Optimization Of The Liquid Fermentation Technology Of Cordyceps Militaris For Cordycepin Production

As one of the important active ingredients of Cordyceps militaris, cordycepinhas been reported to have a large spectrum of pharmacological functions, including theinhibition of inflammation, elimination of germs, immune regulation, anti-tumor, andtreatment of leukemia and trypanosomiasis. In this study, cordycepin wasproducted by submerged liquid culture of Cordyceps militaris, the optimization ofliquid fermentation medium composition, the supplement of additives and therepeated batch fermentation were investigated in order to increasing the yield.To obtain a high cordycepin yield, the single factor experiment and theresponse surface methodology （RSM） were used for optimizing the mediumcomponents, including the concentration of glucose, yeast extract, KH₂PO₄andMgSO₄. The results showed that the linear dependence of yeast extractconcentration, the quadratic dependence of glucose, yeast extract and MgSO₄concentration have extremely significant effect on the cordycepin yield; the lineardependence of MgSO₄concentration, the interaction between yeast extract andMgSO₄concentration have highly significant effect on the cordycepin yield; theoptimum combination was found to be as following:30.16g/L glucose,10.00g/Lyeast extract,2.04g/L KH₂PO₄, and1.46g/L MgSO₄. Under this condition, theyield of cordycepin predicted was63.69mg/L.After comparing the effects of a variety of additives on the cordycepinproduction, three more effective additives were screened out: citric acid, glycineand NH₄Cl. The optimal concentration obtained by single factor test were citricacid0.6g/L, glycine2.0g/L, NH₄Cl0.5g/L, and the best supplement time was thefourth day. Then the combinations of citric acid, glycine and NH₄Cl were jointlyadded to the medium, and the flask culture results showed that the effect of glycineand NH₄Cl combination was most significant; cordycepin yeild and biomass wereas high as154.66mg/L and0.66g respectively, which were23.35%and46.67%more than separately added glycine, and an increase of33.27%and50.00%thanthe single addition of NH₄Cl.RSM was applied to optimize the amount of glycine, NH₄Cl and supplement time, and the analysis indicated that2.08g/L glycine,0.71g/L NH₄Cl, and the4thdays were the optimal combination. Under these conditions, cordycepin yeildpredicted by the response surface model was147.92mg/L. The verification resultsshow that the average yield of cordycepin was146.15mg/L, consisted with thepredicted values, that proved the experimental results was fitted well with themodel, and the response surface model was feasible.The first fermentation dynamics curve illustrated that it was more appropriateto replace the fermentation liquor with fresh medium at the10thday for repeatedbatch fermentation, and the optimal feeding medium optimized by single factortest were glucose10.0g/L, and yeast extract6.0g/L, KH₂PO₄0.5g/L, MgSO₄,0.6g/L.The stability study of repeated batch production of cordycepin showed that thecordycepin yield of2ndand3rdcycles in repeated cycle fermentation kept a relative stablelevels, although a slight decrease in the4thcycle, but were still higher than that ofthe first fermentation; the fermentation time of each batch was shortened to half,the cordycepin yeild raised5.5%-40.8%of the first batch, and the fermentativestrength increased1.3-2.1times; in that period, the mycelial pellets maintainedgood form, fermentation capacity was stable, cordycepin production basicallytrended to linear. and there is no lag phase, which was a more stable and efficientfermentation process.