【作者】 曹莉；

【导师】 黄建新；

【作者基本信息】 西北大学 ， 微生物学， 2007， 硕士

【摘要】 鬼臼毒素类物质因具有显著的抗病毒、抗肿瘤活性，在医药领域具有重要的应用价值。目前该类物质的获得主要是从植物中提取，但天然植物资源有限，无法满足市场需求，利用植物内生真菌可产生与宿主相同代谢物的特性，通过微生物发酵获得该类物质，为获得鬼臼毒素类原料药物质资源提供了新途径。目前这方面的研究多集中于对有价值菌株的筛选。本研究对已获得的可产生鬼臼类物质菌株Md的发酵条件进行研究，以期为用微生物发酵法生产药用植物活性成分提供参考。目的：对可产生鬼臼毒素类物质内生真菌进行发酵研究，为解决鬼臼类抗癌药物短缺提供新的途径；并通过研究获得用微生物发酵生产药用植物活性成分的工艺数据，为实现工业化生产奠定基础。方法：利用薄层层析、薄层色谱扫描、高效液相色谱、氢核磁共振确定菌株Md、Iy发酵培养物中的目的成分；设计了适于菌株Md、Iy生长的半合成发酵培养基并分别优化了其摇瓶发酵条件。L₉（3⁴）正交试验优化了从菌株Md发酵培养物中提取鬼臼类物质的提取条件。结果：确定菌株Md可产生鬼臼毒素，菌株Iy可产生鬼臼毒素葡萄糖甙；经优化后的半合成发酵培养基配方为：2％淀粉、2％蔗糖、0.2％蛋白胨、0.2％硫酸铵，0.1％酵母膏、0.02％K₂HPO₄、0.02％CaCO₃、0.02％NaCl，水100ml，pH 6.8。采用该发酵培养基，Md菌株生物量提高约34.7％，鬼臼毒素产量提高约13.9％。Md菌株优化后的摇瓶发酵条件是：初始pH6.0，装液量100mL/250mL三角瓶，接种量15％，28℃培养7天；菌株Iy优化后的摇瓶发酵条件是：初始pH6.5，装液量80mL/250mL三角瓶，接种量10％，28℃培养6天。菌株Md 50L发酵罐放大试验表明菌体生长较好，产物合成稳定。确定的下游产物提取、分离方法为：菌丝体冷冻4h，组织捣碎机破碎20s，用3倍量的氯仿提取破碎的细胞液3次，每次10h；提取率明显提高。结论：得到用发酵法获得鬼臼毒素类物质和从发酵产物中提取鬼臼类物质的技术路线，该研究具有实现工业生产的潜力。更多还原

【Abstract】 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.更多还原