Hirsutella Sinensis Cultivation Technology Optimization And Extraction,Purification, Structure Performance Elucidation Of A Polysaccharide

Cordyceps sinensis possesses a series of pharmacological effects as a kind of valuable medicinal herbs, the production of natural cordyceps reduce unceasingly. The effective ingredients from liquid deep fermentation of Cordyceps sinensis mycelium are similar to natural ones, which is an effective way to solve the source of natural Cordyceps. The polysaccharides in Cordyceps sinensis is proved to be a kind of key bioactive components which possesses pharmacological activity.In this paper, we optimized the cultivation technology for the submerged fermentation of Cordyceps sinensis (Hirsutella sinensis) by using the advanced detection instruments and multiple parameter correlation analysis in the fermentation process, extracted and purified the Cordyceps polysaccharides from its fermentation product, and then analyzed the molecular structure and oxidation activity of the purified polysaccharides.Firstly, in order to increase the growth rate of mycelium and the active ingredients accumulation, we optimized the cultivation technology for the submerged fermentation of Hirsutella sinensis. The optimal glucose to yeast extract concentration ratio of the fermentation medium was40:33. Through the research of the substrate feeding process, we identified the optimum new technology for adding nitrogen source. Incubation temperature investigation experiments established that the mycelium growth and active ingredients accumulation were most favorable when the temperature was controlled in18°C. Through investigating the relationship between the dissolved oxygen concentration and the growth metabolism, and combining the effecrs of shear stress on the mycelium experiment, we got the control strategy of the rotating speed and the ventilation for the low oxygen fermentation process. Established the relationship between the living cells capacitance, CER and the mycelium growth situation in the fermentation process, realized the process control strategy in supplementary fermentation based on online CER and living cell supplementary. Established the substrate materials hydrolysis procedure and improved the utilization rate.Secondly, we extracted and purified the Cordyceps polysaccharides from the fermentation product of Hirsutella sinensis, and analyzed its molecular structure. The optimal extraction conditions were:the extraction temperature was100°C, the extraction time was120min, and the ratio of solid to liquid was1:15, for four times extraction. The crude polysaccharide was stepwise purified by sevage deproteinization, dialysis, DEAE ion exchange and Sephadex G-100column chromatography, and three kinds of polysaccharides (named HSP-1, HSP-2, and HSP-3) were obtained. After ultraviolet scanning and Sephacryl S-300HR column determination, HSP-1and HSP-2were regarded as homogeneous components while HSP-3was inhomogeneous. The results of phenol-sulfuric acid method determined that the contents of HSP-1were89.42%. The average molecular weight of HSP-1, which was determined by Sephacryl S-300HR column chromatography, was estimated to be1.7x104Da. The results of GC-MS showed that monosaccharide compositions of HSP-1contain D-glucose, D-mannose and D-galactose in which their molar ratio was4.522:1:1.378. The FT-IR detection showed that the a-configuration glycosylic bond consisted in HSP-1. The structural characteristics determination with a combination of chemical and instrumental analysis methods (including periodate oxidation-Smith degradation, methylation analysis, partial hydrolysis, GC-MS and FT-IR) revealed that HSP-1was a branched polysaccharide possessing a backbone of (1â†'4)-α-D-glucose residues(～70%),(1â†'4)-a-D-mannose residues(～15%) and (1â†'4)-α-D-galactose residues(～15%). The branches were at the (1,2,4,6â†')-α-D-glucose residues(-8%) of the backbone, mainly composed of (1â†'4-α-D-glucose residues,(1â†'4)-α-D-galcatose residues,(1â†'4)-α-D-mannose residues, and terminated with a-D-galactose residues.Lastly, the oxidation activity of the purified polysaccharides HSP-1was analyzed. The in vitro antioxidant assay proved HSP-1possessed the hydroxyl radical-scavenging activity and the DPPH radical-scavenging activity with an IC50value of0.834mg/mL and0.714mg/mL, respectively.