Extraction,Purification And Immunological Activity Of Exopolysaccharide Of Cordyceps Militaris

Cordyceps militaris(C.militaris)is an entomogenous fungus with the same medicine and food,and is classified in the Ascomycota,Hypocrea,and Ergotaceae.It is rich in a variety of active ingredients,which can prevent oxidative free radicals,enhance immunity,lower blood sugar and blood lipids,prevent cancer and radiation,inhibit pathogenic bacteria,protect the nervous system,and calm and sleep well.In recent years,C.militaris has been widely used in the food field.Many healthy foods such as Cordyceps beverage,Cordyceps wine,Cordyceps bread,Cordyceps soy sauce,Cordyceps porridge,Cordyceps polysaccharide yogurt,etc.have been developed.Studies have shown that polysaccharides are the primary active component of C.militaris and have rich effects,the most prominent of which is immune regulation.However,the current research on C.militaris polysaccharides is mostly focused on intracellular polysaccharides.There are few reports on the physiological activity of C.militaris exopolysaccharides,and its immunological activity and its mechanism of action are still unclear.Therefore,this study conducted a detailed study on the structural characteristics,immune activity and mechanism of C.militaris exopolysaccharide.It laid a theoretical foundation for the rational development of C.militaris exopolysaccharides,and provided a scientific basis for the rational development and efficient utilization of C.militaris resources in the food field.The specific research content is as follows:1.C.militaris strain(CCIC 14014)was cultivated by liquid fermentation technology,and the content of C.militaris exopolysaccharide was used as an indicator.The single factor test method combined with the response surface method was used to optimize the C.militaris strain fermentation medium formula,and then the anion exchange column(DEAE-Sephacel)combined with the dextran gel column(Sephadex G-200)was used to separate and purify the C.militaris exopolysaccharide.The optimization results of the fermentation medium showed that the best ratio is:potato juice 200 g/L,sucrose 31 g/L,peptone 20.5 g/L,KH₂PO₄1.5 g/L,Mg SO₄1.5 g/L,Vitamins B₁10.5 mg/L.Under these conditions,the extracellular polysaccharide content of C.militaris was 2.57 mg/m L,which was 85.27%higher than before optimization.The crude exopolysaccharide of C.militaris was extracted in large quantities and named as EPS.The purity was determined to be 64.32%,and the EPS was further separated and purified by column chromatography,and then the polysaccharide fraction with the highest component content was collected and named AEPS-?.Its purity is as high as 92.26%,which can be used for subsequent polysaccharide structure analysis.2.The structure analysis of AEPS-?utilizes Ultraviolet Visible spectrum(UV),High Performance Liquid Chromatography(HPLC),Fourier Transform Infrared Spectroscopy(FT-IR)and X-ray diffraction(XRD)techniques.Among them,there were no absorption peaks at 260 nm and 280 nm in the UV,which proves that there was no protein and nucleic acid in AEPS-?;the molecular weight of AEPS-?was 61.52 k Da determined by HPLC;FT-IR results show that the peaks of AEPS-?at 1516.05 cm^-1,1080.14 cm^-1 and 1024.20 cm^-1 are characteristic peaks of the pyranose ring,and the peaks at 844.82 cm^-1 belong to the?-glycosidic bond.It is proved that AEPS-?is a pyranose with?-glycosidic bond;after further hydrolysis of AEPS-?,its monosaccharide composition was analyzed by 1-Phenyl-3-Methyl-5-Pyrazolone(PMP)-HPLC.The results showed that AEPS-?was composed of Mannose(Man),Glucuronic acid(Glu A),Rhamnose(Rha),Galacturonic acid(Gal A),Galactosamine(Gal N),Glucose(Glu),Galactose(Gal)and Arabinose(Ara)are composed of a molar ratio of 1.07:5.38:1:3.14:2.23:15:6.09:4.04,indicating that AEPS-?is an acidic polysaccharide;The XRD results of the ray diffractometer showed that AEPS-?belonged to the amorphous form.3.To explore the impact of EPS on the immune function of mice by measuring the common indicators used to evaluate the immune-enhancing health food in the"Health Food Inspection and Evaluation Technical Specification Implementation Manual".Divide the test into blank group(NC),model group(MC),positive group(PC)and low,medium and high dose groups of EPS(EPS-L=50,EPS-M=100,EPS-H=200 mg/kg).Three experiments of delayed allergy,serum hemolysin and carbon clearance were carried out to evaluate the effects of EPS on the function of T,B and macrophages in immunosuppressed mice.The results of the study:(1).The results of the delayed allergic reaction experiment show that EPS can improve the immune function of T cells by increasing the swelling rate of the ears of mice;(2).The results of the serum hemolysin experiment show that EPS can improve the mouse's Half of the hemolysis value can improve the immune function of B cells;(3).The results of carbon clearance experiments show that EPS can improve the phagocytic function of macrophages by increasing the phagocytic index of mice.4.Explore the immune regulation mechanism of AEPS-?on cyclophosphamide-induced immunosuppressive mice.The test was divided into NC,MC,PC and low,medium and high dose groups of AEPS-?(AEPS-L=50,AEPS-M=100,AEPS-H=200 mg/kg).The immunomodulatory effect of AEPS-?is studied by measuring body weight,spleen index,thymus index,spleen and thymus tissue morphology,splenic lymphocyte proliferation,cytokine and immunoglobulin content of mice.The results of the study showed that the AEPS-?dose group could significantly regulate the body weight,spleen index and thymus index of immunosuppressed mice(P<0.01),at the same time,it can restore the abnormal morphology of the spleen and thymus tissue slices induced by cyclophosphamide;AEPS-?can effectively increase the proliferation of T and B lymphocytes,which was found in the splenic lymphocyte proliferation experiment.The results of flow cytometry showed that AEPS-?had a more pronounced effect on the proliferation of B lymphocytes;Among them,the cytokines IL-2 and INF-?secreted by T lymphocytes showed a tendency to first increase to a normal level and then slightly decrease.It shows that AEPS-?exerts its immune activity on the body mainly through humoral immunity.Further extract the spleen lymphocyte proteins of each group of mice to explore its effect on the expression levels of JNK,ERK and p38 proteins in the MAPKs signaling pathway.The results showed that the immunoregulatory mechanism of AEPS-?is to increase the phosphorylation level of the main proteins JNK,ERK and p38 in the MAPKs signaling pathway,thereby restoring the mouse's immune hypoxia induced by cyclophosphamide.Conclusion:1.In this study,the fermentation medium of C.militaris strain(CCIC 14014)was optimized,and the best ratio was obtained:potato juice 200 g/L,sucrose 31 g/L,peptone 20.5g/L,KH₂PO₄1.5 g/L,Mg SO₄1.5 g/L,Vitamins B₁10.5 mg/L;under these conditions,the content of C.militaris exopolysaccharide(EPS)is 2.57 mg/m L,and the purity is 64.32%;2.Column Chromatographic separation and purification yielded a single polysaccharide component with a purity of 92.26%,named AEPS-?,which belongs to the acidic?-pyranose with a molecular weight of 61.52 k Da;3.This study shows that AEPS-?has a good immunomodulatory function.Effectively promote the proliferation of B lymphocytes,increase the secretion of immunoglobulin,and then exert humoral immune function.4.The mechanism by which AEPS-?restores the immunodeficiency caused by cyclophosphamide in mice is to increase the phosphorylation levels of the main proteins JNK,ERK and p38 in the MAPKs signaling pathway.