Study On Optimize Fermentation Conditions For Producing Podophyllotoxin From Endophytic Fungui

Podophyllotoxin is of great important in the field of medicine because of its antivirus and anti-cancer activities. It was derived mainly from the podophyllum plants. But the natural sources were limited. Endophytic fungi have the activity that can produce same compounds to the plant. It is a new method by fermentation of endophytic fungui. So far, many research focused on separating valuable endophytic fungi which can produce podophyllotoxin.Through study the optimizational ferrnen tation conditions of producing-strains, in the hope of providing some theoretic reference on producing botanic activity compounds by microbiology fermentation.Objectives: Study on the fermentation conditions of producing-strains in order to provide a new method obtaining anti-cancer material——podophyllotoxin. and the technical data obtained from research on producing botanic activity compounds by microbiology fermentation. This will be lay the foundation for industrialization. Methods: The components were identified by thin layer chromatographic(TLC),Thin layer chromatographic scanning,high performance liquid chromatographic(HPLC) and ¹H nuclear magnetic resonance spectral(¹H-NMR) that consist in fermentation culture of Md and Iy strains; The thesis designed semi-synthesized medium compatible to Md and Iy strains, and optimized conditions with flake shaker respectively. the conditions that extract the podophyllotoxin materials from the cultures were screened by the orthogonal L₉ (3⁴) experiment. Results: This thesis confirm that the one strain Md can produce podophyllotoxin and the other strain Iy can produce podophyllotoxin glycoside. The optimizational ingredient of the semisynthesized medium were as follows: 2%soluble starch,2%sucrose,0.2% (NH₄)₂SO₄,0.2% peptone,0.1% yeast extract,0.02% K₂HPO₄,0.02%CaCO₃, 0.02% NaCl, pH6.8. The biomass increase 34.7% and the production-biomass ratio increase 13.9% by using the optimizational medium. The optimized culture conditions of Md strain were: initial pH6.0, liquid capacity 100mL in 250mL flask, 15% inoculating concentration, at temperature of 28℃for 7d. That of Iy strain were: initial pH6.5, liquid capacity 80mL in 250mL flask, 10% inoculating concentration, at temperature of 28℃for 6d. The under-stream extract conditions were: The mycelium freeze, for 4h, break up for 20 seconds with tissue triturator, then extracted with chloroform triplicity volume for 3 times, 10h each time.The extraction rate increased obviously. The scale-up trial in 50L fermentor showed that mycelium grew well and products produced steadily.Conclusions: The technics that obtain podophyllotoxin with fermentation and the technique for extraction were gained. This research has the potential of industrialization.