Study On Fermentation, Extraction, Purification And Biological Activities Of Pachyman From Fermentation Liquid Of Poria Cocos

Objective:The optimum fermentation liquid fermentation medium and the optimal culture conditions were determined by the liquid fermentation test;Extract and isolate pachyman from fermentation liquid of Poria cocos. Study on the purification of exocellular polysaccharide , the structure of exocellular polysaccharide, and biological activityin vitro.Methods:(1)The six Poria cocos strains from different sources were rejuvenatived in test tubes equipped with PDA culture medium(2)According to the fermentation broth pH, reducing sugar content and biomass,the growth line of Poria was drew ;Observe and record the influences of different spawn age of solid medium slope(3)Single factor tests were done to analyze the influence of each factor including temperature ,rotation speed ,inoculum size,medium volume,carbon source and nitrogen source.Then the optimum conditions for the fermentation were obtained by using orthogonal design L933.(4)Classical hot water extracting and ethanol precipitating method was employed to isolate polysaccharides and the content of total sugar was determined by phenol-sulfuric acid method. Extract and isolate exocellular polysaccharide and endocelluar polydaccharide from the fermentation broth..After the exocellular polysaccharide was extracted with hot water and precipitated with ethanol, protein and pigment were also removed. DEAE-cellulose chromatography was used to separate exocellular polysaccharide. Finally,it's homogeneity was proved by HPLC.(5)The chemical composition of exocellular polysaccharide was analyzed by using TLC , gas chromatography ,Uv spectrum, IR spectrum . The monosaccharide and linkage of exocellular polysaccharide were investigated through chemical analyzation.(6)Antioxidant activity of exocellular polysaccharide in vitro are determined hydroxyl free radical,superoxide anion Lipidperoxidation,the restraining oxididation of erythrocytes, were carried out to examine exocellular polysaccharide's anti-oxidation .Results:(1)All six strains were successfully rejuvenatived;ACCC50864 which has high survival rate was selected for the standard strains.(2)fermentation growth lineof Poria was drew ; determine the optimum spawn age (25d) of solid medium slope(3)The results of single factor tests and orthogonal experiments demonstrated that the optimum fermentation conditions are as follows:temperature is 26℃,rotation speed is 140r/min,inoculum size is 10%,medium volume is 100ml/250ml;2.5% glucose,2% extractum carnis, KH₂PO₄/MgSO₄ is 0.6%/0.3%,VB1 is 4mg/100ml(4)The exocellular polysaccharide was fractionated by column chromatography DEAE -52, and LLPSâ… was obtained. It was proved to be homo- geneous polysaccharide on molecular weight.(5)IR revealed the main chain of LLPSâ… mostly linked byβ-glycosidic bond;IR,UV and the element analysis showed that N and S is absent,it's not a aminosugar.The results of GC and TLC analysis showed the monosaccharide of is Glc.( 6 ) The results of the vitro-anti-oxidation suggested that exocellular polysaccharide has good anti-oxidation action,with the dosage of 3.5 mg/mL the inhibitory rate on hydroxyl free radical was 56.2%,each dosage can inhibite superoxide anion ,with the dosage of 5.0 mg/mL the inhibitory rate on the restraining auto-oxididation of erythrocytes was 43%,with the dosage of 5mg/mL the inhibitory rate on lipidperoxidation was 68.2%.Conclusion:The optimum fermentation liquid fermentation medium and the optimal culture conditions were optimized; Study on the structure of LLPSâ… and it has good antioxidant function in vitro.