CN114591879B - 一种抑制幽门螺杆菌的发酵乳杆菌及其应用 - Google Patents
一种抑制幽门螺杆菌的发酵乳杆菌及其应用 Download PDFInfo
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Abstract
本发明提供一种抑制幽门螺杆菌的发酵乳杆菌及其应用，该发酵乳杆菌保藏编号为CGMCC NO.23590，其具有小鼠感染幽门螺杆菌的抑制作用。发酵乳杆菌LFE‑5的能够通过降低幽门螺杆菌感染小鼠胃组织有害菌幽门螺杆菌、拟杆菌属、脱硫弧菌的相对丰度，增加有益菌AKKermansia及乳酸菌的相对丰度来抑制幽门螺杆菌的生长定植，并能够显著降低幽门螺杆菌毒力因子CagA及VagA的mRNA表达水平。
Description
技术领域
本发明属于益生菌技术领域，特别涉及一种对幽门螺杆菌具有抑制作用的发酵乳杆菌及其应用。
背景技术
幽门螺杆菌(Helicobacter pylori，Hp)，是定植于人类胃黏膜的革兰阴性微需氧杆菌，是慢性胃炎、消化性溃疡、胃黏膜相关淋巴组织淋巴瘤及胃癌的主要致病因素。有研究表明，Hp与胃溃疡的发生及发展具有明显的相关性，80％以上的胃溃疡由Hp感染所致。胃溃疡是临床常见的慢性消化系统疾病，病程长，复发率高且难彻底治愈。根除Hp是治疗胃溃疡的关键，并且可以促进胃溃疡治愈、显著降低溃疡的复发率。
随着用药种类的增加，疗程延长、相关不良反应亦增多，尤以腹泻、腹胀等消化道不良反应最为常见。受Hp耐药性的增加及药物不良反应等影响，Hp根除率逐年下降，其主要原因与菌株变异、继发性耐药、交叉耐药、不同菌株交叉感染等诸多因素导致的Hp对抗菌药物耐药性增加有关。与此同时，不规范地抗生素联用对胃肠微生态系统产生相关不良影响，破坏胃肠道微生态平衡及微生物屏障，使肠道中抗菌药物敏感菌株逐步减少，耐药菌株增加，导致部分患者出现上腹部不适、腹痛、恶心、呕吐、腹泻等不良反应，且发生呈现增高趋势。
因此，如何提供一种可以抑制幽门螺杆菌的产品，既不会增加幽门螺杆菌耐药性，同时，在治疗过程中不会导致患者产生不良反应，提高幽门螺杆菌临床治疗的效果，已成为亟待解决的问题。
发明内容
针对现有技术的不足和实际需求，本发明提供一种具有抑制幽门螺杆菌功能的发酵乳杆菌及其应用，研究发酵乳杆菌及其发酵液对小鼠感染幽门螺杆菌的抑制作用，通过动物实验，检测幽门螺杆菌的毒力因子和胃部菌群，评价菌株及其发酵液对幽门螺杆菌的抑制效果。
本发明的目的是通过以下技术方案实现的：
本发明提供一种分离自乳制品中的发酵乳杆菌LFE-55，该菌株于2021年10月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，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，邮编100101，分类命名为发酵乳杆菌Lactobacillus fermentum，保藏编号为CGMCC NO.23590。
本发明提供上述发酵乳杆菌的培养方法，其特征在于，将发酵乳杆菌接种在MRS液体培养基中，在30～37℃下培养18～24h。
另一方面，本发明提供发酵乳杆菌在制备抑制幽门螺杆菌产品中的应用。
优选的，所述产品中发酵乳杆菌的活菌数不低于1×106CFU/mL。
优选的，所述产品为药品或幽门螺杆菌抑制剂中的任意一种。
另一方面，本发明提供一种含有上述发酵乳杆菌的组合物、幽门螺杆菌抑制剂或相关产品。
上述产品中含有的发酵乳杆菌可使宿主体内幽门螺杆菌毒力因子CagA和VacA表达降低或被抑制。
优选的，其中发酵乳杆菌的活菌数不低于1×106CFU/mL。
本发明相比现有技术的有益效果为：
(1)本发明通过平板涂布及划线纯化，成功筛选到一株可抑制幽门螺杆菌的发酵乳杆菌，保藏编号为CGMCC NO.23590。
(2)发酵乳杆菌LFE-5能够通过改变胃部菌群的结构及幽门螺杆菌毒力因子CagA及VagA的表达来抑制幽门螺杆菌的持续感染。
(3)发酵乳杆菌制备的益生菌产品不会使幽门螺杆菌产生耐药性，同时，在治疗过程中不会导致患者产生不良反应，不会破坏患者的胃肠道微生物生态平衡，为根除幽门螺杆菌创造了条件，并且可以避免抗生素滥用造成的环境污染，应用前景广阔。
附图说明
图1各组CagA mRNA表达水平
图2各组VagA mRNA表达水平
图3各组幽门螺杆菌相对丰度
图4各组拟杆菌属相对丰度
图5各组梭菌属相对丰度
图6各组脱硫弧菌相对丰度
图7各组AKKermansia相对丰度
图8各组乳酸菌相对丰度
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
以下将通过实施例对本发明进行详细描述。应当能够理解的是，以下实施例仅用于示例性地进一步详细解释和说明本发明的内容，而不用于限制本发明。
实施例1微生物培养准备
本发明的发酵乳杆菌菌体制备：发酵乳杆菌在MRS培养基内活化两代后，以2％接种量接种于MRS液体培养基内培养12h，离心(4000rpm，10min)去除上清液，用PBS清洗菌体两次后，灌胃时，以0.9％生理盐水重悬浮并调整其菌悬液浓度为1×109CFU/mL。
幽门螺杆菌于哥伦比亚血琼脂平板上进行活化，将菌体刮下并用PBS清洗菌体两次后转移至含5％胎牛血清的脑心浸液培养基(BHI)中，于微需氧条件下继续培养48h后调整其菌液浓度为1×109CFU/mL，并立即进行灌胃实验。
发酵乳杆菌上清液：取活化两代后的发酵乳杆菌，以2％的接种量接种于MRS液体培养基内培养12h后，离心(4000rpm，10min)收集上清液，再用0.22μm无菌滤膜过滤后得到乳酸菌无菌发酵上清液，立即用于小鼠灌胃实验。
实施例2动物模型
实验动物选用SPF级C57BL/6N(5周龄，雌性)小鼠，购自于北京维通利华实验动物技术有限公司。动物实验共分为6组，分别是空白组、模型组、药物组、LFE-55高剂量组、LFE-55低剂量组、LFE-55上清液组，每组8只小鼠，共48只。
小鼠饲养条件：温度24℃，控制光照12h，黑暗12h，每三天更换一次垫料。适应性饲养一周后，灌胃混合抗生素(每只小鼠每天灌胃5mg氨苄青霉素，1mg硫酸庆大霉素，5mg阿奇霉素)3天进行排空，每周称重1次。
空白组先灌胃0.25mL NaHCO3(0.2mol/L)，1h后，再灌胃0.3mL BHI培养基，隔天灌胃，共5次；
模型组及各干预组：先灌胃0.25mL NaHCO3(0.2mol/L)，1h后，再灌胃0.3mL菌液浓度为1×109CFU/mL的幽门螺杆菌菌悬液，隔天灌胃，共5次。造成幽门螺杆菌感染模型后，空白组和模型组灌胃生理盐水，药物组灌胃三联药物，各干预组灌胃高低剂量的菌液(高剂量5×108CFU/kg，低剂量5×107CFU/kg)和上清液，灌胃量为0.2mL，灌胃6-8周。
干预实验结束后禁食24h，通过摘取眼球的方式收集小鼠血液，低温离心后收集血清并冻存于-80℃待测。采用无菌操作摘取鼠胃，切去胃底，沿胃切迹剪切，将胃分为胃体和胃窦。沿胃大弯将胃体和窦剖开，用无菌PBS洗去内容物后，检测组织中毒力因子CagA和VacA的表达和胃部菌群的16S rDNA。
实施例3毒力因子CagA和VacA的检测
毒力因子CagA和VacA的表达量：利用试剂盒提取胃组织RNA，并利用反转录试剂盒将RNA逆转录为cDNA，以cDNA为模板，进行PCR扩增，引物序列为：CagA上游引物ATAATGCTAAATTAGACAACTTGAGCGA，下游引物TTAGAATAATCAACAAACATCACGCCAT；VacA上游引物CTGGAGCCGGGAGGAAAG，下游引物GGCGCCATCATAAAGAGAAATTT。PCR反应体系共20μL：cDNA模板2μL，SYBR Green PCR Master Mix 10μL,上下游引物各0.5μL，无核酸水7μL；反应条件为：95℃5min，95℃15s，60℃1min，共35个循环。以GADPH为内参，采用2-△△Ct法计算目的基因的相对表达量，重复三次。
各组小鼠胃组织CagA mRNA表达水平结果如图1所示，各干预组均能显著降低幽门螺杆菌小鼠毒力因子CagA蛋白表达水平，且发酵乳杆菌LFE-55高剂量组的干预效果显著好于药物组、发酵乳杆菌LFE-55低剂量组及上清液组；药物组、发酵乳杆菌LFE-55低剂量组及上清液组CagA蛋白表达水平无显著差异。
各组小鼠胃组织VagA mRNA表达水平结果如图2所示，各干预组均能显著降低幽门螺杆菌小鼠毒力因VagA蛋白表达水平，且发酵乳杆菌LFE-55高剂量组的干预效果显著好于药物组、发酵乳杆菌LFE-55低剂量组及上清液组；发酵乳杆菌LFE-55低剂量与药物组之间无显著差异；发酵乳杆菌LFE-55上清液组VagA mRNA表达水平显著低于LFE-55低剂量组。以上结果表明，发酵乳杆菌LFE-55可抑制幽门螺杆菌毒力因子的表达，并成剂量依赖性，且发酵上清液也有抑制效果。
实施例4小鼠胃部菌群多样性的测定
根据
soil DNA kit(Omega Bio-tek,Norcross,GA,U.S.)说明书进行微生物群落总DNA抽提，使用1％的琼脂糖凝胶电泳检测DNA的提取质量，使用NanoDrop2000测定DNA浓度和纯度；使用338F(5’-ACTCCTACGGGAGGCAGCAG-3’)806R(5’-GGACTACHVGGGTWTCTAAT-3’)对16S rRNA基因V3-V4可变区进行PCR扩增，扩增程序如下：95℃预变性3min，27个循环(95℃变性30s，55℃退火30s,72℃延伸30s)，然后72℃稳定延伸10min，最后在4℃进行保存(PCR仪：ABI
9700型)。PCR反应体系为：5×TransStart FastPfu缓冲液4μL，2.5mM dNTPs 2μL，上游引物(5uM)0.8μL，下游引物(5uM)0.8μL,TransStart FastPfu DNA聚合酶0.4μL，模板DNA 10ng，ddH2O补足至20μL。每个样本3个重复。
将同一样本的PCR产物混合后使用2％琼脂糖凝胶回收PCR产物，利用AxyPrep DNAGel Extraction Kit进行回收产物纯化，2％琼脂糖凝胶电泳检测，并用QuantusTMFluorometer对回收产物进行检测定量。使用NEXTflexTM Rapid DNA-Seq Kit进行建库:(1)接头链接；(2)使用磁珠筛选去除接头自连片段；(3)利用PCR扩增进行文库模板的富集；(4)磁珠回收PCR产物得到最终的文库。利用Illumina公司的Miseq PE300/NovaSeqPE250平台进行测序(上海美吉生物医药科技有限公司)。
使用fastp软件对原始测序序列进行质控，使用FLASH软件进行拼接，使用UPARSE软件，根据97％的相似度对序列进行OTU聚类并剔除嵌合体。利用RDP classifier对每条序列进行物种分类注释，比对Silva 16S rRNA数据库(version 138)，设置比对阈值为70％。
结果如下：
各组小鼠胃组织幽门螺杆菌(Helicobacter)的相对丰度结果如图3所示，药物组、发酵乳杆菌LFE-55高剂量组、低剂量组及发酵上清液组小鼠胃窦组织幽门螺杆菌的相对丰度显著低于模型组，且干预组之间无显著差异；发酵乳杆菌LFE-55高剂量组；发酵乳杆菌LFE-55高剂量组小鼠幽门螺杆菌相对丰度少于高剂量组及上清液组，但并无显著差异。说明药物及发酵乳杆菌LFE-55的干预能够显著降低幽门螺杆菌感染小鼠胃组织幽门螺杆菌的定植量，且菌体及上清液均有显著效果。
通过组间差异性检验分析与幽门螺杆菌感染及乳酸菌干预相关菌群变化。各组小鼠胃组织拟杆菌属(Bacteroides)的相对丰度结果如图4所示，发酵乳杆菌LFE-55高剂量组与低剂量组能够显著降低幽门螺杆菌感染小鼠胃内拟杆菌属的相对丰度。各组小鼠胃组织梭菌属(Lachnoclostridium)的相对丰度结果如图5所示，发酵乳杆菌LFE-55高剂量组能够显著降低幽门螺杆菌感染小鼠胃内梭菌属的相对丰度。各组小鼠胃组织脱硫弧菌(Desulfovibrio)的相对丰度结果如图6所示，模型组脱硫弧菌相对丰度显著高于空白组；药物组、发酵乳杆菌LFE-55高剂量组与低剂量组脱硫弧菌相对丰度显著低于模型组。各组小鼠胃组织AKKermansia的相对丰度结果如图7所示，模型组AKKermansia的相对丰度显著低于空白组；发酵乳杆菌LFE-55高剂量组AKKermansia相对丰度显著高于模型组。各组小鼠胃组织乳酸菌(Lactobacillus)的相对丰度结果如图8所示，发酵乳杆菌LFE-55高剂量组乳酸菌相对丰度显著高于空白组、模型组、药物组及低剂量组。
实验结果表明，幽门螺杆菌感染小鼠后能够升高胃内有害菌幽门螺杆菌、拟杆菌属、脱硫弧菌的相对丰度，降低有益菌AKKermansia及乳酸菌的相对丰度；发酵乳杆菌LFE-5的能够通过降低幽门螺杆菌感染小鼠胃组织有害菌幽门螺杆菌、拟杆菌属、脱硫弧菌的相对丰度，增加有益菌AKKermansia及乳酸菌的相对丰度来抑制幽门螺杆菌的生长定植，并能够显著降低幽门螺杆菌毒力因子CagA及VagA的mRNA表达水平。
综上所述，发酵乳杆菌LFE-5能够通过改变胃部菌群的结构及幽门螺杆菌毒力因子CagA及VagA的表达来抑制幽门螺杆菌的持续感染。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种抑制幽门螺杆菌(Helicobacter pylori)的发酵乳杆菌(Lactobacillus fermentum)，其特征在于，所述发酵乳杆菌命名为发酵乳杆菌(Lactobacillus fermentum )LFE-55，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.23590，保藏日期为2021年10月13日。
2.一种权利要求1所述的发酵乳杆菌的培养方法，其特征在于，将发酵乳杆菌接种在MRS液体培养基中，在30~37℃下培养18~24 h。
3.权利要求1所述的发酵乳杆菌在制备抑制幽门螺杆菌的产品中的应用。
4.根据权利要求3所述的应用，其特征在于，所述产品中发酵乳杆菌的活菌数不低于1×106 CFU/mL。
5.根据权利要求3所述的应用，其特征在于，所述产品为药品或幽门螺杆菌抑制剂中的任意一种。
6.一种含有权利要求1所述发酵乳杆菌的组合物。
7.一种幽门螺杆菌抑制剂，其特征在于，所述幽门螺杆菌抑制剂中含有权利要求1所述的发酵乳杆菌。
8.一种含有权利要求1所述发酵乳杆菌的产品，其特征在于，所述产品为药品或幽门螺杆菌抑制剂中的任意一种。
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200530395A (en) * | 2004-03-12 | 2005-09-16 | Univ Nat Chunghsing | Lactobacillus strain with anti-H. pylori function and composition containing the same |
| WO2015077794A1 (en) * | 2013-11-25 | 2015-05-28 | Seres Health, Inc. | Synergistic bacterial compositions and methods of production and use thereof |
| WO2015095241A2 (en) * | 2013-12-16 | 2015-06-25 | Seres Health, Inc. | Bacterial compositions and methods of use thereof for treatment of immune system disorders |
| CN105358141A (zh) * | 2013-05-01 | 2016-02-24 | 尼奥酷里私人有限公司 | 治疗细菌感染的方法 |
| WO2016172658A2 (en) * | 2015-04-23 | 2016-10-27 | Kaleido Biosciences, Inc. | Microbiome regulators and related uses thereof |
| CN107667173A (zh) * | 2015-05-06 | 2018-02-06 | 斯尼普技术有限公司 | 改变微生物种群和改善微生物群 |
| WO2018165765A1 (en) * | 2017-03-16 | 2018-09-20 | Microsintesis Inc. | Propiotic molecules for reducing pathogen virulence |
| WO2018165764A1 (en) * | 2017-03-16 | 2018-09-20 | Microsintesis Inc. | Compositions and methods involving probiotic molecules |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100449597B1 (ko) * | 2000-12-08 | 2004-09-18 | (주) 피엘바이오 | 헬리코박터 필로리의 활성을 억제하는 유산균 |
| WO2002045727A1 (en) * | 2000-12-08 | 2002-06-13 | Plbio Co., Ltd. | Lactic acid bacteria inhibiting adhesion of helicobacter pylori to gastric mucosa |
| EP1512408A4 (en) * | 2002-06-13 | 2008-01-23 | Hououdou Co Ltd | ANTIBACTERIAL AGENT AND ANTIBACTERIAL COMPOSITION |
| JP2016509998A (ja) * | 2013-02-22 | 2016-04-04 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | 疾患を治療または予防する、あるいは寿命を延ばすのに有益な微生物の増殖促進性組成物及び方法 |
| US10413577B2 (en) * | 2013-02-22 | 2019-09-17 | The Regents Of The University Of California | Compositions and methods for promoting growth of beneficial microbes to treat or prevent disease or prolong life |
| EP3520799A1 (en) * | 2018-02-06 | 2019-08-07 | European Molecular Biology Laboratory | In-vitro model of the human gut microbiome and uses thereof in the analysis of the impact of xenobiotics |
-
2022
- 2022-05-11 CN CN202210508860.0A patent/CN114591879B/zh active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200530395A (en) * | 2004-03-12 | 2005-09-16 | Univ Nat Chunghsing | Lactobacillus strain with anti-H. pylori function and composition containing the same |
| CN105358141A (zh) * | 2013-05-01 | 2016-02-24 | 尼奥酷里私人有限公司 | 治疗细菌感染的方法 |
| WO2015077794A1 (en) * | 2013-11-25 | 2015-05-28 | Seres Health, Inc. | Synergistic bacterial compositions and methods of production and use thereof |
| WO2015095241A2 (en) * | 2013-12-16 | 2015-06-25 | Seres Health, Inc. | Bacterial compositions and methods of use thereof for treatment of immune system disorders |
| WO2016172658A2 (en) * | 2015-04-23 | 2016-10-27 | Kaleido Biosciences, Inc. | Microbiome regulators and related uses thereof |
| CN107667173A (zh) * | 2015-05-06 | 2018-02-06 | 斯尼普技术有限公司 | 改变微生物种群和改善微生物群 |
| WO2018165765A1 (en) * | 2017-03-16 | 2018-09-20 | Microsintesis Inc. | Propiotic molecules for reducing pathogen virulence |
| WO2018165764A1 (en) * | 2017-03-16 | 2018-09-20 | Microsintesis Inc. | Compositions and methods involving probiotic molecules |
| CN110621332A (zh) * | 2017-03-16 | 2019-12-27 | 微合成有限公司 | 用于降低病原体毒力的益生菌分子 |
Non-Patent Citations (3)
| Title |
|---|
| Characterization of Lactobacillus fermentum UCO-979C, a probiotic strain with a potent anti-Helicobacter pylori activity;Apolinaria García等;《Electronic Journal of Biotechnolog》;20161126;第25卷;75-83 * |
| 乳酸杆菌CL22菌株治疗Balb/c小鼠Hp感染性胃炎模型的有效性研究;王学红等;《中南大学学报(医学版)》;20070415;第32卷(第02期);341-346 * |
| 益生菌在实验动物胃感染幽门螺杆菌中的保护作用;王蕊;《国际消化病杂志》;20130425;第33卷(第02期);122-127 * |
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