Research On The Liquid Fermentation Conditions Of Coprinus Atramentarius And Its Biological Activities

Coprinus atramentarius, a typical species belonging to the genus Agaricus, has been designated as a kind of pharmaceutical fungi with favorable health care function. Solid state fermentation of C. atramentarius has been reported, and most research on this macrofungus at present has been concentrated on the aspect of basic biological characteristics investigation. Few reports regarding its biological active components and other culture method have been obtained.In this thesis, liquid fermentation has been acquired and researches on the substance with biological activities have been carried out. At the stage of liquid fermentation study, biomass and the yield of extracellular polysaccharide have been taken as the index. Extensive one-factor experiments revealed the key factors from various culture medium components and fermentation conditions. RSM (response surface methodology) was taken advantage of to detect the relationship between the key factors. CCD (central composite design) and Box-Behnken experiment design were adopted for culture medium and fermentation conditions optimization, respectively. By solving the quadratic regression model equation using appropriate statistic methods, the optimum concentrations for the submerged fermentation medium were determined: glucose, corn powder, wheat bran, KH₂PO₄ and MgSO₄·7H₂O were 13.51, 17.07, 7.62, 2.29 and 2 g/L, respectively, then the predicted yield of the extracellular polysaccharide was 293.73mg/L. When the above values were 10.07, 18.02, 11.63, 1.58 and 4 g/L, the maximum biomass was 6.24 g/L. And the second RSM analysis disclosed the optimum fermentation conditions for the submerged fermentation were: temperature28.4℃, shaking rate134.5 r/min, culture time 119.28 h, volume of medium 186.5 mL/500 mL, then the predicted yield of the extracellular polysaccharide was 415.58 mg/L. When the above values were 20.45℃,129 r/min,75.12 h,194.5 mL/500 mL, the maximum biomass was 7.26 g/L.Fractional alcohol precipitation was made use of to divide the C. atramentarius broth into six intra- and extra-cellular components. Free radicals scavenging efficiency is one reliable index meaning to the antioxidant activities as well as the inhibition rate of non-enzymatic glycation reaction to the hypoglycemic effect. Hydroxyl radicals and super-oxide anion scavenging efficiency were measured, and the non-enzymatic glycation reaction inhibition rate of the six components was detected.Biological activities determination proved the extra-cellular protein and the extra-cellular polysaccharide to be the components with most satisfactory antioxidant activity and hypoglycemic effect. Further separation and purification were essential to acquire more information about the active substances, as well as to make use of them. The extra-cellular polysaccharide was purified by DEAE and Superdex 200, and TOYOPEARL SP-650M was utilize to purify the extra-cellular protein. From the extra-cellular protein, one protein named EPP was determined to possess both antioxidant activity and hypoglycemic effect.