CN115404182B - 一种益生菌和噬菌体的组合物及其应用 - Google Patents
一种益生菌和噬菌体的组合物及其应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域，具体提供了一种益生菌和噬菌体的组合物及其应用，所述益生菌和噬菌体的组合物包括：溶藻弧菌VA3、Halocynthiibacter、Psychrobacter、Ruegeria中的至少一种和噬菌体VpC‑1；其中，所述溶藻弧菌VA3的保藏编号为CCTCC NO:M 20211703，分类命名为Vibrio alginolyticus；所述噬菌体VpC‑1的保藏编号为CCTCC NO:M20211704，分类命名为Myoviridae。益生菌组合物从对虾肠道中筛选，解决了益生菌对水产环境的不耐性，当选择多菌株进行组合制备复合益生菌制剂时，可以增加菌群的多样性，有助于水产动物肠道的菌群稳定，使菌群保持一个稳定的健康状态，提高非特异性免疫能力，噬菌体能靶向杀灭目标弧菌，两者联合应用对弧菌引起的鱼虾类疾病产生预防效果。
Description
技术领域
本发明属于微生物技术领域，具体涉及一种益生菌和噬菌体的组合物及其应用。
背景技术
我国是水产养殖大国。近年来，我国的水产养殖业获得迅猛发展。随着海水养殖规模的日益扩大和集约化养殖方式的不断推广，疾病的频繁发生对水产规模化养殖的健康发展造成了严重的威胁。水产养殖的病原体主要包括细菌、病毒、寄生虫等，其中细菌性疾病是对危害程度较为严重的一类疾病。
弧菌是能运动的芽孢短杆状细菌，广泛分布于近海岸、河口海区海水及生物体内，是海洋中最常见的细菌类群之一，同时也是引起海水养殖鱼虾细菌性疾病最重要的病原菌。弧菌的致病强度与宿主的生理状态及水质环境条件等综合因素有关，属于条件性致病菌，其主要通过口或伤口感染，产生毒素，致使伤口肌肉溃烂化脓以及内脏器官的严重病变从而导致鱼虾类的死亡。由弧菌引起的疾病又称弧菌病，具有“流行面积广、发病率高、致死性强”等特点，是水产养殖动物的主要疾病，同时也被确定为是阻碍水产养殖业发展的重要限制性因素，给养殖业造成了巨大的危害。其中鳗弧菌(Vibrio anguillarum)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)、副溶血弧菌(Vibrioparahaemolyticus)和杀鲑弧菌(Vibrio salmonicida)等弧菌引起的弧菌病被认为是鱼虾类养殖中最为严重的病害之一。早期死亡综合征，也称作肝胰腺急性坏死综合征影响的主要对虾种类包括凡纳滨对虾、斑节对虾和中国明对虾在内的多个主养品种，其中凡纳滨对虾和斑节对虾的死亡率最高。由于该病发病急、造成的死亡率高、流行范围广，对对虾养殖业危害严重，备受关注。亚利桑那大学的研究已经确定，该疾病是由细菌剂副溶血弧菌引起的，副溶血弧菌通过口腔传播并定殖于虾的胃肠道，并产生毒力蛋白，导致虾消化器官的组织破坏和功能障碍。
目前针对水产养殖相关动物疾病的预防和控制方法主要以化学抗生素法和免疫疫苗法，其中以化学抗生素使用为主。作为当前控制水产动物疾病的主要手段，化学抗生素法具有使用方便、见效快和疗效好等优点。但是，大批量频繁使用抗生素在控制病原菌的同时，极大了助长了病原菌的耐药性，并加速了广谱耐药菌的产生。这使得致病菌的耐药性问题日益严重。此外，化学药物残留也是导致人类食源性疾病产生的重要原因之一，对人类健康造成严重危害。
益生菌可在水产动物肠道或水产环境中对有害菌进行竞争性抑制，减少弧菌的浓度，使其低于发病浓度。近年来抗生素的滥用使得益生菌的优势一直未能完全发挥。由于抗生素对细菌抑制的非特异性，有害菌在被大量清除的同时，有益菌也被抗生素杀灭，从而无法发挥益生菌的作用。现在大众逐渐了解到抗生素滥用的危害，开始寻找能够取代抗生素的抑菌和杀菌药物，益生菌就是其中一个有效措施。然而，传统益生菌例如乳酸菌、芽孢杆菌，在水产养殖上的使用效果并不理想，这些益生菌并不能适应水产养殖的环境，例如过低的温度(＜37℃)，过高的含盐度(～30％)。为了解决这个问题，本发明从对虾肠道中筛选出能有效提高对虾存活率的益生菌。
噬菌体又称细菌性病毒，是一类专一性裂解细菌的病毒，在环境中分布广泛、种类多样、结构简单，具有很强的宿主特异性。最重要的一点，噬菌体裂解细菌的机制不受细菌耐药性的影响，其通过受体识别从而吸附在特定宿主菌表面，将自身遗传物质注入宿主体内进行自我复制组裝，并最终通过裂解宿主菌释放子代从而实现自我的生长繁殖。因此，噬菌体疗法是一种很有前景的预防和治疗弧菌疾病方法。
发明内容
本发明的目的是克服现有技术中抗生素使用受限、耐药菌泛滥、传统益生菌不能适应水产环境的问题。
为此，本发明提供了一种益生菌和噬菌体的组合物，包括：溶藻弧菌VA3、Halocynthiibacter、Psychrobacter、Ruegeria中的至少一种和噬菌体VpC-1；其中，所述溶藻弧菌VA3于2021年12月30日保藏于中国典型培养物保藏中心(地址：中国.武汉.武汉大学)，保藏编号为CCTCC NO:M 20211703，分类命名为Vibrio alginolyticus；所述噬菌体VpC-1于2021年12月30日保藏于中国典型培养物保藏中心(地址：中国.武汉.武汉大学)，保藏编号为CCTCC NO:M 20211704，分类命名为Myoviridae。
本发明还提供了一种包括上述益生菌和噬菌体组合物的抑制副溶血弧菌的复合抑菌制剂。
具体的，上述复合制剂中活菌数不少于1010CFU/mL，具有活性的噬菌体数不少于1011PFU/mL。
本发明提供的这种益生菌和噬菌体组合物和抑制副溶血弧菌的复合抑菌制剂可用于水产养殖领域。具体的，以养殖水体体积计，所述复合抑菌制剂中益生菌终浓度不少于5×106CFU/mL，噬菌体不少于5×107PFU/mL。
与现有技术相比，本发明具有以下优点和有益效果：
本发明提供的这种益生菌和噬菌体的组合物一方面通过从对虾肠道筛选出益生菌的方式，解决了益生菌对水产环境的不耐性，易于在水产环境中繁殖，不会对水产动物及环境造成污染，而且其对弧菌的抑制不会受到耐药菌的影响；当选择多菌株进行组合时，可以增加菌群的多样性，有助于水产动物肠道的菌群稳定，使菌群保持一个稳定的健康状态，提高对有害菌的预防能力，增强肠道的微生物屏障，提高非特异性免疫能力，对弧菌引起的鱼虾类疾病产生一定的预防效果，提高水产养殖的存活率，有效性高，制备成本低；另一方面，噬菌体特异性识别并杀灭致病性弧菌，靶向清除使用病原体，上述两者通过非特异性清除和特异性清除相结合的方式，降低水产动物养殖环境中的致病菌浓度，减少动物弧菌感染的几率。
以下将结合附图对本发明做进一步详细说明。
附图说明
图1是本发明实施例1中13株菌的系统进化树。
图2是本发明实施例2中各备选菌对对虾存活率的影响。
图3是本发明实施例3中试验期内各组水体中VP-6浓度检测结果。
图4是本发明实施例3中各组对虾的生存曲线。
图5是本发明实施例3中致病性弧菌VP-6感染后第5天各组对虾存活率检测结果。
具体实施方式
下面将结合实施例对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。尽管已经详细描述了本发明的代表性实施例，但是本发明所属技术领域的普通技术人员将理解，在不脱离本发明范围的情况下可以对本发明进行各种修改和改变。因此，本发明的范围不应局限于实施方案，而应由所附权利要求及其等同物来限定。
下面通过具体实施例对本发明的益生菌组合物及其应用的效果进行研究。
实施例1：
对健康对虾进行解剖，用75％的乙醇擦拭对虾表面后中，取出对虾肠道，放入 1mL无菌生理盐水中，充分匀浆，将匀浆液稀释至不同浓度梯度(100，10-2，10-4， 10-6，10-8)后在2216E固体培养基上涂板，平板于30℃的培养箱中倒置过夜。挑选颜色形状大小不同的清晰菌落，三次划线纯化细菌直至平板上菌落形态一致，挑单克隆至3mL的2216E液体培养基中，在30℃条件下以220rpm的转速震荡培养过夜。
用细菌基因组提取试剂盒提取分离培养的菌株基因，并进行16s PCR，得到菌株的16S序列，最终筛选得到13株菌作为益生菌的备选菌，13株菌的系统进化树如图 1所示。
实施例2：
本实施例针对由致病性弧菌引起的水产养殖上鱼虾类疾病大肆传播的问题，从患病鱼虾病灶中分离含有Pir基因的弧菌VP-6，并通过实验确定了该弧菌的致病性，本实施例以弧菌VP-6为病原体模拟对虾弧菌病感染。
用抗生素处理对虾，处理期两天，将无菌实验对虾随机分成15组，每组两个重复，每个重复16尾，使用人工海水饲养，具体分组如下：
13个实验组：将13株备选菌分别以5×106CFU/mL的终浓度加到水体中，饲养两天，使对虾肠道菌得到恢复后，以5×106CFU/mL的终浓度加入致病菌VP-6进行副溶血弧菌感染；
阴性对照组：不进行菌群恢复，不进行VP-6感染；
阳性对照组：不进行菌群恢复，饲养两天后，以5×106CFU/mL终浓度加入致病菌VP-6进行副溶血弧菌感染。
计算VP-6感染后第5天各组存活率，结果如图2所示，VA3，Halocynthiibacter、Psychrobacter可将对虾存活率提升15％-45％，Ruegeria虽然没有显著提高对虾存活率，但使用该菌可以增加菌群的多样性，有助于水产动物肠道的菌群稳定，使菌群保持一个稳定的健康状态，增强肠道的微生物屏障作用。
实施例3
本实施例根据实施例2筛选出的有益肠道菌(包括VA3，Halocynthiibacter、Psychrobacter和Ruegeria，菌量比为1:1:1:1)进行组合，并与噬菌体VpC-1共同加入养殖水体，测试该组合对实际养殖中对虾副溶血弧菌的感染是否有显著的治疗效果。
180尾经21天饲养的南美白对虾随机分成4组，每组3个重复，每个重复20尾，第1组为阴性对照组，第2组为阳性对照组，第3组为实验组。实验组加入益生菌和噬菌体组合物，使水体中益生菌终浓度达到5×106CFU/mL，噬菌体终浓度达到 5×107PFU/mL，继续饲养两天后，以5×106CFU/mL的终浓度加入致病菌VP-6进行副溶血弧菌感染，感染后噬菌体与饲料(109PFU/克)混合饲喂；阴性对照组不加入益生菌和噬菌体，不加入致病菌VP-6进行感染；阳性对照组不加入益生菌和噬菌体，与实验组同时加入终浓度为5×106CFU/mL的致病菌VP-6进行副溶血弧菌感染。以加入VP-6进行感染为试验第0天，试验期共5天，每天取水样于弧菌选择性平板 TCBS上涂板计数水体环境中VP-6的浓度，每天记录各组对虾的死亡情况。
结果表明，有益肠道菌和噬菌体联合应用对水体中的VP-6起到杀灭作用，加速其在水体中浓度的降低，在感染后的第3天，实验组水体的VP-6浓度即低于检测下限，阳性对照组在第5天水体中仍有不低于104CFU/mL的VP-6(图3)。对应的，在实验组中，对虾的死亡速度低于阳性对照组(图4)，感染后第5天，阳性对照组对虾存活率为27.7％，实验组对虾存活率提高至56.7％(图5)。
以上例举仅仅是对本发明的举例说明，并不构成对本发明的保护范围的限制，凡是与本发明相同或相似的设计均属于本发明的保护范围之内。
Claims (3)
1.一种溶藻弧菌VA3，其特征在于，所述溶藻弧菌VA3的保藏编号为CCTCC NO:M20211703，分类命名为Vibrio alginolyticus。
2.权利要求1所述的溶藻弧菌VA3在抑制副溶血弧菌中的应用。
3.权利要求1所述的溶藻弧菌VA3在水产养殖中的应用。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101356927A (zh) * | 2008-03-31 | 2009-02-04 | 华南理工大学 | 蛭弧菌在消除海产品及其养殖水体中致病性弧菌的应用 |
| CN101993842A (zh) * | 2010-08-17 | 2011-03-30 | 中国水产科学研究院黄海水产研究所 | 一种溶藻弧菌菌株及其应用 |
| CN107904212A (zh) * | 2017-10-13 | 2018-04-13 | 深圳先进技术研究院 | 溶藻弧菌噬菌体及包含该噬菌体的杀菌组合物 |
| CN109207440A (zh) * | 2018-10-10 | 2019-01-15 | 江苏省农业科学院 | 弧菌噬菌体及其杀菌组合物制备方法和应用 |
-
2022
- 2022-07-26 CN CN202210885250.2A patent/CN115404182B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101356927A (zh) * | 2008-03-31 | 2009-02-04 | 华南理工大学 | 蛭弧菌在消除海产品及其养殖水体中致病性弧菌的应用 |
| CN101993842A (zh) * | 2010-08-17 | 2011-03-30 | 中国水产科学研究院黄海水产研究所 | 一种溶藻弧菌菌株及其应用 |
| CN107904212A (zh) * | 2017-10-13 | 2018-04-13 | 深圳先进技术研究院 | 溶藻弧菌噬菌体及包含该噬菌体的杀菌组合物 |
| CN109207440A (zh) * | 2018-10-10 | 2019-01-15 | 江苏省农业科学院 | 弧菌噬菌体及其杀菌组合物制备方法和应用 |
Non-Patent Citations (4)
| Title |
|---|
| Assessment of synergistic combination potential of probiotic and bacteriophage against antibiotic‑resistant Staphylococcus aureus exposed to simulated intestinal conditions;Jihoon Woo 等;Arch Microbiol;第196卷;719-727 * |
| Characterization and Genomic Analysis of a Bacteriophage with Potential in Lysing Vibrio alginolyticus;Jingyun Fu，等;Viruses;1-23 * |
| Vibrio alginolyticus influences quorum sensing-controlled phenotypes of acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus;Panida Paopradit 等;Peer J;1-22 * |
| 噬菌体微生态疗法与一体化健康: 现状、挑战与机遇;韦中 等;中国科学;第53卷(第5期);698-710 * |
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