CN106987543B - 一种水虻肠道来源的粘质沙雷氏菌bsfl-6及其应用 - Google Patents
一种水虻肠道来源的粘质沙雷氏菌bsfl-6及其应用 Download PDFInfo
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Abstract
本发明属于农业微生物学技术领域，具体公开了一种昆虫水虻肠道来源的粘质沙雷氏菌BSFL‑6及其应用，所述的粘质沙雷氏菌为粘质沙雷氏菌（Serratia marcescens）BSFL‑6，保藏编号：CCTCC NO:M 2017073。本发明提供的粘质沙雷氏菌（Serratia marcescens）BSFL‑6可以有效的降解抗生素四环素，增强水虻在转化物料过程中抵抗和降解四环素的能力，并对有害菌金黄色葡萄球菌（S.Aureus）及大肠杆菌（E.coli）有抑菌活性，是一株水虻肠道益生菌，可以用于促进水虻对含四环素污染的废弃物的转化。
Description
技术领域
本发明属于农业微生物学技术领域，具体涉及一种昆虫水虻肠道来源的粘质沙雷氏菌 BSFL-6及其应用，该粘质沙雷氏菌BSFL-6分离自转化含四环素污染物料的水虻肠道，还涉及该粘质沙雷氏菌与水虻协同降解四环素的应用，还涉及该粘质沙雷氏菌抑制有害菌金黄色葡萄球菌及大肠杆菌生长的应用。
背景技术
随着集约化现代畜禽养殖业的迅速发展，抗生素在其生产过程中使用量越来越大，高剂量的抗生素可以用于治疗各种疾病，而低剂量的则可用于促进畜禽生长和增产，在全球范围内几乎所有地区都采用抗生素来实现追求产量、提高经济效益的目的。其中，四环素在药品市场中占有重要地位，是世界上应用最为广泛的抗生素之一。相关统计显示，2014年我国四环素产量达到4000余吨，此外，全国还有4万吨左右的四环素衍生物及其盐的生产能力。虽然在国内外医药市场上，四环素已退出抗感染的主流药物品种，但因其价格低廉，抗菌谱广，多年来大量用于畜禽用药及添加到饲料中。它们进入动物或人体内后大约70％不能被吸收，而是以母体化合物的形式直接被排出体外。
环境中的四环素对微生物、植物和动物存在着一定的生态毒性，并诱使微生物产生抗性基因增加生态风险。它能抑制微藻蛋白质合成和叶绿体的生成；通过抑制叶绿体合酶的活性，从而对植物生长产生抑制作用；抑制水生动物多种酶的活性，如乙氧基试卤灵-O-去乙基酶、β-半乳糖苷酶等，并可能造成鱼类较严重的DNA损伤；引起小鼠肝细胞基因表达的改变，涉及包括能量代谢的抑制、蛋白质的合成以及药物代谢相关酶系基因的抑制等多个分子机制。同时，由四环素等导致的抗性基因的积累也是耐受细菌——“超级细菌”产生原因之一，是十分严重的生态威胁。
在针对畜禽粪便的各项处理技术中，水虻处理技术是一项新型高效、低成本高产值技术。水虻是一种资源昆虫，其幼虫类似于蝇蛆能以畜禽粪便为食，但比蝇蛆个头更大，生长周期更长，预蛹期会自动爬出，更易实现操纵控制，相应的处理效率和生物质产出更高；其成虫相对苍蝇更洁净，不趋近人类；许多研究者将水虻直接接种到堆积的畜禽粪便中，发现其同时具有趋避蝇蛆、除臭、抑制和杀死病原微生物的作用，至水虻预蛹时期(2-3周)还可以直接收集从畜禽粪便中爬出的已脱皮的干净幼虫，形成更高附加值的副产品(包括虫体蛋白、昆虫油脂、残留肥料等)，用于饲料形成整个生态养殖循环，因此受到了世界研究者的关注。
针对上述问题，申请人在对含四环素的鸡粪和猪粪的水虻转化研究中，发现水虻可以有效分解四环素等抗生素。通过构建无菌水虻体系、比较水虻肠道微生物在分解四环素过程中的贡献，研究发现，水虻肠道微生物是水虻分解四环素的关键参与者。本发明从水虻肠道中分离得到了一株水虻肠道共生菌粘质沙雷氏菌BSFL-6，具备增强水虻在转化物料过程中抵抗和降解四环素的能力，并对有害菌金黄色葡萄球菌(S.Aureus)及大肠杆菌(E.coli)有抑菌活性，是一株水虻肠道益生菌，目前，国际上还没有从昆虫肠道分离具有降解抗生素并可以与昆虫水虻共生降解抗生素的昆虫肠道细菌的报道。
发明内容
本发明的目的在于提供了一种粘质沙雷氏菌BSFL-6，该菌株能与水虻良好共生，本身在好氧环境中具有一定的降解四环素的能力，同时能有效抑制有害菌金黄色葡萄球菌(S. Aureus)及大肠杆菌(E.coli)，该菌株已于2017年2月28日送至中国典型培养物保藏中心保藏，分类命名：粘质沙雷氏菌(Serratia marcescens)BSFL-6，保藏编号：CCTCC NO:M2017073，地址：中国武汉武汉大学。
本发明的另一个目的在于提供了粘质沙雷氏菌BSFL-6的应用，包括利用该菌株制备抵抗和/或降解四环素的水虻，或是利用该菌制备成四环素的降解剂，或是制备同时降解四环素和抑制大肠杆菌和金黄色葡萄球菌的制剂。
为了达到以上目的，本发明采取以下措施：
通过构建无菌水虻体系、比较水虻肠道微生物在分解四环素过程中的贡献，申请人发现，水虻肠道微生物是水虻分解四环素的关键参与者，通过分离，申请人自转化含四环素污染物料的水虻肠道中分离得到一株粘质沙雷氏菌BSFL-6，该菌株已于2017年2月28日送至中国典型培养物保藏中心保藏，分类命名：粘质沙雷氏菌(Serratia marcescens)BSFL-6，保藏编号：CCTCC NO：M 2017073，地址：中国武汉武汉大学。
粘质沙雷氏菌BSFL-6在28℃下培养产生红色色素，该菌具有纤维素水解、蛋白水解、淀粉水解、脂肪水解、解磷、解钾和固氮等活性，并能拮抗有害菌金黄色葡萄球菌(S.Aureus) 及大肠杆菌(E.coli)。
粘质沙雷氏菌BSFL-6的培养：在LB培养基中，28℃下培养。
粘质沙雷氏菌BSFL-6的应用，包括利用该菌株制备抵抗和降解四环素的水虻，或是利用该菌制备成四环素的降解剂，或是制备同时降解四环素和抑制大肠杆菌和金黄色葡萄球菌的制剂。
与现有技术相比，本发明具有以下优点：
1.粘质沙雷氏菌BSFL-6具有纤维素水解、蛋白水解、淀粉水解、脂肪水解、解磷、解钾和固氮等活性，可与水虻共生，促进水虻生长；
2.粘质沙雷氏菌BSFL-6能拮抗有害菌金黄色葡萄球菌(S.Aureus)及大肠杆菌(E.coli)，是一株水虻肠道益生菌；
3.粘质沙雷氏菌BSFL-6可以离体降解四环素，而且可以与水虻联合降解四环素，是一株具有抗生素降解应用潜力的水虻肠道益生菌。
附图说明
图1为粘质沙雷氏菌BSFL-6在含不同浓度四环素的LB培养基中的生长曲线示意图。
图2为粘质沙雷氏菌BSFL-6回接于无菌水虻处理含四环素物料时四环素降解率比较图。
具体实施方式
下述实施例中的实验方法，如无特别说明，均为报道的微生物学常规操作方法。所用试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
从水虻肠道分离和鉴定粘质沙雷氏菌BSFL-6
申请人采用含40mg/L四环素的LB培养基，从转化含四环素污染物料的水虻肠道研磨液中分离得到一株粘质沙雷氏菌BSFL-6，产红色色素，该菌株已于2017年2月28日送至中国典型培养物保藏中心保藏，分类命名：粘质沙雷氏菌(Serratia marcescens)BSFL-6，保藏编号：CCTCC NO：M 2017073，地址：中国武汉武汉大学。粘质沙雷氏菌BSFL-6 的生理生化特性和培养特性如表1所示。
表1粘质沙雷氏菌BSFL-6的生理生化特性和培养特性
“+”为阳性，“-”为阴性。
实施例2：
粘质沙雷氏菌BSFL-6的离体培养：
将粘质沙雷氏菌BSFL-6在平板上划线活化，取平板上单菌落接种到装100mL有LB培养基的三角瓶中，培养温度为28℃、摇床转速180rpm、培养时间12h，得到粘质沙雷氏菌BSFL-6发酵菌悬液(本发明或简称BSFL-6菌悬液)，该菌悬液的有效菌浓度为108cfu/mL(OD600值1.5)。
实施例3：
粘质沙雷氏菌BSFL-6的体外降解四环素：
将500μL BSFL-6菌悬液(有效菌浓度是108cfu/mL )分别接种至装有200mL含20、40、 80和160mg/L四环素的LB培养基的三角瓶中，培养温度为28℃、摇床转速180rpm、培养时间96h，检测内容及结果如下：
1)检测其生长曲线，结果如图1所示，随着四环素浓度的增高，粘质沙雷氏菌BSFL-6 的适应期逐渐延长，但在适应期之后即进入对数生长期，并在进入对数生长期后的24小时内达到108cfu/mL (OD600值1.5)；即使在160mg/L四环素的浓度条件下，粘质沙雷氏菌BSFL-6均能耐受生长，说明其对高浓度的四环素有很好的耐受作用。
2)检测其培养基中的四环素浓度变化，至72h时，含20、40、80和160mg/L四环素的培养基中四环素降解率分别为：29.9±0.8％，42.1±0.5％，57.3±1.2％，61.7±0.9％。
实施例4：
粘质沙雷氏菌BSFL-6在水虻体内促进水虻转化四环素的应用：
本实施例所用的水虻为亮斑扁角水虻武汉品系。
1)将含40mg/L四环素的无菌人工物料(固含量50g，含水率65％)装入无菌三角瓶中，成为物料三角瓶；
2)实验设置3组，每组3个平行：
其一，在物料三角瓶中接种1mL BSFL-6菌悬液(有效菌浓度是108cfu/mL )以及100 头无菌水虻幼虫(不含肠道微生物)，标记为BSFL-6回接处理组；
其二，在物料三角瓶中接入100头无菌水虻幼虫(不含肠道微生物)，标记为无菌水虻处理组；
其三，在物料三角瓶中接种1mL BSFL-6菌悬液(有效菌浓度是108cfu/mL )，标记为 BSFL-6离体处理组；
3)将以上3个实验组置于28℃培养箱，保持通风及无菌环境，培养12d，取初始物料及12d的各组物料，测试其四环素含量，计算各组四环素的降解率，同时测定水虻重量；
4)实验结果如图2所示，BSFL-6回接处理组的四环素降解率为59.6±7.9％，要显著高于无菌水虻处理组(其降解率为38.2±7.3％)和BSFL-6离体处理组(其降解率为30.8±3.2％) 近两倍，说明粘质沙雷氏菌BSFL-6可以有效帮助水虻提高其四环素的降解效果，并且其与水虻共同处理的效果要优于离体处理的效果；12d时BSFL-6回接处理组的水虻鲜重为185±6 mg/条，比较于正常水虻同等条件培养12d的鲜重170±6mg/条的数据，结果说明BSFL-6回接处理有利于水虻自身的生长增重，因此，BSFL-6是水虻生长和降解四环素的肠道益生菌。
Claims (1)
1.一种分离的粘质沙雷氏菌(Serratia marcescens)在制备降解四环素、同时抑制大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphyloccocus aureus)的制剂中的应用；所述的粘质沙雷氏菌的保藏编号为CCTCC NO:M2017073，分类命名为：粘质沙雷氏菌(Serratia marcescens)BSFL-6。
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| Title |
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| "Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens";Stuart A. Thompson et al.,;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20070430;第73卷(第7期);第2200页，左栏第2-3段 * |
| "亮斑扁角水虻卵携带细菌的分离鉴定及其对成虫产卵行为影响";杨森等;《昆虫学报》;20170228;第60卷(第2期);第163页摘要部分 * |
| "粘质沙雷氏菌 （Serratia marcescens）的研究1-Serratia marcescens 9-2 菌株分离、分类鉴定和形态特征";黄文芳等;《华南师范大学学报》;20030831(第3期);第108-111页 * |
| Stuart A. Thompson et al.,."Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens".《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.2007,第73卷(第7期),第2200页，左栏第2-3段. * |
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