【作者】 邓墨渊；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 药物化学， 2007， 硕士

【摘要】 植物内生菌是一个庞大的具有普遍存在性的微生物特殊类群。近年来,研究表明植物中内生菌能产生抗肿瘤、杀菌等特殊活性物质,这使得植物内生菌的应用范围不断扩展,同时也成为药物研究一个新的热点。本论文是在抗结核分枝杆菌的植物内生菌筛选的基础上,根据“一菌多筛”的原则,扩展为具有抑菌活性的植物内生菌筛选及其活性菌株发酵优化。论文进行了植物样本表面消毒、内生菌分离纯化、发酵及发酵产物预处理、抗菌活性菌株筛选、活性菌株鉴定及其发酵条件优化等研究。获得的主要结果如下: 1植物内生菌的分离纯化采用已优化的表面消毒方法对30种植物样本进行处理,分离得到46株内生菌。但9株(3株真菌,6株细菌)不能在体外培养,另有7株内生真菌在本实验特定的人工培养条件下不产生有性分生孢子。2抗菌活性植物内生菌的筛选①得到菌丝预处理丙酮样本37份,乙酸乙酯样本37份;采用酒精沉淀法对发酵液进行预处理,得到样本37份。②采用定量的二倍稀释法(培养基采用改良罗氏斜面培养基)对111份预处理样本进行初筛,得到来源于商陆(Phytolacca acinosa)的EP-S23,狭叶十大功劳(Mahonia fortunei)EM-L40、香樟(Cinnamomum camphora (Linn.)Presl)EC-S44的3株活性内生真菌,其发酵液提取物浓度为10mg/ml时对人型结核标准菌株(H37Rv)有抑制作用。其中,EC-S44的发酵液提取物浓度在15mg/ml时对人型结核耐药菌株有抑制生长作用。③采用KB纸片法对以上3株菌进行复筛,得到香樟内生菌EC-S44发酵液提取物浓度为10mg/ml时对金黄色葡萄球菌、枯草芽孢杆菌、铜绿假单孢菌、大肠杆菌均有不同程度抑制作用,其中对金黄色葡萄球菌的抑菌圈到达16mm,同时用琼脂二倍稀释法测定最小抑菌浓度(MIC值)为6.5mg/ml。3活性菌株的鉴定和发酵条件优化①通过初步形态学鉴定:香樟(Cinnamomum camphora (Linn.)Presl)EC-S44为镰孢霉属(Fusarium),将其命名为Fusarium sp.菌株EC-S44。②对Fusarium sp.菌株EC-S44发酵条件进行优化,得到其在本实验中的最优发酵条件:土豆汁培养基(3%的葡萄糖、0.3%的牛肉膏),4%发酵接种量,20%的装液量,8.0的初始pH值,26℃培养温度,150rpm转速。在此条件下,菌株对结核分枝杆菌耐药株的抑制能力增强,MIC值降低到12.5mg/ml。更多还原

【Abstract】 Endophytes are one kind of large, ubiquitous fungus group. Recent years, studies on Endophytes show that some antitumor and antibacterial products made by Endophytes. This allows applications of Endophytes enlarge more and more, and Endophytes become the one new hot point of medicine research.Based on searching for anti-Mycobacterium tuberculosis Endophytes, according to the principle of using much more Tested bacteria for searching active Endophytes, this study is extended to be as‘Screening for antibacterial Endophytes’. Such the following items were carried through: plant sample surface sterilization, Endophytes isolation and purification, pretreatment for Endophytes fermentation products, screening for antibacterial Endophytes, identification and ferment optimization of active strain. The results obtained are following:1 Isolation and purification of Endophytes30 sorts of plant were dealt by the optimal surface sterilization method, and 46 strains were isolated. But 9 of them (3strains fungi, 6strains bacterium) can’t be cultivated in vitro. And 7 of the left don’t have fruiting body under our experimental condition.2 Screening for antibacterial Endophytes①the Endophytes fermentation pretreatment samples,37 acetone samples of hypha,37 ethyl acetate samples of hypha,and 37 fermentation broth samples dealt by ethanol deposition method were gotten.②Using the double dilution method (Rhodes incline solid medium) to deal with 111 samples, the anti-Mycobacterium tuberculosis Endophyts are obtained that are Strain EP-S23 deriving from Phytolacca acinosa, Strain EM-L40 deriving from Mahonia fortunei, and Strain EC-S44 deriving from Cinnamomum camphora (Linn.) Presl, which can inhibit the growth of Mycobacterium tuberculosis standard strain (H37Rv) with the fermentation compound density of the 10mg/ml. And strain EC-S44 deriving from Cinnamomum camphora (Linn.) Presl with the fermentation compound density of the 15mg/ml can inhibit the growth of Mycobacterium tuberculosis drug-resistance strain.③Using the Agar dilution method to screen the former three active Endophytes again, the extraction compound solution of Cinnamomum camphora (Linn.) Presl strain EC-S44 can inhibit 5 Tested pathogens with different density, and the inhibition zone of active strain EC-S44 against Staphylococcus aureus (ATCC 25923) is 16mm, and MICs is 6.5mg/ml.3 Identification and ferment optimization of active strain①Active Strain EC-S44 is identified by morphology as the one of Fusarium, named as Fusarium sp. Strain EC-S44.②The optimum ferment condition of Fusarium sp. strain EC-S44 is: PDA (3% glucose, 0.3% beef extract), 4% fermentation inoculation amount, 8.0 initial pH,26℃, 150rpm.Under this condition, for Fusarium sp. strain EC-S44, the capability of anti- Mycobacterium tuberculosis drug-resistance strain enhances, and the MICs is 12.5 mg/ml.更多还原