【作者】 潘晓东；

【导师】 吴天星；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2006， 硕士

【摘要】 随着水产养殖的规模化和集约化，水产动物的健康养殖面临着挑战。而抗生素和抗菌药物长期使用，造成诸如细菌抗药性、耐药基因的转移、水产品中药物残留等不良后果，直接或间接威胁人类健康。益生菌即微生态制剂，是一类能够调节水产动物肠道微生态平衡，改善水体环境，增强机体免疫，促进消化、抵御病原菌侵袭的活的微生物。益生菌的使用是安全有效环保的，因此研究和开发水产用益生菌菌株对促进水产养殖有着重要的意义。丁酸梭菌(Clostridium butyricum)，即丁酸梭菌，常作为益生菌用来防治由于多种因素引起的人和动物的腹泻、肠炎等疾病。本次实验研究丁酸梭菌在体外粘附抑制鱼常见病原菌的情况，及其对鮸鱼肠道生理的影响。试验分三部分：(一)分离和培养鮸鱼肠道上皮细胞(intestinal epithelial cell，IEC)，建立肠道上皮细胞的研究模型；(二)丁酸梭菌在IEC模型上的粘附，抑制病原菌粘附作用，及细胞安全性评价；(三)通过饲喂实验，观察其对鮸鱼肠道组织形态、消化酶、肠内短链脂肪酸、肠壁微生物的影响。主要研究结果如下：1采用Ⅳ型胶原酶、Ⅰ型胶原酶和EDTA消化鱼肠道中段分离IEC细胞，以贴壁时间的不同纯化IEC，并缩短处理时间，即可获得有活力的、较纯的IEC，呈单层生长，可作为试验的模型。2丁酸梭菌能够粘附于肠上皮细胞，粘附率8.03％，且不侵袭细胞，粘附后对细胞损伤相较低。丁酸梭菌能降低鳗弧菌的粘附率，其中竞争试验，粘附率降低6.71％；排斥试验，粘附率降低8.27％；替代试验，粘附率降低4.47％。3通过饲喂丁酸梭菌，能提高鱼的消化酶活性，影响肠内短链脂肪酸，其中丁酸尤为明显，中后肠浓度分别增加了2.09mmol／kg、2.12mmol／kg。肠绒毛高度增高，中肠提高了28.81μm，后肠提高了34.23μm。上皮细胞上微绒毛高度增高，中肠和后肠分别增高了1.89μm，1.09μm。电镜下观察，可见后肠肠壁上细菌定植。结论：丁酸梭菌具有粘附能力，不存在致病因素，能竞争的抑制肠内致病原微生物，改善肠道消化生理，增加肠内SCFA，增强肠内消化酶活性，提高肠道绒毛高度。采用IEC模型，体外研究丁酸梭菌的粘附及抑制，在一定程度上能够反映体内的菌群互作，具有一定的参考价值。更多还原

【Abstract】 With the development of aquaculture cultivation, aquatic animal heath faces greatchanllenges. The long-term use of antibiotics and anti-bacteria substance causes muchharmful effect, such as anti-medicine ability of bacteria, translocation of anti-medicinegenes and medicine remnants in muscle. Probiotics, the same meaning of microbialecology products are various live bacteria, which can modulate intestinal microbialecology of aquatic animals, improve water environment, enhance the body immunity,advance the food digestion and inhibit the pathogenic bacteria. It is proved that use ofprobiotics is safe and effective to animal body, so it appears valuable significance tostudy and develop probiotic for aquaculture.Clostridium butyricum which is usually used as human and animal medicine, cancure diarrhea and enteritis. Our research focuses on the cell adhesion and anti-pathogenof Clostridium butyricum, and effect to intestinal physiology of Miichthys miiuy. Theresearch contains three parts: (Ⅰ) Separate and cultivate intestinal epithelial cell(IEC),develop the research model of cell; (Ⅱ) Study Clostridium butyricum adhesion to cellmodel, and inhibition to pathogenic invasion and cell safety evaluation; (Ⅲ) By take ofClostridium butyricum, we observe the fish intestine morpha, gut digestion enzymeactivity, intestinal short chain fatty acid (SCFA) and the ecology of intestinal innerwallThe main results are as follows:1. We can separate the IEC withⅣcollagen enzyme、Ⅰcollagen enzyme and EDTA.Purifying the IEC in line with the time difference of adhesion to wall and reducingthe treatment time. In this way, IEC can be obtained lively, and appear monolayergrowth.2. Clostridium butyric can adhere to IEC, adhesion rate 8.03%, and will not invade orharm cells. It can reduce the adhesion of Vibrio anguillarum in different treatmentgroups. On competition experiment, adhesion rate of Vibrio anguillarum candecrease 6.71%; repulsion experiment, decrease 8.27%; substitution experiment,4.47%. 3. Feeding Miichthys miiuy with Clostridium butyric, it can increase its intestinaldigest enzyme activity and improve SCFA quantity, especially butyric acid withthe increase of 2.09mmol/kg in mid-gut and 2.12mmol/kg in hinder gut. Intestinalvillus height increases 28.81μm in mid-gut and 34.23μm in hinder gut; microvillusheight increases 1.89μm, 1.09μm separately. With the electronic lens, we canobserve bacteria adhering to intestinal inner wall.Conclusion:Clostridium butyricum has cell adhesion ability, and has no pathogenic property.It can inhibit intestinal adhesion of pathogenic bacteria, and improve the quantity ofintestinal SCFA, digest enzyme activity, intestinal villus height. In vitro, the researchon bacteria adherence and inhibition can reflect the mutual action of fish intestinalbacteria in certain extent.更多还原