Studies On The Submerged Fermentation And Purification Of Cordycepin

Cordycepin is an important active ingredient of Cordyceps and efficient in the treatment of cancer and leukemia. So Cordycepin was attracting more and more attentions nowadays in the world. It is confirmed that Cordyceps militaris is the predominant strain to produce cordycepin and the concentration of cordycepin in fermentation broth was higher than that in the mycelium and fruitingbody, Cordycepin production by fermentation has become a trend today. At the same time, the macroporous resin had a more widespread application in the extraction and separation of natural products, so that it is worthy of study for using macro porous resins on cordycepin extraction and purification from fermentation broth. In this thesis, a cordycepin-producing Cordyceps strain JN168 was isolated for the fermentation, the concentration of cordycepin under different conditions was studied, the HPLC analytic methods for cordycepin was established as while as macro porous resins were studied to separate cordycepin from Cordyceps militaris. The results are as follows:(1) The white mutant fungus was identified as Cordyceps militaris JN168 based on the analysis of its 26S rRNA gene sequence.(2) The fermentation conditions for cordycepin production of Cordyceps militaris JN168 were optimized. The optimum fermentation medium comprised: 40 g/L sucrose, 10 g/L peptone, 0.5 g/L MgSO₄ and the optimum cultivation conditions were: under natural conditions of pH, temperature of 25℃, rotation speed of 200 r/min, fermentation period of 7 d. Under the optimized condition 90 mg/L of cordycepin could be obtained.(3) The concentration of cordycepin was determined using an SHIMADZU LC-20AB HPLC system (JAPAN) equipped with a Media C18 column (0.46mm×250mm, 5μm). The HPLC was performed at 260 nm using water: methanol (85:15, v/v) as the elutant at a flow rate of 1.0 mL/min.(4) Ten kinds of macro porous resins were applied to separate cordycepin from Cordyceps militaris broth in order to select the best resin, and the resin of H103 was selected as the best one. Then the adsorption conditions and elution conditions of cordycepin with H103 were studied. The results showed that H103 resin had the better adsorpting capacity when pH value was 9.0, concentration of sample was 0.2 g/L, and the flow rate was 2 BV/h. And the cordycepin could be eluted from H103 resin with 40% ethanol.