| Objective: The optimum liquid submerged fermentation culture medium and the optimal culture conditions were determined by the liquid fermentation test;Extract and isolate Polyporus umbellatus polysaccharide(PUPS)from fermentation liquid of Polyporus umbellatus. Study on the purification of exocellular polysaccharide,and its biological activity in vitro.Methods:(1)The seven Polyporus umbellatus strains from different sources were rejuvenatived in test tubes equipped with PDA culture medium(2) Observe and record the influences of different microbial strain age of solid medium slope on the effects of liquid fermentation;According to the fermentation broth pH,reducing sugar content and biomass,the growth line of Polyporus umbellatus mycelium was drew ;(3)Single factor tests were done to analyze the influence on biomass, including temperature,rotation speed,noculum size,medium volume,carbon source,nitrogen source,carbon consistency,nitrogen consistency. Then the optimum macroelement and microelement for the fermentation were obtained by using L423 and L934 orthogonal design.(4)Optimize the ultrasonic extraction condition of Polyporus umbellatus mycelium.Hot water extracting and ethanol precipitating method was employed to isolate Polyporus umbellatus endocelluar polydaccharide of coenocytic mycelium and sub-entities between which polydaccharide extraction ratio is compared. Hot water extracting and ethanol precipitating method was employed to isolate exocellular waterbase polysaccharide,endocelluar water base polydaccharide and endocelluar alkali soluble polydaccharide from the fermentation broth. The content of total sugar was determined by phenol-sulfuric acid method.(5)The basic physical and chemical property would be tested. Antioxidant activity of exocellular polysaccharide in vitro were determined by cleaning hydroxyl free radical,cleaning superoxide anion,restraining oxididation of erythrocytes,restraining liver cells Lipidperoxidation.Results:(1) Seven strains were rejuvenatived successfully;number 10 strains whose survival rate and growth rate were high were selected for the experimental strains.(2)Determine the optimum microbial strain age of solid medium slope was 25d ; fermentation growth line of Polyporus umbellatus mycelium was drew.(3)The single factor tests demonstrated that the optimum fermentation conditions are as follows: temperature is 26℃,rotation speed is 130r/min, inoculum size is 10%,medium volume is 100mL/250mL;4.0% potato starch, 2.0%Yeast extract;Orthogonal experiments showed that the optimum macro- element for the fermentation are MgSO4.7H2O 1.0g,KH2PO4 2.0g,CaCl2 0.1g, and the optimum microelement for the fermentation are ZnSO4 1.5mg,MnSO4 1.5mg,FeSO4 0.5mg and Vb1 2.0mg。(4)The optimum extraction condition is as follow: ultrasonic frequency 16kHz,liquid-to-solid ratio 20,breaking time 30min; Polyporus umbellatus endocelluar polydaccharide extraction ratio of coenocytic mycelium and Sub-entities is 3.9% and 1.7%; exocellular waterbase polysaccharide,endocelluar water base polydaccharide and endocelluar alkali soluble polydaccharide from the fermentation broth are 6.3mg/mL,0.8mg/mL,1.7mg/mL.(5)The basic physical and chemical property experiment shows that Polyporus umbellatus polysaccharide are possessed of basic polysaccharide property. The results of the vitro-anti-oxidation suggested that exocellular polysaccharide has good anti-oxidation action,with the dosage of 8.0mg/mL the clearance rate on hydroxyl free radical was 60.3%; with the dosage of 6.0mg/mL the clearance rate on superoxide anion was 49.8%; with the dosage of 3.0mg/mL the inhibition rate on auto-oxididation of erythrocytes was 35.5%; with the dosage of 4.0 mg/mL the inhibition rate on liver cells Lipidperoxidation was 56.8%.Conclusion:The optimum liquid fermentation medium and the optimal culture conditions were optimized;Polyporus umbellatus polydaccharide were extracted,and the exocellular polydaccharide are demonstrated have good antioxidant function in vitro.
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