Exploration Of The Effect On The In Viro Bioactivity Of Agaricus Bisporus Polysaccharides Caused By Autolysis

Agaricus bisporus is a common edible fungus in China,with high polysaccharide content and various biological activities.Agaricus bisporus is susceptible to bacterial infection and mechanical damage due to their delicate tissues,high water content,and no obvious protective structure,leading to autolysis.Autolysis is the hydrolysis of intracellular biopolymers into low molecular weight products under the action of autohydrolytic enzymes,which is susceptible to external temperature,p H and other conditions.During autolysis,polysaccharides are degraded to various degrees due to the action of autolysin.At present,the degradation of Agaricus bisporus polysaccharides（ABPs）by autolysis is not well understood,and the research on the change of polysaccharide activity of Agaricus bisporus before and after autolysis is even rarer.In this study,we first screened the polysaccharides of Agaricus bisporus which were autolysed at a better temperature and time,then analyzed the changes of its antioxidant and antitumor activity in vitro,and finally separated and purified the polysaccharides with better activity in vitro and determined its molecular weight.The results are as follows:（1）After autolysis of fresh Agaricus bisporus at 45℃and 50℃for different times（4 h,8h,12h,16h）,the content of high molecular weight（MW）polysaccharides from 70%ethanol precipitation decreased significantly（P<0.05）,and the content of medium MW polysaccharide from 80%ethanol precipitation increased significantly（P<0.05）,the content of low MW polysaccharide from 90%ethanol precipitation have no significantly difference.which indicated that the polysaccharides of Agaricus bisporus degraded after autolysis.There was no significant difference in the content of each polysaccharide component after autolysis for different time,indicating that autolysis mainly occurred in the first 4 hours.（2）The results showed that the anti-oxidant activity of high MW polysaccharide of Agaricus bisporus was weaker than that of medium MW and low MW polysaccharide after ABPs were autolyzed at 45℃and 50℃for 4 hours;the scavenging ability of low MW polysaccharide to DPPH,·OH and ABTS radicals increased significantly（P<0.05）,and the scavenging ability of medium MW polysaccharide to three radicals decreased to some extent.after 4 hours autolysis at45℃,the scavenging ability of high MW polysaccharides to DPPH and ABTS radicals was significantly enhanced（P<0.05）,while the scavenging ability to OH radicals was significantly weakened（P<0.05）.The scavenging ability of high MW polysaccharides to three kinds of free radicals decreased in different degrees after 4hours autolysis at 50℃;and the ability of high MW polysaccharides to scavenge three free radicals is weaker than that of medium MW and low MW polysaccharides.Besides,the inhibition effect of high MW polysaccharides on Hela and Hep G2 cells was weak before and after autolysis;after autolysis at 45℃and 50℃,the inhibition effect of high MW polysaccharide on both cells was significantly enhanced;after autolysis at 50℃,the inhibition effect of low MW polysaccharides on Hela cells was no significant change,and after autolysis at 45℃,the inhibition effect of low MW polysaccharides on Hela cells was enhanced;but the inhibitory effect on Hep G2 cells was significantly enhanced（P<0.05）.（3）After purification of Abp-50-A,two eluting components,Abp-50-A11 and Abp-50-A22,were obtained,with molecular weight of 77375.42 Da and 88861.78 Da respectively;after purification of Abp-45-B,Abp-45-C and Abp-Con-C,only one eluting components was obtained for each.They are Abp-45-B11,Abp-Con-C11 and Abp-45-C11,with MW of 1281.70 Da,625.11 Da and 619.72 Da respectively.The molecular weight of Abp-50-A was larger than that of other polysaccharides,and its activity in vitro was weaker than that of other polysaccharides.The molecular weight of Abp-45-C was smaller than that of other polysaccharides,but its activity in vitro was the strongest.In conclusion,after autolysis,the anti-oxidation in vitro of low MW polysaccharides of Agaricus bisporus increased significantly（P<0.05）,but that of medium MW polysaccharides decreased,among which Abp-45-C had the best antioxidant capacity in vitro,and the EC₅₀values of DPPH,·OH and ABTS free radical scavenging experiments were 0.04±0.01 mg/m L、0.07±0.01 mg/m L、0.24±0.01mg/m L respectively.In addition,the enhanced effect after autolysis at 45℃was stronger than that after autolysis at 50℃.The best inhibitory effect was Abp-45-C.the IC₅₀values of Abp-45-C on Hela and Hep G2 cells in vitro is 1.42±0.18 mg/m L、1.65±0.16 mg/m L.