Submerged Fermentation Of Polysaccharides, Extraction, Purification And Antioxidant Effects Research

Grifola frondosa is a large drug edible fungus, belonging to the the phylum Basidiomycotina, class layer Basidiomycetes, order Aphyllopherales and family Polyporeceae. Its fruiting body contains much nutrition, such as Amino acids, polysaccharides, and trace elements and so on. Grifola frondosa polysaccharides have many biological effects of immune regulation, tumor, diabetes, improving fat metabolism and antioxidant. The study of submerged fermentation polysaccharides were relatively short at present.In this study, we investigated the Grifola frondosa submerged fermentation, Optimization of medium composition and its expanding culture, Different extraction methods extract mycelium polysaccharide, purified polysaccharide, the structure of information representation and oxidation of polysaccharides. The results are as follows:1. The carbon sources and the nitrogen sources of Grifola frondosa fermentation medium are determined by single factor experiments .Three significant factors were selected on the polysaccharides synthesis: glucose, yeast extract and rapeseed oil. Response surface analysis showed when the optimal conditions of medium include glucose 27.15g/L, yeast extract 5.9g/L, and rapeseed oil 3.66g/L,the mycelium maximum amount reached 4.93g /L.And the optimal conditions of medium include glucose 25.85g/L, yeast extract 5.56g/L, and rapeseed oil 3.34g/L, The maximum amount of exopolysaccharide maximum reached 1.89 g/L. Expansion of cultured the mycelia reached the maximum yield of 5.5g/L, the maximum amount of extracellular polysaccharide reached 2.1g/L in the 5 L fermenter under the optimization of medium composition;they were higher than the yield of before optimization.2. The methods of the hot water, ultrasonic and microwave extraction were studyed to extract the Grifola frondosa mycelium polysaccharide.The water ratio, extraction time, extraction temperature of three kinds of Grifola frondosa mycelium polysaccharide extraction methods were studyed. To the basis of a single factor, hot water extraction, ultrasonic extraction and microwave extraction orthogonal test were studied. The results reveals ,the maxium of extraction rate reached 3.98%, when hot water extraction conditions were solid to water ratio 1:15,time 4 hour,temperatuer 95℃. The maxium of extraction rate reached 3.98%, when ultrasound extraction conditions were solid to water ratio 1:10, time 45 minute, temperature 70℃. The maxium of extraction rate reached 4.74%, when the microwave extraction conditions were solid to water ratio 1:20,time 12 minute, power 600W.Comparison of three extraction process, microwave extraction is the best method.The maxium of extraction rate reached 4.74% when microwave extraction conditions were solid to water ratio 1:20,time 12 minute, power 600W.3. Mycelium polysaccharide GFPⅠ-Ⅰand surbmerged fermentation polysaccharide WGFPⅠ-Ⅰwere obtained by water extraction, deproteinized, dialysis, ethanol precipitation, DEAE-52 and Sephadex G-75 chromatography. According to GC analysis, GFPⅠ-Ⅰand WGFPⅠ-Ⅰwere major composed by glucose. The structure of polysaccharides were analyzed using UV-visible spectroscopy, infrared spectroscopy, gas chromatography and nuclear magnetic resonance, GFPⅠ-Ⅰpossible structure was [α-D-Glc(1→4)-]n type glucan, average molecular weight 7.3×104. WGFPⅠ-Ⅰpossible structure was [α-D-Glc(1→4)-]n type glucan, and the branched-chain of [α-Glc(1→3)-]n, average molecular weight 5.66×104.4. Antioxidant effects of the mycelium polysaccharides and the extracellular polysaccharides were studyed in vitro, in vivo respectively;The major experiments contained polysaccharides of reducing power, DPPH? anti-oxidation system, scavenging superoxide anion and hydroxyl radicals, and anti-D-galactose induced aging mice test.The polysaccharides reducing power have a higher increasing with the concentration increasing.When the mycelium polysaccharide concentration was 0.4mg/mL ,its clear O2?- the ability to reach 80%, and the same concentration exopolysaccharide reached 90%. Mycelium and extracellular polysaccharides inhibition hydroxyl radicals reached 70%, when the concentration was 1.0mg/mL. Polysaccharides scavenging DPPH? increased with polysaccharide concentration increasing, especially mycelium polysaccharide, its concentration 1.0mg/mL, its clear the DPPH? ability to reach 67%. Experiments in mice show that mycelium polysaccharides and exopolysaccharide can significantly improve T-AOC, GSH-Px, SOD activity in the brain, liver, heart tissue and serum,comparing with model group(P<0.05),especially exopolysaccharide. Grifola frondosa polysaccharides could significant reduce MDA in liver, heart tissue and serum (P<0.01), no significant in brain.