Structures And Antioxidant Activities Of Extracellular Polysaccharides Produced By Marine Microorganisms

The unique living environments of marine microorganisms, makes them have theability of generate various exopolysaccharides with novel chemical structural andparticular biological activities. Therefore the marine microorganisms-derivedexopolysaccharides(EPSs) are the new resources for development of marinepolysaccharide drugs. As the natural biological macromolecules, exopolysaccharideshave increasing more and more attention in the biological medicine and daily chemicalindustry. In this paper,16kinds of extracellular polysaccharides were isolated fromdifferent sources of marine microbial fermented liquid. The yield, basic physicochemicalproperty, monosaccharide composition and antioxidant activity of the EPSs wereanalyzed. Based on the analysis results ifve kinds of marine microorganisms wereobtained, including the coral symbiotic fungus Penicillium commune518#, mangrovesymbiotic fungus Fusarium oxysporum Y24-2，mangrove forest root Mud fungiTrichoderma.sp ghq-198，the jiaozhou bay sea mud fungus Penicillium.sp ghq-18andmoss symbiotic fungus Penicillium purpurogenum ZZ05-4.The crude extracellularpolysaccharides were further isolated and puriifed, and there structures characteristicsand antioxidant activities were investigated. Results of the study are as follows:1. The yield of16species of microorganism extracellular polysaccharides werebetween0.43?3.56g/L. The total sugar content and protein content were55.45%?92.30%and0.82%?13.65%. After deproteinized with Sevag method the binding proteinstill existence. All the EPSs samples were not detected the presence of sulfate. In the16kinds of EPSs, seven kinds of them were mainly composed of Man and Glc，two kindsof them were composed of Man and Gal, and three kinds of them were compose ofsimilar proportion of Man, Glc and Gal. In addition two EPSs produced by strainsAH17-3and GW3-13contains a higher proportion of GlcA and GlcN. The results showed that different sources of microorganisms EPSs varying in yield, monosaccharides composition, physicochemical property and DPPH radicals scavenging activities.2. After separation and purification with Q Sepharose Fast Flow anion exchange column chromatography and gel permeation chromatography on Superdex75prep, three purified EPSs were obtained from gorgonian Muricella abnormalis symbiotic fungi Penicillium commune518#. The main component F1P-2S with a molecular weights of18.6kDa, and the total sugar content and protein content were about96.4%and1.23%, respectively. After HPLC monosaccharide composition analysis, F1P-2S was mainly composed of Man, Glc and Gal in a ratio of27.9%,14.7%and57.4%. Structure analysis indicated that F1P-2S was mainly composed with a-D-Glcp-(1→,→6)-β-D-Galf-(1→,→2)-β-D-Galf-(1→,→2,6)-α-D-Manp-(1→and→2)-α-D-Manp-(1→in a molar ratio of2:6:2:1:1. The EPSs structure of F1P-2S with a→2)-α-D-Manp(1→as backbone chain and substituted at O-2position with a long branch chain composed of galactofuranose and glucopyranose. EPSs with this structure was isolated from coral source fungus for the first time.3. Two EPSs TW and TS were isolated from Fusarium oxysporumY24-2with a receiving rate about17%and36%, respectively. TS was further purified by Sephacryl S-100HR gel column chromatography and a purified EPS named TS-1withmolecular weight about61.2kDa was obtained. The total sugar and protein contents of TS-1were about91.3%and0.79%, respectively. TS-lwas mainly composed of Man, Glc and Gal in a molar ratio of1.33:1.30:1.00. Methylation, IR, GC-MS and NMR analysis results indicated that TS-1had a [→-6)-β-D-Galf-(1→] glycosyl main chain with a branch structure at C-2position of every [→-6)-β-D-Galf-(1→]glycosyls. The branch structure fragments of TS-1was mainly consist of a-D-Glcp-(1→, β-D-Manp-(1μ2)-β-D-Manp-(1→2)-α-D-Glcp-(1→and β-D-Manp-(1→2)-α-D-Glcp-(1→in a ratio of2:1:1. In vitro antioxidant activity tests showed TS-1possessed good scavenging abilities on hydroxyl radicals and DPPH radicals asevidence by their low EC50value of1.15mg/mL and2.11mg/mL. With regard to superoxide radicals scavenging ability, the EC50value was about2.17mg/mL. This research provide basis for the discovery and application of novel structure exopolysaccharides from marine epiphytes.4. Two purified EPSs WHW-1and WHS-1were isolated from moss endophytic fungi Penicillium purpurogenum ZZ05-4, and their molecular weights were about28.69kDa and43.91kDa, respectively. The total sugar content and protein content were at90.30%-87.74%and2.69%-4.35%. Structure analysis showed WHW-1was a mannan, with [→6)-a-D-Manp(1→] glycosyls as its bachbone and branched at C-2position of the main chain. The branches of WHW-1were major composed of→-2)-a-D-Manp-(1→and a-D-Manp-(1→. WHS-1was a heteropolysaccharide mainly constituted by Man, Glc and Gal in a ratio of4.20:1.30:0.02. After partial acid hydrolysis, methylation and and GC-MS analysis, WHS-1maybe with a→2)-a-D-Manp-(1→constitute the main chain, and the branches were major of→6)-a-D-Manp-(1→and Manp residue.→5)-Galf-(1→and Galf residues were connected to the outermost peripheral portion of the whole sugar structure.5. After isolation and purification with Q Sepharose Fast Flow and Superdex75prep column chromatography, two purified EPSs HQW1and HQS1with a molecular weight of about12.87kDa and29.82kDa were obtained.The sugar and protein contents of HQS1were88.84%and3.49%, respectively. HQS1was mainly composed of Man, Glc and Gal in a molar ratio of5.92:0.87:3.98. Methylation analysis showed HQS1mainly composed of Manp-(1→, Galf-(1→,→2)-Manp-(1→,→3)-Manp-(1→,→2)-Galf-(1→,→6)-Manp-(1→,→6)-Galf(1→,→5,6)-Galf-(1→,→2,6)-Manp-(1→,→3,6)-Manp-(1→and→2,3,6)-Manp-(1→. After partial acid hydrolysis and GC-MS analysis, the mainly connection type were1-2,1-3,1-6,1-2,6, and the non-reducing terminal mannose, wherein the→6)-Manp-(1→and→2)-Manp-(1→connections increaced significantly. This results indicating both connections constitute the main chain structure of HQS1. The oligosaccharides of HQS1with DP2-5was prepared by mild acid hydrolysis and purified by gel-permeation chromatography. HQS1showed certain antioxidant activity in vitro antioxidant experiments, but the activity is not significant. 6. Four polysaccharide fraction HW-1, HW-2, HS1-1and HS2-1were obtained after purification from the jiaozhou bay sea mud fungus Penicillium sp. ghq-18and the yield of the four polysaccharide were53.21%,9.22%,61.08%and37.59%, respectively. The molecular weight of the four polysaccharide were16.9,11.3,20.7and26.6kDa. HS1-1was mainly composed of Man, Glc and Gal in a molar ratio of14.76:1.00:6.53. Structure analysis indicated HS1-1was a galactomannan, the backbone of mannose core composed of a-Manp(1→,→2)-a-Manp(1→、→6)-a-Manp(1→。→2,6)-a-Manp(1→The galactofuranose had a (3-(1→5)-Galf backbone with a branch structure of β-Galf-(1→at O-6position of the main chain. In vitro antioxidant activity tests showed HS1-1has a certain scavenging activities on DPPH, hydroxyl radicals and superoxide radicals.Different kinds of EPSs with novel structure were isolated and from different sources of marine microbial fermentation broth. The determination of in vitro antioxidant activities of EPSs enrich the "Marine sugar library", which provide a certain theoretical basis for the research of marine carbohydrate drugs and functional foods. This thesis has important guiding significance for further enrich microbial extracellular polysaccharide products market.