【作者】 单晓枫；

【导师】 钱爱东；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 在水产动物疾病的防控中，由于抗生素等药物的大量使用，导致了细菌多重抗药性、药物残留和危害环境等问题的出现，使得人类致力寻求对自身和环境无毒、无害的药物和产品，以期能够部分或全部替代抗生素的使用。而益生菌的出现和使用，则相应的缓解了以上问题。本研究就是基于以上理念，在国内首次从鲤鱼肠黏膜分离筛选出具有抗逆性、对病原菌具有拮抗性、相对安全的益生乳酸菌。为其在水产养殖业的应用打下了良好的基础。本研究主要分以下两部分试验进行：试验一主要做的是益生乳酸菌株的筛选。首先通过酸化胆盐培养基，从鲤鱼肠黏膜固有菌群中共分离到8株乳酸菌。然后通过耐受胆盐、耐受pH值性、耐受蛋白酶和耐高温等试验，筛选出2株抗逆性较强的菌株，其中在胆盐耐受性试验中，YL-1和YL-7在胆盐浓度0.4％、培养4h时仍有30％以上的存活率；在pH值耐受性试验时，2株乳酸菌均可在酸性和碱性条件下存活；胰蛋白酶对2株菌的存活率没有影响；在70℃的高温中，2株乳酸菌均有一定的耐受性。在体外抑菌试验中，通过共培养和上清液抑菌试验的结果发现：YL-1对嗜水气单胞菌的抑菌效果优于YL-7，因此选用YL-1做为益生菌的候选菌株。通过对YL-1上清液抑菌物质分析可以发现，其抑菌物质主要是有机酸和对胰蛋白酶敏感的一类物质。在YL-1的药物敏感性试验中发现，该菌株存在着一定的耐药性。对小白鼠和鲤鱼的接种，初步认定菌株YL-1具有安全性。最后，通过一系列的生理生化试验，初步鉴定YL-1是粪肠球菌。试验二为YL-1增菌培养基的优化。在前半部试验中，主要是通过对最佳碳源的筛选、最佳氮源的筛选、最佳无机盐离子的筛选以及最佳刺激因子的筛选等单因子试验，筛选出对YL-1生长有利的各个因子。之后在SAS软件的辅助下，通过Plackett-Burman试验设计在培养基的各个因子中筛选出对YL-1生长有利的重要因子，再进行最陡爬坡试验逼近最大响应区域，最后在Box-Behnken试验设计下确定最大响应值，并通过优化的结果验证，表明预测值和实际值有良好的拟合性，优化模型可靠。最后确定的YL-1培养的最佳培养基为：蛋白胨1.365％、牛肉膏1.25％、酵母浸粉0.625％、麦芽糖2.5％、吐温-80 0.1％、KH₂PO₄0.2％、柠檬酸三铵0.16％、MgSO₄0.25％、初始pH值6.4。更多还原

【Abstract】 In the prevention and control for Aquatic Animals diseases, many problems, such as multipleantibiotic resistance, residues and pollutions, has been caused, due to the abuse of antibiotics. This madepeople dedicate to seek medicines and products which are harmless to human and environment, so thatwe can take the place of antibiotics completely or partially. However the occurrence and application ofProbiotic Bacteria can solve these problems. On account of ideas mentioned above, we isolate Probioticstrains of Lactobacillus which is of Anti-Resistance, Antagonistic to Pathogen, and relatively safe,from Intestinal mucosa of Cyprinus carpio firstly in China。This research sets a good base for theapplication of it in aquaculture.This research is mainly divided into two experiments as follows:The first experiment is mainly the screening of Probiotic strains of Lactobacillus. In the first place,isolate 8 strains of Lactic Acid Bacteria from Intestinal mucosal flora of Cyprinus carpio using bile saltsculture. Then, screen two strains of Bacteria of strong Anti-Resistance using the tests of tolerance of bilesalts, tolerance of pH, tolerance of protease and tolerance of heat. In the test of tolerance of bile salts,over 30％of YL-1 and YL-7 can survive after 4-hour culture in the bile salts of 4％concentration, in thetolerance of pH, the 2 strains of Lactic Acid Bacteria can survive both in acid environment and alkalineenvironment; Trypsin didn’t affect the survival of these two strains; and the bacteria show a certaintolerance in the heat condition of 70℃. In bacteriostastic test in vitro, we find that YL-1 is better thanYL-7 in the inhibition to Aeromonas hydrophila, thus YL-1 is selected as the candidate bacteria ofprobiotics. Through the analysis to the cultured supernatant of YL-1, we can find that the bacteriostasticsubstance is mainly Organic Acid and substance sensitive to Trypsin. In sensitivity test of drugs forYL-1, we find that this strain has certain drug resistance. Through the inoculation on mice and Cyprinuscarpio, we identify the safety of YL-1. Finally, we identify YL-1 as Enterococcus faecalis by a series ofphysiological and biochemical tests.The second experiment is the optimization of culture medium for YL-1. The first half ofexperiment is mainly the screening of the best Carbon source, the best Nitrogen source, the bestInorganic Salts Ion, and the best stimulating factors. After the screening of various favorable factors forYL-1, with the help of SAS software, through Plackett-Burman design, we can screen the importantfactors favorable to the growth of YL-1, then with Steepest Ascent experiments, approach the maximum response region. Finally, with the help of Box-Behnken design, we can identify the maximum responsevalue. Through verification, it shows good fitting between the estimated value and the real value, andthe model is reliable. At last, the optimization of cultured medium of YL-1 is as follows: 1.365％peptone, 1.25％extractum carnis, 0.625％yeast powder, 2.5％maltose, 0.1％Tween-80, 0.2 KH₂PO₄,0.16％ammonium citrate, 0.25％MgSO₄, incipient pH 6.4.更多还原