Studies On Exopolysaccharide Of Agaricus Bisporus By Submerged Fermentation

Agaricus bisporus is produced mainly for wild and artificial cultivation. However, thesemethods of production have disadvantages of long production cycle, covering an area of largeand high labor intensity, affecting the production and application of Agaricus bisporus. As oneof excellent edible fungus developed in recent years, Agaricus bisporus has caused growingattention. However, there is little research on extracellular polysaccharide of Agaricusbisporus at present.1)In this study, Agaricus bisporus was used as the research object, using submergedfermentation technology, and different fermentation conditions of extracellularpolysaccharide from Agaricus bisporus were studied and optimized. Also, the extractionprocess of extracellular polysaccharides was studied and optimized. Purification, structuralfeature and hypoglycemic effect of extracellular polysaccharide from Agaricus bisporus wasalso investigated. The main research results were as follows: A Box-Behnken factorial designwas employed to optimize the medium composition for the production of extracellularpolysaccharides from Agaricus bisporus. The optimized media was as follows: glucose35.7g/L, KH₂PO₄2.1g/L, peptone3.1g/L, KH₂PO₄2.1g/L, corn steep liquor15g/L. Under theseoptimum parameters, the maximum yield of extracellular polysaccharide from Agaricusbisporus was1.87g/L. After shaking flask test, fermentation temperature was set at25°C andthe initial pH was6.0. The strategy of controlled pH and dissolved oxygen were as follows:pH7.0was controlled on0-40h, pH6.5was controlled on40-62h, pH6.0was controlled on62-120h, agitation rate160r/min, aeration rate0.9vvm. Under the optimum condition, themycelial biomass reached a maximum of23.64g/L and exopolysaccharides reachedmaximum of3.71g/L respectively, which were increased by49.62%and33.45%than that ofuncontrolled fermentation.2)Based on the single factor experiment, a Box-Behnken central composite design wasapplied to optimize the extraction process of extracellular polysaccharides from Agaricusbisporus. After response surface analysis, the optimized extraction parameters were as follows:ethanol concentration85%（V/V）, pH8, sedimentation time22h. And the maximumconcentration of extracellular polysaccharides was2.69g/L under this optimized extractionparameters, which was consistent with the predicted value （2.71g/L）.3)Dialysis bag, the molecular weight cut-off of which was8000-14000, was used in thisstudy. And the excellent effect of dialysis was proved with ultraviolet spectrophotometry. Anovel Agaricus bisporus extracellular polysaccharides ABPS1purified by Sepharose CL-6B gel chromatography after DEAE-Sephadex A25chromatography. ABPS1was showed as asingle peak on high-performance gel-permeation chromatography （HPGPC） and the averagemolecular weight was5.7×10⁵Da. By means of gas chromatography, infrared and NMR, thestructure of ABPS1was investigated. The analysis results showed that ABPS1was aheteroglycan which was composed of glucose, mannose and xylose with a molar ratio of6.2:1:2.5. There were two glycosidic configurations （and β） in structure of Agaricusbisporus extracellular polysaccharides. It was also revealed that the structure of ABPS1contained pyranride, with with1â†'3glycosidic bonds as the main chain and1â†'6glycosidicbonds as branched chain.4) The extracellular polysaccharides from Agaricus bisporus have strong scavengingeffect of hydroxyl radicals, superoxide anion and DPPH radicals, and therefore it hasoutstanding performance in antioxidation. Also, in vitro model was employed to investigatethe effect of extracellular polysaccharides on alpha-glucosidase activity. It was found thatextracellular polysaccharides from Agaricus bisporus could inhibit the activity ofalpha-glucosidase. In addition, it was found that extracellular polysaccharides from Agaricusbisporus could enhance the islet β-cell viability and increase the secretion of insulin protein.5)Agaricus bisporus extracellular polysaccharides can play an active role in reversingsymptoms of diabetes （weight loss, darker hair） in mice. And it can reduce blood glucoseconcentration in diabetic mice, promote the conversion of glucose synthesis of hepaticglycogen. At the same time, ABPS1was effective in improving the activities of SOD andother antioxidant enzyme, scavenging free radical.