Submerged Cultivated Mycelium Of Lepista Sordida And Studies On Its Purification,Structural Analysis And Biological Activities

Lepista sordida is a kind of mushroom which is not only delicious but also nutritious.In this study,submerged cultured mycelia of Lepista sordida were used,and fermentation yield and extraction and purification of polysaccharides LMWDcl,LMADal,LMWDb,LMADc and LFDS from Lepista sordida fermentation were optimized,and their structures and biological activities were determined.Setting Mycelial fermentation yield as response value,through screening using a single factor in the system,Plackett-Burman experiment,steepest ascent design and Box-Behnken design,the optimal fermentation conditions of Lepista sordida were determined as follows:7.5%inoculum size,24oC,PH 6.5,0.06%MgSO4,0.05%K2HPO;,8.20%lactose,3.4%soybean meal,160 r/min and 29 days.Under these conditions the fermentation yield of Lepista sordida mycelium elevated 37%than before.To enhance polysaccharide extraction rate,single factor experiment and Box-Behnken design were carried out.The optimal polysaccharide extraction conditions were as follows:material-water ratio 1:18,extraction temperature 90oC,ultrasonic treatment time 20 min,extraction time 3 h,ethanol concentration 100%,Precipitation time 7.14 h,and ethanol volume ratio 5:1.Under these conditions,the yield of mycelial polysaccharides obtained was 76.9375 mg/g,which was 29%higher than before.The polysaccharide content of alkali-soluble LMADalwas 91.24%after the purification process which included deproteinization,DEAE anion exchange resin chromatography,and molecular exclusion elution.The Mw and Mn of LMADal was 1442 and 605 kDa respectively,and the distribution was 2.384,which indicated the efficiency of the purification.LMADal was composed of glucose(57.9%),xylose(31.8%),and small amounts of rhamnose,arabinose,galactose,glucuronic acid,and galacturonie acid(1.2-3.1%).The construction of LMADal was verified and further aialyzed by FT-IR and 2D NMR.The units of saccharides were linked by p-glycosidic linkages.Besides cc-xylose and(1?4)-?-D-Glcp as the main components,4-O-Me-?-D-GlcpA and a-Araf were also detected?LMADal and LMWDcl exhibited notable l,l-dephenyl-2-picryhydrazyl(DPPH)radical scavenging and hydrogen peroxide scavenging effects,lipid peroxidation inhibitory activity,reducing activity,and Fe2+ chelating activity.Administration of LMADal and LMWDcl to mice prior to CCl4 treatment significantly prevented the CCl4-induced elevation in serum alanine aminotransferase and aspartate aminotransferase activities and hepatic malondialdehyde concentration.Mice treated with LMADal and LMWDcl demonstrated increased activities of superoxide dismutase and glutathione peroxidase in the liver.LMADal and LMWDcl also prevented CCl4-induced oxidative histological alteration in the liver.The results suggested that LMADal and LMWDcl protected the liver from CCl4-induced damage via antioxidant mechanisms.Polysaccharides LMWDb and LMADc from Lepista sordida mycelia suppressed the proliferation of melanoma B16 cells in vivo with an inhibition rate of 50%via elevating IL-2 and IFN-y and by reducing IL-10 levels in the serum and promoting the immune organs including the spleen and thymus.The activity of LMADal and LMWDcl in strengthening the immune system of the body led to better antitumor activity in mice.The freshness-preserving effects of Lepista sordida mycelial polysaccharides LMWDel and exopolysaccharides LFDS were also determined in the current study.Both of them could keep button mushroom fresh for 14 days at 4?.Button mushrooms in the group with LFDS as additive had a better appearance and weight loss ratio than the LMWDcl group,but the group soaked in plasma activated water(PAW)produced the best freshness-preserving effect.