CN104388337B - 一种短小芽孢杆菌菌株及其应用 - Google Patents
一种短小芽孢杆菌菌株及其应用 Download PDFInfo
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- CN104388337B CN104388337B CN201410582638.0A CN201410582638A CN104388337B CN 104388337 B CN104388337 B CN 104388337B CN 201410582638 A CN201410582638 A CN 201410582638A CN 104388337 B CN104388337 B CN 104388337B
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- bacillus pumilus
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Abstract
本发明的目的是提供一种短小芽孢杆菌菌株，其保藏编号为CCTCC M2013263。本发明的短小芽孢杆菌能够改善对虾消化道优势菌群的组成和数量，形成并维持良好的消化道微生态平衡，通过营养竞争、空间竞争或分泌细菌素等抵御病毒在消化道的黏附和增殖。能够激活对虾酚氧化酶原、超氧化物歧化酶、溶菌酶、热休克蛋白、脂多糖‑葡聚糖结合蛋白、信号传导及转录激活因子等与抗病原感染相关分子的表达，减缓病毒在对虾体内的增值速率。本发明的短小芽孢杆菌可以添加到对虾饲料原料中，也可以直接施用于对虾养殖水环境中。
Description
技术领域
本发明属于益生菌筛选技术领域，具体涉及一种短小芽孢杆菌菌株及其应用。
背景技术
短小芽孢杆菌(Bacillus pumilus)在细菌分类上隶属于厚壁菌门(Firmicutes)(低G+C革兰氏阳性菌)、芽孢杆菌纲(Bacilli)、芽孢杆菌目(Bacillales)、芽孢杆菌科(Bacillaceae)、芽孢杆菌属(Bacillus)。短小芽孢杆菌普遍存在于土壤及植物体表，也是人、动物肠道内常见的共生菌之一。短小芽孢杆菌是1989年美国食品药物管理局(FDA)和美国饲料公定协会(AAFCO)公布的44种可直接饲喂且通常认为是安全的微生物之一。短小芽孢杆菌分泌蛋白能力强且胞外蛋白酶活性低，因此具有分泌表达外源蛋白的天然优势，利用它们可望建立高效的原核分泌表达系统。
短小芽孢杆菌和其它芽孢杆菌一样，具有营养体-芽孢-营养体的独特生活周期史，抗逆能力强、繁殖速度快、营养要求简单，具有可抵抗酸、高温、干燥等不良环境条件等优势，适宜大规模产业化发酵生产。短小芽孢杆菌在生长过程中可分泌蛋白酶、纤维素酶、淀粉酶、几丁质酶、脂肪酶、麦芽糖酶、多聚糖降解酶、碱性蛋白酶、β-半乳糖苷酶、果聚糖酶等多种酶类，其丰富的胞外酶系不仅有助于饲料的消化吸收，可降解内外环境中的有机残余物，促进良性生态循环。短小芽孢杆菌的防病能力源于对养殖对虾体内外生态环境的改善和机体免疫力的提高，将其应用到水产养殖环境改良剂、饲料添加剂等有广阔的前景。
但目前对虾养殖生产中应用的短小芽孢杆菌菌株较少，且存在着菌种生物学性状平平，活菌数量偏低，作用效果不稳定等不足，特别是还没有抗对虾病毒病菌株的应用。病毒性疾病是危害对虾养殖生产的主要病害，约占对虾病害总损失率的六成；特别是对虾白斑综合征病毒(White Spot Syndrome Virus，简称WSSV)引起白斑病，由于宿主范围广、感染死亡率高，是我国乃至全世界影响最大、危害最为严重的病毒性疾病。本发明涉及的短小芽孢杆菌菌株，优先考虑菌株的抗病毒活性，通过反复养殖实验证实该菌株可提高对虾溶菌酶、过氧化氢酶、酚氧化酶、磷酸酶、超氧化物歧化酶等相关免疫酶活性，增强对虾非特异免疫功能，减少病毒感染的机会，从而有效阻断病毒病的发生与流行。
发明内容
本发明的目的是提供一种短小芽孢杆菌菌株及其应用，该菌株来源于对虾消化道，具有增强对虾细胞和体液的免疫水平，特别是提高对虾抗病毒感染能力的生物学活性。
本发明的短小芽孢杆菌LV012(Bacillus pumilus LV012)菌株于2013年6月16日保藏在武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC M 2013263。
上述的短小芽孢杆菌(Bacillus pumilus)LV012菌株是从对虾消化道中分离的。
短小芽孢杆菌LV012菌株可用于制备短小芽孢杆菌LV012发酵菌液。
短小芽孢杆菌LV012发酵菌液制备方法如下：从-80℃保存的菌种挑取少量划线于2216E固体培养基，在37℃培养24h获得单菌落，取一单菌落接种于2216E培养液中，37℃摇瓶培养24h获得种子液，再把种子液接种于地衣芽孢杆菌发酵液中发酵培养，发酵温度控制在37℃；当发酵液OD600值达到1.2～1.4时，终止发酵，获得发酵菌液；发酵液中短小芽孢杆菌LV012活菌浓度≥109CFU/mL。
短小芽孢杆菌LV012的发酵液成分质量百分比为：蛋白胨，0.5％；酵母粉，0.1％；蔗糖，1.5％；氯化钠，2.5％；硫酸铁，0.05％；硫酸镁，0.004％；硫酸锰，0.01％。
本发明的短小芽孢杆菌LV012筛选自对虾消化道，具有：①改善对虾消化道优势菌群的组成和数量，形成并维持良好的消化道微生态平衡，通过营养竞争、空间竞争或分泌细菌素等抵御病毒在消化道的黏附和增殖；②通过提高对虾溶菌酶、过氧化氢酶、酚氧化酶、磷酸酶、超氧化物歧化酶等相关免疫酶活性，增强对虾非特异免疫功能，减少病毒感染的机会；③激活对虾酚氧化酶原、超氧化物歧化酶、溶菌酶、热休克蛋白、脂多糖-葡聚糖结合蛋白、信号传导及转录激活因子等与抗病原感染相关分子的表达，减缓病毒在对虾体内的增值速率；④提高对虾蛋白酶、脂肪酶和淀粉酶的活性，促进对虾消化能力和饲料养分的消化，提高对虾抗应激反应能力等多种生物学功能。本发明的短小芽孢杆菌LV012可以添加到对虾饲料原料中，也可以直接施用于对虾养殖水环境中。
具体实施方式
下面结合具体实施例对本发明的菌株进行详细的描述。
实施例1：短小芽孢杆菌的筛选
本发明的短小芽孢杆菌(Bacillus pumilus)LV012菌株筛选自凡纳滨对虾消化道。将采集的健康对虾样品放在盛有海水的充气袋中运回实验室。无菌操作台上用75％的酒精擦拭活虾体表进行消毒，无菌操作将活虾解剖，取出对虾肠道组织。将取出的肠道组织用无菌的PBS(pH＝7.0)缓冲液冲洗3-4次后，用无菌研磨棒研磨均匀。之后用无菌PBS缓冲液适当稀释，取0.1ml涂布于2216E固体培养基上，倒置于28℃恒温培养箱中培养16h-20h。观察2216E平板上细菌的菌落形态，对菌落形态一致的细菌进行编号，无菌条件下用接种环挑取优势菌单菌落接种于2216E海水固体培养基中进行反复纯化培养。纯化后的单菌落接种于2216E海水液体培养基，在转速为180rpm/min，28℃振荡培养箱中培养16h-20h，取0.8ml菌悬液放入保种管中，再加入0.2ml的70％灭菌甘油，混匀，用封口膜封好，放于-80℃冰箱保存。
筛选的短小芽孢杆菌在2216E培养基上的菌落形态：菌落直径约1-2mm，菌落呈圆形，乳白色、不透明，表面呈蜡状，有凸起且边缘整齐。
碳源利用情况为α-环式糊精、β-甲基-D-葡萄糖苷、D－阿糖醇、腺苷、D-丙氨酸、2'-脱氧腺苷、β-环式糊精、松二糖、L-丙氨酸、麦芽三糖、D-阿洛酮糖、木糖醇、D-苹果酸、D-甘露醇、D-蜜三糖/棉子糖、D-木糖、L-天门冬酰胺、琥珀酸单甲基酯、D-甘露糖、L-鼠李糖、丙酮酸甲酯、5'-磷酸腺苷、D-半乳糖、D-松三糖、吐温80、龙胆二糖、γ-羟丁酸、p-羟基苯乙酸、β-甲基-D-半乳糖苷、α-酮戊二酸、2,3-丁二醇、6-磷酸-D-葡萄糖、苦杏仁苷在Biolog碳源消耗中呈现阳性。
其它生理生化特性：淀粉水解呈阴性，明胶液化阴性，氧化酶试验阳性，过氧化氢酶试验阳性。
短小芽孢杆菌LV012菌株可用于制备短小芽孢杆菌LV012发酵菌液。
短小芽孢杆菌LV012发酵菌液制备方法如下：从-80℃保存的菌种挑取少量划线于2216E固体培养基，在37℃培养24h获得单菌落，取一单菌落接种于2216E培养液中，37℃摇瓶培养24h获得种子液，再把种子液接种于地衣芽孢杆菌发酵液中发酵培养，发酵温度控制在37℃；当发酵液OD600值达到1.2～1.4时，终止发酵，获得发酵菌液；发酵液中短小芽孢杆菌LV012活菌浓度≥109CFU/mL。
短小芽孢杆菌LV012的发酵液成分质量百分比为：蛋白胨，0.5％；酵母粉，0.1％；蔗糖，1.5％；氯化钠，2.5％；硫酸铁，0.05％；硫酸镁，0.004％；硫酸锰，0.01％。
实施例2：
将制得的100mL短小芽孢杆菌LV012溶液与10Kg基础对虾饲料原料(含鱼粉30％、虾粉10％、豆粉25％、花生粉25％、面粉3％、玉米粉3％、复合维生素1％)混合，制成含短小芽孢杆菌LV012的免疫饲料。各种原料分别粉碎过60目筛，准确称重后逐级充分混匀，以褐藻酸钠作为粘合剂，加适量水用小型绞肉机挤压制成颗粒饲料，在40℃条件下烘干至水分含量10％以下，分袋包装，于4℃冰箱中保存备用。
健康凡纳滨对虾300余尾，实验对虾初始平均体长为6.3cm，随机为实验组和对照组2个处理组，每个处理组有3个平行，每个平行组养殖对虾50尾。实验组连续4天投喂免疫饲料，3天投喂基础饲料，7天一个循环，直至实验结束。对照组在整个实验阶段一直投喂基础饲料。水温24-26℃，连续充气；每日换水1次，日换水量为1/3；每日投喂饲料4次，日投饵量约为1.2-1.4克/10尾，投喂1h后吸去剩余饲料，并根据残饵的多少调节投饵量。免疫饲料养殖21天后，进行白斑综合征病毒(White Spot Syndrome Virus，WSSV)人工注射感染实验。感染前1天停止投喂饲料，次日每尾对虾在第2腹节处注射50μL WSSV粗提液(浓度为0.16g/mL)，以PBS溶液为阴性对照组。攻毒后各组继续投喂相应饲料，每天及时捡出死虾，记录死亡数量，14d后结束感染实验。实验组和对照组每日平均累计死亡率结果见下表，实验结果显示在感染4d内，实验组与对照组累积死亡率为差异不显著(P>0.05)；从第5d始各组均呈明显的上升趋势，对照组累积死亡率较实验组差异显著(P<0.05)；至感染实验结束前1天对照组的累积死亡率为100％，实验组的累积死亡率为63.3％。
实施例3：
含1％(V/W)短小芽孢杆菌LV012溶液的对虾免疫饲料(活菌总浓度≥107CFU/mL)的制备方法同实施例1。
健康凡纳滨对虾300余尾，实验对虾初始平均体长为6.3cm，随机为实验组和对照组2个处理组，每个处理组养殖对虾150尾。实验组连续4天投喂免疫饲料，3天投喂基础饲料，7天一个循环，直至实验结束。对照组在整个实验阶段一直投喂基础饲料。免疫饲料养殖21天后，进行WSSV人工注射感染实验。感染前1天停止投喂饲料，次日每尾对虾在第2腹节处注射50μL WSSV粗提液(浓度为0.16g/mL)，以PBS溶液为阴性对照组。免疫期间分别在1、3、7和15d进行取样，攻毒期间分别在6h，18h，96h，192h取样，采用实时荧光定量PCR法测定复合芽孢杆菌制剂对凡纳滨对虾肠道硫氧还蛋白(Thioredoxin，Trx)基因表达量的影响。以对照组Trx表达量为1，实验组Trx的相对表达量见下表。从表中数据可以看出实验组Trx相对表达量在整个免疫期间呈上调再下调趋势，不同时刻的实验组的Trx相对表达量较对照组有显著差异；WSSV感染期间实验组Trx相对表达量呈上调再下降的趋势，不同时刻都可看出免疫饲料组较对照组有显著差异。结果表明投喂含复合芽孢杆菌饲料后，凡纳滨对虾肠道Trx基因的表达水平可显著提高。
硫氧还蛋白是一种分子量为12KD的酸性蛋白，它们在多种反应中通过可逆的双硫键和硫醇变化起氧化还原载体的作用，是重要的酶活力调节蛋白。对虾的硫氧还蛋白在抗白斑病毒中起到重要作用，其Trx基因的表达水平与机体抗WSSV感染能力密切相关。本株短小芽孢杆菌可以显著提高凡纳滨对虾肠道Trx基因的表达水平的特征是该菌株提高对虾抗WSSV感染能力的原因之一。
实施例4：
含1％(V/W)短小芽孢杆菌LV012溶液的对虾免疫饲料(活菌总浓度≥107CFU/mL)的制备方法同实施例1。
健康凡纳滨对虾300余尾，实验对虾初始平均体长为6.3cm，随机为实验组和对照组2个处理组，每个处理组养殖对虾150尾。实验组连续4天投喂免疫饲料，3天投喂基础饲料，7天一个循环，直至实验结束。对照组在整个实验阶段一直投喂基础饲料。免疫饲料养殖21天后，进行WSSV人工注射感染实验。感染前1天停止投喂饲料，次日每尾对虾在第2腹节处注射50μL WSSV粗提液(浓度为0.16g/mL)，以PBS溶液为阴性对照组。分别在病毒感染后的6、18、96和192h取样，采用实时荧光定量PCR法测定虾鳃的病毒拷贝数，结果见下表。
由表中数据可知，在感染实验的8d内实验组的病毒拷贝数先上升后下降趋势，而对照组的病毒拷贝数一直呈现上升趋势，且在8d内除96h时刻外实验组的病毒拷贝数均低于对照组1个数量级。结果证实饲料中添加复合芽孢杆菌制剂可有效降低虾鳃组织内的病毒拷贝数，延缓病毒在虾体内的复制速度。
Claims (2)
1.一种发酵菌液，其特征在于，是通过下述方法制备而成的：
从-80℃保存的短小芽孢杆菌的菌种中挑取少量划线于2216E固体培养基，在37℃培养24h获得单菌落，取一单菌落接种于2216E培养液中，37℃摇瓶培养24h获得种子液，再把种子液接种于短小芽孢杆菌发酵液中发酵培养，发酵温度控制在37℃；当发酵液OD600值达到1.2～1.4时，终止发酵，获得发酵菌液，菌液中短小芽孢杆菌活菌浓度≥109CFU/mL；
所述的短小芽孢杆菌发酵液成分质量百分比为：蛋白胨0.5％；酵母粉0.1％；蔗糖1.5％；氯化钠2.5％；硫酸铁0.05％；硫酸镁0.004％；硫酸锰0.01％；
所述的短小芽孢杆菌的保藏编号为CCTCC M 2013263。
2.一种对虾饲料，其特征在于，所述的对虾饲料中添加有权利要求1所述的发酵菌液。
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