CN110157645B - 一种唾液乳杆菌y4及其应用 - Google Patents
一种唾液乳杆菌y4及其应用 Download PDFInfo
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- CN110157645B CN110157645B CN201910458213.1A CN201910458213A CN110157645B CN 110157645 B CN110157645 B CN 110157645B CN 201910458213 A CN201910458213 A CN 201910458213A CN 110157645 B CN110157645 B CN 110157645B
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- lactobacillus salivarius
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- pathogenic bacteria
- lactobacillus
- salivarius
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Abstract
本发明提供了一种唾液乳杆菌Y4及其应用，所述唾液乳杆菌Y4保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC NO.17459；其可应用于抑制病原菌，该病原菌包括大肠杆菌、沙门氏菌、绿脓杆菌、金黄色葡萄球菌、肺炎克雷伯菌、单增李斯特菌等；其还可应用于制备食品、饲料等。本发明唾液乳杆菌Y4耐酸耐胆盐能力强，体外培养繁殖快，可用于微生态制剂的开发，可有效抵抗各病原菌的感染，对动物体具有有效的保护作用。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一种唾液乳杆菌Y4及其应用。
背景技术
菌株(Strain)又称品系，表示同种微生物不同来源的纯种培养，从自然界中分离得到的每一个微生物纯培养都可称一个菌株。如培养伤寒杆菌时可从血液中、骨髓中、粪便中、胆汁中进行分离培养，而以胆汁中者最为良好，称为标准株。益生菌是一类对宿主有益的活性微生物，是定植于人体，已被广泛应用于预防和治疗多种疾病，并且它们的效力在某些临床情境中有有力的证据。例如，WO 2007/043933记载了将益生菌用于制造食品、饲料产品、膳食补充剂，以控制体重增加、预防肥胖、增加饱腹感、延长饱腹感、降低食物摄入量、降低脂肪沉积、改善能量代谢、增强胰岛素敏感性、治疗肥胖和治疗胰岛素抵抗。
唾液乳杆菌是乳杆菌属的重要成员之一，大多数为动物体表和内部黏膜表面的益生菌之一。存在于胃肠道黏膜、口腔呼吸道黏膜及生殖道表面的益生菌作为一种活的常驻菌对肠道黏膜及呼吸道黏膜具有双重的保护作用，一方面它可以在黏膜表面内附着定植，维护胃肠道及呼吸道黏膜微生物菌群平衡；另一方面益生菌可直接与宿主的免疫***尤其是宿主的黏膜免疫***发生相互作用诱发黏膜免疫；并能够刺激脾脏、胸腺及法氏囊等免疫中枢器官的发育，还可以促进巨噬细胞发挥佐剂作用；通过影响黏膜***细胞因子等的合成和分泌，从而增强T、B细胞对抗原刺激的反应性能，有效增强特异性免疫作用；黏膜表面常驻益生菌可以活化黏膜内的相关淋巴组织，增强sIgA生物合成，提高消化道黏膜及呼吸道黏膜免疫功能，通过诱导淋巴细胞和巨噬细胞产生细胞因子，发挥免疫调节作用，从而增强机体免疫功能。
发明内容
本发明的目的是提供了一种唾液乳杆菌Y4及其应用，该唾液乳杆菌Y4耐酸耐胆盐能力强，体外培养繁殖快，可用于微生态制剂的开发，可有效抵抗各病原菌的感染，对动物体具有有效的保护作用。
为实现以上目的，本发明通过以下技术方案予以实现：
本发明菌株分离自健康儿童的粪便，经MRS培养基接种培养、分离纯化后获得一株唾液乳酸杆菌(菌株号为Y4)，其微生物保藏号为CGMCC NO.17459；保藏日期：2019年03月29日；其分类命名为：乳杆菌属唾液乳酸杆菌(Lactobacillus salvarius)；保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；保藏地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
本发明主要通过以下方法从来自于健康儿童的粪便样品中分离得到上述唾液乳杆菌菌株。
将采集的儿童粪便直接接种于MRS固体培养基上，挑选特征明显的菌落反复环线分离，直至分理处形态、大小一致的菌落，进行革兰氏染色，对菌体形态进行显微观察；反复接种纯化后，即得。
经法国梅里爱公司API鉴定***进行菌株判定，在进行分子生物学鉴别，本发明所分离得到的唾液乳酸杆菌菌株Y4的分类地位属于：乳酸杆菌属唾液乳酸杆菌(Lactobacillus salvarius)。
该菌株在MRS平板上的菌落为半球形，表面光滑湿润，呈乳白色，直径约为1-2mm，大小均一；革兰氏染色观察，该菌株为革兰氏阳性菌，菌体杆状，宽：0.5-0.6um，长：1.2-3.0um；在MRS液体培养基(pH 6.2)中生长良好，呈均匀浑浊生长，在37℃，120 rpm转速下，8小时达到对数生长末期。
本发明唾液乳杆菌Y4在抑制病原菌中具有较好的应用效果，应用于制备病原菌抑菌剂，该病原菌包括大肠杆菌、沙门氏菌、绿脓杆菌、金黄色葡萄球菌、肺炎克雷伯菌、单增李斯特菌等。特别是对沙门氏菌的抑制效果最好。同时，本发明唾液乳杆菌Y4还可应用于制备食品、饲料等。
本发明取得的有益效果如下：
本发明菌株Y4经16S rDNA鉴定为唾液乳杆菌(Lactobacillus salvarius)。本发明的唾液乳杆菌具有较强的耐酸耐胆盐能力，能分别在pH＝3的培养液和含0.35g/L脱氧胆酸钠的培养液中生长。
该菌株体外培养繁殖快，可用于微生态制剂的开发。该菌株体外抑菌试验表明，本发明的唾液乳杆菌Y4对大肠杆菌、沙门氏菌、绿脓杆菌、金黄色葡萄球菌、肺炎克雷伯菌、单增李斯特菌均有明显的抑制作用，可以有效抵抗各病原菌的感染，对动物体具有有效的保护作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为唾液乳杆菌Y4菌株菌落形态图；
图2为唾液乳杆菌Y4菌株16S rDNA分子进化树；
图3为唾液乳杆菌Y4生长曲线及pH值的变化图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：唾液乳杆菌的筛选与鉴定
1、唾液乳杆菌Y4的分离筛选
以健康儿童的粪便为样品，用无菌木棒取粪便样品于无菌生理盐水中，混合均匀并进行梯度稀释，MRS培养基(补加X-gal)倾注培养筛选“蓝色”的菌落，多次纯化后得到单菌落，命名为Y4，保存。所述MRS培养基为培养乳酸杆菌的通用培养基，其配方为：蛋白胨10g/L，牛肉膏10g/L，酵母粉5g/L，葡萄糖20g/L，吐温- 80 1mL/L，磷酸氢二钾2g/L，三水乙酸钠5g/L，柠檬酸三铵2g/L，七水硫酸镁0.58g/L，一水硫酸锰0.05g/L，pH 6.2。
所述菌株Y4在MRS平板上菌落的主演形态特征如下：如图1 所示。菌落圆，光滑，在含X-gal的MRS培养基上呈蓝色，不透明。革兰氏染色显示阳性，菌体杆状，宽：0.5-0.6um，长：1.2-3.0um。
2、生理生化指标测定鉴定菌株
采用法国梅里埃公司碳水化合物鉴定试剂条鉴定分离获得的Y4 菌株的生化图谱。依据生化图谱对菌株进行种属鉴定，根据鉴定试剂条说明，将分别加入规定浓度(混浊度相当于2McFarland的菌悬液)的菌悬液的试剂条置于37℃培养箱培养进行孵育，孵育24小时记录反应结果，根据24小时反应结果的发酵反应生化图谱，菌株生化图谱为：木糖、葡萄糖、甘露糖、山梨醇、七叶灵、柳醇、纤维二糖、麦芽糖、乳糖、蔗糖、海藻糖、棉子糖为阳性；甘油、***糖、鼠李糖、甘露醇、松叁糖为阴性；应用法国梅里埃公司 API鉴定***的软件进行菌种的判定，该菌株鉴定结果为唾液乳杆菌(Lactobacillus salvarius)。
3、Y4菌株的16S rDNA序列测定
以所述菌株Y4的DNA为模板，16S rDNA通用引物进行扩增，扩增片段进行序列测定；Blast比对显示该菌株序列于GenBank基因库中Lactobacillus salivarius的16S rDNA序列同源性最高，同源率为99％。通过MEGA 4.0对RDP库中已有乳杆菌属模式菌株的16SrDNA序列进行遗传进化分析，如图2结果显示，菌株Y4与 Lactobacillus salivarius同源性最高，判定该菌株Y4为唾液乳杆菌。
将筛选到的菌株Y4进行菌株保藏，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)；保藏日期：2019年 03月29日；唾液乳杆菌Lactobacillussalivarius的保藏编号为 CGMCC NO.17459。
实施例2：唾液乳杆菌Y4的生长测定
将活化的Y4菌液以2％(约107CFU/mL)的接种量接种于所述MRS液体培养基中，置于37℃摇床中培养，220rpm培养24h，分别在0、2、4、6、7、8、10、12、24h取样，然后以培养时间为横坐标，以pH和OD600nm值为纵坐标，绘制生长曲线及pH变化图。如图3所示，实验结果表明，Y4培养2h即进入对数生长期， 8h进入稳定期。培养24h时培养液pH下降至3.86。
实施例3：唾液乳杆菌Y4对酸、胆盐的耐受性测定
将活化的Y4菌液以2％(约107CFU/mL)的接种量分别接种于pH为2.0、2.5、3.0、3.5、4.0、6.2的MRS液体培养基中，在 37℃、220rpm条件下培养2h，稀释适当比例后涂平板，培养24h 计数。唾液乳杆菌Y4对酸耐受结果如表1所示。
用同样的方法处理菌株，将菌液接种于脱氧胆酸钠浓度为0％、 0.20％、0.25％、0.30％和0.35％的培养基中，在37℃、220rpm条件下培养3h，稀释适当比例后涂平板，培养24h计数。唾液乳杆菌 Y4对脱氧胆酸钠盐耐受结果如表1所示。
表1唾液乳杆菌Y4对酸耐受结果
表2唾液乳杆菌Y4对脱氧胆酸钠盐耐受结果
由表1和表2可知，唾液乳杆菌Y4菌株在pH 2.5是其对酸耐受的临界点，pH 4.0时不影响菌株正常生长的临界点，pH在2.5-4.0 之间菌株生长，但生长速度较正常缓慢；唾液乳杆菌Y4菌株对脱氧胆酸钠盐的耐受临界点大于0.35％，0％-0.3％胆盐浓度时不影响菌株正常生长的临界范围。
实施例4：唾液乳杆菌Y4菌株体外抑菌试验
体外抑菌试验采用牛津杯法测定益生菌的抑菌活力。将甘油保存的唾液乳杆菌Y4划线接种到MRS固体培养基于37℃培养24h，再挑取菌落接种至MRS液体培养基中过夜培养，取37℃培养过夜的唾液乳杆菌Y4菌悬液作为抑菌液备用。将甘油保存的大肠杆菌、沙门氏菌、绿脓杆菌、肺炎克雷伯菌、金黄色葡萄球菌和单核细胞增多性李斯特菌划线接种到TSB固体培养基于37℃培养24h，再挑取菌落接种至TSB液体培养基培养至OD600＝1.0，用生理盐水1： 103稀释，各取200uL稀释后的菌悬液“米”字划线至TSB固体培养基，晾干后备用。
以MRS液体培养基作为空白对照，平均每个TSB平板上均匀放置3个牛津杯，每个牛津杯加入200uL抑菌液，至于37℃恒温培养箱培养24h，观察牛津杯杯底的抑菌圈情况，并测定抑菌圈直径。实验结果如表3所示。
表3唾液乳杆菌Y4对6种致病菌的抑菌结果
由表3可知，唾液乳杆菌Y4对大肠杆菌、沙门氏菌、绿脓杆菌、肺炎克雷伯菌、金黄色葡萄球菌和单核细胞增多性李斯特菌均有一定的抑制效果，且唾液乳杆菌Y4对沙门氏菌的抑制效果最好。
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (6)
1.一种唾液乳杆菌Y4，其特征在于，所述唾液乳杆菌(Lactobacillus salvarius)Y4保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC NO.17459。
2.如权利要求1所述的唾液乳杆菌Y4，其特征在于，培养所述唾液乳杆菌的培养基为MRS固体培养基，MRS固体培养基pH值为6.2。
3.如权利要求1所述的唾液乳杆菌Y4，其特征在于，所述唾液乳杆菌的培养温度为37℃。
4.如权利要求1所述的唾液乳杆菌Y4在制备病原菌抑菌剂中的应用。
5.如权利要求4所述的应用，其特征在于，所述病原菌为大肠杆菌、沙门氏菌、绿脓杆菌、金黄色葡萄球菌、肺炎克雷伯菌、单增李斯特菌。
6.如权利要求1所述的唾液乳杆菌Y4在制备食品、饲料中的应用。
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