Study On The Fermentation Conditions And Extracellular Polysaccharide Of Coprinus Comatus

In the present thesis, liquid fermentation conditions in shake flask culture and theextraction techniques of extracelluar polysaccharide (EPS) of RCEF0986, a strain ofCoprinus comatus, were studied. The liquid fermentation conditions included nutritionaland non-nutritional factors, which affected the synthesis of EPS. The extraction techniqueof EPS, which was based on the biological activities, included the effect of temperature onthe yields of EPS under different pH values and effect of different concentration times onthe yields of EPS. Furthermore, the separation and purification of EPS were studied. There are three strains of Coprinus comatus, from which RCEF0986 was chosen asthe test strain for the high yield. The fermented broth of RCEF0986 was concentrated and precipitated at a final 15%ethanol at 4℃ for 24 hours. The broth and precipitation (EPS) were harvested respectivelyafter ten minutes centrifuge for 5000rpm. The precipitation was washed several times with95% ethanol and 100% ethanol, before dried by vacuum refrigerator. By the same methodsand processes, the broth was adjusted to the final concentration of 30% ethanol, and thenthe precipitation and broth were obtained. Again the same procedure was done with thefinal concentration of 50% ethanol and 70% ethanol. At last, we got four precipitationsamples. The assay of life span on Drosophila melanogaster suggested that the products(EPS) from 70% ethanol fractionation contain active components, which could prolong lifespan of Drosophila melanogaster by utmost 17.2% and average 8.2% for males, and byutmost 17.9% and average 13.0% for females. Therefore, the prolonging effect was evident,especially for the females. The nutritional source experiments showed that corn powder, glucose and yeastpowder were most favorable for EPS formation. The optimum carbon source wascompound, corn powder and glucose. And the glucose concentration is usually 2.0%, thecorn powder concentration was set up 4 concentration (1.0%, 2.0%, 3.0%, 4.0%). Theoptimum nitrogen source was yeast powder among five nitrogen sources, and threeconcentrations were set up. Through an orthogonal test the optimum inorganic salt andgrowth factor were decided, and the optimal medium culture was determined: corn powder3.0%, glucose 2.0% , yeast powder 1.0%, KH₂PO₄ 0.20%, MgSO₄·7H₂O 0.15% and VB115mg/l. Non-nutritional factors, including inocula amount, temperature, seed culture age andliquid medium capacity, were studied through an orthogonal test. The result showed thatthe optimal culture conditions were: the inocula amount 10%, the temperature 22℃, theseed culture age 6 days and the medium volume 2/5. Extraction technique included the effect of temperature on the yields of extracellularpolysaccharide under different pH values and effect of different concentration times on theyields of extracellular polysaccharide. The results showed that the yield and content of EPSwere tend to concurrent under pH7, and the yield of EPS reached its max. when the filtratewas concentrated to 1/3 volume. The suitable procedure for the extraction of EPS wasdetermined as follows: first, concentrated the filtrate to 1/3 volume at 65℃ and pH7.Second, precipitated at a final 50% ethanol at 4℃ for 24 hours. Third, adjusted to the finalconcentration of 70% ethanol after centrifuge and precipitated at 4℃ for 24 hours. Theprecipitation was harvested by centrifuge, washed several times with 95% ethanol and100% ethanol, then dried by vacuum refrigerator. The raw EPS was further separated into three fractions (EPSâ… , EPSâ…¡and EPSâ…¢) byDEAE-DE52(CL-) chromatography. The yield rates of EPSâ… , EPSâ…¡and EPSâ…¢ were12.1%, 16.0%, and 8.7%, which contained 85.2%, 68.8%EPSâ…¢ and 38.8% of totalpolysaccharide respectively. EPS â…  was further purified by Sephadex G-150 gelchromatography to obtain the homogeneous component, which was proved bypolysaccharide gel electrophoresis. EPSâ…¡ and EPSâ…¢ were further separated into threefractions by Sephadex G-150 gel chromatography. The protein content of EPSâ… , EPSâ…¡and EPSâ…¢ was 14.3%, 21.