【作者】 王文君；

【导师】 田相利；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2015， 硕士

【摘要】 近年来,伴随着水产养殖业的迅速发展,各种养殖生物受到的病害威胁也越来越严重。病害的发生不仅带来巨大的经济损失,也给环境带来较大的影响。而为防治水产生物病害而滥用抗生素等化学药物,则会对环境和人类产生严重的影响。益生菌作为抗生素的替代品,可以抑制病原菌,为养殖动物提供营养,提高养殖动物的免疫力,改善水质条件,修复微生态环境,因此益生菌的在水产养殖中使用日渐受到人们的重视。益生菌的作用机理之一是通过产生抑菌物质来抑制病原菌的生长。因此,选择具有较高抑菌活性的细菌作为潜在的益生菌进行研究开发具有重要的实践意义。本研究对从对虾养殖环境中分离出一株对弧菌病原菌具有拮抗作用的菌株进行了鉴定、产物性质分析和应用的初步研究,旨在为将其最终应用于对虾养殖实践提供理论基础。研究获得的具体结果如下：1.一株弧菌拮抗菌的分类鉴定及其抑菌作用研究本研究从对虾养殖池塘分离筛选出一株对病原菌有拮抗作用的菌株,编号为BL1。采用十字交叉法、点种法以及牛津杯法对其进行了体外拮抗试验,以确定其抑菌谱。研究结果显示,菌株BL1对溶藻弧菌(Vibrio alginolyticu)、创伤弧菌(V. vulnificus)及副溶血弧菌(V. parahaemolyticus)具有明显拮抗作用。根据细菌形态、生理生化特征和16S rDNA序列分析,初步鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis)。采用单因素分析法进一步研究了菌株BL1的最佳培养条件,确定其在培养温度28℃、初始pH7.0、接种量1%(V/V)、转速190rpm/min的条件下生长状况最好。2.菌株BL 1抑菌产物的初步分离与性质分析本研究以优化培养条件后的枯草芽孢杆菌BL1(Bacillus subtilis)发酵液为研究对象,以创伤弧菌(Vibrio vulnificus)为指示菌,采用牛津杯法作为检测方法,对菌株BL1发酵液中起抑菌作用的抑菌活性物质的性质进行了初步研究。培养时间对抑菌效果影响的实验显示,培养24h后的菌液抑菌活性最好；采用过滤、饱和硫酸铵沉淀及超滤等方法对培养24h的菌株BL1的发酵液进行初步分离纯化处理,结果显示枯草芽孢杆菌BL1发酵液中的抑菌物质为胞外分泌型、非蛋白类、分子量大于10kDa的物质；对经过初步分离纯化的抑菌物质进行稳定性的研究,发现抑菌物质对高温(60-100℃)和pH(3.0-11.0)的耐受能力比较强,对蛋白酶K不敏感。可以看出,菌株BL1所产生的抑菌物质具有很好的抑菌作用及稳定性,表明该菌株可能具有作为益生菌应用于海水养殖的潜力。3.泼洒和投喂拮抗菌BL1对凡纳滨对虾生长和水体弧菌数量的影响本研究对潜在益生菌BL1进行了菌株安全性检验后,以泼洒和投喂两种方式将菌株BL1应用于凡纳滨对虾(Litopenaeus vannamei)养殖中,以期了解其对凡纳滨对虾生长及水体中弧菌数量的影响。两种方式均设置3个不同的浓度梯度,其中,投喂组添加量分别为107、109和1011cfu/kg(以活菌量计),泼洒组添加量分别为106、108和1010cfu/m3,以投喂基础饲料组为对照组,泼洒周期为5天,实验共持续42天。实验过程中任选1个泼洒周期按泼洒后12h以及1、2、3、4和5d定时采样测定水体弧菌数量。实验结束时测量对虾的生长情况,饵料利用率及存活率。实验结果显示：(1)生长方面,在两种施用方式下,除了泼洒组106cfu/m3,其他处理组中菌株BL1均能显著促进对虾生长,提高对虾的特定生长率(SGR)和饵料利用率(FER)。其中,投喂组以109cfu/m3为最适浓度,泼洒组中108和1010cfu/m3两个浓度均能显著提高SGR,但是两组之间对SGR的影响差异不显著(P>0.05)。(2)实验期间投喂组不同浓度的三个处理组均能有效降低弧菌数量,效果持续并且三组之间的作用规律相似；泼洒处理组在试验期间水体的弧菌数量均低于对照组,水体弧菌数量随泼洒浓度的提高而降低,以泼洒浓度为1010cfu/m3的实验组弧菌数量最低水平。但研究也发现,泼洒组弧菌数量在3-5d时会呈现一定反弹趋势。更多还原

【Abstract】 In recent years, with the rapid development of aquaculture, the animals are threatened by diseases more serously. The occurrence of diseases brings not only huge economic losses but also great impact on the environment at the same time. The abuse of chemical drugs such as antibiotics which are used for preventing the disease has caused serious harm on the environment and human. As substitutes for antibiotics, probiotics can inhibit pathogenic bacteria, provide nutritions for animals, enhance the ability of immunity of animals, improve the water quality conditions and repair the ecological environment. So the usage of probiotics in aquaculture is attracting more attention gradually. One of the inhibitory mechanisms of probiotics is to restrain the growth of the pathogen bacteria by producing inhibitory substances. Therefore, it is important to choose some bacterias which have great inhibitory activity as potential probiotics.In this study, we did some researches on the indentification, inhibitory substances and application of an antagonistic bacterium strain against vibrio. It is aimed to provide a theoretical basis for its applicaton in aquaculture. The results of the study are as follows:1. Identification of an antagonistic bacterium strain against vibrio and study of its inhibitory activityIn this experiment, we separated and screened an antagonistic bacterium strain, named it as BL1. After tested by cross-streaking method, spot inoculation and well test, it is found that strain BL1 has obvious inhibitory activity against Vibrio alginolyticus, V. vulnificus and V. parahaemolyticus. According to the results of the morphological, physiological and 16S rDNA sequence analysis, strain BL1 is identified as Bacillus subtilis. The single-factor analysis further helps to confirm the best culture conditions of strain BL1 is 28℃, pH7.0, inoculation quantity of 1% (V/V) and 190rpm/min.2. Separation and characterization of inhibitory substances of strain BL1Taking the fermentation liquor of BL1 as the subject, Vibrio vulnificus as the indicator bacteria, the characterization of inhibitory substances are premilinary studied through well test. First, the experiment about effect of time on the inhibitory activity shows that 24h is the best. Second, after preliminary purification including filter, ammonium sulfate precipitation and ultra filtration, it is found that the inhibitory substances are extracellular, non-protein and have greater molecular weight than 10 kDa. At last, the inhibitory substances have great ability of enduring the changes of environment, such as temperature (60-100℃) and pH (3.0-11.0). And they are not sensitive to proteinase K. The results show the inhibitory substances produced by strain BL1 have great inhibitory ability and stability, which indicates strain BL1 can be used as potential probiotic in aquaculture.3. Effects of strain BL1 on the growth of Litopenaeus vannamei and the quantity of vibrio in water.The safety inspection proves that strain BL1 has no negative effects on litopenaeus vannamei, therefore, strain BL1 is applied to the water to study its impact on growth and the quantity of vibrio in water. The additive concentrations of living bacillus are 107,109 and 1011 cfu/kg for supplement in feed (T1, T2 and T3)and 106,108 and 1010 cfu/m3 for addition to water (P1, P2 and P3) respectively. The control was feed with basic bait without BL1 (A). The experiment lasts 42 days. The quantity of vibrio in water is determined at the time of 0.5,1,2,3,4 and 5d of the cycle. At the end of the experiment, measure the weight growth rate (WGR), specific growth rate (SGR), feed utilization rate (FER) and survival rate (SR). The results show that:(1) in the respect of growth, strain BL1 in all the treatment groups except P1 can significantly increases SGR, promotes the growth of the shrimps. While the T2 (109 cfu/kg) is the best, P2 and P3 (108 and 1010 cfu/m3) have no significantly different effect on SGR and FER (P>0.05); (2) all the different dose of strain BL1 for supplement in feed can reduce the quantity of vibrio effectively and permanently, and they have the similar change rule. The quantity of vibrio of P1, P2 and P3 are also lower than that of the control group during experiment, and the effect is better with the concentration increasing. Finally, in the water of the P3 (1010 cfu/m3), the quantity of vibrio falls to the lowest level. But, the study also finds that the quantity of vibrio in addition to water group will partly rebound on 3-5d.更多还原