Study On The Fermentation Of Nucleosides In Cordyceps Militaris

Cordyceps militaris also called north cordyceps, is the type species of the genus cordyceps and are widely distributed in China. So far Cordyceps militaris is the only cordyceps that be able to cultivation mass in China, and cordycepin production capability of it is the most powerful.In this paper, To optimize the ultrasonic extraction conditions and to develop a method by HPLC for simultaneously determination of four nucleotides （uridine, guanosine, adenosine and cordycepin） in Cordyceps militaris. optimization of the ultrasonic extraction conditions was conducted by orthogonal layout. The HPLC analysis working conditions: column is Amethyst C18-H（4.6 mm×250 mm, 5μm）; solvent is methanol-water （15:85）, retention time is 18 min; flow rate is 0.8 mL·min^-1; temperature is 35℃; detective wavelength is 260 nm; injection volume is 10μL. The optimum extraction conditions were confirmed as follows: the ratio of sample to water is 1:30; solvent is pure water; extraction temperature is 25℃; extraction time is 45 min. The HPLC method used in this experiment is fast, simple and convenient, and it is valuable for quality evaluation of Cordyceps militaris. The content of 4 nucleotides （uridine、guanosine、adenosine、cordycepin） in fermented broths of 3 kinds of Cordyceps militaris strains were analyzed by HPLC, and it was found that the strain Cordyceps militaris gaoyou was the highest in 4 nucleotides content. Optimization of the fermentation conditions for strain Cordyceps militaris gaoyou by orthogonal layout showed that the optimal medium was 25 g·L^-1glucose, 15 g·L^-1maltose, 10 g·L^-1peptone, 1 g·L^-1yeast powder, K2HPO4 1 g·L^-1, and KH2PO4 1 g·L^-1. Furthermore, the investigation of the culture conditions showed that the pre-shaking combined with late static culture could significantly improve the nucleoside yield and 1.8 times higher than those with simply shaking culture. In addition, late static culture combined with illumination could further increase nucleosides yield by 30%.The sduty on the dynamic changes of 4 main nucleoside components（uridine, guanosine, adenosine, cordycepin） in fermentation of Cordyceps militaris strain gaoyou showed that ,both in the mycelium or in broth, uridine, guanosine, adenosine levels were presented downward trend with the fermention time extended, with the cordycepin content with increasing fermentation time extended the contrary. The first 2 days of fermentation, 4 nucleosides were detected in mycelium. Uridine, guanosine, adenosine levels first increased and then decreased. Starting from the fermentation, cordycepin content was showed an upward trend, rising after the 8th day especially, until the end of fermentation.Presence of 4 nucleosides was detected in broth in next day fermentation. Uridine, guanosine content has been showing a downward trend, and we can not detect both in the 10th. High levels of adenosine in broth with the beginning of fermentation, and in the former 6 days, adenosine levels was detected showing rapid downward trend, after the 6th day, adenosine levels continued to decline but the trend slowed down until the 12 days culture could not be detected in the broth. Cordyceps has been accumulating, 8 days after the content has become very clear upward trend continued until the end of fermentation.In this study, we first found a certain amount of light can promote the production of cordycepin in fermentation of Cordyceps militaris, and first studied dynamic changes of 4 nucleosides during the fermentation of Cordyceps militaris. These results provide reference for the industrial production of Cordyceps militaris, and also provides a possible entry point on the the mechanism of the biosynthesis.