Studies On Isolation,Identification And Optimization Of Fermentation Conditions Of Endophytic Fungi Producting Artemisinin Derivatives

Artemisinin and its derivatives are widely used as first-line anti malaria drugs,which have the characteristics of good activity, small side effects, narrow sources and so on.Until now the methods of manufacturing of them are still rely on the extraction of Artemisia annua L. in international and domestic. There are problems such as limited source of plant, low content and instability.Many studies have showed that endophytic fungi not only can promote the growth of plants and induce the formation of secondary metabolites in the process of long-term coexistence and interaction with the host plant,and it can also produce the same or similar biological activity with the host plant.The endophytic fungi IS928 were identified by using morphological characteristics and molecular biological methods,which was screened from Artemisia annua L. by using morphological characteristics and molecular biological methods.The culture medium composition and fermentation conditions for the production of artemisinin compounds were studied by liquid-state fermentation,and the fermentation medium was optimized..The main results are as follows:(1)The six endophytic fungi of the different colony morphology were isolated from the stem of the healthy Artemisia annua L..A strain,which named fungi IS928 was found that the mycelium of the ethanol extract reacted with 0.2%NaOH solution and absorbed at 292nm. It was identified as Aspergillus oryzae according to the results of the morphology, the structure of the mycelium and the conidium head structure, and the rDNA ITS sequence analysis and phylogenetic tree construction.(2) The effects of optimal medium composition for the production artemisinin derivatives in liquid-state fermentation including carbon sources,nitrogen sources and inorganic salt were studied by using single-factor experiment and the optimal fermentation medium was optimized by using response surface method.The results showed that the optimal fermentation medium composition were as follows:sucrose 3.15%, yeast powder 2.27%, MgSO4·7H2O 0.173%, KH2PO40.254%, NaCl 0.05%. The optimum fermentation conditions were:the initial pH value 6.5, inoculation quantity 8%, seed age 60h, liquid loading for 100mL/250mL triangle flask, fermentation temperature 30?, shaking speed 170 r/min, fermentation time 5d. Under these conditions, the average yield of artemisinin reached 1831.619?g/100mL.