Isolation Of Cordycepin From Submerged Liquid Fermentation With Cordyceps Militaris

Cordycepin,also known as 3'-deoxyadenosine,is one of the important active component in Cordycepin militaris,which has attracted worldwide attention and research because of its pharmacological value of treating leukemia,anti-bacterial,anti-inflammatory and enhancing immunity and effect of inducing apoptosis of lung cancer,breast cancer and brain cancer.However,the shortcomings of low yield,long production cycle and difficulty in isolation of cordycepin have become the bottleneck restricting its further research and application.The submerged liquid fermentation by Cordyceps militaris is the most effective method to produce cordycepin at present,but its yield and production intensity are still unable to meet its large-scale production and application.Therefore,how to obtain cordycepin throught submerged liquid fermentation by Cordyceps militaris is the key of research.In view of this in situ product removal was proposed to weaken the feedback inhibition during fermentation process and enhance the production of cordycepin.The results are as followed:(1)The carbon source,nitrogen source and spore concentration in the medium were optimized.The highest cordycepin yield could be reached 660.99 mg/L with the synthetic medium contained 30 g/L glucose,5 g/L(NH4)2SO4 and 106 spores/mL,which was 47.6%higher than that of the control group without optimization,and 340%higher than that of the control group using natural medium.(2)The separation effect of cordycepin from fermentation broth by macroporous resin adsorption method was studied.The macroporous resin NKA-II with better adsorption effect was screened out through static adsorption experiment,and the factors affecting its adsorption and desorption efficiency were optimized.The optimum adsorption and desorption conditions were obtained as follows:the optimum sampling velocity was 2 BV/h,the sample volume was 17 times,the adsorption rate was 94.93%,the optimum desorption velocity was 3 BV/h,the eluent was 10 times the column volume of 100%anhydrous ethanol,and the desorption rate was 98.18%.The inhibition of cordycepin on Cordyceps militaris growth and cordycepin synthesis was confirmed by adding cordycepin in vitro.In the fermentation process,adding macroporous resin NKA-II in situ could effectively weaken the inhibition and increase cordycepin production.On the 9th day of fermentation,adding 2%macroporous resin NKA-II,the cordycepin production reached 741.9 mg/L,which was 16.02%higher than that of the control group.Cordycepin extraction rate was 93.8%,further,the yield of cordycepin increased by 32.07%and the removal rate of cordycepin was 75.64%by ectopic coupled fermentation.(3)Two distinct salting-out systems for cordycepin extraction from fermentation broth were screened out,ethanol/(NH4)2SO4 system and ethanol/K2HPO4 system.When the mass fraction of inorganic salt was 15%and the organic soluble mass fraction was 27%,the recovery of cordycepin was the highest.The recovery of cordycepin was 98.73%in ethanol/(NH4)2SO4 system and 93.79%in ethanol/K2HPO4 system.The extraction efficiency was the highest when the uncentrifuged fermentation broth was used to carry out salting-out extraction system,ethanol/(NH4)2SO4 system can reach 99.56%,ethanol/K2HPO4 system can reach 94.06%.A preliminary attempt was made to couple batch fermentation with salting-out extraction to increase cordycepin content in fermentation broth.The cordycepin yield could reach 793.85 mg/L,which was 25.83%higher than that of the control group when the culture medium was fed into fermentation broth of cordycepin.Cordycepin extraction rate was 95.37%The strategy of submerged liquid fermentation coupled with separation was proposed to product cordycepin innovatively in this study.And two types of system were established.Both of them could efficiently weaken the feedback inhibition of cordycepin,and dramatically enhance the productivity of product,providing novel ideas for industrial cordycepin production.