Study On The Extracellular Polysaccharides Produced By The Fermentation Of Paecilomyces Cicadae

Cordyceps cicadae is Chinese traditional medicine and food fungus,similar to cordyceps sinensis,it contains polysaccharides,nucleosides,proteins,amino acids,fatty acids and other active substances and it has many pharmacological effects,such as nourishing health,improving renal function sedation and hypnosis,immune regulation,and anti tumor,so it has broad space of development and application.Polysaccharide is the main bioactive substance of Cordyceps cicadae,and it has anti-tumor,anti-aging,antioxidant and many other effects.Due to the scarcity of natural Cordyceps cicadae,microbial liquid fermentation technology has the advantages of easy operation,low cost,short fermentation cycle,high yield,and high product quality,and it has been reported that the fermentation broth of Paecilomyces cicadae contained a large amount of hyaluronic acid,so it can be used to extract polysaccharides through liquid fermentation of Paecilomyces cicadae and replace natural Cordyceps cicadae.In this article,liquid fermentation technology was used to produce extracellular polysaccharides from Paecilomyces cicadae,the fermentation process conditions were optimized,the extracellular polysaccharide was separated and purified and its structure and component were analyzed which layed foundation for the development and application of Paecilomyces cicadae exopolysaccharides.The main contents and results of the study were listed as follows:（1）The liquid fermentation process was optimized for the target of improving polysaccharide content and microbial biomass.The best fermentation medium(g·L^-1):sucrose 80,beef extract 7.5,yeast extract 1.25,MgSO₄·7H₂O 3,KH₂PO₄ 2,bran 5 was obtained.The best liquid fermentation conditions was temperature 24°C,rotation speed 180 r·min^-1,inoculum dose 10%,loaded liquid 75mL.Under this condition,the highest exopolysaccharides yield was 5.96 g·L^-1,and the highest biomass was 42 g·L^-1.（2）In this paper,a small 5 L fermentation tank was used to expand the culture of Paecilomyces cicadae.The optimum initial sugar concentration was 5%,under the condition,the highest exopolysaccharides yield was 5.6 g·L^-1,and the highest biomass was 38 g·L^-1.Under the condition of fed-batch fermentation,exopolysaccharides yield was 5.89 g·L^-1,the biomass was 40 g·L^-1,so the effect of fed-batch fermentation was better than batch fermentation.（3）The separation and purification of extracellular polysaccharide.The ethanol precipitation method was used to extract extracellular polysaccharide,sevag method was used to remove protein,DEAE anion exchange chromatography and gel filtration chromatography was used for the separation and purification of extracellular polysaccharide,and two polysaccharide components with high purity F1,F2 were obtained.（4）The component and structure analysis of the purified polysaccharide components F1,F2,and comparison with hyaluronic acid.The High performance liquid chromatography（HPLC）method was used to analyze monosaccharide composition and molecular weight.The results showed that F1 was a neutral polysaccharide,its monosaccharide composition included arabinose,glucose and galactose,and mannose;F2 was an acid polysaccharide containning uronic acid,and contained glucose and galactose,mannose in addition.The molecular weight of F1 and F2 was 27338.54 kDa,32574.10kDa respectively.The infrared spectra showed that F1 and F2 had tipical absorption peaks of polysaccharides,but the infrared absorption was different with hyaluronic acid.The results showed that Paecilomyces cicadae extracellular polysaccharides was a macromolecular polysaccharide containing uronic acid,and it had certain water retention,which was somewhat similar to hyaluronic acid,but it was different with hyaluronic acid in terms of monosaccharide composition and molecular weight.（5）The intracellular polysaccharides was extracted from the mycelia of Paecilomyces cicadae,and the extraction yield was 60 mg·g^-1.The antioxidant capacity of exopolysaccharides and intracellular polysaccharides of Paecilomyces cicadae was determined.The results showed that extracellular polysaccharides and intracellular polysaccharides both had certain DPPH scavenging capacity,OH scavenging capacity and reducing ability,and had good in vitro antioxidant activity.（6）The detection of the content of intracellular cordycepic acid.The cordyceps acid was extracted by hot water reflux extraction method,and the content of cordyceps sinensis was 116.3mg·g^-1 tested by sodium periodate oxidation method.