Optimization Of Extraction Technology,structure Identification And Biological Activity Analysis Of Polysaccharide(LDG-M) From Lactarius Deliciosus Gray From Barkam City

Lactarius deliciosus Gray belongs to Lactarius of Basidiomycotina,Hymenomycetes,Agaricales,Russulaceae.It has a lot of common name,such as delicious pine mushrooms,pine bacteria,black pine bacteria,and milk paste bacteria and so on.Lactarius deliciosus Gray has an obvious morphological characteristic,which is identifted.Under normal circumstances it is orange,and partial white or milky yellow in part.Their distribution is very extensive and they are mainly distributed in the middle and lower reaches of the Yangtze River and coastal areasin China.These places like Yunnan,Sichuan,Hunan and other places are the main production place of L.deliciosus Gray.At present,the research on L.deliciosus Gray is mainly focused on artificial domestication cultivation and genetic diversity research,but the research on extraction,purification,structure analysis and biological activity of polysaceharides of L.deliciosus Gray is rarely reported.Therefore,the crude polysaceharides from L.deliciosus Gray was extracted by hot water extraction method and the crude polysaccharide was purified by DEAE cellulose column.The structure of polysaccharide from L.deliciosus Gray was analyzed by optical chromatography,and its biological activity was studied to provide a theoretical basis for the research of L.deliciosus Gray.This paper is divided into three parts,namely,extraction and isolation of polysaceharides from L.deliciosus Gray,structural analysis of polysaceharides from L.deliciosus Gray and immunoregulatory activity of polysaceharides from L.deliciosus Gray,including the following:1 Extraction and Isolation and Purification of Polysaccharide from LDG-MIn this chapter,the crude polysaceharides of L.deliciosus Gray were obtained by hot water extraction.Ethanol was used to elute the protein.DEAE-cellulose ionexchange column was used to purify the crude polysaccharide.The orthogonal experiment was used to study the purification process of polysaccharide,and found that the optimum process of selecting 180 g of column packing,0.5 g of sample loading and0.01 mol/L of elution concentration would gain the maximum amount of polysaccharide,with a polysaccharide yield of 12%.2 Structural Analysis of polysaceharides from LDG-MThe average molecular weight of LDG-M was 573689 Da by high performance gel permeation chromatography.The characteristic groups of polysaceharides were analyzed by infrared spectroscopy and nuclear magnetic resonance spectroscopy.Thepolysaccharide was mainly composed of fructose and galactose at a ratio of about 2: 3compositions by high performance liquid chromatography.3 Studying on the Immunoregulatory Activity of Polysaccharide from LDG-MThe biological activity was investigated by a series of cell text,such as cell proliferation assay of mouse monocyte-macrophage Raw264.7 cell,neutral red phagocytosis test,tumor killing assay in vitro,TNF-? and IL-6 ELISA assay,B cell proliferation and B cell cycle experiment.The results showed that LDG-M could significantly increase the efficiency of monocyte-macrophage RAW264.7 cells,which was 87.8% when LDG-M concentration was 5 ?g/ml.And it is significantly promoted the phagocytic activity of neutral red.When the concentration of LDG-M was 2.5 ?g/ml,it could increase the phagocytic efficiency of 61.4%.The killing activity of L929 was increased by 49.8% when the LDG-M concentration was 5 ?g/ml.When the concentration of LDG-M was 2.5 ?g/ml,TNF-? could increase 13.4 ratio of the secretion.When the concentration of LDG-M was 5 ?g/ml,the secretion of IL-6 was increased by 23.5 times;LDG-M significantly increased the proliferation of B lymphocytes.When the concentration of LDG-M was 2.5 ?g/ml,the proliferation rate of B lymphocytes was increased by 39.9%.Through the above research,we obtain the conditions for the extraction of Barkam L.deliciosus polysaccharides,identified the polysaccharide is a novel polysaccharide structure by fructose and galactose approximately to the ratio of 2:3.And the polysaccharides have good in vitro immunological activity.This study provides a theoretical basis and scientific basis for the development and utilization of Barkam pine mushroom.