导　　师： 田相利

学科专业： I0801

授予学位： 硕士

作　　者： ;

机构地区： 中国海洋大学

摘　　要： 刺参（Apostichopus japonicus）属于棘皮动物门（Echinodermata）、海参纲（Holothroidea），是我国北方经济效益较高的、重要增养殖种类。刺参养殖主要分布在山东、辽宁、河北、江苏北部等地，2009年全国刺参养殖面积达到15.5万公顷，产量达到10.22万吨，产值超过200亿元，刺参养殖业已成为我国海水养殖的重要的特色支柱产业。然而，相对于刺参养殖业迅速发展的养殖规模，与刺参养殖技术相关的基础研究相对滞后，制约了该产业的健康发展，导致刺参养殖相继出现了一系列问题，造成了较大的经济损失。例如，刺参病害问题日趋突出，出现了多种明显病症和大规模死亡现象，制约了刺参养殖产业的持续、健康、稳定发展，给养殖业者造成了巨大的经济损失。目前，我国水产养殖病害控制还以抗生素和化学消毒药物为主，养殖水体中长期使用抗生素和化学药物导致了一系列的环境和社会问题。为了缓解这一矛盾，近年来人们一直在寻找一种既能有效控制疾病同时又不会对养殖环境造成破坏的方法。益生菌因其在抑制病原微生物、调节养殖动物肠道微生态环境、增强养殖动物免疫力、促进生长，且无污染、无残留、无毒副作用的特点，目前在水产养殖业中的应用正得到越来越多的关注。本论文研究了几株分离自养殖池塘的刺参潜在益生菌的生物学特性，并对其进行了分类鉴定。进一步的养殖实验效果验证了，2菌株均能明显促进刺参的生长，并提高刺参肠道消化酶和体腔液非特异性免疫酶活性，对刺参养殖环境微生物数量也有较大影响，是应用价值较高的刺参益生菌材料。具体研究结果如下： 1两株刺参益生菌的鉴定及安全性检验 结合形态观察、生理生化指标测定以及16S rDNA序列分析等方法对BC-1、BC-2两菌株进行了分类学鉴定。结果表明，2菌株均为蜡样芽孢杆菌（Bacilluscereus）。其中，菌株BC-1的适宜生长温度为18～39℃，最适生长温度为24℃；菌株BC-2适宜生长温度为18～39℃，最适生长温度为27℃。菌株BC-1的适宜生长盐度范围为0～30；菌株BC-2适宜生长盐度范围为0～35。菌株BC-1、BC-2适宜生长的pH值均为5.5～9.0之间，BC-1菌最适宜生长pH为7.5；菌株BC-2最适宜生长pH为6.5。而采用肌肉注射和浸浴两种方式对2菌株进行安全性检验均未发现其对刺参明显的负面影响，因此，该2菌株均可以作为进一步养殖效果研究的材料。 2BC-2对刺参生长以及消化和非特异性免疫相关酶活性的影响 研究了一株蜡样芽孢杆菌对刺参生长、消化和免疫相关酶活性的影响。芽孢杆菌的添加量分别为10~7、10~9和10~/(11/)cfu//kg（以活菌量计），以投喂基础饲料组作为对照，实验时间为56d。实验过程中分别于实验中期和结束时测量刺参生长情况，并于实验结束时测定刺参肠道消化酶和体腔液非特异性免疫相关酶类的活性。结果表明：添加10~7和10~9cfu//kg芽孢杆菌可以显著提高刺参的生长速度，降低刺参体重变异系数（p<0.05）。刺参肠道蛋白酶活性与投喂的菌浓度呈明显的正相关，而淀粉酶活性则呈明显的负相关，但添加该芽孢杆菌对脂肪酶活性影响不大（p>0.05）。添加芽孢杆菌对刺参非特异性免疫相关酶类影响有所不同。在本研究的添加量范围内，刺参体腔液超氧化物歧化酶（SOD）活性显著提高（p<0.05）；适量添加该芽孢杆菌可以显著提高刺参体腔液中酸性磷酸酶（ACP）、碱性磷酸酶（AKP）和溶菌酶（LSZ）活性（p<0.05）。综合比较表明，芽孢杆菌BC-2的适宜添加量为10~9cfu//kg，对刺参的生长、消化功能和非特异性免疫能力均具有显著的促进作用。 3BC-1不同施用方式对刺参生长以及消化和免疫功能相关酶活性影响的对比研究 通过拌饵投喂及水体泼洒两种施用方式，研究了一株蜡样芽孢杆菌对刺参生长，消化、免疫相关酶活性及环境微生物数量的影响。两种投喂方式下各设3个不同的施用浓度，拌饵投喂实验组芽孢杆菌添加量分别为10~7、10~9、10~/(11/)cfu//Kg（以活菌量计），水体泼洒实验组芽孢杆菌添加量分别为10~6、10~8、10~/(10/)cfu//m~3，以投喂基础饲料为对照组，实验时间为56d。实验过程中每2周测定一次水体异样菌数量，实验中期和结束时测量刺参生长情况，并于实验结束时测定刺参肠道消化酶和体腔液非特异性免疫相关酶类的活性。实验结果显示：（1）整个实验期间泼洒组水体异养菌数量与对照比较一直处于较低水平，而拌饵投喂组水体异养菌数量前期显著高于对照组（p<0.05），随着实验进行两者之间的差距逐渐缩小直至消失；（2）生长方面，泼洒10~6cfu//m~3、拌饵投喂10~9cfu//Kg的BC-1菌能显著促进刺参的生长降低刺参体重变异系数（p<0.05）；（3）消化酶活性方面，投喂10~9、10~/(11/)cfu//Kg的BC-1菌能显著提高刺参肠道淀粉酶及蛋白酶活性，而投喂浓度为10~7cfu//Kg、泼洒浓度为10~8cfu//m~3两个实验组脂肪酶活性及蛋白酶活性均显著高于对照组（p<0.05）。（4）两种施用方式下，BC-1菌对刺参体腔液免疫酶活性的提高均有明显的促进作用，其中投喂浓度为10~9、10~/(11/)cfu//Kg两个实验组SOD、AKP、ACP以及溶菌酶活性均显著高于对照组，泼洒浓度为10~/(10/)cfu//m~3的实验组除SOD活性外，其它几种免疫酶活性均高于对照组（p<0.05）。综合比较表明，芽孢杆菌BC-1水体泼洒和拌饵添加的适宜浓度分别为10~8cfu//m~3和10~9cfu//Kg，对刺参的生长、消化功能和非特异性免疫能力均具有显著促进作用。 1The identification and safety inspection of two probiotics forApostichopus japonicus By the means of morphological, physiological and biochemical tests, and16S rDNAsequence analysis, two bacterial strains /(BC-1, BC-2/) isolated from sea cucumberculture pond were identified. The bacterial strain BC-1and BC-2were identified asBacillus cereus. The effects of temperature, salinity and pH on growth of strains weretested. The results show that, The suitable growth temperature of the strain BC-1was18~39℃, and the optimum growth temperature was24℃; the suitable growthtemperature of the strain BC-2was18~39℃, the optimum growth temperature was27℃. The suitable growth salinity range of the strain BC-1was0~30; the suitablegrowth salinity range of the strain BC-2was0~35. The suitable growth pH valuerange of the strains BC-1and BC-2were5.5~9.0. The optimum growth pH for BC-1was7.5and the BC-2was6.5. Water purification study found that content ofharmful materials in breeding wastewater, such as ammonia nitrogen and nitritenitrogen, was significantly reduced by addition of the two strains /(p<0.05/).Meanwhile, COD level in water was also decreased. Safety of these two strains wasevaluated by muscle injection and extraction. Results showed that these strains had nosignificantly negative influence on Apostichopus japonicus growth under appropriateadditive concentrations. Therefore, they could be used as further research materialsfor breeding effect study. 2Effects of addition of Bacillus cereus on the growth, digestive andnon-specific immune enzymes activities of Apostichopus japonicus Effects of Bacillus cereus on the growth, activities of digestive and non-specificimmune enzymes in Apostichopus japonicus were studied over56days. The additivedoses of living bacillus cells in feed were0,10~7,10~9and10~/(11/)cfu//kg, respectively.Results showed that the growth of A. japonicus enhanced significantly /(p<0.05/),while the coefficient of variation in body weight decreased significantly /(p<0.05/)when B. cereus was added in feed at the dose of10~7and10~9cfu//kg. Intestinal proteaseactivity exhibited a positive correlation with Bacillus concentration in feed whileamylase activity showed a negative correlation /(p<0.05/). The activities ofnon-specific immune enzymes were affected by various addtive doses of Bacillus infeed. With the increase of Bacillus concentration in feed, superoxide dismutase /(SOD/)activity in coelomic fluid of A japonicus enhanced significantly /(p<0.05/). And theactivities of acid phosphatase /(ACP/), alkaline phosphatase /(AKP/) and lysozyme /(LSZ/)in A japonicus when Bacillus was added in feed at the dose of10~9cfu//kg significantlyenhance /(p<0.05/). According to the results in the present study, it could be concludedthat the optimal additive concentration of B. cereus BC-2in feed was10~9cfu//kg, atwhich the growth rate, activities of digestive enzyme and the capacity of non-specificimmunity in A. japonicus could be significantly improved. 3Effects of a probiotic on the growth, environmental microorganism，digestive and non-specific immune enzymes activities of Apostichopusjaponicus Two feeding ways of mixing with bait and splashing water out were chosen toevaluate the effect of a Bacillus cereus on Apostichopus japonicus growth, digestiveand non-specific immune enzymes activities and environmental microbial amount.The additive concentrations of living bacillus in two feeding ways were10~7、10~9、10~/(11/)cfu//Kg in mixture group and10~6、10~8、10~/(10/)cfu//m~3in splashing group. In thecontrol, basic bait was added. The whole experiment was finished in56days. Strangebacterium quantity in water was determined every two weeks. Growth ofApostichopus japonicas was measured both in the mid-stage and at terminated period. Activities of digestive enzymes in the digestion gut and non-specific immuneenzymes in coelomic fluid were assayed at the end of the experiment. Results were asfollows:/(1/) Strange bacterium quantity in splashing group kept lower lever comparedwith the control, while mixture group showed significant higher level /(p<0.05/). Thedivergence narrowed with time going on until almost disappeared./(2/)The growth ofApostichopus japonicus was promoted and the weight coefficient of variation wasdecreased obviously with bacterium BC-1concentration of10~9cfu//Kg in mixturegroup and10~6cfu//m~3in splashing group./(3/)When the additive concentrations of BC-1were10~9or10~/(11/)cfu//Kg in mixture group, amylase and protease activities in intestinalwere enhanced prominently. The lipase and protease activities were higher undermixing feed concentration of10~7cfu//Kg and splashing concentration of10~8cfu//m~3compared with the control /(p<0.05/)./(4/)Under the two feed ways, bacterium BC-1showed obvious stimulative effect on immune enzymes activities in coelom. Themixture group with bacterium concentrations of10~9、10~/(11/)cfu//Kg exhibited higherSOD、AKP、ACP and lysozyme activities than that in the control. When the splashingconcentration was10~/(10/)cfu//m~3, except SOD, other immune enzymes were higher thanthat in the control. Considering all the results above, it could be conclude that thegrowth rate, digestive and non-specific immune enzymes activities could be improvedsignificantly when the mixing concentration was10~9cfu//Kg and splashingconcentration was10~8cfu//m~3.

关 键 词： 刺参 益生菌 蜡样芽孢杆菌 非特异性免疫 酶活性

分 类 号： [S9]

领　　域： [农业科学]