularity and other parameters of humoral immunity (p<0.05) compared to control. SPPS, PPS and SPS treatment (400 mg/kg B.W.) significantly (p<0.05) can relieve immunodepression and significantly (p<0.05) increase the spleen and thymus index, decrease CD8+ cells and stimulate the proliferation of T and B lymphocytes compared to CP-treated animals alone. SPPS and PPS treatment (400 mg/kg B.W.) significantly increased (p<0.05) IL-2, TNF-αlevels in serum and spleencyte culture. In quantitative hemolysis of sheep red blood cell (SRBC) (QHS) assay and PFC response in CP-treated animals, SPPS, PPS and SPS treatment (400 mg/kg B.W.) showed protection in CP-treated animals. Additionally, treatment with SPPS (400 mg/kg B.W.) was more effective than other treatment groups. SPPS treatment itself produced no toxicity. The administration of SPPS to CP-exposed animals resulted in improved humoral and cellular responses. SPPS may be developed into a new kind of immunomudulation agent.5. Study on the Antioxidative Activity of Enriched-Selenium ExpolysaccharidesThe experiment was conducted to investigate the protective effect of selenium exopolysaccharide produced by E. cloacae Z0206 against H2O2-induced Raw 264.7 murine macrophages oxidative damage and the possible mechanism of action. Results showed that H2O2 addition reduced the viability of cells by MTT analysis compared to control (p<0.05). Addition of SPPS (200μg/ml and 400μg/ml), and SPS (400μg/ml), PS (400μg/ml) increased (p<0.05) superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, reduced (p<0.05) the production of intracellular peroxide (MDA), DNA ladders, reactive oxygen species(ROS) and the release percentage of Lactate Dehydrogenase (LDH), and protected cells from H2O2-induced a decrease in the mitochondrial membrane potential, activation of Caspase-3 (p<0.05) compared to H2O2-treated cells only. SPPS, SPS and PS (200μg/ml and 400μg/ml) upregulated Bax, downregulated Bcl-2 mRNA and proteins levels compared to H2O2-treated cells only. Additionally, treatment with SPPS, SPS, and PS (200μg/ml and 400μg/ml) significantly increased HO-1 mRNA and protein levels compared to H2O2-treated cells only (p<0.05). Moreover, treatment with SPPS (200μg/ml) was more effective than other treatment groups. These data suggested that induction of HO-1 protein, increase of antioxidant enzyme activities, reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events may participate in the protective mechanism of enriched-selenium polysaccharide on H2O2-induced apoptosis.