Study On The Process Of Crude Polysaccharides Extraction And Their Antioxidant Activity Of Lepista Nuda Mycelium

Objective: In this experiment,Lepista nuda mycelium as the test object,to study the optimum technology for extracting crude polysaccharide from mycelium of liquid fermentation culture,the extracellular enzyme activity during different training stages,mycelium protein Sevag methods,the capacity of scavenging free radicals of polysaccharides and so on.Methods: An orthogonal test was used in screening of culture conditions for liquid fermentation of mycelium;The extraction process of crude polysaccharide from mycelium was optimized by microwave assisted method;Sevag method was used to remove protein from mycelium;Mycelium extracellular enzyme activities were determined by spectrophotometric method;The free radical scavenging ability of crude polysaccharide was studied respectively by DPPH method,Prussian blue method,salicylic acid colorimetric method and adjacent benzene three phenol self oxidation method.Results: 1.Dry weight of mycelium and extracellular polysaccharide content as evaluation index,the single factor and orthogonal experiments were carried out on the mycelium liquid fermentation culture medium,p H value and culture temperature,the liquid fermentation medium components which were most suitable for the growth of mycelia were soluble starch 2%?w/v?and yeast extract powder 0.5%?w/v?,KH₂PO₄ 0.3%?w/v?,Mg SO₄ 0.05%?w/v?,Ca Cl₂ 0.001%?w/v?,VB₁ 0.001%?w/v?,p H=7,the optimum culture temperature was 25?.2.In the experiment of extracting crude polysaccharide from mycelium,hot water extraction,alkali extraction,microwave assisted extraction,ultrasonic assisted method tests were carried out to compare.The results showed that the polysaccharide extraction rate was 19%,10.67%,28.67% and 23%,so the microwave assisted method was the optimum extraction method;Then it was optimized by single factor test and orthogonal test to determine the optimum extraction parameters: extract 4 times,extract for 15 min,material to liquid ratio?the ratio of extract and 95% ethanol solution?1:5,the extraction rate of crude polysaccharide from mycelium was 28.67%.3.In crude polysaccharide purification test,the protein was removed by sevag method,by determining the loss rate of polysaccharide and protein removal rate,we found that sevag volume ratio 5:1,removal of protein for 3 times,and the polysaccharide,protein content,respectively 29.07% and 6.28%.4.Respectively by DPPH method,Prussian blue method,salicylic acid colorimetric method,adjacent benzene three phenol self oxidation method,the scavenging free radicals ability of crude polysaccharide was determined.Results show that,DPPH free radical scavenging rate: deproteinized mycelium polysaccharide > mycelium crude polysaccharide > Polysaccharide of fermentation broth;The clearance rates were 82%,71.44% and 52.61%;Reduction ability: deproteinized mycelium polysaccharide > mycelium crude polysaccharide > fermentation broth polysaccharide,the OD700 values were 1.421,0.966 and 0.493.?OH free radical scavenging rate: deproteinized mycelium polysaccharide > mycelium crude polysaccharide > fermentation broth polysaccharide,scavenging rate respectively was 82.11%?81.63% and 65.97%;In adjacent benzene three phenol self oxidation method,the free radical scavenging rate: the free radical scavenging rate of O₂?—free radical scavenging rate: deproteinized mycelium polysaccharide > mycelium crude polysaccharide > fermentation broth polysaccharide,the clearance rate was 54.67%,44.83% and 31.77%.5.By UV spectrophotometry,researched on mycelial extracellular enzyme activity of different culture days,the results were as: CMC enzyme activity in culture to 10 d reached maximum,the enzyme activity 10.28U;the enzyme activity of amylase in culture to 13 d,the enzyme activity was 11.23U;the maximum enzyme activity of peroxidase in cultured 10 d,the enzyme activity was 6.6U;the enzyme activity of filter paper enzyme in culture to 13 D,the enzyme activity was 6.7U.Conclusion: Lepista nuda mycelium crude polysaccharide,deproteinized mycelium polysaccharide and fermentation broth polysaccharide all show good antioxidant capacity,but antioxidant abilities of three kinds of crude polysaccharide are different;the free radical scavenging rate of deproteinized mycelium polysaccharide is greater than that of mycelia polysaccharide and fermentation liquid crude polysaccharide;Of the three kinds of polysaccharides,free radical scavenging rate is less than that of VC;The concentration of polysaccharide in 0.5¹0.0mg/m L range,with the increase of the concentration,the antioxidant capacity also subsequently enhances.