Study On The Extraction Technology Of Crude Polysaccharide Of Mussel Thick Shell And Its Relieving Effect On Mice With Colitis

Mytilus coruscus is a bivalve mollusk that is widely distributed in the waters of Zhoushan,Zhejiang Province.It is also known as shell vegetables and mussels.It is rich in nutrition and is an economical shellfish for food and medicine.Mytilus coruscus polysaccharides(MP)has been proven to have immunomodulatory,anti-influenza virus and antioxidant effects,but there are few studies on the improvement of inflammation and its mechanism.Therefore,it is necessary to optimize the extraction process of MP and Carry out further research on the effect of improving inflammation.In this experiment,through response surface experiment,colitis mouse model and RAW264.7 cell inflammation model,the extraction process of MP was optimized and the improvement effect of MP on inflammation was studied.The research method is as follows:(1)Using thick-shelled mussel as raw material,MP is extracted from it by enzymatic hydrolysis.After the single factor experiment,five experimental factors were selected as the material-to-liquid ratio,pH,enzyme amount,enzymolysis temperature and enzymolysis time,and the crude polysaccharide content was the response value to determine the optimal extraction process of MP.(2)Four-week-old male ICR mice were divided into blank group,Dextran Sulfate Sodium Salt(DSS)group,and DSS+Mytilus coruscus crude polysaccharides administration group(low and high dose groups).Except for the blank group,all the other groups changed their drinking water to water containing 3.5% DSS in the last 7 days of the experimental period.After eyeball blood was collected,each intestinal tissue was collected,and the disease activity index(DAI)of each group of mice was scored,and the weight change of colon mice and the degree of colon shrinkage were evaluated,combined with hematoxylin-eosin staining method(HE staining),scanning electron microscope and transmission electron microscope results to observe the changes of mouse colon tissue.Detection and analysis of mouse colon and serum glutathione oxidase(GSH-Px),superoxide dismutase(SOD),inflammation-related indicators tumor necrosis factor(TNF-?),interleukin-6(IL-6),Intestinal loss indicators lipopolysaccharide(LPS),diamine oxidase(DAO)expression and changes in flora.(3)Lipopolysaccharide(LPS)was used to treat macrophage RAW264.7 to cause inflammation in vitro.The proliferation ability of MP on RAW264.7 cells,TNF-?,interleukin-1?(IL-1?),IL-6 and IL-10 were studied by CCK8 method,enzyme-linked immunosorbent method,real-time fluorescent quantitative PCR method and Western blot method.The secretion of 10 and its mRNA expression level and the expression of P-p65/p65 and P-I?B?/I?B? protein in the NF-?B pathway.The experimental results are as follows:(1)Using single factor to optimize the extraction process of enzymatic hydrolysis,the results showed that the MP extraction volume reached the maximum at a material-to-liquid ratio of 1:6,pH of 6.50,1.5% enzyme addition,and extraction temperature of 50 ? for4.5 h.The results of response surface experiments show that the extraction temperature has a great influence on the content of crude polysaccharides,and the interaction term has no significant effect on the content of crude polysaccharides.The interaction effects are ignored.The optimal conditions are obtained by the extreme value analysis of the regression model of the crude polysaccharide content value as: feed liquid The ratio is 1:8,the pH is 6.47,the amount of enzyme added is 1.0%,the extraction time is 4.0 hours,and the extraction temperature is 55 ?.Considering the actual production operation,the optimal extraction conditions are adjusted to 1:8,pH It is 6.50,the amount of enzyme added is 1.0%,the extraction time is 4.0 h,and the extraction temperature is 55 ?.(2)DSS-induced colitis mice lose weight,shorten their colon,reduce DAI score,break intestines,arrange the microvilli disorderly,decrease the expression levels of GSH-Px and SOD in the colon,and decrease the expression levels of TNF-?,IL-6.The expression levels of,LPS and DAO increased,the permeability of the intestine increased,the abundance of Firmicutes in the intestine decreased,and the abundance of Bacteroidetes increased.The expression levels of antioxidant enzymes GSH-Px and SOD in the intestinal tissue homogenate and serum of mice given different concentrations of MP in advance were significantly increased,and the expression levels of inflammatory factors TNF-?,IL-6,LPS and DAO decreased.The abundance of Firmicutes in the intestinal tissues increased,while the abundance of Bacteroidetes decreased.It is proved that MP administration can improve colitis in mice.(3)This experiment proved that the relative proliferation rate of RAW264.7 cells increased after MP treatment,proving that MP has no toxic effect on cell growth.After LPS treatment,the levels of TNF-?,IL-6 and IL-10 increased significantly,and the mRNA expression of TNF-?,IL-1? and IL-6 increased,P-p65/p65 and P-I?B?/The ratio of I?B?increased.The MP pretreatment can reduce the levels of TNF-?,IL-6 and IL-10,the mRNA expression of TNF-?,IL-1? and IL-6,and the ratio of P-p65/p65 and P-I?B?/I?B?.Prove that MP administration can improve cell inflammation.In combination with the above,the material-to-liquid ratio is 1:8,pH is 6.50,the amount of enzyme added is 1.0%,the extraction time is 4 hours,and the extraction temperature is 55 ?.The extraction conditions are the optimal process for MP and can improve acute colitis in mice caused by DSS.And LPS stimulated cell inflammation.This article provides an experimental basis and research ideas for the in-depth study of the medicinal value of MP and the development of it into a natural anti-inflammatory product.At the same time,it provides some theoretical basis for the subsequent development of the marine resource of thick-shelled mussel.