【作者】 张玲；

【导师】 麦康森；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2007， 博士

【摘要】 以益生性和安全性为筛选标准,从白斑病毒耐过中国明对虾肠道中分离到一株对虾专用益生菌,并对其作用机理和应用效果进行了研究。在此基础上探讨了该菌株的多种配伍及其应用效果。研究了从白斑病毒(WSSV)耐过中国明对虾(Fenneropenaeus chinensis)肠道中分离的一株海洋细菌B12的益生特性和安全性,并结合形态观察、生理生化特征和16S rDNA序列分析对菌株B12进行了分类鉴定。研究结果表明:菌株B12是一株安全的益生菌株。鉴定结果证实B12为嗜盐单胞菌属Halomonas sp.。研究了不同给予方式(浸浴或口服),不同给予时间(间隔或连续),不同添加剂量和不同添加形式(活菌或灭活菌)的益生菌B12对中国明对虾幼体,中国明对虾稚虾或养成期日本囊对虾(Marsupenaeus japonicus)成活率、免疫力、抗病力、肠道菌群组成、中肠组织结构或养殖水体水质的影响。并分别从免疫学、肠道微生物学和组织学的角度,探讨益生菌B12对对虾的作用机理。研究结果表明,益生菌B12可以有效地调节对虾肠道微生物菌群组成,激活对虾免疫反应,改善对虾养殖水体水质。提高中国明对虾幼体的成活率和对副溶血弧菌的抗病力,提高了中国明对虾对白斑病毒的抗病力。饲料中添加7.18×1010 cfu g-1浓度的益生菌B12更有利于改善中国明对虾的健康状况。107 cfu ml-1浓度的益生菌B12浸浴处理,能够提高中国明对虾幼体的成活率,调节幼体的免疫反应,改善培育水体的水质,从而在中国明对虾育苗中发挥益生作用。益生菌B12和植物乳杆菌(Lactobacillus plantarum)激活日本囊对虾免疫反应需要的时间和对日本囊对虾免疫反应影响的程度均有种属特异性。这两株益生菌的活菌和热灭活形式均能显著激活日本囊对虾的免疫反应,益生菌B12的活菌形式能够延长激活日本囊对虾免疫反应的时间,灭活乳酸菌添加组日本囊对虾的免疫水平会逐渐高于活的乳酸菌添加组。饲料中添加不同类型的益生菌改变了日本囊对虾肠道中的微生态菌群组成,不同种类益生菌添加组日本囊对虾肠道优势菌群的数量和种类存在显著差异。同种细菌的活菌和热灭活菌对日本囊对虾肠道优势菌群的数量和种类的影响也不同。饲料中添加不同种类的益生菌的活菌和灭活形式均有利于维护日本囊对虾肠道粘膜结构的完整性。饲料中持续添加益生菌B12能够维护日本囊对虾中肠组织学结构的完整性。水体中添加复合益生菌或合生元有利于提高中国明对虾幼体的成活率、改善中国明对虾幼体培育水体水质和加强中国明对虾幼体的免疫反应,复合益生菌或合生元在中国明对虾育苗中发挥了较好的益生作用。饲料中添加复合益生菌与果寡糖和商品复合酶制剂的复合形式显著提高了中国明对虾幼体的成活率(且显著高于其他处理组)(P<0.05),显著激活了中国明对虾幼体的免疫反应(P<0.05),而且显著降低了幼体培育水体中氨氮的浓度(P<0.05),表现了较好的复合益生效果,是本研究中最优的功能性复合制剂的组合。总之,本研究从免疫学、肠道微生物学和组织学的角度,探讨益生菌B12的作用机理,并对益生菌B12的投喂方式(口服或浸浴),投喂时间(间隔或连续),投喂形式(活菌或灭活菌)和有效配伍进行了研究,为益生菌B12在对虾养殖中的研制和生产推广奠定了基础。更多还原

【Abstract】 The study was conducted to screen a specific probiotic for shrimp from the midgut of the WSSV (White Spot Syndrome Virus) tolerance shrimp (Fenneropeneaus Chinensis) according to its probiotic and safe characteristics, explore its application effects and functional mechanisms, and explore its effective compound probiotic preparation and evaluate their application effects based on the above-mentioned research results.An experiment in vitro or in vivo was conducted to screen a potential probiotic B12 (PP B12), which was isolated from the midgut of the WSSV (White Spot Syndrome Virus) tolerance shrimp (F. chinensis). The antagonism of PP B12 to pathogens (Vibrio harveyi and V. parahaemolyticus) and its safety for shrimp (F. chinensis) larvae were evaluated. PP B12 was identified according to its morphological, physiological and biochemical characteristics as well as homology analysis of 16S rRNA gene sequence. The results showed that strain B12 was a safe and specific probiotic for shrimp and was identified as Halomonas sp.The experiments was conducted to investigate the effects of different administration manners (oral or immersion), different continuing administration time, different doses and different supplementation forms (viable or heat-inactivated) of probiotic B12 on the survival rate, immune response, resistance to pathogenic infection, intestinal microflora component and histological structure or water quality of culturing marine water of shrimp (F. chinensis) larvae or juvenile or shrimp (Marsupenaeus japonicus) at grower stage, and explore the functional mechanism of probiotic B12 due to the studies on the immunology, intestinal microbiology and histology of the shrimp. The results showed that probiotic B12 could modulate the intestine microflora, and stimulate shrimp immune system, consequently enhance its effectiveness in preventing WSSV infections in shrimp. The oral administration of probiotic B12 at 7.18×1010 cfu g-1 was more beneficial to shrimp F. chinensis health. probiotic B12 with an immersion concentration at 107 cfu ml-1 is beneficial to shrimp (F. chinensis) postlarvae through enhancing its survival rate, modulating its immune response and improving the culturing water quality. Immune response of shrimp both in Lactobacillus plantarum and probiotic B12-supplemented treatment with viable or heat-inactivated form were significantly enhanced (P<0.05) compared to those of the control. The positive immunostimulant time in viable probiotic B12-supplemented treatment was longer than that in heat-inactivated probiotic B12-supplemented treatment. The immune level in heat-inactivated L. plantarum-supplemented treatment was higher than that in viable L. plantarum-supplemented treatment gradually. The immunostimulant mechanism of different probiotic strains depended on their species. The intestinal microflora components of shrimp were obviously changed with supplementation of different kinds of stains with different forms compared to those of the control. The number and variety of intestinal dominant microflora were obviously different in shrimp fed diets supplemented with different kinds of stains with different forms. The number and variety of intestinal dominant microflora were obviously different in shrimp fed diets supplemented with the same strains but with different forms (viable or heat-inactivated). The number and variety of intestinal dominant microflora were obviously different in shrimp fed diets containing viable V. parahaemolyticus with that in heat-inactivated V. parahaemolyticus treatment, and those probiotic (probiotic B12 and L. plantarum) treatment with different forms. The shrimp in probiotic (probiotic B12 and L. plantarum) treatment with different forms had better integrity of midgut mucosae than those of the control. The integrity of midgut of shrimp fed diets supplemented with viable V. parahaemolyticus was damaged, and permeability of midgut in this treatment was obviously increased than those of the other treatments. The integrity of shrimp midgut in viable probiotic B12 treatment with continuing administration was better than that in viable probiotic B12 treatment with changing into basal diet during the feeding experiment. Multispecies probiotic or synbiotic immersed into the culturing water were beneficial to enhance the survival rate and immune response of shrimp larvae, and improve the water quality of shrimp culturing water. The survival rate was significantly higher (P<0.05) (maximal level), and the immune responses significantly enhanced (P<0.05) in shrimp fed diet containing combination of 0.01% multi-enzyme preparation with 0.02% FOS and multispecies probiotic. Moreover, the ammonium concentration in shrimp larvae culturing water in this group (T14) was significantly lower than that of the control in this group (P<0.05). However, no significant differences were found in ammonium concentration between the other groups and the control (P>0.05). Therefore, combination of 0.01% multi-enzyme preparation with 0.02% FOS and multispecies probiotic (T14) was the optimal combination in the present study.In summary, the present study explore the functional mechanism of probiotic B12 based on its immunological, intestinal microbiological and histological characteriastic, and evaluate the application effects of probiotic B12 administered with different feeding manners (oral or immersion), different administration time, different forms (viable or heat-inactivated) and effective compound probiotic preparation. Therefore, the results in the present study were the theoretical and applied basement of development and manufacture of probiotic B12 for shrimp culture.更多还原