CN101384700A - 新的乳杆菌属菌株及其用于对抗幽门螺杆菌的用途 - Google Patents
新的乳杆菌属菌株及其用于对抗幽门螺杆菌的用途 Download PDFInfo
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Abstract
本发明涉及新的分离的乳杆菌属细胞，其能够在人体消化道、特别是胃的培养条件下聚集幽门螺杆菌，以及涉及这种细胞的用途。
Description
技术领域
本发明涉及新的乳杆菌属菌株及其用途，特别用于药物和/或食疗组合物。
现有技术和发明背景
益生微生物包括存活或具有存活能力的细胞，其在其生命形态内在人体或动物体中表现出有益的作用。益生组合物包含这类微生物。有益作用特别是在于可以改善消化道微生物***。特别地，可以通过益生微生物和不期望的微生物之间的直接的相互作用，通过基于由益生微生物的表达产物来抑制不期望的微生物代谢的直接的相互作用，或者通过加强天然的免疫***来抑制微生物***中不期望的其他微生物。通常认为，主要的机制是，胃肠道的竞争性繁殖是一个重要的作用因素，由此，不期望的微生物也就不再能以干扰的程度繁殖于粘膜上或遭到排斥。
例如通过乳酸菌素可形成一组益生微生物。这里一般指的是格兰氏阳性的微需氧菌或厌氧菌，其会将糖发酵形成酸，特别是乳酸。
文献US-5716615中公开了一种药物组合物，其特别含有乳酸菌素(Lactobazillen)。其还特别用于治疗胃肠道疾病。
文献US2005/0186190A1公开了一种食疗或药物组合物，其包含鞘磷脂酶或含鞘磷脂酶的乳酸菌素。其适于治疗幽门螺杆菌(HeliobacterPylori)感染。
文献WO2004/087891公开了一种乳杆菌属菌株，其适于制备用以治疗胃肠道幽门螺杆菌感染的药物或食疗组合物。
文献WO2005/060937A1中公开了含有具存活能力的乳杆菌属细胞的片剂形式的制剂。该试剂适于口服给药和治疗胃肠道的病菌感染。
文献WO2004/031368A1公开了适于治疗与幽门螺杆菌感染有关的炎症的乳杆菌属菌株。
乳酸菌素与幽门螺杆菌的相互作用还公开于文献Wang等，Am.J.Clin.Nutr.80:737-41(2004)，Felley等，Best Practice & Research ClinicalGastroenterology 17(5):785-791(2003)，Cazzato等，Scandinavian Journal ofNutrition 48(1):26-31(2004)和Sgouras等，Applied and EnvironmentalMicrobiology 70(1):518-526(2004)中。
幽门螺杆菌是繁殖于胃部的螺旋形的细菌，并且通过产生脲酶而提高胃内的pH值，而所以所述细菌被保护以防止胃酸细菌渗透进粘膜并堆积于胃的上皮细胞上。这种感染会激活体内自身的免疫***，但是免疫反应又不足够有效以消除感染，结果便是加强的免疫反应。最后导致慢性炎症和胃炎或胃溃疡(Magengeschwüren)疾病。迄今还未知，幽门螺杆菌采用何种机理来抵抗免疫***。
对于已知的乳杆菌属菌株对抗幽门螺杆菌的作用机理，在前述那些文件中都已阐述了不同的理论。但是有关机理方面的认知还没有。
总之，颇为希望开发出能保持胃中幽门螺杆菌细胞数很低并没有生理副作用的乳杆菌属菌株。
发明内容
本发明技术问题
因此，本发明的技术问题在于提供能抑制幽门螺杆菌繁殖于胃粘膜上的乳杆菌属菌株。
另外，本发明的技术问题还在于提供食疗(
)和/或药物组合物，其特别在预防幽门螺杆菌感染方面具有很高作用。
本发明的基本特征和优选实施方式
为解决该技术问题，本发明教导了分离的、优选具有存活能力的乳杆菌属细胞，其能够在人体消化道、特别是胃的培养条件下聚集幽门螺杆菌。
本发明基于如下令人惊奇的认识，即经特定选择的乳杆菌属菌株能够结合游离的幽门螺杆菌并形成聚集体。这类较大的聚集体不再能渗透进粘膜并且幽门螺杆菌细菌也因此不再能够到达并感染胃的上皮细胞。最后完全不再能引发免疫***的慢性发炎反应并可靠地避免了胃炎或胃溃疡疾病。聚集体穿过胃肠道并以自然方式离开躯体。对于已经进行的感染本身，本发明的乳杆菌属菌株的这种作用机理也是非常有帮助的，因为这能够避免由另外的幽门螺杆菌细菌引起另一感染并因此通过杀死所存在的幽门螺杆菌细菌而更为简单地对抗当前的感染。通常，这甚至对于患者的天然免疫***来说是成功的。另外，本发明的乳杆菌属菌株或许也能够抑制幽门螺杆菌的脲酶活性，从而使得幽门螺杆菌细菌在聚集体中丧失它们对于胃酸侵蚀的防护能力。就此而言也就获得了协同效应。
人体胃管道的基本培养条件包括1.8至4.5范围内的pH值和存在胃蛋白酶以及NaCl。用以表征这些培养条件的参比介质由以下成分组成:水、5g/l NaCl以及3g/l胃蛋白酶，且其中用HCl将pH值调节到2.0。
术语聚集表示形成大小为至少1μm至1000μm和更大的细胞聚集体，其在例如根据以下实施例的悬浮液中，特别是在如上所述的参比介质中含有乳杆菌属细胞和幽门螺杆菌细胞。
在本发明范畴内，实验不同乳杆菌属菌株的聚集幽门螺杆菌的能力和鉴别出以下菌株并作为本发明的菌株保存在德国微生物和细胞培养物保藏中心GmbH(DSMZ)，Mascheroder Weg1b，D-38124Braunschweig，德国:DSM 17646，DSM 17647，DSM 17648，DSM 17649，DSM 17650，DSM 17651，DSM 17652和DSM 17653。其中DSM 17646，DSM 17649，DSM 17652和DSM 17653涉及的是短乳杆菌(Lactobacillusbrevis)菌株。其中DSM 17647，DSM 17648和DSM 17651涉及的是发酵乳杆菌(Lactobacillus fermentum)菌株。其中DSM 17650涉及的是戊糖乳杆菌(Lactobacillus pentosus)菌株。
本发明还涉及一种含有生理有效剂量的本发明的、优选具有存活能力的乳杆菌属细胞以及生理上可接受的载体的药物和/或食疗组合物。药物组合物涉及的是只是提供治疗或预防目的并且除了活性成分之外在盖伦制剂中只是添加常规助剂和/或载体物质的组合物。食疗组合物涉及的是除了活性成分之外还含有营养品和营养补充物的组合物。
本发明还涉及本发明的、优选具有存活能力的乳杆菌属细胞的用途，用于制备特别是用以预防和/或治疗由幽门螺杆菌感染引起的疾病，例如胃肠疾病的药物或食疗组合物。属于此类的特别是胃炎，胃溃疡和胃癌。
本发明的药物组合物的特征可以在于，其含有10^2至10^15、优选10^6或10^8至10^12，特别是10^8至10^10的乳杆菌属细胞。在此，起始数值是剂量单位，例如片剂。优选使该组合物被制备为用于口服给药的。合乎目的地使乳杆菌属细胞冷冻干燥。
本发明药物组合物的盖伦制剂的制备可以按照本领域常规的方式进行。合适的固体或液体的盖伦制剂形式是例如粒剂、粉剂、糖锭剂、片剂、(微)胶囊、栓剂、糖浆、汁液、悬浮液或乳液，在它们的制备过程中可以使用常规的助剂如载体物质、***剂、粘结剂、镀层剂、溶胀剂、润滑剂或滑动剂、增味剂、甜味剂和增溶剂。作为助剂提及的有碳酸镁，二氧化钛，乳糖，甘露醇和其他的糖，滑石，乳蛋白，明胶，淀粉，纤维素及其衍生物，动物和植物的油如鱼肝油、葵花油、花生油或芝麻油，聚乙二醇和溶剂如消毒水和一元或多元醇，如甘油。本发明的药物组合物可通过以下方法制备，即将至少一种本发明所用的乳杆菌属菌株的细胞以特定剂量与特定剂量的药物学上合适的和生理学上可接受的载体和任选的其他合适的活性成分、添加剂或助剂相混合并制成所期望的给药形式。作为载体特别可以考虑选自“麦芽糖糊精、微晶纤维素、淀粉特别是玉米淀粉、果糖(Levulose)、乳糖、葡萄糖和这些物质的混合物”的那些物质。组合物可以含有以细胞和载体总量为基准计的0.1至95重量％的载体和5至99.9重量％的冷冻干燥的乳杆菌属细胞或者由它们组成。
若是食疗组合物，则可以设计使组合物含有10^2至10^15、优选10^6至10^9，特别是10^7至10^9的乳杆菌属细胞。在此，起始数值是剂量单位，例如售卖于终端消费者的食品的包剂单位。生理上可接受的载体通常是食品，其特别选自“乳制品，发酵的乳制品，奶，酸奶，奶酪，谷物，混合麦片条(Müsliriegel)和儿童营养制剂”。
本发明还涉及一种制备本发明药物和/或食疗组合物的方法，其中将冷冻干燥或非冷冻干燥的、优选具有存活能力的乳杆菌属细胞与生理上可接受的载体相混合并制备成用于口服给药的。
最后，本发明涉及一种预防或治疗患由幽门螺杆菌感染引起的疾病，特别是胃炎或胃溃疡或者面临患这种疾病的人的方法，该方法中以每日一次至五次给人给药生理有效剂量的本发明的药物和/或食疗组合物。给药可以在一个随时间限制的时间段内进行，例如1至30周，或者不受时间限制地进行。特别地，后一情况适于持续的预防以及防治旧病复发。
以下根据仅是由实施方式描述的实施例来更详尽地阐述本发明。
具体实施方式
实施例1:所用菌株的存放
在冷冻状态下进行乳杆菌属菌株的存放。将1ml在MRS介质(55g/l，pH 6.5；Difco，USA)中培养到稳定期(OD600/ml 4-8)的培养物与500μl的50％浓度(v/v)消毒的甘油溶液混合并将混合物冷冻到-80℃。
以冷冻状态进行幽门螺杆菌的存放。将1ml在布鲁氏发酵液(28g/l，pH 7.0；BD，USA)中补充有5％(v/v)defillibriert的马血(Oxoid)培养至稳定期的培养物与500μl的50％浓度(v/v)消毒的甘油溶液混合并将混合物冷冻到-80℃。马血在使用前冷冻并在20℃下分解(aufgeschlossen)以破坏血细胞。
实施例2:通过本发明的乳杆菌属菌株聚集幽门螺杆菌
在封闭的Falcon小管中于MRS介质中在37℃下培养乳酸菌素24-48小时。
幽门螺杆菌在爱伦美氏瓶中在微需氧菌条件下和其余如实施例1中所述的条件下培养5至6天。
在培养之后，用显微镜观察细胞形态。用由具有∑形态的细胞和具有球形形态的细胞组成的培养物进行分析。也检测具有混合形态的培养物。
各细胞通过在3200g且10min条件下离心分离进行收集并舍弃上层物。将细胞在5ml缓冲液中洗涤一次并再悬浮于5ml缓冲液(PBS-缓冲液，含1.5g/l Na2HPO4*2H2O，0.2g/l KH2PO4和8.8g/l NaCl)中。用HCl调节pH值到7.0。测量OD600值并通过添加缓冲液调节到数值2。
混合各个如此所得的细胞悬浮液(幽门螺杆菌/乳杆菌属)2.5ml并涡旋混合物10min。用显微镜检测所得结果。通过分别试验培养物通过分别采用乳杆菌属和幽门螺杆菌而单独进行基于自身聚集的对比实验。
图1中可知，在具有乳杆菌属和幽门螺杆菌的混合物的悬浮液中已形成了大的聚集体，而这种聚集体在对比实验中不存在。对于所有的本发明菌株都能获得该结果，图1中只是示例性地描述了乳杆菌属菌株DSM 17648。
图1A所示为通过菌株DSM 17647形成的典型的幽门螺杆菌聚集体。图1B是单独的菌株DSM 17648。图1C所示只是幽门螺杆菌。放大倍率是1000倍。聚集体大小一般为1μm，通常为5μm至50μm，或如图1A所示甚至最高达1000μm和更大(最大延长值)。
原则上可以以这种聚集试验来如下测试可考虑的乳杆菌属菌株，即是否受试的菌株能诱导由乳杆菌属和幽门螺杆菌形成的这种聚集。
实施例3:模拟胃管道的条件
为模拟体内的行为，重复实施例2的实验，但是其中在模拟的胃液(5g/l NaCl和3g/l胃蛋白酶(Sigma))中进行乳杆菌属细胞的再分散。用HCl将pH调节到2。在37℃下培养进行30min。因为幽门螺杆菌可以在直接的细胞环绕下将pH值调节到pH4，所以如实施例2中所述收集细胞并接着将其再分散于pH为4的醋酸盐缓冲液。为调节缓冲液，用含0.2Mol/l的醋酸钠溶液将41ml含0.1Mol/l的醋酸的溶液调节到pH为4。用水将最终体积补充到100ml。
根据实施例2培养幽门螺杆菌细胞。
在根据实施例2收集细胞之后，与实施例2不同地，将细胞再分散于醋酸盐缓冲液(见上)中。由此根据实施例2实施聚集实验。结果示于图2中。图2A显示了在模拟的胃通道之后通过菌株DSM 17648所形成的幽门螺杆菌的典型聚集体。图2B显示了仅仅菌株DSM 17648经过模拟的胃通道之后的情况。图2C显示了仅仅幽门螺杆菌的情况。放大倍率是1000倍。人们可以看到，混合物中所得的聚集体尺寸在2μm至1000μm和更大的范围内。
聚集实验的这些变量也适于鉴定本发明的乳杆菌属菌株。
实施例4:乳杆菌属的冷冻干燥的效果
根据实施例1培育细菌。通过在3200g条件下离心10min收集1ml的乳杆菌属培养物的等份试样。舍弃上层物并在真空条件下使丸片冷冻干燥2小时。将如此得到的每个本发明乳杆菌属菌株的干燥丸片再悬浮于1ml的pH7.0的PBS缓冲液中。将经再悬浮的乳杆菌属细胞按照1:1的体积比与新鲜培养的幽门螺杆菌培养物混合并如实施例2和3中所述测定聚集过程。乳杆菌属细胞诱导幽门螺杆菌聚集的能力不会受到冷冻干燥的影响，就如根据前述实施例的试验所示的那样(但是没有进行拍照留证)。
实施例5:菌种的测定
本发明的乳杆菌属菌株的分类确定根据碳水化合物发酵模式进行。采用API 50 CH***(bioMerieux，法国)测定菌种并采用APILAB PLUS软件(相同生产商发布的3.3.3版(Release))进行分析。根据生产商说明进行测定。
实施例6:制备含有本发明乳杆菌属菌株的药物组合物
根据实施例4培育本发明乳杆菌属菌株或多种乳杆菌属菌株的细胞并将其冷冻干燥。接着将丸片研磨到粒度为最大约1mm的直径。所得粒料按照以下用量比(重量％)与载体或助剂混合:
20％ 粒料
2％ 二氧化硅(Syloid AL-1FP，GRACE Davidson)
1％ 硬脂酸镁(MF-2-V，Ackros)
77％ 微晶纤维素(Avicel PH112，FMC)
在Quintech微量混合器中在位置70等级II处进行混合。同时添加所有成分。进行混合约120s。接着将所得混合物在常规条件下、但可能在较低压力(<10kN)下置于商业通用的片剂压机内压制成重量为约500mg的片剂。每片片剂含有约10^8至10^10个乳杆菌属细胞。
实施例7:制备含有本发明乳杆菌属菌株的食疗组合物
根据实施例4培育本发明乳杆菌属菌株或多种乳杆菌属菌株的细胞并将其冷冻干燥。含有约10^7至10^8个乳杆菌属细胞的冷冻干燥物分别与约11的商业通用的巴氏杀菌的奶(Milch)混合并在5℃下短时均化。经均化的奶接着以常规方式装瓶并封装。
Claims (14)
1、分离的乳杆菌属细胞，其能够在人体消化道、特别是胃的培养条件下聚集幽门螺杆菌。
2、如权利要求1所述的分离的乳杆菌属细胞，并且保藏号为DSM17646、DSM 17647、DSM 17648、DSM 17649、DSM 17650、DSM 17651、DSM 17652或DSM 17653。
3、药物和/或食疗组合物，其含有生理有效剂量的如权利要求1或2所述的乳杆菌属细胞以及生理学上可接受的载体。
4、如权利要求1或2所述的乳杆菌属细胞用于制备药物或食疗组合物的用途。
5、如权利要求1或2所述的乳杆菌属细胞的用途，用于制备用以预防和/或治疗由幽门螺杆菌感染引起的疾病、特别是胃肠疾病如胃溃疡的药物或食疗组合物。
6、根据权利要求3至5之一所述的药物组合物或用途，其中所述组合物含有10＾2至10＾15、优选10＾6或10＾8至10＾12、特别是10＾8至10＾10个乳杆菌属细胞。
7、如权利要求3至6之一所述的药物组合物或用途，其中所述组合物被制备为用于口服给药。
8、如权利要求3至7之一所述的药物组合物或用途，其中将乳杆菌属细胞进行冷冻干燥。
9、如权利要求3至8之一所述的药物组合物或用途，其中所述载体选自“麦芽糖糊精、微晶纤维素、淀粉特别是玉米淀粉、果糖、乳糖、葡萄糖和这些物质的混合物”。
10、如权利要求3至9之一所述的药物组合物或用途，其中所述组合物含有0.1至95重量％的载体和5至99.9重量％的冷冻干燥的乳杆菌属细胞。
11、如权利要求3至5之一所述的食疗组合物或用途，其中所述组合物含有10＾2至10＾15、优选10＾6至10＾9、特别是10＾7至10＾9个乳杆菌属细胞。
12、如权利要求3至5或11之一所述的食疗组合物或用途，其中所述的生理学上可接受的载体是食品，特别选自“乳制品，发酵的乳制品，奶，酸奶，奶酪，谷物，混合麦片条和儿童营养制剂”。
13、制备如权利要求3至12所述的药物和/或食疗组合物的方法，其中所述的生理有效量的冷冻干燥的或非冷冻干燥的乳杆菌属细胞与生理学上可接受的载体相混合，并制备成口服给药的。
14、预防和/或治疗患由幽门螺杆菌感染引起的疾病或者面临患这种疾病的人的方法，其中以每日一次至五次给人给药生理有效剂量的如权利要求3至12所述的药物和/或食疗组合物。
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| PCT/DE2006/001842 WO2007073709A1 (de) | 2005-12-22 | 2006-10-12 | Neue lactobacillus stämme und deren verwendung gegen helicobacter pylori |
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| WO (1) | WO2007073709A1 (zh) |
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| CN103648511A (zh) * | 2011-06-08 | 2014-03-19 | 有机平衡有限公司 | 喷雾干燥的乳杆菌属菌株/细胞及其对抗幽门螺杆菌的用途 |
| CN108403725A (zh) * | 2018-05-22 | 2018-08-17 | 台州市劢康生物科技有限公司 | 用于治疗消化道溃疡的组合物及其应用 |
| CN111991429A (zh) * | 2020-08-19 | 2020-11-27 | 湖南天根乐微君科技有限公司 | 罗伊氏乳杆菌在制备抗口腔细菌感染的制品中的应用 |
| CN113164533A (zh) * | 2018-10-24 | 2021-07-23 | 诺维信公司 | 包含乳杆菌属的代谢健康用益生菌补充剂 |
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| WO2004087891A1 (en) * | 2003-04-02 | 2004-10-14 | Axcan Pharma S.A. | Lactobacillus fermentum strain and uses thereof |
| WO2005060937A1 (en) | 2003-12-23 | 2005-07-07 | Chr. Hansen A/S | Compressed tablets comprising viable probiotic microorganisms |
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2005
- 2005-12-22 DE DE102005062731A patent/DE102005062731A1/de not_active Ceased
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- 2006-10-12 CA CA2639961A patent/CA2639961C/en active Active
- 2006-10-12 CN CN2006800532967A patent/CN101384700B/zh active Active
- 2006-10-12 US US12/158,592 patent/US20120020942A1/en not_active Abandoned
- 2006-10-12 JP JP2008546085A patent/JP5392672B2/ja active Active
- 2006-10-12 WO PCT/DE2006/001842 patent/WO2007073709A1/de active Application Filing
- 2006-10-12 PL PL06805445T patent/PL1963483T3/pl unknown
- 2006-10-12 EP EP06805445.1A patent/EP1963483B1/de active Active
- 2006-10-12 ES ES06805445T patent/ES2425390T3/es active Active
- 2006-10-12 KR KR1020087017949A patent/KR101349452B1/ko active IP Right Grant
- 2006-12-22 US US11/645,363 patent/US7846711B2/en active Active
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI392498B (zh) * | 2010-07-19 | 2013-04-11 | Synbio Tech Inc | 戊糖乳桿菌ｌｐｓ１６及其在抑制幽門螺旋桿菌之用途 |
| CN103596452A (zh) * | 2010-09-30 | 2014-02-19 | 普利茅斯大学 | 以产乳酸菌发酵的食品 |
| CN103648511A (zh) * | 2011-06-08 | 2014-03-19 | 有机平衡有限公司 | 喷雾干燥的乳杆菌属菌株/细胞及其对抗幽门螺杆菌的用途 |
| CN103648511B (zh) * | 2011-06-08 | 2018-12-07 | 诺沃奇梅兹有限公司 | 喷雾干燥的乳杆菌属菌株/细胞及其对抗幽门螺杆菌的用途 |
| CN108403725A (zh) * | 2018-05-22 | 2018-08-17 | 台州市劢康生物科技有限公司 | 用于治疗消化道溃疡的组合物及其应用 |
| CN113164533A (zh) * | 2018-10-24 | 2021-07-23 | 诺维信公司 | 包含乳杆菌属的代谢健康用益生菌补充剂 |
| US11963986B2 (en) | 2018-10-24 | 2024-04-23 | Novozymes A/S | Probiotic supplement for metabolic health |
| CN111991429A (zh) * | 2020-08-19 | 2020-11-27 | 湖南天根乐微君科技有限公司 | 罗伊氏乳杆菌在制备抗口腔细菌感染的制品中的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101349452B1 (ko) | 2014-01-08 |
| EP1963483A1 (de) | 2008-09-03 |
| CN101384700B (zh) | 2012-12-05 |
| DE102005062731A1 (de) | 2007-06-28 |
| JP2009520470A (ja) | 2009-05-28 |
| PL1963483T3 (pl) | 2013-10-31 |
| US7846711B2 (en) | 2010-12-07 |
| EP1963483B1 (de) | 2013-05-22 |
| US20120020942A1 (en) | 2012-01-26 |
| US20070148149A1 (en) | 2007-06-28 |
| KR20080081186A (ko) | 2008-09-08 |
| WO2007073709A1 (de) | 2007-07-05 |
| ES2425390T3 (es) | 2013-10-15 |
| CA2639961C (en) | 2015-10-06 |
| JP5392672B2 (ja) | 2014-01-22 |
| CA2639961A1 (en) | 2007-07-05 |
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