导　　师： 杨博;杨亚勇

学科专业： 071010

授予学位： 硕士

作　　者： ;

机构地区： 华南理工大学

摘　　要： 本论文从药用植物中分离内生菌,进行了植物样本表面消毒、内生菌分离纯化、抗菌活性菌株筛选、活性菌株鉴定以及其摇瓶发酵条件优化等研究,为寻找天然抗菌物质提供资源。获得的主要结果如下: 1、植物内生菌的分离纯化 从3种药用植物样本中分离得到8株内生菌/(丝状真菌5株,细菌2株,放线菌1株/),但有3株/(2株丝状真菌,1株放线菌/)不能在体外培养。 2、抗菌活性植物内生菌的筛选 内生菌发酵液经离心取上清液,进行抑菌试验。在本实验条件下,小叶榕内生细菌EF-B01菌株发酵处理液对铜绿假单胞菌和大肠杆菌有很强的抑制作用,其中对铜绿假单胞菌的抑菌圈直径达23.28mm,对金黄色葡萄球菌有较弱的抑制作用。EF-B01菌株发酵处理液对黑曲霉菌丝的生长有很强的抑制作用,培养5天,抑制率达82.3/%,且EF-B01菌株发酵处理液能强烈抑制黑曲霉孢子的形成。 3、抗菌活性内生菌EF-B01菌株的鉴定 对抗菌能力较强的内生细菌EF-B01菌株进行了培养特征、形态观察、部分生理生化特性鉴别以及16S rDNA测序,综合鉴定该菌株为枯草芽孢杆菌/(B. subtilis/),将其命名为Bacillus subtilis EF-B01。 4、抗菌活性内生菌EF-B01菌株发酵条件的优化 为了增加发酵液中生物量和抗菌活性物质的量,对内生细菌B. subtilis EF-B01菌株进行了最适碳源和氮源、初始pH值、装液量、发酵接种量、摇床转速和培养温度的研究。结果表明,通过单因子试验得到的最适的碳源、氮源及其含量分别为3/%玉米粉、4/%豆饼粉,初始pH值8.0,装液量30/%,发酵接种量1/%,摇床转速160rpm,培养温度34℃。 在最优发酵条件下,内生细菌B. subtilis EF-B01菌株培养48h后发酵液的OD值为7.95,比优化前的5.23提高了52.0/%。培养72h后,发酵液对铜绿假单胞菌的抑菌圈直径达26.42 mm,比活性菌株初筛时的23.28mm提高了13.5/%。 The following items were carried through in this research: surface sterilization of plant samples, isolation and purification of endophytes, screening for antibacterial endophytes,identification and ferment optimization of the active strain,which can be resources for natural antimicrobial products.The results obtained were as follows: 1. Isolation and purification of endophytes Eight endophyte strains were isolated from three sorts of plants, including five fungal strains, two bacterial strains and one actinomycete strain. But three of them /(two fungal strains ,one actinomycete strain/) can’t be cultivated in vitro. 2. Screening for antibacterial endophytes Endophyte fermentation fluid was disposed by centrifugation. Supernatant liquid was used for antibacterial tests. The processed fermentation fluid of endophytic bacteria EF-B01 strain derived from Ficus microcarpa showed strong inhibition to Pseudomonas aeruginosa and Escherichia coli , and the inhibition zone dimeter of the processed fermentation fluid of endophytic bacteria EF-B01 against Pseudomonas aeruginosa was 23.28mm. The processed fermentation fluid of EF-B01 strain showed weak inhibition to Staphylococcus aureus. The processed fermentation fluid of EF-B01 strain showed strong inhibition to the growth of Aspergillus niger, and the rate of restrain reached 82.3/% after 5 days. Moreover, the processed fermentation fluid of EF-B01 strain showed strong inhibition to the spore formation of Aspergillus niger. 3. Identification of active endophyte strain EF-B01 Antibacterial endophytes strain EF-B01 was identified by cultivation characters, morphologic observations, physiological and biochemical characters and sequence analysis of the 16S rDNA as the one of Bacillus subtilis, named as Bacillus subtilis EF-B01. 4. Optimization of fermentation conditions of active endophyte strain EF-B01 With the purpose of finding out the optimum fermentation conditions which could be used to increase the biomass and the yield of active substances ,optimum carbon and nitrogen source, initial pH, medium loading quantity, inoculation amount, rotary rate and fermentation temperature were researched. The results indicated that the optimal carbon source was 3/% corn flour, the optimal nitrogen source was 4/% bean flour, initial pH was 8.0, medium loading quantity was 30/%, inoculation amount was 1/%, rotary rate was 160rpm and fermentation temperature was 34℃. Under the optimal conditions, the OD value of the fermentation fluid of B. subtilis EF-B01 strain was 7.95 after 48h, increasing 52.0/% comparied with the initial conditions. The inhibition zone dimeter of the processed fermentation fluid against Pseudomonas aeruginosa was 26.42mm, increasing 13.5/% comparied with the initial conditions.

关 键 词： 植物内生菌 分离纯化 抗菌活性 鉴定 发酵优化

分 类 号： [S476.1]

领　　域： [农业科学] [农业科学]