CN112322544B - 一种用于对虾养殖的鲁杰氏菌 - Google Patents
一种用于对虾养殖的鲁杰氏菌 Download PDFInfo
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Abstract
本发明提供一种从对虾中分离的鲁杰氏菌，所分离的鲁杰氏菌具有降氨氮的效果，并且具有更高的安全性；其保藏编号为CGMCC No.20973。本发明所提供的鲁杰氏菌应用于对虾养殖中。本发明所提供的鲁杰氏菌BD15‑5对凡纳滨对虾不具有致病性，可以在凡纳滨对虾养殖生产中大剂量应用；在水产养殖水质调控中具有较大的巨大潜力。
Description
技术领域
本发明属于水产养殖益生菌筛选技术领域，具体涉及一种用于对虾养殖的鲁杰氏菌。
背景技术
益生菌在养殖系统中定植并形成优势菌群是生物絮团发挥高效性能的关键。利用环境适应能力强并具有特定功能的菌株培育生物絮团，有助于提升絮团的稳定性和功效。在生物絮团养殖模式下，外源添加益生菌能显著提升异育银鲫的消化酶活性，增加肠道肌层厚度和黏膜下层厚度，促进异育银鲫生长。
外源益生菌常因不适合特定养殖环境难以定植形成优势菌群，而经筛选的具有特定功能土著益生菌更能适应当地的养殖环境，更容易定植并形成优势菌群。从海南热带海洋分离的土著乳酸菌与现有市售某复合菌株产品拌料投喂凡纳滨对虾，土著乳酸菌能显著提升凡纳滨对虾的增重率和成活率。
目前对于益生菌的研究主要集中在益生菌的益生作用及机制和益生菌作为生物工程中基因载体的潜能，而对益生菌安全性的关注相对较少。在水产养殖中被认为安全的芽孢杆菌也存在引起水产动物腐皮病、败血症等病害的潜在风险。益生菌在使用过程中主要存在致病性和感染能力、有害的代谢活动、过度的免疫反应和可能的基因转移等四个方面的安全问题。通过安全性评价可以评估益生菌的使用风险，更加规范、安全地使用益生菌，避免造成不可挽回的损失。目前，安全性评价尚没有统一的标准，主要包括益生菌的溶血性、毒力因子、抗生素抗性和产有毒代谢产物检测的体外安全性评价和体内安全性评价两方面。
发明内容
本发明的目的是提供一种从对虾中分离的鲁杰氏菌，所分离的鲁杰氏菌具有降氨氮的效果，并且具有更高的安全性；从而弥补现有技术的不足。
本发明所提供的鲁杰氏菌(Ruegeria sp.)BD15-5菌株，于2020年10月29日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.20973；保藏地址为北京市朝阳区北辰西路1号院3号。
本发明所提供的鲁杰氏菌在对虾养殖中的应用；
本发明还提供筛选的鲁杰氏菌在制备对虾养殖用的制品中的应用；
所述的制品，为活菌制剂。
本发明还提供所筛选的鲁杰氏菌在对虾养殖中调节水质中的应用。
本发明所提供的鲁杰氏菌BD15-5对凡纳滨对虾不具有致病性，可以在凡纳滨对虾养殖生产中大剂量应用；在水产养殖水质调控中具有较大的巨大潜力。
附图说明
图1：BD15-5的系统分类图；
图2：菌株BD15-5在LB固体培养基上的菌落形态图；
图3：碳源对菌株BD15-5的TAN降氮性能的影响图；
图4：碳氮比对菌株BD15-5的TAN降氮性能的影响图；
图5：盐度对菌株BD15-5的TAN降氮性能的影响图；
图6：pH对菌株BD15-5的TAN降氮性能的影响图；
图7：菌株BD15-5的溶血现象图；
图8：菌株BD15-5注射感染实验中凡纳滨对虾的存活率图。
具体实施方式
下面结合实施例和附图对本发明的菌株进行详细的描述。
实施例1：筛选鲁杰氏菌菌株
1)筛选样品来源与筛选用培养基
2017年9月于威海文登泰裕水产良种繁育有限公司获得的对虾生物絮团(盐度为30‰)，该生物絮团是以甘蔗渣、稻壳粉和芽孢杆菌为元件，通过养殖系统中的絮团发生装置培育形成的，在养殖过程中发现该生物絮团能有效降低养殖对虾水体的氨氮水平，且大量培养投放后，对虾不会产生应激反应。
在无菌状态下，取0.2mL涂布于固体LB平板上，4℃保存，带回实验室进行进一步的筛选。
筛选过程中使用的培养基的组分配比如下：
富集培养基：NaNO2 1g，CH3COONa 4.8g，过滤海水1L，pH 7.5；
筛选培养基：NaNO2 1g，CH3COONa 4.8g，过滤海水1L，琼脂15g，pH 7.5；
测试培养基A：NaNO2 0.3g，C6H1206 0.225g，KH2PO4 0.05g,Na2HPO4 0.5g,MgSO40.2g,蒸馏水1L，PH 7.5；
测试培养基B：NH4Cl 1g，CH3COONa 4.8g，过滤海水1L，pH 7.5；
LB液体培养基：蛋白胨10g，酵母浸粉5g，氯化钠30g，纯水1L；
LB固体培养基：在液体培养基的基础上加15g琼脂粉。
2)高效降解菌的筛选与鉴定
富集：用无菌海水冲刷LB固体培养基上的菌苔形成菌液，然后按5％的比例接种到富集液体培养基中，以没有接菌的培养基作为对照，于恒温振荡培养箱(28℃,180r/min)中培养24h，取5mL菌液离心(8000r/min,10min)，检测上清液的亚硝态氮浓度；然后选取亚硝态氮降解程度较大的菌液以10％的比例转接至空白液体培养基中，于恒温振荡培养箱(28℃,180r/min)中培养24h，连续转接三次以淘汰非目标菌。
分离纯化：将所得的菌液稀释至10-3，取10-2和10-3的稀释菌液0.1mL涂布于LB固体平板，28℃培养24h。用无菌接种环从平板上挑选颜色、形状、大小和透明度不同的菌落,采用平板划线法分离纯化,直至得到纯的单菌落。采用甘油保种法将菌种于-80℃保存。
复筛：将上述分离纯化得到的菌株接种至LB液体培养基(28℃,180r/min)中培养24h，离心(8000r/min,10min)，弃上清，生理盐水重悬，重复3次，制备得到菌悬液。将菌悬液按5％比例接种至测试培养基(28℃,180r/min)培养24h，取样测定OD600，离心(8000r/min,10min)后取上清测定亚硝态氮浓度，筛选降解性能较高的菌株供下一步研究。
3)筛选菌株的降氮性能
分别以TAN(10mg/L)、NO2 --N(10mg/L)和TAN+NO2 --N(5+5mg/L)替代测试培养基中的NO2 --N，检测复筛所得的高效降氮菌的降氮特性。
初步分离纯化得到109株菌，然后挑选生长优势明显、菌落差异较大的菌株以亚硝态氮和氨态氮作为唯一氮源进行复筛，得到11株菌。
在以亚硝态氮为唯一氮源时，菌株亚硝态氮降解的效果是BD15-5>AC3-2>14a>BD15-8>2b>BD15-1。以氨态氮和亚硝态氮为混合氮源时，菌株亚硝态氮降解率：BD15-5＝14a>BD15-8>AC3-2>2b>BD15-1。在综合考虑总氮去除率、菌株生长以及安全因素，最终选择AC3-2、BD15-5和BD15-8作为后续研究的菌株。
表1：菌株对不同氮源的利用效果表
4)菌株分子生物学鉴定
将BD15-5菌株送至上海生工生物工程技术有限公司进行16S rDNA的提取及测序，鉴定结果通过NCBI序列比对，然后用mega 5根据邻接法构建进化树。
根据16S rDNA测序及系统发育分析，确定菌株BD15-5与鲁杰氏菌属菌株具有最高的相似度，判定BD15-5为鲁杰氏菌。
本发明所提供的鲁杰氏菌(Ruegeria sp.)BD15-5菌株，于2020年10月29日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.20973；保藏地址为北京市朝阳区北辰西路1号院3号。
实施例2：BD15-5菌株的理化性质
LB固体培养基在28℃的恒温培养箱中培养24h后，菌落的表观特为：菌落呈圆形，边缘整齐，表面光滑，乳黄色半透明(图2)。
菌株的生长温度范围为20～40℃，最适温度为30℃；生长pH范围为7～9，最适pH为8；生长盐度范围为10～40，最适盐度范围为20～30。
实施例3：BD15-5菌株的降氮性能
1)碳源对菌株生长和降解性能的影响
菌株BD15-5分别以蔗糖和柠檬酸钠为唯一碳源时，总氨态氮去除率为84.6％和83.1％，总氮去除率为65.4％和63.7％；而以蔗糖为唯一碳源时，羟胺的积累量仅为0.7mg/L(图3)。因此，蔗糖为菌株BD15-5的最适碳源。
2)碳氮比对菌株生长和降氮性能的影响
鲁杰氏菌BD15-5株对总氨态氮的去除率和OD值随碳氮比的升高而显著升高，当C/N≥20时，BD15-5对总氨态氮的去除率和OD值达到最大值(图4)。
3)盐度对菌株生长和降氮性能的影响
BD15-5菌株对总氨态氮的去除率和OD值随盐度的升高呈先升高后下降趋势，当盐度为30‰时，BD15-5对总氨态氮的去除率和OD值均达到最大值(图5)。BD15-5在盐度为20‰条件下总氨态氮的去除率和OD值显著高于盐度为40‰，表明菌株对低盐的耐受性高于高盐。
4)pH对菌株生长和降氮性能的影响
菌株BD15-5在pH为8时，总氨态氮的去除率和OD值均达到最大值；且pH大于8时的菌株的生长量和总氨态氮降解率显著大于pH小于8菌株的生长量和总氨态氮降解率(图6)。
综上，以蔗糖或柠檬酸钠为碳源时，菌株BD15-5的总氨态氮和总氮去除率显著比葡萄糖和乙酸钠为碳源时高。BD15-5在碳氮比为20时，总氨态氮的去除率和OD值达到最大值并不随碳氮比的升高而继续增加；说明菌株BD15-5对高C/N的耐受性强。
因此，在对虾生物絮团养殖系统中筛选到的氨氮降解菌鲁杰氏菌BD15-5株在水产养殖水质调控中具有较大的巨大潜力。
实施例4：鲁杰氏菌BD15-5菌株的安全性评价
1)实验材料
凡纳滨对虾(Litopenaeus vannamei)购于山东乳山对虾养殖场体长为(5.0±0.2)cm，平均体重(1.55±0.16)g。将对虾暂养于室内，水温26℃，盐度26，持续充气，早晚各投喂1次，每天吸残饵粪便，换水1/3。暂养15d后开始实验。
试验菌株：鲁杰氏菌BD15-5菌株
副溶血弧菌由中国海洋大学病害与免疫实验室提供。
血琼脂培养基(BZW12005)购于贝瑞特生物技术(郑州)责任有限公司，抗菌药物药敏纸片(S1099)购于杭州微生物试剂有限公司。
注射感染实验在水族箱(规格为长21cm×宽31cm×高19cm)中进行，水体积大约为10.4L。
实验数据用平均值±标准差(mean±SD)表示，通过SPSS 22软件对实验数据进行统计分析，采用单因子方差分析(One-way ANOVA)和Duncan多重比较法进行差异显著性分析，取P<0.05为差异显著。
1)溶血性实验
将实验菌株活化后接种至LB液体培养基，在恒温震荡培养箱(28℃、180r/min)中培养24h(菌液浓度约为108CFU/mL)；然后将菌液点种(10μL)在血平板上，每株菌在同一个血平板上点种3次(即3个平行)，在恒温培养箱(30℃)中培养24h，观察是否有溶血现象产生。以副溶血弧菌作为阳性对照。
菌株溶血实验结果如图7所示，副溶血弧菌组血平板上的滤纸片周围有草绿色圆环，为α溶血；而鲁杰氏菌BD15-5菌株组平板上的滤纸片周围没有圆环，为γ溶血。结果表明鲁杰氏菌BD15-5菌株不产生溶血素，对养殖动物相对安全。
2)抗生素敏感性实验
采用药敏片琼脂扩散法’将实验菌株活化后接种至LB液体培养基，在恒温震荡培养箱(28℃,180r/min)中培养24h(菌液浓度约为108CFU/mL)；然后取0.2mL菌液在LB固体培养基上均匀涂布，贴上药敏纸片(每种抗生素3个平行)，扩散30min后，在恒温培养箱(30℃)中培养24h后测量并记录待测菌株的抑菌圈直径。测试菌株对20种抗生素的药敏性参照CLSI 2017版本的标准进行。
菌株药敏实验结果如表2所示，鲁杰氏菌BD15-5株对诺氟沙星、氧氟沙星、环丙沙星、头孢曲松、头孢他啶、头孢呋辛、苯唑西林、哌拉西林、羧苄西林、多粘菌素B、复方新诺明、万古霉素等12种抗生素敏感；对头孢哌酮、头孢唑林、头孢氨苄、氨苄西林、呋喃吡酮、青霉素、麦迪霉素等7种抗生素中度敏感；对头孢拉定等1种抗生素耐药。
表2菌株BD15-5的药敏实验结果
注：R为耐药，I为中度敏感，S为敏感。
3)注射感染实验
将实验菌株活化后接种至LB液体培养基，在恒温震荡培养箱(28℃、180r/min)中培养24h(菌液浓度约为108CFU/mL)；然后取适量培养液离心(8000r/min,5min)收集菌体，用无菌生理盐水冲洗三次，将菌体密度调整为1.46×107CFU/mL，4℃暂存备用。
实验分为5组，每组设3个平行，每个平行10尾虾。向对虾第四、五腹节分别注射50μL生理盐水(生理盐水组)、50μL(107CFU/mL)嗜碱盐单胞菌AC3-2株菌体(AC3-2组)、50μL(107CFU/mL)鲁杰氏菌BD15-5株菌体(BD15-5组)、50μL(107CFU/mL)美丽盐单胞菌BD15-8株菌体(BD15-8组)、50μL(107CFU/mL)副溶血弧菌菌体(副溶血弧菌组)；其中，生理盐水组作为阴性对照组，副溶血弧菌组作为阳性对照组。定期观察并记录对虾存活数量。注射感染实验结果显示AC3-2组、BD15-8组和BD15-5组与生理盐水组的对虾存活率无显著性差异(P>0.05)，均显著高副溶血弧菌组(P<0.05)。由此可见，向对虾体内注射107CFU/mL浓度的鲁杰氏菌BD15-5株对对虾没有产生毒害作用(图8)。
溶血素即细菌分泌的能够作用于细胞膜而使细胞溶解的毒素，是动物细菌性疾病的重要病源’通过血平板检测菌株的溶血性可以反映细菌是否产生溶血素。实验结果表明鲁杰氏菌BD15-5株没有溶血特征。
对益生菌菌株抗生素耐药性的测定是体外安全性评价的重要内容，益生菌菌株抗性基因易在环境中细菌间进行转移，具有巨大的安全隐患，在实际应用中应避免选择具有抗性基因的菌株；益生菌菌株对抗生素的敏感性可以在一定程度上反映其是否携带抗生素抗性基因。另外，益生菌对抗生素的敏感性可以在细菌爆发无法控制时，为抗生素的合理使用提供参考数据。本发明的鲁杰氏菌BD15-5株对大部分常见抗生素敏感或者中度敏感，具有较高的安全性。
对养殖生物的安全性试验是益生菌安全评估中不可或缺的一项。目前，大多数学者将菌株通过浸浴、拌料投喂和肌肉注射等方式作用于动物，以动物的存活率、体内生理生化指标甚至基因表达的变化情况为依据，评判实验菌株的安全性。本实施例中将10CFU/mL的鲁杰氏菌BD15-5株注射感染凡纳滨对虾，结果表明对凡纳滨对虾无明显的毒害作用。
综合溶血性实验、抗生素敏感性实验和注射感染对虾实验结果证明鲁杰氏菌BD15-5株对凡纳滨对虾不具有致病性，可以在凡纳滨对虾养殖生产中大剂量应用。
Claims (6)
1.一种鲁杰氏菌，其特征在于，所述的鲁杰氏菌(Ruegeria sp.)的保藏编号为CGMCCNo.20973；保藏地址为北京市朝阳区北辰西路1号院3号。
2.权利要求1所述的鲁杰氏菌在对虾养殖中的应用。
3.权利要求1所述的鲁杰氏菌在制备对虾养殖用的制品中的应用。
4.如权利要求3所述的应用，其特征在于，所述的制品为活菌制剂。
5.权利要求1所述的鲁杰氏菌在对虾养殖中调节水质中的应用。
6.一种调控对虾养殖水质的方法，其特征在于，所述的方法是使用权利要求1所述的鲁杰氏菌来处理水质。
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