产黄酮类物质银杏内生真菌菌株的初步研究

【作者】 王梅霞；

【导师】 陈双林；

【作者基本信息】 南京师范大学 ， 微生物学， 2003， 硕士

【摘要】 植物内生真菌是近年深受重视的一类重要微生物资源，一般认为它们可产生与宿主植物相同或相似的生物活性物质。本研究以传统药用植物银杏为对象，寻找能产生黄酮类物质的内生真菌，为黄酮类物质开辟微生物发酵生产的途径。结果在银杏组织中分离得到了85株内生真菌，初步鉴定发现了分别属于刺盘孢属Colletotrichum、球壳孢属Sphaerosi、镰刀菌属Fusarium、匐柄霉属Stemphylium、盘长孢属Gloeosporium、蜜孢霉属Sphacelia、球座菌属Guignardia等属的菌种。抑菌试验表明其中4个菌株的代谢物对某些细菌有明显的抑制效应；显色反应表明以上4株菌均能够产生黄酮类物质，且其中一个菌株EG₄的总黄酮产率稳定。EG₄培养液经TLC与HPLC分析证明存在与银杏黄酮对照品相同和相似的组分。UV谱分析显示该组分可能具有黄酮母核结构，且具有4’—羟基、A环酚羟基、5—羟基、4—羰基、7—OH、6位、8位同时具有含氧取代基、不具有邻二酚羟基。对该菌株的培养生物学特性研究表明，EG₄在马铃薯葡萄糖培养基上生长较好，最佳生长碳、氮源分别为葡萄糖和NH₄⁺；乳糖、NO₂^-和尿素则能够促进其产生黄酮类物质；最适培养温度为28℃，最佳pH值为自然pH值6.8；100mL／250mL的装液量、100r/min的转速较利于生长；维生素B₁、B₂、C和叶酸都能不同程度的促进其生长，叶酸还能显著提高黄酮产率；该菌产黄酮代谢受Ca（2+）的调节，Ca（2+）能明显促进其产黄酮能力的提高。正交优化试验表明该菌较适宜的发酵条件为：20g/L乳糖、3.2g/L尿素、28℃的培养温度、自然pH值、100mL/250mL的培养基装液量、100r/min、0.5g/L的CaCl₂和微量叶酸。更多还原

【Abstract】 In this paper, 85 strains of endophytic fungi were isolated from Ginkgo biloba. They were identified as Colletotrichum sp., Sphaerosis sp., Fusarium sp., Stemphylium sp., Gloeosporium sp., Sphacelia sp., Guignardia sp. based on morphological taxonomy. The fermentation liquid of 4 strains can inhibit some bacteria. They were proved to produce flavone-like compounds by color reaction analysis. One strain of which the strain EG4 (Colletotrichum sp.) was excellent with steady properties and high production. It was proved that there were similar and same components with the ginkgo flavonoids in the fermentation liquid of EG4 by TLC and HPLC. It could be predicted by UV analysis the components bore the common structure of flavonoids, 4’-carboxyl, 5-carboxyl, 4-carbonyl, A-phenol hydroxyl group. 7-carboxyl,oxygenie substituting groups in 6 and 8 sites, without o-phenol hydroxyl group. Then the cultural characteristics of EG4 was preliminarily studied. The results showed that it could grow well in potato dextrose liquid medium. Glucose and NH4+ were very suitable for its growth, lactose, urea and NO2" could distinctly improve its ability to produce flavone-like compounds, the optimum temperature for mycelia growth is 28 C, the optimum pH is natural pH, 100mL media volume in 250mL flasks and 100r/min were propitious to develop. Vitamin B1 B2, C and FH4 could promote the growth rate of EG4 in different degree, FH4 and Ca2+ could clearly improve its ability to produce flavonoids. The optimum condition for fermentation is 20g/L lactose, 3.2g/Lurea, 28 C, natural pH, lOOmL media volume in 250mL flasks, 100 r/min, 0.5g/L Ca2+ and minimum FH4.更多还原