Screening Of Strains With High Production Of Exopolysaccharide Isaria Cidada And Preliminary Study On Polysaccharide Properties

Cordyceps Cidadae,as a traditional medicinal and edible fungus in China,the active ingredients of Cordyceps Cidadae is mainly composed of polysaccharides,with multiple effects such as anti-tumor,improving immunity,anti-oxidation and anti-aging.In recent years,wild cidadae is affected by the environmental factors and human factors limited resources,unable to meet the market demand,and the quality of cidadae from different sources varies,thus affecting their effectiveness.Liquid fermentation culture has the characteristics of short fermentation period and controllable product quality.Therefore,samples of Isaria cidada were collected from the field,several strains of cordyceps were isolated by spore separation method,and a strain of Isaria cidada in the article.with short growth cycle and high extracellular polysaccharide yield was screened out.The fermentation technology was optimized to extract extracellular polysaccharide,and the physical and chemical properties and activities of the polysaccharide were studied.The specific aspects of the research are as follows:(1)Forty-three strains were isolated from wild cidadae collected by spore separation method.By comparing the content of exopolysaccharide in liquid deep fermentation,one strain of Isaria cidada with short growth and high yield was obtained.ITS sequence was further used for PCR amplification,and NCBI sequence alignment analysis was performed to identify Isaria cidada named as C1.(2)The fermentation medium and culture conditions of C1 were optimized by using the method of deep liquid fermentation and taking the yield of extracellular polysaccharide as the screening condition.The optimum technological conditions were determined as follows:glucose 6%,peptone 0.4%,yeast extract 0.2%,KH₂PO₄ 0.15%,Mg SO₄·7H₂O 0.075%.With a natural p H,a rotating speed of 160 r/min,a temperature of 28?,an inoculant volume of 6%,and fermentation of 4 days,the maximum yield of C1 exopolysaccharide could reach 4.99 g/L,4.2 times higher than that before.(3)Classification using ethanol precipitation for separation and purification of extracellular polysaccharide,eventually get CEPS30E polysaccharide components,the purified polysaccharide components CEPS30E with further scans the ultraviolet spectrum do not contain proteins and nucleic acids,with infrared spectrometer scanning,preliminary determination CEPS30E for polysaccharide substance.Ultrasonic degradation can increase the solubility of CEPS30E.HPSEC-MALLS-RI was used to detect the CEPS30E presenting a single symmetrical peak.The weight average MOLECULAR weight Mw and the number average molecular weight Mn were 7.668×10⁶ Da,6.075×106 Da and the dispersion coefficient PD(Mw/Mn)were 1.144.The narrow molecular weight distribution suggested that the CEPS30E was well homogenized.(4)The monosaccharide composition of CEPS30E is glucose and galacturonic acid by anion chromatography,and the ratio of glucose and galacturonic acid is 92:2,respectively.The glycoside bond connection mode and relative molar ratio of CEPS30E are analyzed by hypermethylation,reduction and acetylation.CEPS30E is mainly composed of T-Glcp,1,3-?-D-Glcp,1,2,3-?-D-Glcp,1,3,6-?-D-Glcp,1,2,6-?-D-Glcp,with a small amount of1,4-?-D-Glcp,1,6-?-D-Glcp.The molar ratios of glycosidic bond types were40.3:13.5:10.7:16.6:13.3:3.6:2.0.(5)The biological activity of producing extracellular polysaccharide from Isaria cidada C1was studied preliminarily.Cell experiments showed that CEPS30E at 25?200?g/m L could activate the production of NO in mouse monocyte macrophage RAW 264.7.With the increase of polysaccharide concentration,the release of NO gradually increased,showing a certain dose-effect relationship.When CEPS30E concentration is 200?g/m L,the release of NO is 33mol/L,which shows good immune activity.