Mutagenesis And Fermentation Conditions' Optimization Of A Sipeimine-Producing Endophytic Fungus

The strain P7 is a sipeimine-producing endophytic fungus isolated from the bulbus of a medicinal plant Fritillaria ussuriensis.To solve the degeneration problem of P7 during the course of preservation, the strain was rejuvenated by adding different concentration of Bulbus F. ussuriensis into culture medium. The results showed that, the biomass and sipeimine yield of the strain rejuvenated by adding 3% bulbus were effectively improved, while the bulbus concentration exceeded 6%, the biomass was also improved, but the sipeimine yield reduced.In order to get mutant strain with higher sipeimine yield, different physical and chemical mutagens were used to mutagenize the initial strain P7, including UV, He-Ne laser and HNO2. Then comparing the positive mutation frequency within selected dosage, the results showed that the best single mutagen for P7 was UV, the second was HNO2, and He-Ne laser was the last. Then the dominant strains mutagenized by single mutagen as the second initial strains were further mutagenized, and a dominant strain P-UH-3 was selected, its sipeimine yield and biomass were 0.0881mg/L and 2.80g/L, respectively,89.9% and 18.6% higher than those of the initial strain P7. The yield was constant after successive culture of 10 generations.In order to further improve the product yield, the culture conditions of the mutant strain P-UH-3 were then optimized. Based on the growth rate and the yield of sipeimine, the optimal solid culture medium was PDA and the liquid culture medium was bean sprouts medium. The suitable fermentation conditions for sipeimine accumulation of P-UH-3 were then further optimized by single-factor and the orthogonal experiment. The results showed that bean sprouts liquid medium as a basic culture medium, adjusted the glucose concentration to 10%, peptone 0.15%, pH 7.0, age of inoculum 36h, inoculation volume 10%, the temperature 26℃and culture period 7d. Comparing with the initial condition, the sipeimine yield was 0.1258mg/L, improved by 42.8%, but the change in biomass was not obvious.