乳酸菌对凡纳滨对虾益生机理的研究

IOCAS-IR > 实验海洋生物学重点实验室

	乳酸菌对凡纳滨对虾益生机理的研究
	沙玉杰1,2
学位类型	博士
导师	王雷
	2016-05-13
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	水产养殖
关键词	凡纳滨对虾益生菌乳酸菌益生机理
摘要	从商业益生菌产品、中国对虾（Fenneropenaeus chinensis）和矛尾虾虎鱼（Acanthogobius hasta）的肠道中共分离得到53株乳酸菌，并对这些菌株进行了溶血活性、抑菌性和粘附性的测定。实验结果表明，53株乳酸菌均没有溶血活性，有两株菌在所筛选得到的菌株中分别表现出了较好的抑菌性和粘附性。基于16S rRNA分子生物学鉴定和生理生化实验，确定以上两株菌分别为戊糖乳杆菌HC-2和粪肠球菌NRW-2。探讨和比较了HC-2、NRW-2和HC-2发酵上清液对凡纳滨对虾的益生作用及其益生机理。首先，在基础饲料中分别添加HC-2、NRW-2、HC-2的发酵上清液，最终每克基础饲料中含有1.0×10⁷ CFU益生菌或此菌量所对应的发酵上清液，共配制3组实验饲料，进行为期四周的投喂实验。结果表明凡纳滨对虾投喂含有HC-2或NRW-2的饲料时，其特定生长率高于对照组；而投喂HC-2发酵上清液时，其特定生长率低于对照组但是差异不显著。同时，利用实时定量PCR测定了对虾中肠和肝胰腺中相关免疫基因的表达情况。实验结果表明，饲料中添加NRW-2两周和四周时显著促进了凡纳滨对虾中肠中PEN-3α、proPO和lysozyme的表达；而HC-2及其发酵上清液对肝胰腺中相关免疫基因的表达具有较高的诱导作用。此外攻毒实验显示所有处理组中凡纳滨对虾对病原菌的抵抗力均显著高于对照组。其次，研究了饲料中添加益生菌及其发酵上清液对凡纳滨对虾肝胰腺和肠道消化酶活性的影响。实验结果如下：与对照组相比，各实验组对虾肠道中蛋白酶活力显著提高（P < 0.05），但肝胰腺中蛋白酶活力无显著差异；添加NRW-2的实验组与对照组相比，肠道和肝胰腺中淀粉酶和脂肪酶活力均显著提高（P < 0.05），HC-2组对虾肝胰腺中淀粉酶及肠道中脂肪酶的活力显著提高（P < 0.05），而HC-2发酵上清液的添加仅显著提高了对虾肠道蛋白酶和淀粉酶的活性。结果表明，饲料中添加NRW-2、HC-2及其发酵上清液可以提高凡纳滨对虾肠道及肝胰腺中消化酶的活力，但在不同组织中提高消化酶活性的种类是不同的。此外，利用Illumina Miseq高通量测序技术分析了凡纳滨对虾肠道中的细菌组成。高通量数据显示Proteobacteria，Verrucomicrobia和Actinobacteria是各个处理组中的优势菌群，但各个处理组中均存在其特有的细菌种类，并且饲料中添加HC-2发酵上清液后，与对照组相比，显著增加了对虾肠道中Actinobacteria的丰度。以上实验结果说明，益生菌及其发酵上清液的添加改变了对虾肠道细菌的组成及其丰富。但是不同实验饲料的投喂没有明显改善对虾肠道的形态结构，由此推测对虾肠道中微生物群落之间复杂的相互作用或许在保持或促进对虾健康方面发挥着重要的作用。再次，为了探讨HC-2和副溶血弧菌E1（VPE1）在对虾体内外的相互作用，利用DAPI和CFDA-SE分别对HC-2和VPE1进行荧光标记后，用荧光显微镜观察两株菌在对虾肠道中的分布情况。当对虾同时投喂这两株菌时，在对虾肠道内HC-2对VPE1具有一定的竞争排斥作用。此外，在体外跟踪测定了HC-2与VPE1共培养或者纯培养时两株菌的生长情况和菌中与群体感应或毒力因子相关的免疫基因的表达。结果得知，HC-2与VPE1共培养8小时后，培养液中VPE1的菌浓度显著下降而HC-2的菌浓度变化趋势与纯培养时相一致。转录数据显示，共培养8小时后，HC-2中luxS基因的表达量逐渐上升，说明LuxS或许参与到HC-2对VPE1的竞争排斥过程。有趣的是，热失活的HC-2菌体可以显著促进VPE1中的aphA、opaR和tlh的表达，该实验结果表明，HC-2的胞内成分在共培养时可以促进VPE1毒力相关因子的短期表达，这或许会增加对虾感染弧菌的风险。最后，探讨了戊糖乳杆菌HC-2在凡纳滨对虾感染弧菌前后的短期投喂对凡纳滨对虾存活率和肠道中相关免疫基因表达的影响。实验结果显示，凡纳滨对虾投喂益生菌后再浸浴在弧菌中的存活率高于对虾感染弧菌后再投喂益生菌的存活率，但是差异不显著。并且对虾肠道相关免疫基因的表达情况也说明，当凡纳滨对虾受到弧菌的感染后，益生菌的添加对凡纳滨对虾内在免疫具有一定的调节作用，在一定程度上可以增强其对病原菌的抵抗力，但益生菌的短期投喂不能较好的提高凡纳滨对虾抵抗病原菌的能力。
其他摘要	Fifty-three lactic acid bacteria (LAB) strains were respectively isolated from commercial probiotics, and the intestinal tracts of shrimp (Fenneropenaeus chinensis) and fish (Acanthogobius hasta). The antibacterial and adhesive activities of these strains were examined, the results showed that Lactobacillus pentosus HC-2 (HC-2) and Enterococcus faecium NRW-2 (NRW-2) had higher antibacterial and adhesive activities, respectively, the identification of strains was based on the 16S rRNA analysis. The aim of this study was to investigate and compare the probiotic effects and mechanisms of NRW-2, HC-2 and the corresponding supernatant on the shrimp (Litopenaeus vannamei). Firstly, the following four trails (three treated and one control) were carried out for four weeks: T1, shrimp fed with a commercial diet + strain HC-2 (1×10⁷ CFU g feed^-1); T2, shrimp fed with a commercial diet + strain NRW-2 (1×10⁷ CFU g feed^-1); T3, shrimp fed with a commercial diet + the supernatant of strain HC-2 (the corresponding supernatant of 1×10⁷ CFU g feed^-1 of HC-2); and T4, shrimp fed with a commercial diet alone as a control. The results showed that the specific growth rates of shrimp fed with selected strains were higher than the control group; whereas that of the supernatant of the HC-2-containing group was lower than the control even though it was not significant. Real-time PCR was used to determine the expression levels of immune-related genes in the midgut and hepatopancreas. The results suggested that NRW-2 had higher stimulation to PEN-3α、proPO and lysozyme in the midgut at two weeks and four weeks post-feeding, respectively, while HC-2 and its corresponding supernatant had higher induction of genes in the hepatopancreas. According to the challenge tests, all of the treatment groups had significantly higher survival rate than the control group. Secondly, the effects of probiotics and the corresponding supernatant on activities of digestive enzymes in Litopenaeus vannamei were investigated. The results showed that the protease activities were significantly (P < 0.05) increased in the intestine of three treatment groups compared with the control group, but they were no significant in the hepatopancreas. The amylase and lipase activities were all increased significantly (P < 0.05) in intestine and hepatopancreas of shrimp fed with NRW-2 compares with the control group, the amylase and lipase activities were increased significantly (P < 0.05) in hepatopancreas and intestine respectively in the group of shrimp fed with HC-2, whereas the protease and amylase activities were just increased significantly in the intestine of shrimp fed with the supernatant of HC-2. This study suggested that activities of digestive enzymes in L. vannamei can be increased by NRW-2, HC-2 and the corresponding supernatant, but varied in different tissues. In addition, a microbial identification technique based on the 16S rRNA gene sequenced with Illumina Miseq was used to thoroughly investigate the intestinal bacterial composition of L. vannamei after four dietary regimens with different probiotics or probiotic supernatant. Proteobacteria, Verrucomicrobia, and Actinobacteria were dominant in the intestines of L. vannamei, regardless of its diet. However, unique species existed in all subsets of each dietary group and the abundance of Actinobacteria was significantly increased in the intestinal bacterial community of shrimp fed with the bacteria-free supernatant of an HC-2 culture compared with the control. These results suggested that the composition and richness of bacterial species in the intestines of shrimp were influenced by dietary supplementation with probiotics or probiotic supernatant. In addition, the histology of intestines of the shrimp from four dietary groups were also described, but no obvious improvements in the intestinal histology were observed. Therefore it can be speculated that bacterial interactions in complex microbial communities may play important roles in maintaining or promoting the health of the host. Thirdly, the interaction between HC-2 and Vibrio parahaemolyticus E1 (VPE1) when cocultured in Litopenaeus vannamei in vivo and in vitro was investigated. Fluorescence microscopy was used to observe the distribution of these two strains, which were labeled with DAPI and CFDA-SE, respectively, in the gut of the shrimp. Fluorescent imaging revealed that HC-2 competitively excluded VPE1 in the intestine of L. vannamei when the shrimp diet contained both these strains. The growth of both bacteria and the expression of quorum-sensing- and virulence-related genes of HC-2 or VPE1 were monitored when they were cultured alone or together in vitro. In contrast to HC-2, the population density of VPE1 decreased significantly after coculture for 8 h. A transcriptional analysis showed that the expression of the luxS gene in HC-2 increased after 8 h when it was cocultured with VPE1, suggesting that LuxS was involved in the competitive exclusion of VPE1 by HC-2. Interestingly, the aphA, opaR, and tlh genes of VPE1 were significantly upregulated in response to heat-killed HC-2 cells. This suggested that the intracellular components of HC-2 induced the expression of toxicity-related factors in VPE1 for a short time in coculture, and these may increased the risk of shrimps infected by vibrios. Lastly, the effects of the short-term addition of HC-2 on the survival rate and expression of immune-related genes of L. vannamei were investigated before or after L. vannamei infected by VPE1. The results showed that the survival rates were higher when HC-2 was added before shrimps infected than after, even though it was not significant. The transcriptional profiles of immune-related genes in the guts of L. vannamei suggest that the addition of HC-2 after shrimps infected can enhance the immunity at a certain degree, but the short-term addition of HC-2 can not significantly enhance the disease resistance of L. vannamei.
学科领域	水产养殖学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112564
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	沙玉杰. 乳酸菌对凡纳滨对虾益生机理的研究[D]. 北京. 中国科学院大学,2016.

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