CN1420781A - 能够预防腹泻的双歧杆菌 - Google Patents
能够预防腹泻的双歧杆菌 Download PDFInfo
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- CN1420781A CN1420781A CN00811244A CN00811244A CN1420781A CN 1420781 A CN1420781 A CN 1420781A CN 00811244 A CN00811244 A CN 00811244A CN 00811244 A CN00811244 A CN 00811244A CN 1420781 A CN1420781 A CN 1420781A
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Abstract
本发明涉及属于双歧杆菌的微生物的应用于制备一种载体,这种载体可用于治疗或预防腹泻。本发明也涉及含有这样的微生物的食物或药品的组合物。
Description
本发明涉及将非病原性微生物双歧杆菌类用于制备一种载体和加入食品或药物来治疗或预防轮状病毒引起的腹泻,以及涉及含有此微生物的食品或药物组合物。
很早以来人们就知道产生主要代谢组合物乳酸的生物体。在牛奶或牛奶处理工厂中,活着或腐烂的植物中,也可在人和动物的肠子中发现这些细菌。这些被概括为术语“乳酸细菌”的微生物,代表了一组相当不同类的细菌包括例如乳球菌属(Lactococcus)、乳杆菌属(Lactobacillus),链球菌(Streptococcus)、双歧菌属(Bifidobacterium)、片球菌属(Pediococcus)。
乳酸细菌作为低pH值条件下保存食物的发酵剂使用,发酵产物的作用抑制有害细菌的生长。另外,乳酸细菌也用于制作不同的乳制品例如奶酪、酸奶酪和其他发酵的奶制品。
近期,乳酸细菌的一些菌株显示出对人和动物的吸收颇有价值的特性,引起了人们的密切关注。特别是,特殊的乳酸菌或双歧菌的菌株被发现能够在肠内膜上定居并且有助于维持人和动物的健康。
在这方面,欧洲专利EP0768375公开了双歧菌的特异性菌株,这种菌株能够灌输进入肠内并且粘附在肠细胞上。报道说这些双歧细菌有助于免疫功能,能够竞争性抑制病原性细菌对肠内细胞的粘附,有助于维持生物个体的健康。
最近几年的科研集中在乳酸细菌作为益生菌试剂的潜在应用。益生菌被认为是能通过保持肠内自然微生物区系促进生物个体健康的可生长的微生物制剂。微生物制剂通常被认为是益生菌也就是这种有效的微生物并且人们已经知道了它们作用的模式。益生菌贴附在肠粘膜上,在肠道内建群并且防止有害微生物在其上粘附。对于它们在肠内发挥作用的至关重要的先决条件是它们必须以一种适当的能存活的方式粘附到内脏的粘膜上而且在胃肠上部不被消灭,特别是不受胃内低pH区域的影响。
在这方面,作为这样的一种益生菌,WO97/00078公开了一种特异性菌株,称为乳酸菌GG(ATCC53103)。在防止或治疗食物诱发的过敏反应的方法中特定的使用这种微生物,也就是将其和已经经过用胃蛋白酶和/或胰岛素以及进行水解处理的食品给药于受体。所选择的乳酸菌菌株显示了粘附和生长性能及蛋白酶体系,以至于含在将被给药的食物的蛋白质通过被这种特殊的乳酸菌分泌的蛋白酶进一步水解。这份文件所讨论的方法最终导致蛋白质被内脏消化,没有显示出存在任何引起过敏的物质。
更进一步,在EP 0 577 903参考资料中采用了这种乳酸菌,它具有取代幽门螺杆菌的能力,幽门螺杆菌公认是引起溃疡的原因,上述制备可以治疗和预防幽门螺杆菌引起的溃疡。
乳酸菌特殊菌株显示出有价值的特性,现有技术目的在于希望发现细菌菌株能够有益于人类和/或动物的健康的其它的特性。
因此,本发明要解决的问题是提供另外的乳酸菌,它对生物体的摄食机能产生有益的作用。
在导致本发明的研究过程中,双歧杆菌类微生物没有被确定的性质现在被令人惊奇的发现了。本发明提供了属于双歧菌且能够基本上防止轮状病毒感染肠内细胞的微生物在用于腹泻的治疗或护理的载体中的应用。
所用的双歧杆菌优选从青春双歧杆菌(Bifidobacterium adolescentis)的或长双歧杆菌(Bifidobacterium longum)中选择组成,优选青春双歧杆菌,更优选双歧杆菌CNCM I-2168。
微生物可以用于一种可吸收的载体制备,例如牛奶、酸奶酪、凝乳、发酵奶、奶基发酵产品、发酵谷类产品、奶粉(milk based powders)、婴儿食品或宠物食品,且以大约105cfu/g到1011cfu/g的量包含在载体中。本发明采用的缩写cfu的意思是“菌落形成单位”,定义为琼脂板上的微生物计数所表示的细菌细胞的数目。
本发明也提供一种食品或药物组合物,含有至少一种双歧杆菌菌株,能够基本上防止肠细胞被轮状病毒感染。
本发明制备的食物组合物至少使用一种双歧杆菌菌株并以大约105cfu/g到1011cfu/g的含量,和合适的载体混合在一起,优选含量为大约106cfu/g到1010cfu/g,更优选大约为107cfu/g到109cfu/g.。
药品可以以片剂、液体细菌悬浮液、口服补充剂(oral supplement)、湿口服补充剂、干管饲或湿管饲等形式制备,所用的双歧杆菌菌株的含量高达1012cfu/g,优选大约107cfu/g到1011cfu/g,更优选107cfu/g到1010cfu/g。
微生物可以进一步被配制于载体内,以获得所希望的释放模式如胶囊剂等等。依据本领域技术人员的目的,可以选择适当的赋形剂和/或添加剂。
微生物在生物体肠内的活性当然取决于剂量。也就是分别通过上述食品或药物组合物摄入的微生物越多,保护和/或治疗的作用越高。由于所用的微生物对于人类或动物无害,实际上是从自然环境中分离出来的,也就是从婴儿的粪中分离出来的,加入的量高,生物体肠内微生物建群的比例也就高。
在这些图中
图1显示用于评价细菌菌株对轮状病毒的保护特性的细胞培养物的图例说明。
在本发明的广泛研究时,发明人调查了从婴儿粪便中分离的不同细菌菌株或从美国组织及细胞保藏中心获得不同的细菌菌株(ATCC15704)。检验这些不同的菌株防止肠细胞被轮状病毒引起腹泻的能力。
由于具有抑制轮状病毒的性能,几种细菌包括双歧杆菌属、乳球菌属、链球菌属类被筛选。用三种轮状病毒的血清型进行抑制性能试验,这三种轮状病毒血清类型代表了人类病毒腹泻的主要病原因子(血清型G1、G3和G4)。
各种乳酸细菌在适当的培养基中生长,例如在它们适合的培养基MRS、Hugo-Jago或M17培养基中,最佳的生长温度30到40℃。在这些细菌稳定生长后采用离心分离和在生理食盐溶液中重新悬浮收集这些细菌。这些不同试验之间中的细菌细胞被冷冻储存(-20℃)。
通过融合细胞单层的感染制备各种轮状病毒原种。在感染之前培养这些轮状病毒。这些细胞用20个组织培养感染剂量感染。
为了评估抗轮状病毒的特性采用了两种不同的方案。一个方案直接检验和轮状病毒直接作用的这些不同细菌菌株,而同时另一个方案使这些菌株和细胞轮状病毒的受体交互作用并筛选这些细菌。
第一个方案涉及采用不同的轮状病毒菌株与各自的细菌悬浮液相接触并在在合适的培养基中培养。然后将这种病毒-细菌混合物施加在人类未分化的结肠腺瘤细胞HT-29的单层细胞上(肠内上皮细胞系)并连续培养。然后检验病毒复制。
第二个方案涉及首先将各自的细菌悬浮液和人类未分化的结肠腺瘤细胞HT-29一起培养然后再加入这种病毒。在连续培养后检验病毒复制。
轮状病毒的复制很容易采用感染细胞中的轮状病毒蛋白质的组织免疫着色法来检测。
当和轮状病毒加上指定的细菌一起培养的细胞培养基中的感染细胞与仅与轮状病毒一起培养的细胞相比时感染细胞的数目减少到90%时,显然轮状细胞的抑制作用被施加于给定的细菌。
总数超出260种的不同的细菌菌株中只有4种具有抑制轮状病毒复制的作用。这些不同细菌属于双歧杆菌属的亚种青春双歧杆菌或长双歧杆菌。属于青春双歧杆菌的已命名为Bad4,根据布达佩斯条约被保存并获得保存号CNCM I-2168。这种菌株证明能够极其有效的防止人类细胞被轮状病毒感染。本发明将采用举例的方法描述。
培养基和溶液
MRS(Difco),
Hugo-Jago(蛋白胨Difco 30g/l,酵母提取物Difco 10g/l,乳糖Difco 5g/l,KH2PO45g/l,牛肉汁Difco 2g/l,琼脂Difco 2g/l)
M17(Difco)
M199(Seromed)
Ringer溶液(Oxoid)
PBS(NaCl 8g/l,KCl 0.2g/l,Na2HPO4 1.15g/l,KH2PO4 0.2g/l))
蛋白胨磷酸盐肉汤(流体)
胰酶-乙二胺四乙酸溶液(黏液)
从美国费城儿童医院P.A.Offit获得人类轮状病毒Wa(G1血清型)和猿的轮状病毒SA-11(G3血清型)。从美国国立卫生研究所Bethesda,A.Kapikian获得DS-1xRRV重排列体(reassortant)病毒。从德国慕尼黑大学P.Bachmann获得血清型人类轮状病毒Hochi。
实施例1
从婴儿粪便中分离乳酸菌
从15到27天大的健康婴儿的尿布收集新鲜的粪便。1克新鲜粪便在无氧的条件下送到实验室在Ringer溶液系列稀释样品之后,在2小时内进行微生物分析,并涂在选择性培养基上。在37℃温度下在加入抗菌素的MRS琼脂(磷霉素80μg/ml,磺胺甲基异噁唑新明磺93pg/ml,甲氧苄胺嘧啶5ug/ml)培养48小时,用于分离乳酸细菌。随机提取并提纯菌体。进行分离株的生理学和遗传学特征的分析。在这项试验中也使用了获自ATCC(ATCC15704)的另一种菌株,对应于本发明优选的菌株Bad4。
实施例2
细胞中抗轮状病毒活性的菌株试验
由双歧杆菌属、乳球菌属和链球菌属组成的几种属的细菌被选出并检测其中显示出在细胞培养基抑制试验中具有抗轮状病毒活性的成员(见下述1st和2st方案)。乳球菌属采用一个种(乳酸乳球菌)由两个亚种组成(乳酸乳球菌乳亚种和乳酸乳球菌乳脂亚种)(Lc.lactis supsp.lactis and cremoris)。试验了总共30个菌株。链球菌属采用一个种(嗜热链球菌(S.thermophilus))的45个菌株。明串珠菌属(Leuconostoc)和丙酸菌属(Propionibacterium)由一个种(6个菌株)代表,而肠球菌和葡萄球菌属(Staphylococcus)每一种采用两个种类代表,总共17个菌株。
总共260个细菌菌株用于试验轮状病毒的活性。
1 st 方案:
30ul的各自细菌悬浮液(含平均3×106细菌)和70u.l M199培养基补充了10%的蛋白胨磷酸盐肉汤(流体)和5%胰酶-EDTA溶液(黏液)混合,再加入100μl补充M199培养基中的病毒。这种病毒-细菌混合物在4℃培养1小时,37℃培养1小时。作为融合单层细胞在96-well微量滴定板中(在M99培养基中用10%蛋白胨磷酸盐肉汤(流体)和5%胰岛素-EDTA溶液(黏液)用PBS冲稀1∶4补充)生长的人类未分化结肠瘤细胞HT-29用磷酸盐缓冲剂盐水(PBS,Ph7.2)洗三次。上述工艺混合的病毒和细菌混合物被转到这些细胞中并在微量滴定板中在CO2培养箱中培养18个小时。病毒的复制按照如下描述进行检验。
2 nd 方案:
30ul细菌悬浮液和加有10%的蛋白胨磷酸盐肉汤(流体)、5%胰酶-EDTA溶液(黏液)的70ul M199培养基混合,直接加到生长的HT-29细胞上并按照1st方案描述的方法在微量滴定板中进行预处理。在37℃下培养1小时后,将M199培养基中的100μl的病毒加入到微量滴定板中的细胞上。在CO2培养箱(Heraeus)中连续培养18个小时。复制病毒按照如下所述进行检验。
复制轮状病毒采用在后面将描述的感染细胞的轮状病毒蛋白质组织免疫着色进行检验。
在感染一天后,从微量滴定板去除细胞培养基并用无水乙醇固定细胞10分钟。去除乙醇并用PBS缓冲剂洗这些板三次。(主要直接抗VP6蛋白质)兔产生的50ul抗轮状病毒血清(从洛桑大学ISREC获得)在PBS中稀释1∶2000倍加入每个孔并用盖子盖上防止干燥,在37℃培养1小时。然后清除抗体血清并用PBS清洗三次。然后抗兔免疫球蛋白G(IgG)的山羊抗血清和过氧物酶(GAR-IgG-PO;Nordic)稀释1∶500倍的PBS加入到每个孔中,这些滴定板在37℃培养1小时。血清清除,滴定板再用PBS清洗三次。然后100pLI的下述混合物加入每一个孔中:0.05M的Tris盐酸(pH7.8),1ml H2O2(30%suprapur,用水冲稀1∶600倍,Merck)和200μl 3-氨基-9-乙基咔唑(0.1g/10ml的乙醇在-80℃储存在200μl aliquots;A-5754;Sigma)。这些滴定板在室温至少培养30分钟。去除培养基然后注入200ul的H2O来停止反应。用倒置显微镜(Diavert;Leitz)计数感染细胞灶。
只有非常少的细菌菌株和轮状病毒发生作用。260个细菌细胞的4种至少在第一个方案中抑制轮状病毒复制。青春双歧杆菌CNCM I-2168(Bad4)有极强的抗血清型1轮状病毒、血清型3轮状病毒SA-11和血清型4轮状病毒Hochi的活性。
根据在"Genera of lactic acid bacteria",Ed.B.J.B.Wood and W.H.Holzapfel,Blackie A & P.公开的方法,Bad4是革兰氏阳性和过氧化氢酶阴性菌,在发酵时不产生CO2并产生L(+)乳酸。
134表
申请人或代理人挡案号 | 国际申请号 |
关于微生物保藏的说明
(细则13之二)
A.对说明书第 4 页,第10 行所述的微生物的说明 |
B.保藏事项 |
保藏单位名称 国立巴斯德研究所微生物培养物保藏中心 |
保藏单位地址(包括邮政编码和国名)25,Rue du Docteur RouxF-75724 Paris Cedex 15 |
保藏日期 1999年3月15日 保藏编号 NCC 251-I-2168 |
C.补充说明(必要时) |
D.本说明是为下列指定国作的(如果说明不是为所有指定国而作的) |
E.补充说明(必要时) |
下列说明将随后向国际局提供(写出说明的类别,例如:“保藏的编号”) |
PCT/RO/134表(1992年7月)
Claims (9)
1.属于双歧杆菌属的能够防止轮状病毒感染肠细胞的乳酸细菌在制备一种可以治疗和预防腹泻的载体中的应用。
2.权利要求1的应用,其中双歧杆菌选自由长双歧杆菌或青春双歧杆菌组成的组。
3.权利要求1的应用,其中双歧杆菌是双歧杆菌CNCM I-2168。
4.上述任一权利要求的应用,其中双歧杆菌含在可吸收的载体中。
5.权利要求5的应用,其中双歧杆菌含在载体中的量从大约105到大约1012cfi/g载体。
6.按照权利要求4或5的应用,其中载体是选自奶、干酪、凝乳、酸奶酪、奶基发酵产品、冰激凌、发酵谷类产品、奶粉、婴儿食品或宠物食品的食物组合物。
7.食品或药物组合物,至少含有一种能够防止轮状病毒感染肠细胞的双歧杆菌菌株。
8.按照权利要求7的组合物,选自奶、干酪、凝乳、酸奶酪、奶基发酵产品、冰激凌、发酵谷类产品、奶粉、婴儿食品或宠物食品、片剂、液体细菌悬浮液、干口服补充剂、湿口服补充剂、干管饲或湿管饲。
9.按照权利要求7组合物为片剂、液体细菌悬浮液、干口服补充剂、湿口服补充剂,干管饲或湿管饲的形式。
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CN103082294A (zh) * | 2013-01-21 | 2013-05-08 | 广州知光生物科技有限公司 | 脆弱拟杆菌在制备治疗腹泻组合物中的应用 |
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ITBO20020564A1 (it) * | 2002-09-06 | 2004-03-07 | Alfa Wassermann Spa | Bifidobatteri e preparazioni che li contengono. |
KR100492455B1 (ko) * | 2003-09-26 | 2005-05-30 | 주식회사 비피도 | 로바타바이러스 억제활성을 갖는 신균주 비피도 박테리움 롱검 에이알81 및 이로부터 분리된 활성단백질 |
EP2522358B1 (en) | 2007-06-27 | 2016-11-09 | Laboratorios Ordesa, S.l. | Peptides against rotavirus infection |
EP2206506A1 (en) * | 2008-12-18 | 2010-07-14 | Bracco Imaging S.p.A | Probiotic formulations |
EP2455094A1 (en) | 2010-11-11 | 2012-05-23 | Nestec S.A. | Non-replicating probiotic micro-organisms protect children against gastrointestinal infections |
CN110839698A (zh) * | 2019-11-29 | 2020-02-28 | 内蒙古伊利实业集团股份有限公司 | 一种组合物及其食品用途 |
CN116121119B (zh) * | 2022-11-30 | 2024-01-26 | 天津小薇生物科技有限公司 | 一种治疗腹泻的长双歧杆菌bc012及其应用 |
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DK0768375T4 (da) * | 1992-07-06 | 2002-09-16 | Nestle Sa | Mælkesyrebakterier |
DE69223615T2 (de) | 1992-07-06 | 1998-04-09 | Nestle Sa | Lactobacillus Acidophilus enthaltende Antigastritis-Mittel |
AUPM823094A0 (en) * | 1994-09-16 | 1994-10-13 | Goodman Fielder Limited | Probiotic compositions |
FI104465B (fi) | 1995-06-14 | 2000-02-15 | Valio Oy | Proteiinihydrolysaatteja allergioiden hoitamiseksi tai estämiseksi, niiden valmistus ja käyttö |
US5902578A (en) | 1996-03-25 | 1999-05-11 | Abbott Laboratories | Method and formula for the prevention of diarrhea |
EP0904784A1 (en) | 1997-09-22 | 1999-03-31 | N.V. Nutricia | Probiotic nutritional preparation |
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- 2000-07-26 CA CA2378426A patent/CA2378426C/en not_active Expired - Fee Related
- 2000-07-26 AU AU66980/00A patent/AU6698000A/en not_active Abandoned
- 2000-07-26 EP EP00954568A patent/EP1353681B1/en not_active Expired - Lifetime
- 2000-07-26 DE DE60037343T patent/DE60037343T2/de not_active Expired - Lifetime
- 2000-07-26 WO PCT/EP2000/007207 patent/WO2001010453A2/en active IP Right Grant
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- 2000-07-26 DK DK00954568T patent/DK1353681T3/da active
- 2000-07-26 PT PT00954568T patent/PT1353681E/pt unknown
- 2000-07-26 ES ES00954568T patent/ES2296635T3/es not_active Expired - Lifetime
- 2000-07-26 TR TR2002/00300T patent/TR200200300T2/xx unknown
- 2000-07-26 BR BRPI0012973-9B1A patent/BR0012973B1/pt not_active IP Right Cessation
- 2000-07-26 AT AT00954568T patent/ATE380033T1/de active
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2003
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101884782A (zh) * | 2010-06-29 | 2010-11-17 | 南昌大学 | 长双歧杆菌粘附抑制蛋白Aip在防治腹泻中的应用 |
CN101884782B (zh) * | 2010-06-29 | 2015-09-16 | 南昌大学 | 长双歧杆菌粘附抑制蛋白Aip在防治腹泻中的应用 |
CN103082294A (zh) * | 2013-01-21 | 2013-05-08 | 广州知光生物科技有限公司 | 脆弱拟杆菌在制备治疗腹泻组合物中的应用 |
CN103798391A (zh) * | 2014-03-06 | 2014-05-21 | 吉林修正修元生物科技有限公司 | 一种治疗婴童乳糖不耐症、腹泻的食品及其制备方法 |
Also Published As
Publication number | Publication date |
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EP1353681A2 (en) | 2003-10-22 |
PT1353681E (pt) | 2008-02-25 |
WO2001010453A3 (en) | 2003-08-21 |
US6998119B1 (en) | 2006-02-14 |
DE60037343D1 (de) | 2008-01-17 |
ES2296635T3 (es) | 2008-05-01 |
TR200200300T2 (tr) | 2003-03-21 |
CA2378426C (en) | 2010-03-23 |
WO2001010453A2 (en) | 2001-02-15 |
HK1056315A1 (en) | 2004-02-13 |
CN1198631C (zh) | 2005-04-27 |
EP1353681B1 (en) | 2007-12-05 |
ATE380033T1 (de) | 2007-12-15 |
BR0012973A (pt) | 2002-04-30 |
AU6698000A (en) | 2001-03-05 |
DE60037343T2 (de) | 2008-11-27 |
DK1353681T3 (da) | 2008-03-10 |
BR0012973B1 (pt) | 2013-12-17 |
CA2378426A1 (en) | 2001-02-15 |
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