CN114874945A - 一株短乳杆菌及其应用 - Google Patents
一株短乳杆菌及其应用 Download PDFInfo
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Abstract
本发明主要涉及微生物技术领域，具体涉及一株短乳杆菌及其应用。该菌株GforU‑11寄存于中国典型培养物保藏中心，保藏编号为CCTCC NO:M20211557。此菌株GforU‑11具有维持及修护皮肤屏障、促进细胞修复、减轻皮肤炎症等功能，可用于食品、药品、化妆品等。
Description
技术领域
本发明主要涉及微生物技术领域，具体涉及一株短乳杆菌及其应用。
背景技术
皮肤作为人体最大的器官生活着广泛的微生物，皮肤构成了陷入部、经特化的间隙等多样形态的栖息地，帮助分布广泛的微生物能够生存。微生物与作为宿主的人构成了共生关系，皮肤微生物群在宿主中发挥重要而有用的功能，不仅因为它有能力抵抗皮肤病原体的粘附和发展，还因为它有能力与免疫系统对话和相互作用。微生物和皮肤环境之间的平衡一旦被打破，本来无害的微生物将由健康状态变为致病状态，就会引起各种皮肤疾病；反之，皮肤疾病也会导致皮肤微生物结构异常和菌群失调，所以很多皮肤问题都与皮肤微生态失衡有着密切关系。
益生菌外用可以抑制皮肤病原菌的生长，使皮肤表面的微生物菌群达到平衡，促进皮肤屏障的修复。同时，益生菌在维持宿主机体稳态，激活免疫系统、维持机体免疫平衡等方面具有重要作用，，研究发现益生菌能通过调节巨噬细胞的吞噬能力以及细胞因子的释放来控制全身免疫状态，从而预防和治疗各种炎症疾病。本发明旨在采用益生菌方式解决上述现有技术问题。
发明内容
为实现上述技术目的，本发明从发酵苏子叶中分离鉴定的短乳杆菌，将其编号为GforU-11，该菌株及其相关代谢产物兼具皮肤抗炎和抑菌，以及屏障修护功效，在食品、药品、化妆品等领域具有极大的市场潜力。
以上目的是通过以下技术方案来实现的：
本发明首先提供一株短乳杆菌(Lactobacillus brevis)，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 20211557。
该短乳杆菌来源于发酵苏子叶，编号GFORU-11,经16S rRNA鉴定为短乳杆菌(Lactobacillus brevis)。该菌株革兰氏阳性，显微镜下呈杆状；在MRS平板上生长，可形成表面光滑不透明的圆形菌落，白色，边缘整齐；在MRS液体培养基中呈均匀浑浊生长，久置菌体呈白色沉淀，最适生长温度37℃。
本发明进一步提供上述短乳杆菌的用途，用于皮肤抗炎、抑菌、皮肤屏障修复等。
根据上述用途，本发明更进一步的提供该短乳杆菌的另一用途，用于制备皮肤抗炎或/和抑菌或/和皮肤屏障修复等相关的食品、药品、化妆品。
所述的抑菌是指用于抑制金黄色葡萄球菌或/和人葡萄球菌或/和溶血葡萄球菌或/和铜绿假单胞菌的生长。
用于制备相关食品、药品、化妆品时，所采用的短乳杆菌可以是其菌体或/和其衍生物或/和其代谢产物。其中，所述的菌体可以是活菌或/和灭活菌体。
所述的衍生物为短乳杆菌的裂解物或/和提取物，包括但不限于细胞壁、胞内蛋白、胞内多糖等。
所述的代谢产物应当理解为短乳杆菌的初级代谢产物或次级代谢产物，例如益生素、含有免疫原性成分的化合物，胞外多糖等，也可以直接是发酵液的上清液。
本发明的有益效果为：
本发明的GforU-11经16S rRNA鉴定为短乳杆菌(Lactobacillus brevis)。
体外细胞实验表明，本发明的短乳杆菌GforU-11具有降低炎症因子释放的作用，能够降低脂多糖(LPS)诱导小鼠巨噬细胞Raw264.7的一氧化氮(NO)生成量21.93％～52.40％。
体外细胞实验表明，本发明的短乳杆菌GforU-11具有下调金黄色葡萄球菌(Staphylococcus aureus)诱导的HaCaT细胞炎症因子相关基因表达的作用，基因表达量下调13％～96％。
体外抑菌实验表明，本发明的口乳杆菌GforU-11具有抑制包括金黄色葡萄球菌(Staphylococcus aureus)、人葡萄球菌(Staphylococcus hominis)、溶血葡萄球菌(Staphylococcus haemolyticus)和铜绿假单胞菌(Pseudomonas aeruginosa)等皮肤常见病原菌生长的作用，抑制率20.30％～31.90％。
保藏信息
保藏时间：2021年12月06日；
保藏单位名称：中国典型培养物保藏中心；
保藏编号：CCTCC NO:M 20211557；
保藏单位地址：湖北省武汉市武昌区八一路299号武汉大学校内；
分类命名：短乳杆菌(Lactobacillus brevis)。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容做进一步的详细说明，但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围，除特殊说明外，下述实施例中均采用常规现有技术完成。
以下实施例中GforU-11均指短乳杆菌(Lactobacillus brevis)，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 20211557。
实施例一：GforU-11的分离
于发酵苏子叶中采样。将样品适当处理后于生理盐水中震荡混匀，取上清划线于MRS固体平板，37℃恒温培养24～48h后，挑取白色菌落反复接种筛选，直至得到均匀的单个菌落，命名为GforU-11。
革兰氏染色镜检：菌株GforU-11为革兰氏染色阳性，显微镜下呈杆状；在MRS平板上生长，可形成白色、表面光滑圆润不透明圆形小菌落，边缘整齐；在MRS液体培养基中呈均匀浑浊生长，久置菌体呈白色沉淀。
实施例二：GforU-11的核酸鉴定
1、16s rRNA基因序列分析：
挑取单菌落置MRS液体培养基中，37℃培养过夜后，离心收集菌体，按照DNA提取试剂盒步骤进行操作。引物用细菌通用引物27F,1492R，PCR扩增体系为50μL体系，95℃预变性5min；94℃15s，57℃15s，72℃40s，35个循环；72℃延伸10min。
2、结果
PCR产物测序结果与GenBank中已发表的标准序列进行同源性比较(BLASTN)后得出GforU-11菌株为短乳杆菌(Lactobacillus brevis)。
实施例三：GforU-11降低Raw264.7细胞NO生成量
1、GforU-11菌液制备
将GforU-11用MRS培养基培养过夜，检测OD600，并用PBS缓冲液调整菌液浓度至OD600＝0.2，离心后，上清液用0.22μm滤膜过滤,菌体经PBS清洗两次后以PBS重悬至OD600＝0.2,上清液和菌体样品最后以121℃高压灭菌30min灭活。
2、Raw264.7细胞制备
将Raw264.7细胞消化后以2×105cell/孔接种至24孔板，5％二氧化碳培养箱37℃培养过夜。
3、GforU-11添加及LPS刺激
培养过夜的Raw264.7细胞，实验组1加入灭活上清液5％(V/V，相应对照组加等体积PBS)、实验组2加入灭活菌体10％(V/V，相应对照组加等体积PBS)，2h后添加0.5ml浓度为0.2μg/ml的LPS溶液，诱导Raw264.7细胞发炎，20h后取细胞培养上清，NO含量检测试剂盒进行NO含量检测，按照碧云天NO检测试剂盒所述方法进行标准曲线绘制，计算样品中NO的浓度及抑制率。
结果：
GforU-11降低Raw264.7细胞NO生成量
结果显示GforU-11能够降低LPS诱导的Raw264.7细胞NO生成量，与LPS对照组相比上清液降低52.40％，灭活菌体降低21.93％，说明该GforU-11菌体及其发酵上清液均具有抗炎作用。
实施例四：GforU-11下调HaCaT细胞炎症因子相关基因的表达
1、GforU-11样品制备
将GforU-11用MRS培养过夜，检测OD600，并用PBS缓冲液调整菌液浓度至OD600＝0.2，离心后，菌体121℃高压灭菌30min得菌体，离心上清用0.22μm滤膜过滤得上清液。
2、HaCaT细胞制备
将HaCaT细胞消化后以2×105cell/孔接种至24孔板，5％二氧化碳培养箱37℃培养过夜。
3、金黄色葡萄球菌制备及添加
金黄色葡萄球菌接入营养肉汤培养基，37℃摇床培养过夜，用MEM无血清培养基调整菌液浓度至OD600＝6.0，每孔100μl添加入培养过夜的HaCaT细胞中，刺激细胞产生炎症因子，3h后弃去细胞培养基，PBS清洗5次，每孔重新加入1ml的MEM无血清培养基。
4、GforU-11样品添加
将GforU-11上清液以5％加入金黄色葡萄球菌刺激过的HaCaT细胞中，每组3个复孔，培养过夜。
5、qPCR法检测细胞炎症因子mRNA相对表达倍数
将上述细胞弃去培养基后，用RNA提取试剂盒提取RNA，检测RNA浓度及纯度，将所有样品调整至1μg，用反转录试剂盒、SYBRGreen qPCR试剂盒进行RT-PCR及qPCR，计算炎症因子相关基因IL-6、IL-8和COX-2的相对表达倍数F。
公式：F＝2-ΔΔCT
结果：
GforU-11下调炎症因子相关基因的表达
结果显示GforU-11上清液能够下调金黄色葡萄球菌诱导的HaCaT细胞炎症因子相关基因表达，表达量降低13％～96％。因此，GforU-11具有抗炎作用。
实施例五：GforU-11抑制病原菌生长实验-菌液浓度变化
1、短乳杆菌GforU-11菌液制备：
将活化的短乳杆菌GforU-11菌液以MRS液体培养基培养于37℃培养箱静置培养16～18h，检测并并用PBS缓冲液调整OD600＝2.0，离心取上清液，0.22μm滤膜过滤，121℃，30min灭活得待测样品。
2、病原菌菌液制备：
将4种病原菌：金黄色葡萄球菌CGMCC 1.8721、人葡萄球菌CGMCC 1.493、溶血葡萄球菌CGMCC 1.540、铜绿假单胞菌CGMCC 1.1783，分别以BHI培养基37℃培养18h，检测并用BHI培养基调整OD600＝0.2。
3、抑制病原菌实验
将灭活上清液以10％(V/V，对照组加等量MRS培养基)加入病原菌菌液中，37℃培养3h，以菌液浓度(OD600)降低百分比评价对病原菌生长的抑制作用。
4、结果：
GforU-11对皮肤病原菌抑制率
结果显示GforU-11对金黄色葡萄球菌、人葡萄球菌、溶血葡萄球菌和铜绿假单胞菌等常见皮肤病原菌均具有抑制作用。
另外，上述所有实施例中涉及到的发酵上清液或灭活菌体，经验证，对其进行进一步加工、提取等获得的GforU-11的衍生物(裂解物或提取物)，或发酵上清液中的GforU-11的初级代谢产物或次级代谢产物等均具有相应的功效。
Claims (8)
1.一株短乳杆菌(Lactobacillus brevis)，其特征在于，其保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 20211557。
2.一株短乳杆菌的应用，其特征在于，用于修护皮肤屏障、消炎、抑菌，所述短乳杆菌保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 20211557。
3.根据权利要求2所述的短乳杆菌的应用，其特征在于，所述的抑菌是指用于抑制金黄色葡萄球菌或/和人葡萄球菌或/和溶血葡萄球菌或/和铜绿假单胞菌的生长。
4.一株短乳杆菌的应用，其特征在于，该短乳杆菌用于制备具有促进皮肤屏障修复、抗炎、抑菌作用相关的食品、药品、化妆品，所述短乳杆菌保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 20211557。
5.根据权利要求4所述的短乳杆菌的应用，其特征在于，所述的短乳杆菌可以是其菌体或/和其衍生物或/和其代谢产物。
6.根据权利要求5所述的短乳杆菌的应用，其特征在于，所述的菌体可以是活菌或/和灭活菌体。
7.根据权利要求5所述的短乳杆菌的应用，其特征在于，所述的衍生物为短乳杆菌的裂解物或/和提取物。
8.根据权利要求5所述的短乳杆菌的应用，其特征在于，所述的代谢产物应当理解为短乳杆菌的初级代谢产物或次级代谢产物。
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