【作者】 刘佳琳；

【导师】 李筠；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2006， 硕士

【摘要】 有益菌(probiotics)作为生物防治的手段应用于水产养殖体系中,用来改善养殖生态环境并取得了成效。人们在水产养殖中应用有益菌,将其既用作饲料添加剂来调节养殖动物肠道微生态平衡,又用于加入水体起控制养殖池微生物生态区系的作用,此外还用于改善养殖水体的水质条件。有益菌的应用可以调节和改善养殖环境微生态平衡,控制和减少养殖动物爆发性病害的发生,提高养殖动物的的免疫力,增强其抗病能力,因此成为国内外研究的热点。本试验对从健康斑节对虾(Penaeus monodon)幼体分离的菌株L1及从养殖水体中防污附钢片的微生物粘膜中分离的菌株L2和L3,采用琼脂扩散法进行体外拮抗试验,测定其抑菌图谱。这三株海洋细菌均对鲈鱼病原鳗弧菌W1(Vibrio anguillarum)表现出较强的抑菌作用。根据细菌形态和生理生化特征和16S rDNA序列分析,鉴定这三株海洋有益菌菌均属假交替单胞菌属( Pseudoalteromonas ),其中L1是橙色假交替单胞菌(Pseudoalteromonas aurantia) , L2是杀鱼假交替单胞菌(Pseudoalteromonas piscicida), L3与柠檬假交替单胞菌(Pseudoalteromonas citrea)的亲缘关系最近。采用玻璃纸覆盖平板法制备菌株L1、L2和L3的胞外产物,优化培养条件,结果表明:菌株L1在28℃下培养48 h、菌株L2在22℃下培养48 h、菌株L3在25℃下培养24 h制备的胞外产物抑菌活性最高。对其胞外产物进行凝胶层析部分纯化后,进行温度、pH和蛋白酶K处理,结果表明热处理、酸碱处理和蛋白酶消化均可使其活性丧失,表明菌株L1、L2和L3的胞外活性物质含蛋白质。更多还原

【Abstract】 The research of probiotics for aquatic animals is increasing with the demand for environment-friendly aquaculture. The probiotics were defined as live microbial feed supplements that improve health of aquatic animals, accommodate the balance of gastrointestinal microorganisms of fish and shellfish and increase the resistance to diseases of their host. The research on probiotics used as biocontrol in the field of aquaculture has been becoming the focus in present.Marine bacteria L1 was isolated from healthy larvae of Penaeus monodon, L2 and L3 strain was isolated from microbial biofilms of antiseptic steel pieces. By 16S rDNA sequence analysis and the morphological, physiological and biochemical characteristics, marine bacteria L1 was identified to be Pseudoalteromonas aurantia, L2 was identified to be Pseudoalteromonas piscicida and L3 was identified to be Pseudoalteromonas citrea.The extraction of antibacterial extracellular products (ECP) was conducted by the method cellophane plate technique. The optimum incubation condition to obtain ECP with the strongest antimicrobial activity was studied. The results indicated that the optimum condition was 28℃, 48 h for L1 strain; 22℃, 48 h for L2 strain; and 25℃, 24 h for L3 strain.The extracellular products with antimicrobial activity of P. aurantia strain L1, P. piscicida strain L2 and P. citrea strain L3 was purified by Gel filtration of Sephadex and SDS-PAGE. The characters of inhibitory substances of L1、L2 and L3 strain towards pH、thermal、protease K treatment indicated the antibacterial extracellular production was determined to be protein.更多还原