CN115287224B - 一株改善本源动物肠道微生物发育的牦牛源罗伊氏乳杆菌及其应用 - Google Patents
一株改善本源动物肠道微生物发育的牦牛源罗伊氏乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株改善本源动物肠道微生物发育的牦牛源罗伊氏乳杆菌及其应用，该菌株的保藏编号为CCTCC NO:M 2022084，来源于成年健康牦牛，对常见致病菌（大肠杆菌、金黄色葡萄球菌、沙门氏菌）有较强的抑制作用，新生幼畜连续服用安全无害，绿色环保，补饲犊牦牛后显著改善了犊牦牛的血清抗氧化性能（总抗氧化能力、超氧化物歧化酶、丙二醛）、血清生化指标（天门冬氨酸氨基转移酶、总胆固醇、尿素）和生长性能，促进宿主肠道内多种有益菌定植以改善肠道菌群，并且减少了犊牦牛腹泻疾病的发生。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株牦牛源罗伊氏乳杆菌(Lactobacillusreuteri)LR60-2022，可以抑制病原菌生长并改善本源动物肠道微生物群以及肠道代谢。
背景技术
罗伊氏乳杆菌(Lactobacillus reuteri)是存在于动物胃肠道内的重要菌种，对于改善宿主肠道微生态环境、抑制肠道有害菌、维持肠道菌群平衡、进而改善宿主的健康状况具有重要作用。我国农业部发布的第2045号公告《饲料添加剂品种目录(2013)》中，确定了罗伊氏乳杆菌可作为饲料级微生物添加菌种用于临床动物生产。罗伊氏乳杆菌通过以下机制发挥其益生性能：(1)物理竞争与排斥，占据肠上皮细胞的黏附位点，竞争性排斥致病菌的黏附；(2)产生有机酸(多为乳酸)、细菌素等代谢产物，它是重要的抑菌物质，抑制致病菌的繁殖并降低肠道pH；(3)菌体表面成分促进肠道免疫。研究表明，外源添加罗伊氏乳杆菌可以降低仔猪、肉鸡中潜在致病菌的数量、提高肠道有益菌丰度并降低肠道pH，通过调节肠道菌群改善肠道代谢物的组成和数量，从而增强肠道屏障功能，增强宿主抗病性。
乳酸杆菌被用作功能性食品和生物添加剂广泛应用于保健和疾病管理等方面。它们通过调节免疫反应、肠道健康和分泌各种有益代谢物影响宿主多种器官的健康，主要对胃肠道疾病有益。在对益生菌在动物模型中安全性的所有不良反应现有信息进行调查后发现，败血症、真菌血症居首位。由于益生菌具有菌株特异性，因此有必要在临床应用之前确保益生菌产品的安全性。
牦牛(Bos grunniens)是我国青藏高原及毗邻地区的特色优势畜种，主要分布于我国的青海、西藏、四川等地区。作为唯一能在青藏高原高寒地区生存的牛亚科动物，它是藏区牧民的生活和经济来源。牦牛产业已经成为高原地区的特色和经济支柱产业，为当地牧民提供肉、奶、皮毛、役力、燃料等生存必需原料，已经被赋予了重要的社会和生态意义。成年牦牛对于恶劣的生存条件有着极强的耐受性，然而，高寒、缺氧、饲草资源匮乏以及致病菌的感染是犊牦牛面临的巨大挑战。犊牦牛的早期发育健康及有效的饲养管理是发展牦牛产业的重要基础。
牦牛的胃肠道是一个巨大的微生物发酵罐，包含非常复杂的微生态系统，其中微生物数量巨大、种类繁多，与宿主的生理状态息息相关，对宿主的生长发育、生产性能、营养代谢、免疫功能、抗病性能均有重要影响。健康动物胃肠道含有的微生物量约为机体细胞的10-20倍，宿主与微生物菌群之间的密切相互作用高于宿主细胞间相互作用的10倍以上，影响宿主生理学的多种方面。肠道菌群可被视为由微生物组成的完整有机体，与宿主互作互利互惠，包括：(1)可促进宿主营养物质的消化吸收，将宿主无法利用的多糖转化为短链脂肪酸，加强肠道免疫；(2)维持肠道黏膜屏障，阻止致病菌定植，促进肠道稳态内环境；(3)分泌乳酸、细菌素等代谢物，通过全身血液循环参与不同的生理过程。然而，肠道菌群与宿主的相互作用是一把双刃剑，动态平衡的健康菌群给宿主带来诸多的生理益处。当菌群失调时，紊乱的肠道菌群会对宿主产生有害的作用，主要表现为微生物群结构和成分的改变，它已经作为很多疾病的诱发因素，引发了广泛关注，包括炎症性肠病、心血管疾病以及糖尿病等。不同疾病状态下，肠道菌群会发生相对应的变化。科研工作者已经表征了与炎性相关的疾病中(克罗恩病、抗生素相关性腹泻、新生儿坏死性小肠结肠炎)宿主与肠道菌群的特定关联。此外，反刍动物自出生起接触母体产道微生物就逐渐形成特有的肠道菌群，随着年龄的增长、采食、生长环境的变化而变化并逐渐发育成熟。在这一过程中，肠道菌群呈极不稳定的波动状态，与此时犊牦牛较高的疾病易感性密切相关。研究表明，约50％的犊牛都在遭受不同程度的肠病干扰，这也是引起犊牛死亡的重要诱因。因此，促进犊牦牛肠道微生物群的健康发育对于早期生长发育的顺利过渡以及提高生长性能具有重要意义。
发明内容
本发明的目的是提供一株罗伊氏乳杆菌，保藏编号为CCTCC NO:M2022084。该菌株来源于成年牦牛，且新生幼畜服用安全无害。应用于犊牦牛改善生长性能、肠道菌群的早期发育以及代谢，从而降低犊牦牛肠道疾病易感性。
为了达到上述目的，本发明采取以下技术措施：
申请人从西藏林芝牦牛基地采集牦牛的肠道样本，使用MRS琼脂及MRS肉汤培养基集中分离罗伊氏乳杆菌。经过选择性培养基筛选以及基因组测序，将分离鉴定得到的罗伊氏乳杆菌经过体外试验、安全性试验及预防大肠杆菌性腹泻试验、犊牦牛早期补饲试验筛选，得到一株效果良好的罗伊氏乳杆菌LR60-2022。该菌株已于2022年01月18日送至中国典型培养物保藏中心进行保藏，保藏编号：CCTCC NO:M 2022084，分类命名罗伊氏乳杆菌(Lactobacillus reuteri)LR60-2022，可用于制备犊牦牛益生菌添加剂，用于改善犊牦牛生长性能，促进犊牦牛有益菌定植和改善肠道菌群，防治犊牦牛腹泻。
与现有益生菌添加剂相比，本发明具有以下优点：
本发明使用的罗伊氏乳杆菌LR60-2022来源于成年健康牦牛，对常见致病菌(大肠杆菌、金黄色葡萄球菌、沙门氏菌)有较强的抑制作用，新生幼畜连续服用安全无害，绿色环保，补饲犊牦牛后显著改善了犊牦牛的血清抗氧化性能(总抗氧化能力、超氧化物歧化酶、丙二醛)和血清生化指标(天门冬氨酸氨基转移酶、总胆固醇、尿素)，促进宿主肠道内多种有益菌定植以改善肠道菌群，并且减少了犊牛腹泻疾病的发生。为高原地区犊牦牛的养殖提供了重要的技术支撑。
附图说明
图1为罗伊氏乳杆菌LR60-2022在MRS琼脂培养基的菌落图。
图2为罗伊氏乳杆菌LR60-2022革兰氏染色镜检图片。
图3为补饲罗伊氏乳杆菌LR60-2022对犊牦牛抗氧化指标的影响。WTC，WTM和WTL分别代表21天的对照组、代乳粉组和罗伊氏乳杆菌组；WFC，WFM和WFL分别代表42天的对照组、代乳粉组和罗伊氏乳杆菌组。
图4为补饲罗伊氏乳杆菌LR60-2022对犊牦牛血清生化指标的影响。WTC，WTM和WTL分别代表21天的对照组、代乳粉组和罗伊氏乳杆菌组；WFC，WFM和WFL分别代表42天的对照组、代乳粉组和罗伊氏乳杆菌组。
图5为补饲罗伊氏乳杆菌LR60-2022对犊牦牛肠道微生物多样性的影响。A图代表第21天的犊牦牛肠道微生物多样性；B图代表第42天的犊牦牛肠道微生物多样性。WTC，WTM和WTL分别代表21天的对照组、代乳粉组和罗伊氏乳杆菌组；WFC，WFM和WFL分别代表42天的对照组、代乳粉组和罗伊氏乳杆菌组。
图6为补饲罗伊氏乳杆菌LR60-2022对犊牦牛肠道菌群的影响。
图7为补饲罗伊氏乳杆菌LR60-2022对犊牦牛肠道代谢物和代谢途径的影响。A，B代表正电离模式下的差异代谢途径；C，D代表负电离模式下的差异代谢途径；E，F代表正电离模式下显着上调或下调的代谢物数量；G，H代表负电离模式下显着上调或下调的代谢物数量。FC，FM，FW分别代表42天的对照组、代乳粉组和罗伊氏乳杆菌组。
具体实施方式
以下实施例用于进一步解释说明本发明，但并不意味着对本发明的限制。本发明所涉及的实验技术和操作，如未特别说明，均为本领域的常规方案。所涉及到的试剂和耗材，如未特别说明，均来源于正规商业渠道。
实施例1罗伊氏乳杆菌LR60-2022的分离、筛选鉴定和保藏
1、罗伊氏乳杆菌的分离及筛选
从西藏林芝牦牛基地采集牦牛的肠道样本，用于罗伊氏乳杆菌的分离。具体操作方法为：在无菌操作台中使用无菌手术刀刮下肠道内容物及内壁黏膜，用无菌剪刀剪碎并置于装有7ml无菌PBS液的EP管中，涡旋震荡混匀。在37℃摇床中培养1h后，在无菌操作台中吸取50μl混合液于MRS琼脂板表面，然后用无菌涂布棒涂开。倒置于37℃培养箱培养48小时，挑选6个圆润、微凸起的乳白色疑似菌落，使用一次性接种环三区划线法接种到新的MRS琼脂板进行纯化，见于图1。纯化3-4代直到MRS琼脂板上的菌落大小、形态均匀一致，挑选单个菌落到MRS肉汤培养基进行扩大培养，置于37℃恒温摇床培养24小时，转速为120r/min。
体外耐酸、耐胆盐试验：使用1M HCl用于调节MRS肉汤培养基的pH值。用移液枪吸取100微升过夜培养的菌悬液置于pH 2、3、4、5的MRS液体培养基以及含有0.1％、0.2％、0.3％、0.4％和0.5％(w/v)胆盐的MRS液体培养基。不含HCl和胆盐的MRS液体培养基用作对照。置于37℃摇床培养3小时，评估菌株对不同pH和胆盐浓度环境的耐受性。结果显示，6株分离菌株中的2株对pH3和0.3％胆盐浓度有最高的耐受性。这2株分离菌株被用于后续试验以筛选出益生特性良好的益生菌菌株。
体外抑菌实验：使用大肠杆菌、金黄色葡萄球菌和沙门氏菌作为病原指示菌检测分离菌株的抑菌能力。吸取100微升过夜培养的病原指示菌均匀涂布于LB琼脂板表面，琼脂扩散法测量抑菌圈直径以评估分离菌株的抑菌能力(孔径5mm)。结果发现编号为LR60-2022的菌株对大肠杆菌、沙门氏菌的抑菌圈直径>17.00mm，对金黄色葡萄球菌的抑菌圈直径在7.00-17.00mm之间，结果见于表1。
表1菌株LR60-2022对指示病原菌抑制圈直径评分
5mm(-)，5–7mm(+)，7–17mm(++)，>17mm(+++)。
2、罗伊氏乳杆菌的分类学鉴定
对在耐酸、耐胆盐试验和体外抑菌试验中表现最佳的1株分离菌株进行革兰氏染色并镜检，可见蓝紫色、短杆状疑似乳杆菌的菌体，见于图2。挑取单个菌落在MRS肉汤培养基中过夜培养，其菌悬液使用DNA提取试剂盒提取基因组DNA，使用16s rDNA通用引物对基因组DNA进行PCR扩增，然后测序获得16s rDNA的基因序列，输入到NCBI进行BLAST比对，构建系统发育进化树，显示分离菌株与罗伊氏乳杆菌相似性>99％，鉴定分离菌株为罗伊氏乳杆菌。将分离得到的罗伊氏乳杆菌保藏于中国典型培养物保藏中心(武汉大学)，保藏日期为2022年01月18日，保藏编号为CCTCC NO:M 2022084，分类命名罗伊氏乳杆菌(Lactobacillus reuteri)LR60-2022。
实施例2罗伊氏乳杆菌LR60-2022的安全性试验
1、以小鼠为试验动物，进行口服毒性试验验证罗伊氏乳杆菌LR60-2022的安全性。简言之，在标准卫生条件下，选择20只小鼠(15-18克)随机分为两组，试验组(n＝10)的每只小鼠每天灌胃总量为1×109CFU的罗伊氏乳杆菌LR60-2022，对照组的小鼠用等体积的生理盐水灌胃。在整个实验过程中每天观察动物的活动、行为、毛发光泽、食物摄入量、体重和一般健康状况。试验期18天，在整个过程中，没有动物出现疾病、死亡或腹泻等症状，且行为、毛发、精神状态或采食量、器官指数方面没有表现出显着差异。
2、新生幼龄动物普遍被认为免疫系统、肠道微生物等发育不健全，对外来致病因素更为敏感。因此，以新生幼鼠(出生第一天)为试验对象，再次验证罗伊氏乳杆菌LR60-2022的安全性。购买200只出生第一天的小鼠(携带母鼠)，随机分为试验组(n＝100)：每天使用软针灌胃小鼠总量为1×109CFU(浓度2×1010CFU/ml，体积50μl)的罗伊氏乳杆菌LR60-2022，对照组(n＝100)：每天使用软针灌胃小鼠等体积的生理盐水。整个试验期为3周。试验期间所有小鼠都得到母鼠的自然哺乳，密切关注小鼠的精神状态、吮吸母乳、运动行为、被毛、腹围、腹泻、生长发育等状况，结果发现所有小鼠生理行为一致，未出现任何疾病相关症状及死亡。证明了幼龄动物早期连续补饲罗伊氏乳杆菌LR60-2022的安全性。
实施例3预防小鼠大肠杆菌性腹泻试验
以小鼠为试验动物建立大肠杆菌性腹泻模型。30只小鼠随机分为三组，试验组(n＝10)：每天灌胃小鼠1×109CFU的罗伊氏乳杆菌LR60-2022；对照组(n＝10)：每天灌胃小鼠等体积的生理盐水；空白对照组(n＝10)：每天灌胃小鼠等体积的生理盐水。第23天，对照组和试验组的每只小鼠同时腹腔注射大肠杆菌总菌量为1×109CFU，密切观察并记录小鼠的一般健康状况。攻毒2天后，颈椎脱臼处死存活的小鼠，统计每组小鼠的死亡率和腹泻率。结果发现，与空白对照组相比，对照组出现水样便、肛门周围有粪污、精神不振、反应迟钝、皮毛杂乱。对照组腹泻率为60％，死亡率为20％；罗伊氏乳杆菌组腹泻率为30％，无死亡病例出现。
实施例4罗伊氏乳杆菌LR60-2022对犊牦牛生长发育、肠道微生物菌群发育及代谢的影响
该试验在四川省阿坝藏族羌族自治州红原县麦瓦牦牛养殖基地进行，该基地的牦牛自由放牧，无任何饲料补充，自由饮水。共挑选了18头年龄、健康、初始体重和免疫接种程序相近的犊牦牛(0d，24h内出生)，佩戴项圈编号。将小牛随机分配到以下三组：对照组、奶粉组、罗伊氏乳杆菌组。每组6头小牛(一半雄性和一半雌性)，并在3±1日龄实施实验程序。奶粉组每头小牛每天两次(06:00和18:00)喂食0.5L奶粉；罗伊氏乳杆菌组每头小牛每天两次(06:00和18:00)喂食总量为1×109CFU的罗伊氏乳杆菌LR60-2022，用奶粉定容至0.5L；对照组不做任何处理。每天对奶瓶进行冲洗并灭菌。为了与牦牛的自然习性保持一致，所有的犊牦牛都与牦牛群一起自由放牧(从早上06:30到下午05:30)。两个藏牧民协助每天两次(早上和下午)找出带项圈编号的犊牦牛，以执行喂养策略，然后将所有试验犊牛放回牛群。在每天执行饲喂前，使用手持温度计每周(周一、周三、周五和周日)测量直肠温度4次。在试验第1天和第42天饲喂前测量犊牦牛空腹体重作为初始和终末体重。第21天和第42天，使用无菌针头经颈静脉穿刺采血到无抗凝剂的一次性采血管中用于分析血液抗氧化指标(超氧化物歧化酶SOD、过氧化氢酶CAT、谷胱甘肽过氧化物酶GSH-Px、丙二醛MDA、总抗氧化能力T-AOC)和血液生化指标(丙氨酸氨基转移酶ALT、天门冬氨酸氨基转移酶AST、葡萄糖Glu、白蛋白ALB、甘油三酯TG、总胆固醇TC、总蛋白TP、肌酐CREA、尿素Urea、钙、磷、镁)。在第21、42天，通过直肠刺激犊牦牛排便，立即使用无菌粪便采集管收集新鲜粪便并编号，然后立即放入液氮中，通过干冰运输样品至实验室，保存于-80℃冰箱用于16s rDNA高通量测序和非靶向代谢组学分析。
结果发现，罗伊氏乳杆菌对犊牦牛生长性能产生积极影响，平均增重比对照组提高3.16kg；此外，如图3和图4所示，罗伊氏乳杆菌LR60-2022改善了犊牦牛血清天冬氨酸氨基转移酶、总胆固醇、尿素指标，提高了血液抗氧化能力(超氧化物歧化酶，丙二醛，总抗氧化能力)。如图5所示，测量多个α多样性指数以研究微生物群落丰富度和多样性的整体差异，发现早期补饲罗伊氏乳杆菌LR60-2022显著增加了肠道微生物多样性。由图6可知，补饲罗伊氏乳杆菌优化了犊牛肠道微生物组的结构和组成，由多种功能性有益细菌主导，威胁健康的微生物物种丰度降低。补饲LR60-2022后犊牦牛肠道代谢组发生显着变化。具体来说，对照组和罗伊氏乳杆菌组之间共鉴定出150种正离子化代谢物和224种负离子化代谢物丰度显著变化；奶粉组和罗伊氏乳杆菌组之间共鉴定出185种正离子化代谢物和81种负离子化代谢物丰度显著变化。对其进行通路富集分析表明，与蛋白质消化吸收、维生素消化吸收、cAMP信号通路、次生代谢产物的生物合成相关等的代谢模式受到罗伊氏乳杆菌LR60-2022的有益影响，该部分结果见于图7。
Claims (5)
1.罗伊氏乳杆菌（Lactobacillus reuteri）LR60-2022，其特征在于，保藏编号为CCTCCNO:M 2022084。
2.犊牦牛益生菌添加剂，其特征在于，含有罗伊氏乳杆菌（Lactobacillus reuteri）LR60-2022，保藏编号为CCTCC NO:M 2022084。
3.权利要求1所述的罗伊氏乳杆菌LR60-2022在制备改善犊牦牛生长性能的产品中的应用。
4.权利要求1所述的罗伊氏乳杆菌LR60-2022在制备促进犊牦牛有益菌定植和改善肠道菌群的产品中的应用。
5.权利要求1所述的罗伊氏乳杆菌LR60-2022在制备防治犊牦牛腹泻的药物或饲料添加剂中的应用。
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