CN116463248B - 一株抑制水霉生长的乳酸菌及其应用 - Google Patents
一株抑制水霉生长的乳酸菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株抑制水霉的乳酸菌及其应用，属于水产养殖技术及病害防控技术领域。本发明提供了一株植物乳杆菌Lactiplantibacillus plantarum L75a，其保藏号为GDMCC No:63183，对水产致病菌水霉具有很好的抑菌作用，可减少化学药物的使用量，推动水产养殖业绿色、生态发展，在水产养殖水霉病的防控中具有广阔的应用前景。
Description
技术领域
本发明涉及一株抑制水霉生长的乳酸菌及其应用，属于水产养殖技术及病害防控技术领域。
背景技术
水霉是条件致病菌；水霉病发生的前提通常是水温偏低(特别是冬春季节)、水质量恶化、微生物菌群失衡、养殖密度过高、鱼体表存在物理损伤等。水霉的感染一般发生在水生动物的体表或它们的卵表面。生活在淡水环境的水生动物很容易感染水霉病。受侵染的鱼类有鳜鱼、鲤鱼、罗非鱼、加州鲈、鲶鱼、鲑鱼和鳟鱼等。在淡水养殖中，水霉病的主要病原菌是寄生水霉(Saprolegnia parasitica)和异丝水霉(Saprolegnia diclina)。寄生水霉大多感染鱼类，而异丝水霉则多见于感染的鱼卵。水霉归属于卵菌(oomycetes)，虽然卵菌与真菌有许多相似之处，但它不是真正的真菌，归属于不等鞭毛类(Stramenopiles，Heterokonts)。水霉病的症状通常表现为白色或灰色的棉絮状菌丝附着在鱼体表、鱼鳍或鱼卵上。鱼类和鱼卵感染水霉致死的病因不同。对于鱼类而言，大量入侵性水霉菌丝的生长破坏表皮的结构从而破坏鱼的渗透平衡；对于鱼卵而言，水霉菌丝使绒毛膜破裂导致渗透失衡。水霉病的爆发导致鱼卵和幼鱼的损失平均为10％，但是50％的损失也有报道。
少数研究表明有益微生物具有实验室活体抗水霉作用，这些有益微生物包括气单胞菌(Aeromonas)，假单胞菌(Pseudomonas)、放线菌(Frondihabitans)和木霉(Trichoderma)。利用生物防治技术抗病害和提高宿主健康程度已经逐渐成为医药、营养等领域的发展潮流。乳酸菌(lactic acid bacteria)是最常用的益生菌之一，它们改善宿主健康状况的作用机理主要包括调节宿主微生物菌群、产出抑制病害菌的物质、提高宿主对营养物质的消化和吸收功能、提高宿主屏障功能、增强宿主免疫能力等。抗病性益生菌的应用范围包括治疗人类、动物和植物的细菌病、寄生虫病、真菌病和病毒病等。在益生菌中，乳酸菌已经被广泛用作抗菌药物、微生物菌群调节剂、免疫调节剂和食物添加剂等。
发明内容
本发明的第一个目的是提供了一株乳酸菌，为植物乳杆菌Lactiplantibacillusplantarum L75a，其保藏号为GDMCC No:63183。
本发明提供的植物乳杆菌Lactiplantibacillus plantarum L75a，保藏于广东省微生物菌种保藏中心，保藏登记号GDMCC No:63183，保藏日期为2023年2月22日，保藏单位地址：广东省广州市。
本发明的植物乳杆菌Lactiplantibacillus plantarum L75a从中国南海锯缘青蟹(Scylla serrata)体内获得。
本发明的另一个目的是提供一种抑制水霉剂。
本发明提供的抑制水霉剂的活性成分为上述植物乳杆菌Lactiplantibacillusplantarum L75a或其发酵液或其菌悬液或其发酵产物或其发酵除菌上清液。
在本发明的一个实施例中，所述植物乳杆菌Lactiplantibacillus plantarumL75a发酵液的制备方法如下：将L75a菌株接种于MRS液体培养基中，37℃、200r/min培养24h，得到OD600≈0.95的L75a培养液为发酵液；将L75a发酵液5000xg离心5min，收集上清液后用0.22μm过滤器过滤后的上清液即为L75a发酵除菌上清液。
在本发明的另一实施例中，所述L75a菌悬液的制备方法如下：将L75a菌株接种于MRS液体培养基中，37℃、200r/min培养24h，得到L75a发酵液，将L75a发酵液5000xg离心5min，收集菌体，然后用无菌水清洗菌体3次后，加入与原培养基等量的无菌水混匀，即为L75a菌悬液。
本发明又一目的是提供上述植物乳杆菌Lactiplantibacillus plantarum L75a或上述抑制水霉剂的新用途。
本发明提供了上述植物乳杆菌Lactiplantibacillus plantarum L75a或上述抑制水霉剂在抑制水霉生长中的应用。
本发明的有益效果在于：本发明从中国南海锯缘青蟹中筛选出了具有抑制水霉效果特别是能够预防由其引起的水产养殖冬季低温爆发的水霉病的乳酸菌，不仅丰富了乳酸菌菌种资源库，拓宽了乳酸菌在水产养殖水霉病害防控领域的应用范围，也为水产动物水霉病的防治提供了新的解决方法。
附图说明
图1为乳酸菌16S rDNA序列扩增片段电泳结果和植物乳杆菌L75a系统发育树(L43和L80为同批筛选的其他菌株)。
图2为梯度浓度植物乳杆菌L75a发酵液对水霉的离体抑菌效果。图中标记“*”表示与空白对照组相比具有显著差异(P<0.05)，标记“**”表示与空白对照组相比具有极显著差异(P<0.01)。
图3为植物乳杆菌L75a发酵液、植物乳杆菌L75a菌悬液和植物乳杆菌L75a除菌上清液对水霉的离体抑菌效果。图中标记“*”表示与空白对照组相比具有显著差异(P<0.05)，标记“**”表示与空白对照组相比具有极显著差异(P<0.01)。
图4为植物乳杆菌L75a发酵液同其他乳酸菌发酵液对水霉的离体抑菌率对比。
具体实施方式
1乳酸菌分离纯化和测序分析
从广东省江门市、珠海市和广州市多个养殖水体和水生动物采集样品。肠道和环境样品通过MRS乳酸菌选择性培养基筛选出单菌落，挑选单菌落分离纯化后抽提DNA后测序(参见序列表)，对16S rDNA测序后进行生物信息学分析，鉴定多株乳酸菌的种属。将所有已鉴定的乳酸菌建库保存，其中，乳酸菌L75a鉴定为植物乳杆菌(Lactiplantibacillusplantarum)(如图1所示)。
2乳酸菌的酶活性测试
将筛选到的乳酸菌进行蛋白酶、淀粉酶、脂肪酶及运动能力等生物学活性测试，其中，植物乳杆菌L75a(乳酸菌L75a)的生物学活性如表1所示。
表1植物乳杆菌L75a酶活性测试和运动能力测试
注：“+”代表有活性，“-”代表无活性。
3乳酸菌发酵液对水霉的离体抑菌效果
将保存于-80℃的乳酸菌菌种接种于装有液体MRS培养基的离心管(5mL/50mL,V/V)中，在37℃、转速为200r/min的摇床中培养24h后，测量OD600值，转接菌液到装有新鲜MRS培养基的离心管(5mL/50mL,V/V)中，使菌液起始浓度为105cells/mL。将离心管置于37℃、转速为200r/min的摇床中培养24h，形成乳酸菌发酵液。
实验中使用的水霉菌株为寄生水霉Saprolegnia parasitica F26(MW819628)。将保存于矿物油的水霉块用无菌水冲洗后转接于PDA(马铃薯葡萄糖琼脂培养基)平板上，待平板长满后置于4℃冰箱做后续水霉实验的接种源。使用打孔器(直径为3mm)从上述PDA平板中取出一块PDA琼脂以长菌面向下置于新的1/5PDA平板上，于22℃培养至平板长满水霉菌丝待用。
将6孔板中每孔加入2.7ml 1/5PDB(马铃薯葡萄糖水培养基)后，加入300μL用无菌水稀释好的乳酸菌L75a发酵液，使6孔板内发酵液浓度调整至10000、5000、1000、750、500、250、100、75、50、25和10ppm，空白对照加入300μL无菌ddH2O。混匀后用打孔器取待用的水霉块(3mm)正放于6孔板孔中，22℃培养，分别于24h和48h拍照记录并测量水霉生长直径。同时设置乳酸菌P和乳酸菌L做实验效果对比。
实验结果如图2所示，与空白对照相比，实验中10ppm-10000ppm的乳酸菌发酵液浓度对水霉的生长均有显著的抑制作用，随着发酵液浓度越高，抑制能力越强，且10000ppm和5000ppm的乳酸菌发酵液对水霉的生长完全抑制。
同时，根据“生长抑制率＝[1-(处理组菌落直径-菌饼直径)/(对照组菌落直径-菌饼直径)]*100％”计算三种乳酸菌发酵液对水霉的生长抑制率(如图4所示)，结果表明，随着植物乳杆菌L75a发酵液浓度下降，其水霉抑制率逐渐降低，5000ppm及以上浓度抑制率达到100％，10ppm低浓度发酵液的抑制率在培养48h时也达到了26.35％。与乳酸菌P和乳酸菌L的抑菌率对比可得，L75a除了在高浓度104ppm均达到最低抑制浓度外，其他所有测试浓度中的抑制率均显著高于P，在750、500、250、25、10ppm浓度时抑制率显著高于L，植物乳杆菌L75a发酵液在抑制水霉生长能力上同其他乳酸菌相比表现出较大优势。
4乳酸菌发酵液、菌悬液和除菌上清液对水霉的离体抑菌效果
将6孔板中每孔加入2.7mL1/5PDB(马铃薯葡萄糖水培养基)后，加入300μL用无菌水稀释好的乳酸菌L75a发酵液、菌悬液和除菌上清液，使6孔板内浓度调为1000ppm和100ppm，空白对照加入300μL无菌ddH2O。混匀后用打孔器取待用的水霉块(3mm)正放于6孔板孔中，22℃培养，分别于24h和48h拍照记录并测量水霉生长直径。
实验结果如图3所示，结果表明1000ppm和100ppm的乳酸菌发酵液和菌悬液对水霉生长均有显著的抑制作用，高浓度的除菌上清液对水霉有生长抑制作用。同时，通过植物乳杆菌L75a除菌液、菌悬液和除菌上清液的抑制作用两两对比，可得乳酸菌发酵液的抑菌能力显著高于菌悬液和除菌上清液，且菌悬液的抑菌能力显著高于除菌上清液的抑菌能力。
Claims (6)
1.一株抑制水霉生长的乳酸菌，为植物乳杆菌(Lactiplantibacillus plantarum )L75a，其保藏号为GDMCC No: 63183。
2.一种抑制水霉剂，其特征是，其活性成分为权利要求1所述的植物乳杆菌L75a或其发酵液或其菌悬液。
3.根据权利要求2所述的抑制水霉剂，其特征是，所述植物乳杆菌L75a发酵液的制备方法如下：将L75a菌株接种于MRS液体培养基中，37℃、200r/min培养24h，得到OD600≈0.95的L75a培养液为发酵液。
4.根据权利要求2所述的抑制水霉剂，其特征是，所述L75a菌悬液的制备方法如下：将L75a菌株接种于MRS液体培养基中，37℃、200r/min培养24h，得到L75a发酵液，将L75a发酵液离心5min，收集菌体，然后用无菌水清洗菌体3次后，加入与原培养基等量的无菌水混匀，即为L75a菌悬液。
5.权利要求1所述的乳酸菌在抑制水霉生长中的应用。
6.权利要求2-4任一项所述抑制水霉剂在抑制水霉生长中的应用。
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