Production And Isolation Of Cordycepin From Solid State Fermentation

Cordycepin is one of the most active components of Cordyceps militaris,which has important medical value and extensive pharmacological effects.However,cordycepin is very low in natural Cordyceps militaris and is difficult to extract and purify,these factors affect the application and further study of cordycepin.Therefore,improving the production of cordycepin has become an urgent problem.In the early stage of laboratory,a stable and high yield strain was obtained by mutation breeding.In this paper,the solid-state fermentation conditions and isolation conditions of cordycepin were studied by this starting strain.Firstly,the medium compositions and culture conditions of Cordyceps militaris by solid-state fermentation were optimized.Through a series of single-factor experiments and orthogonal experiments,the rice was selected as the fermentation substrate,glucose and yeast extract were respectively the optimal carbon and nitrogen sources,the optimum medium compositions and culture conditions were: rice 30 g?diameter 0.90¹.25 mm?,solid-liquid ratio 1:1.2,glucose 3%,yeast extract 2%,wheat bran 1.5%,inoculation amount 20%,seed age 2 d,fermentation time 10 d,under these conditions cordycepin yield was 4.83 mg·g^-1.Secondly,the extraction conditions were optimized.The optimal extraction method was: alcoholic concentration 40%,solid-liquid ratio 1:20,pH 4,which was extracted at 30 ? for 2 times,extraction time 2 h.After extraction,removed the solid residue,then through rotary evaporation and freeze-drying,the purity of cordycepin was 5.56%.Thirdly,the purification of cordycepin was carried out by macroporous adsorption resin.Through the screening of 20 macroporous adsorption resins,we found that the effect of H103 resin on the separation of cordycepin was better,and then analyzed the factors affecting the separation effect.The optimum adsorption and desorption conditions were: pH 7,cordycepin concentration 2.35 mg·m L^-1,flow rate 2 m L·min^-1,loading amount 8 columne volume,elution solution 40% ethanol,elution flow rate 2 m L·min^-1,elute 5 columne volume.Through rotary evaporation and freeze-drying,the purity of cordycepin could reach 65.37%.Finally,preparative chromatography was used to purify cordycepin.Firstly,the preparation conditions of cordycepin were selected,the optimized condition of ratio of methyl alcohol to water 55:45.Secondly,the analytical conditions of chromatography were amplified linearly,and the preparation conditions were optimized,the optimized conditions were methyl alcohol to water 50:50,flow rate 16 m L·min^-1,upper sample volume 0.2 m L,upper sample concentration 4 mg·m L^-1,retention time 45 min.Under these conditions,cordycepin purity was 98.36%.