【作者】 曹海鹏；

【导师】 吕利群；

【作者基本信息】 上海海洋大学 ， 水产养殖学， 2013， 博士

【摘要】 抗生素的滥用已经引起了嗜水气单胞菌日益严重的耐药性问题，导致嗜水气单胞菌引起的养殖鱼类疾病的有效防治难度加大，因而寻求抗生素的理想替代品迫在眉睫。芽孢杆菌广泛存在于自然界中，能够形成内生孢子，具有很强的抗逆性，不仅能够防病治病，促进动物生长，而且具有无副作用、无残留、无污染、不产生耐药性等特点，是替代抗生素的理想制剂，具有巨大的应用价值。鉴此，本研究筛选了一株抗嗜水气单胞菌解淀粉芽孢杆菌优良菌株G1，在确定了其对嗜水气单胞菌及其引起的草鱼细菌性败血症具有良好抗菌防病效果的基础上，通过比较蛋白质组学和抗菌相关基因检测探讨了其抗菌机制；优化了其微胶囊的实验室生产工艺，并进一步评价了微胶囊的耐酸、耐碱性能及其对水产养殖环境的安全性，为后续开发可替代抗生素的解淀粉芽孢杆菌微胶囊奠定了坚实的基础。本研究主要结果如下：1．抗嗜水气单胞菌解淀粉芽孢杆菌的分离及其对嗜水气单胞菌的拮抗效果从养殖池污泥中分离筛选了一株优良的抗嗜水气单胞菌芽孢杆菌G1，通过API50CH细菌鉴定系统以及16S rDNA序列分析法，菌株G1被鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），其16S rDNA序列（GenBank登录号: HM245965）与基因库中芽孢杆菌属菌株的16S rDNA序列有99%100%的同源性，而且与解淀粉芽孢杆菌Ba-74501的16S rDNA序列（GenBank登录号: DQ422953）的亲缘关系最近。菌株G1对病原性嗜水气单胞菌具有广谱拮抗活性，对嗜水气单胞菌的生长具有良好的抑制作用，对草鱼抗嗜水气单胞菌感染具有良好的效果。2．抗嗜水气单胞菌解淀粉芽孢杆菌的比较蛋白质组学分析以抗嗜水气单胞菌解淀粉芽孢杆菌G1的无抗菌活性突变株作为对照，利用双向凝胶电泳（2-DE）技术建立了解淀粉芽孢杆菌G1及其突变株菌体蛋白2-DE图谱，并进一步通过质谱检测和数据库检索鉴定了解淀粉芽孢杆菌G1的差异蛋白质。实验结果表明，解淀粉芽孢杆菌G1的2-DE图谱蛋白质点为1135个，匹配率为89.37%；以解淀粉芽孢杆菌G1突变株的菌体蛋白2-DE图谱作对照，解淀粉芽孢杆菌G1共有65个差异蛋白质点，其中36个高丰度的差异蛋白质经鉴定与蛋白质合成与修饰、能量代谢、菌体抗逆自我保护、耐药以及抗菌等生理功能有关，揭示解淀粉芽孢杆菌G1除了其产生的抗菌蛋白直接参与抗菌作用外，其对环境的抗逆能力、生长性能对其抗菌作用也具有重要的意义。3．抗嗜水气单胞菌解淀粉芽孢杆菌的抗菌素相关基因分析分别对抗嗜水气单胞菌的解淀粉芽孢杆菌G1的抗菌素相关基因进行了PCR扩增与测序，并对其编码产物的氨基酸序列、跨膜螺旋信号、结构域与二级结构等进行了预测与分析。实验结果表明，菌株G1仅含有伊枯草菌素合成必需基因，该基因与GenBank基因库中芽孢杆菌属其他细菌的伊枯草菌素A、杆菌抗霉素D、抗霉枯草菌素等伊枯草菌素家族基因自然聚类，与枯草芽孢杆菌MH25株和RB14株的伊枯草菌素A基因有98%的高度同源性，而且其编码产物的氨基酸序列与GenBank中芽孢杆菌属其他细菌的伊枯草菌素、伊枯草菌素A、脂肽类化合物bacillorin、芽孢菌素D等抗菌物质的氨基酸序列有高度同源性，与枯草芽孢杆菌MH25的伊枯草菌素A合成酶B（GenBank登录号：ABY89499）的亲缘关系最近。此外，菌株G1伊枯草菌素合成必需基因的编码产物不具有明显的跨膜结构，但其结构域中存在AMP结合位点和PP结合位点，二级结构中存在α螺旋、伸展链、β转角和无规卷曲。4．抗嗜水气单胞菌解淀粉芽孢杆菌微胶囊的制备工艺与特性以明胶为壁材，采用单因素法，考察了明胶浓度、进风温度、进料速度、空气流量等因素对抗嗜水气单胞菌解淀粉芽孢杆菌微胶囊有效含菌量的影响，并进一步通过正交试验设计优化了解淀粉芽孢杆菌微胶囊制剂的喷雾干燥工艺参数，观察了其微胶囊颗粒形态以及对人工模拟胃液和肠液的耐受力。实验结果表明，解淀粉芽孢杆菌微胶囊喷雾干燥的最佳制备工艺条件为：明胶浓度为3.0%，进风温度为155°C，进料速度为8ml/min，空气流量为700L/h，各因素对解淀粉芽孢杆菌微胶囊喷雾干燥工艺的影响程度为：明胶浓度>进料速度>空气流量>进风温度。此外，解淀粉芽孢杆菌微胶囊制剂的颗粒呈球形，表面有凹陷，但没有孔和裂纹，颗粒粒径分布基本均匀，平均大小为9.22μm，对人工模拟胃液和肠液具有较好的耐受力。5.抗嗜水气单胞菌解淀粉芽孢杆菌微胶囊的安全性评价参照《GB/T21805-2008化学品藻类生长抑制试验》、《GB/T13266-91水质物质对蚤类（大型蚤）急性毒性测定方法》、《GB/T13267-91水质物质对淡水鱼（斑马鱼）急性毒性测定方法》、《渔药临床试验技术规范》等国家标准及相关法规，观察了解淀粉芽孢杆菌微胶囊对小球藻生长抑制作用以及对大型蚤、斑马鱼和草鱼的急性毒性，分析了其对养殖水体主要理化因子的影响，实验结果表明，解淀粉芽孢杆菌微胶囊在终浓度为0.2mg/L~2000mg/L时对小球藻生长具有促进作用，对小球藻的半数抑制浓度大于2000mg/L，而且其对大型蚤、斑马鱼和草鱼的半数致死浓度也大于2000mg/L（或mg/Kg体重）。此外，在养殖水体中加入解淀粉芽孢杆菌微胶囊至终浓度为0.2mg/L~2000mg/L后14天内，各浓度组的氨氮含量、硫化物和pH值均缓慢下降，仅亚硝酸盐氮含量稍微升高后逐渐缓慢降低，但这些理化因子的变化与解淀粉芽孢杆菌微胶囊的加入量呈负相关关系。因此，解淀粉芽孢杆菌微胶囊实际无毒，对养殖水中氨氮、亚硝酸盐氮、硫化物和pH等理化因子的影响均控制在虾虎鱼仔鱼、黄颡鱼、白斑狗鱼、克氏原螯虾等水产养殖动物的安全浓度范围内，为其在水产养殖中的安全应用提供了重要的科学依据。综上所述，本研究证实了解淀粉芽孢杆菌在嗜水气单胞菌引起的草鱼细菌性败血症防控中的潜在价值，丰富了解淀粉芽孢杆菌的抗菌机理研究，弥补了水产用解淀粉芽孢杆菌新型制剂创制及其应用安全性评价研究的不足，对我国水产微生态制剂的研发与安全应用体系的建立具有重要的借鉴意义。更多还原

【Abstract】 The wide use of antibiotics has caused the serious drug resistance of Aeromonashydrophila, which in turn makes it difficult to effectively control fish aeromonasis. Thus,it is extremely urgent to develop the substitutes to antibiotics. Bacillus species arewidely distributed in nature and have high stress tolerance as a result of their endosporeproduction. They are reported to exhibit the ability to control fish diseases, promote fishgrowth, and have the merits of no side effects, no residues, no drug resistance and freeof pollution. Therefore, they are considered to be ideal substitutes for antimicrobials andhave tremendous applicable value. In this study, a potential strain G1of B.amyloliquefaciens against A. hydrophila was isolated, its inhibitory effect on A.hydrophila, as well as control efficacy on grass carp (Ctenopharyngodon idellus)aeromonasis, was determined. On this basis, its antibacterial mechanism was exploredusing comparative proteomics and antibiotics-related genes’ analysis. In addition, thelaboratory production process technique of its microcapsules was optimized, itsmicrocapsules’ acid resistance, alkali tolerance and safety to the aquacultureenvironment were further evaluated. The results of this study laid the solid foundationfor the future development of B. amyloliquefaciens microcapsules as a potentialsubstitute for antibiotic additives. The main results are as follows:1. Isolation of B. amyloliquefaciens against A. hydrophila and its antagonisticefficacy on A. hydrophilaA potential Bacillus strain G1against A. hydrophila was isolated from pondsediments, and identified as a Bacillus amyloliquefaciens isolate using API50CHidentification system and16S rDNA sequence analysis. Its16S rDNA sequence wassubmitted to the GenBank with an accession number of HM245965, and found to havethe high homology of99%100%with those of Bacillus species in GenBank, and exhibited most closely related to B. amyloliquefaciens strain Ba-74501(GenBankaccession no.: DQ422953). In addition, the G1isolate showed broad-spectrumantagonism against A. hydrophila pathogens, displayed good inhibitory effects on A.hydrophila, and exhibited good efficacy on the diseased grass carps infected with A.hydrophila.2. Comparative proteomic analysis of B. amyloliquefaciens against A. hydrophilaBased on the successful screening of the non-antagonistic mutant of B.amyloliquefaciens strain G1, two-dimensional gel electrophoresis (2-DE) maps of strainG1and its mutant were imaged by the2-DE technique, and the differentially expressedproteins were further identified using the mass spectrometry and database searching.The experimental results showed that the total protein spots in the G1isolate’s2-DEmap were1135with the matching scores of89.37%. In comparison with the2-DE mapof the G1isolate’s mutant,65different protein spots were detected, and36of them werehighly expressed and successfully identified to participate in physiological functionssuch as protein synthesis and modification, energy metabolism, stress responsetolerance, drug resistance, antibiosis. This revealed that besides the antimicrobialproteins, its self-protection in adverse environments and good growth performanceplayed important roles in the antibacterial action.3. Antibiotics-related gene analysis of B. amyloliquefaciens against A. hydrophilaThe antibiotics-related genes from B. amyloliquefaciens strain G1against A.hydrophila were amplified by PCR and sequenced, the amino acid sequence,transmembrane heices, domain and secondary structure of their coding products werealso analyzed. The experimental results showed that only iturin synthetase essentialgene was present in the G1isolate, naturally clustered with genes of iturin family fromBacillus sp. in GenBank, including iturin A, bacillomycin D, mycosubtilin, and showed98%similarity to the iturin A gene of B. subtilis strain MH25and strain RB14. Theamino acid sequence of its coding product exhibited high similarity to those of iturin,iturin A, bacillorin and bacillomycin D, and was close relative to the iturin A synthetaseB of B. subtilis strain MH25(GenBank accession no.: ABY89499). In addition, noobvious transmembrane structure was present in the coding product of iturin synthetaseessential gene of the G1strain. However, AMP-binding site, PP-binding site werepresent in its domain, and alpha helix, beta turn, random coil, extended strand were alsopresent in its secondary structure. 4. Production process technique and characteristics of microcapsules of B.amyloliquefaciens against A. hydrophilaOn the basis of gelatin as the wall materials, the influences of gelatin concentration,inlet temperature, feeding speed and air flow on the viable cells in the microcapsules ofB. amyloliquefaciens strain G1were assayed using single factor method, the spraydrying processing parameters of its microcapsules were optimized through orthogonalexperimental design, and the morphology and tolerance to artificial gastric and intestinaljuices were further examined. The experimental results showed that the optimum spraydrying processing parameters to prepare B. amyloliquefaciens microcapsules weregelatin concentration of3.0%, inlet temperature of155°C, feeding speed of8ml/minand air flow of700L/h, and the most leading influence factor on the production of B.amyloliquefaciens microcapsules was the gelatin concentration, followed by feedingspeed, air flow and inlet temperature. In addition, B. amyloliquefaciens microcapsuleswere spherical, featured dimpled surface without holes and cracks,uniformly-distributed with an average size of9.22μm. They were also found to havegood tolerance to artificial gastric and intestinal juices.5. Safety evaluation of the microcapsules of B. amyloliquefaciens against A.hydrophilaAccording to Chemicals—Alga growth inhibition test (GB/T21805-2008), Waterquality—Determination of the acute toxicity of substance to Daphnia (Daphnia magnastraus)(GB/T13266-91), Water quality—Determination of the acute toxicity ofsubstance to freshwater fish (Brachydanio rerio Hamilton-Buchanan)(GB/T13267-91)and Clinical experiment technical practice for fishery drugs, the growth inhibition ofthe B. amyloliquefaciens microcapsule on Chlorella sp. and its acute toxicity toDaphnia, zebra fish and grass carps were observed, and its influence on mainphysicochemical factors of aquaculture water was also assayed. The experimentalresults showed that the growth of Chlorella sp. was promoted with B. amyloliquefaciensmicrocapsules at the final concentrations of0.2mg/L~2000mg/L, its IC50to Chlorellasp. was2000mg/L, and its LD50to Daphnia, zebra fish and grass carps were all>2000mg/L (or mg/Kg body weight). In addition, in the period of14days after the input of theB. amyloliquefaciens microcapsule into the farming water at0.2mg/L~2000mg/L, thecontents of the ammonia and sulfide as well as the pH values were gradually reduced,only the nitrite nitrogen concentration first rose slightly and followed by a slow decrease, and the changes of the physiochemical factors related negatively to theconcentration of the B. amyloliquefaciens microcapsules. Thus, the B.amyloliquefaciens microcapsule was actually nontoxic, its effects on the ammonia,nitrite nitrogen, sulfide and pH in the fish faming water were under the control of theirsafe concentrations to aquatic animals, such as the goby fry, Pelteobagrus fulvidraco,Esox lucius, Procambarus clarkia. The present study provided important scientific basison the safe use of B. amyloliquefaciens microcapsules in aquaculture.In conclusion, this study confirmed B. amyloliquefaciens as potential probiotics inthe fish aeromonas control, enriched its antibacterial mechanism, and made up for thedeficiency in the new preparation and application safety evaluation of B.amyloliquefaciens probiotics, and played important significance in the development ofaquatic probiotics and establishment of their safe use systems in aquaculture.更多还原