The Extraction And Isolation Of Lentinus Edodes Residue Polysaccharide And Its Anti-inflammatory Activity

With the gradual increase in the production of shiitake mushrooms in my country,the amount of waste mushroom scraps produced by the cultivation of shiitake mushrooms has also increased sharply and will cause resource waste and environmental pollution.However,there are a lot of bioactive substances such as polysaccharides in mushroom bran.Therefore,it is of great significance to study the high-value comprehensive utilization of mushroom bran.The purpose of this subject is to study the effects of compound enzyme-high temperature and high pressure pretreatment on the extraction rate,structural characteristics and activity of water-soluble polysaccharides in the degradation process of Lentinus edodes residue,and to determine optimal process parameters of enzymatic extraction,so as to provide development and application technical support.Firstly,determine the preparation process of the crude polysaccharide of Lentinus edodes residue,and fractionate the extract.The high temperature and high pressure steam method(121?,30 min)was used to pretreat the mushroom residue,and then the compound enzyme was used to assist the extraction.The optimal extraction parameters were determined through different enzyme ratios,single factor and response surface optimization experiments.The results showed that the enzyme ratio(cellulase:xylanase:?-glucanase)was 3:2:1,the total amount of enzyme added was 660 U/g,pH 5.20,enzymatic hydrolysis temperature was 45?,enzymatic hydrolysis time was 4 h and the material-liquid ratio was 1:20.Under this condition,the extraction rate of total sugar was 13.4%.Compared with direct water extraction(LR-W)and direct high temperature and high pressure extraction(LR-U),the yield of water-soluble polysaccharides in the high temperature and high pressure-complex enzymatic method(LR-UE)is significantly improved.The results of polysaccharide content and molecular weight distribution show that the extract obtained by high temperature and high pressure-complex enzyme method from Lentinus edodes residue can effectively separate and enrich the crude extract after 80%ethanol(v/v)precipitation and dialysis treatment.Secondly,evaluate the in vivo and in vitro anti-inflammatory activities of the crude polysaccharides from Lentinus edodes residue.Lipopolysaccharide(LPS)was used to stimulate macrophage RAW264.7 to establish an in vitro inflammation model.Cell experiments showed that the inhibitory effect of LR-UE on the release of nitric oxide(NO)from RAW264.7 was significantly better than that of LR-W and LR-U.This shows that the use of high temperature and high pressure-complex enzyme method can not only improve the extraction efficiency of crude polysaccharides but also enhance its anti-inflammatory activity;the two components LR-UE3 and LR-UE4 that obtained by LR-UE separation showed good anti-inflammatory activity in vitro and inhibit NO in a dose-dependent manner.Dextran sodium sulfate(DSS)induced acute colitis model in C57 mice were established to further evaluate the anti-inflammatory activity of LR-UE3 and LR-UE4 in vivo.The results showed that LR-UE3 and LR-UE4 could adjust the level of oxidative stress,improve the pathological damage of mice tissues,and reduce the release of inflammatory factors by down-regulating the expression of inflammatory factors,and play a role in the treatment of ulcerative colitis in mice.The therapeutic effect could closely related to the molecular weight of polysaccharides.Finally,isolation,purification,structure analysis and anti-inflammatory in vitro study of the purified components of the crude polysaccharide from Lentinus edodes.Combined with the molecular weight distribution of LR-UE3,Sephacryl S-3 00 gel with a separation range of 2×103-4×105 was selected for separation and purification,and two homogeneous components which were named N-2 and N-3 respectively were obtained.Among them,the polysaccharide content of N-2 is 83.10%,the protein content is 6.47%,and the weight average molecular weight(Mw)is 2.067×104 g/mol;the polysaccharide content of N-3 is 72.96%,the protein content is 15.88%,and the Mw is 1.184×104 g/mol.Analysis of monosaccharide composition showed that N-2 and N-3 are heteropolysaccharides composed of arabinose,galactose,glucose,xylose and mannose;the molar ratios are approximately 25.14:7.02:15.33:35.59:16.91 and 18.53:13.60:20.62:31.62:15.62.Infrared and nuclear magnetic results can infer that N-2 and N-3 are glycoprotein complexes composed of pyranose linked by ? and ? glycosidic bonds.Cell experiments showed that compared with N-3,when low molecular weight N-2 acts on LPS-stimulated RAW264.7 cells,it has a significant inhibitory effect on the release of NO,can significantly reduce the release level of inflammatory factors,and perform better in vitro anti-inflammatory activity.