CN103562381B - 缺失d-乳酸产生并且具有改进的贮藏期限的约氏乳杆菌菌株cncm i-1225的衍生物 - Google Patents
缺失d-乳酸产生并且具有改进的贮藏期限的约氏乳杆菌菌株cncm i-1225的衍生物 Download PDFInfo
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- CN103562381B CN103562381B CN201280025795.0A CN201280025795A CN103562381B CN 103562381 B CN103562381 B CN 103562381B CN 201280025795 A CN201280025795 A CN 201280025795A CN 103562381 B CN103562381 B CN 103562381B
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Abstract
本发明一般地涉及益生菌领域。具体而言,本发明涉及具有改进特性的约氏乳杆菌(Lactobacillus?johnsonii)菌株CNCM?I-1225的天然衍生物。例如,本发明涉及缺失D-乳酸产生并且在产品储存期间表现出改进的存活力的约氏乳杆菌菌株CNCM?I-1225的天然衍生物。
Description
本发明一般地涉及益生菌领域。具体而言,本发明涉及具有改进特性的约氏乳杆菌菌株CNCMI-1225的天然衍生物。例如,本发明涉及缺失D-乳酸产生并且在氧暴露下表现出提高的存活力的约氏乳杆菌菌株CNCMI-1225的天然衍生物。
人类分离物约氏乳杆菌CNCMI-1225,也称为约氏乳杆菌NCC533或者嗜酸乳杆菌(Lactobacillusacidophilus)La1、或者约氏乳杆菌Lj1(Bernet-Camard,M.F.,等人,(1997)Appl.Environ.Microbiol.63,2747-2753)是目前以商标Lc1非常成功地商业化的益生菌。
通常对于含活的益生菌的产品而言,在生产此类产品时要确保细菌在贮藏期限期间仍存活是一个挑战,因为这些微生物对氧暴露是敏感的。因此,期望的是可以得到表现出改进的氧抗性的约氏乳杆菌CNCMI-1225的衍生物。
约氏乳杆菌CNCMI-1225具有几种明确记录的健康益处,其中例如,免疫调节活性(Haller,D.,等人,2000,Infect.Immun.68:752-759;Haller,D.,等人,2000,Gut47:79-87;或Ibnou-Zekri,N.,等人,2003,Infect.Immun.71:428-436)或病原体抑制(Bernet,M.F.,etal.,1994,Gut,35:483-489)以及长期的安全使用历史。
限制约氏乳杆菌CNCMI-1225在一些产品种类,例如在意图用于幼儿和婴儿的产品中的应用的一个方面是从糖的发酵中主要地产生了D-乳酸同分异构体。例如,如果在MRS培养基中培养,那么约氏乳杆菌CNCMI-1225将乳糖发酵成60:40%比率的D-和L-乳酸。
由于3岁以下婴儿有限的D-乳酸清除(这可能导致D-乳酸酸中毒),所以CODEXInfantFormulaDirective建议3岁以下婴儿避免消费D-乳酸和产生D-乳酸的细菌。
支持该结论的证据是有限的,并且主要是基于食物中D-乳酸的存在,并且并没有基于产生D-乳酸细菌(其是胃肠道的天然居民)的施用。
因此,一些出版物挑战该立场(Connolly,E.等人,NTRAfoods3(3),37-49,2004;Haschke-Becher,E.,等人,2008,Ann.Nutr.Metab.53:240-244,Mack,D.R.,2004,Can.J.Gastroenterol.18:671-675),但到目前为止,CODEXInfantFormulaDirective的建议仍没有改变。
CODEX基本上排除了将产生D-乳酸的益生菌作为婴儿制剂中的补充剂,但鼓励只产生L-乳酸的菌株的遗传改造。此类研发的实例是经遗传修饰的生物(GMO),特别是经遗传修饰的约氏乳杆菌菌株的产生,其中分离了d-乳酸脱氢酶(D-LDH)基因(ldhD),并通过基因替换,将ldhD基因的体外截短的克隆拷贝用于失活基因组拷贝(Lapierre,L.,等人,1999,Appl.Environ.Microbiol.65:4002-4007)。
这种经遗传改造的菌株仅产生用于实验室目的,并且从未用于食物产品中,因为使用重组DNA技术已经改变了其遗传物质,并因此认为该菌株是GMO。
然而,期望的是存在这样的可能,其允许使约氏乳杆菌菌株CNCMI-1225的大量健康益处也可用于个体,对于所述个体,目前并不建议消费D-乳酸或者产生D-乳酸的细菌。
因此,本领域存在对天然益生菌株,特别是约氏乳杆菌菌株CNCMI-1225的天然衍生物的强烈需求,所述约氏乳杆菌菌株CNCMI-1225的天然衍生物缺失D-乳酸产生,但尽管如此仍是存活的,并且其在产品中表现出提高的成活率。
本发明人满足了这些需求。
因此,本发明的目的是为本领域提供约氏乳杆菌菌株CNCMI-1225的衍生物,其还可以应用于意图用于婴儿和幼儿的产品,而且考虑了CODEXInfantFormulaDirective的当前规定,所述约氏乳杆菌菌株CNCMI-1225的衍生物表现出改进的贮藏稳定性,并且其是天然的,并不会被认为为GMO。
本发明人令人惊奇地发现,他们可以通过独立权利要求的主题实现本发明的目的。从属权利要求进一步发展了本发明的理念。
本发明人研究分离约氏乳杆菌La1的天然的(非GMO)、活的和遗传稳定的变体的可能性,所述约氏乳杆菌La1的变体只产生L-乳酸,并且其具有改进的贮藏稳定性。
基因组序列的改变以相对低的频率天然地发生,例如由于损伤DNA的错误修复或者DNA复制错误。
为了寻找约氏乳杆菌菌株CNCMI-1225的此类天然衍生物,本发明人筛选了大约21’000个约氏乳杆菌CNCMI-1225的菌落,并且已经鉴定了一株可以实现本发明的目的的天然D-乳酸缺失变体。
本发明人还测定了MRS培养上清液中的D-和L-乳酸浓度,并且显示与培养上清液中总的乳酸浓度比较,相对D-乳酸浓度低于1.5%;即约1.1%。
DNA序列分析鉴定了乳酸脱氢酶基因中主要的点突变,所述点突变改变酶的氨基酸序列,并因此改变其催化特性。
天然衍生物显示出是可存活的并且稳定的,并没有回复至D-乳酸产生的实例。
本发明人还评估了在8℃和在15℃的贮存稳定性,并且已经将鉴定的衍生物与约氏乳杆菌菌株CNCMI-1225比较。与约氏乳杆菌菌株CNCMI-1225相比,鉴定的衍生物表现出显著改进的贮藏期限。本发明人还将该菌株的贮藏稳定性与约氏乳杆菌菌株CNCMI-1225的其他衍生物进行了比较,并且发现该天然衍生物表现出最好的贮藏期限。
因此,本发明的一个实施方案是约氏乳杆菌菌株CNCMI-1225的天然衍生物,其中所述约氏乳杆菌菌株CNCMI-1225的衍生物缺失D-乳酸产生,并且与约氏乳杆菌菌株CNCMI-1225比较,表现出增加的贮藏期限。
据本发明人了解,这是第一次提供了与约氏乳杆菌菌株CNCMI-1225比较,缺失D-乳酸产生并且表现出增加的贮藏期限的约氏乳杆菌菌株CNCMI-1225的天然衍生物。
“缺失D-乳酸产生”意为本发明的目的,与总乳酸产生比较,菌株产生5%以下,优选地2%以下,甚至更优选地1.5%以下,和理想地约1.1%的D-乳酸。可以在无细胞培养上清液中测量D-和L-乳酸浓度。
由约氏乳杆菌菌株CNCMI-1225的天然衍生物产生的乳酸的总量最初与细胞生长相关。当细胞进入稳定期和停止分裂时,它们继续代谢糖并产生更多的乳酸。然而,发现产生的D-和L-乳酸的比率是恒定的。
约氏乳杆菌菌株CNCMI-1225的“天然”衍生物意为不被认为是GMO的菌株。例如可以通过筛选经历了天然发生的基因组序列改变(例如,由于损伤DNA的错误修复或者DNA复制错误)的菌落,来获得此种天然衍生物。通过使菌落经历胁迫条件,例如通过应用甲磺酸乙酯EMS可以增强错误的这种天然发生。
出于本申请的目的,术语“GMO”应当根据2001年3月12日对于故意释放经遗传修饰的生物到环境中的DIRECTIVE2001/18/ECOFTHEEUROPEANPARLIAMENTANDOFTHECOUNCIL并且废除CouncilDirective90/220/EEC进行定义。因此,“经遗传修饰的生物(GMO)”意为除人类之外的这样的生物,其中遗传物质已经以不是通过交配和/或自然重组天然发生的方式改变。
就该定义而言,遗传修饰至少通过以下技术的使用发生
(1)重组核酸技术,其涉及将在生物之外的通过任何手段产生的核酸分子插入到任何病毒、细菌质粒或其他载体系统中,并掺入到宿主生物,它们并非天然存在于所述宿主生物中但是它们能继续增殖,来形成的遗传物质的新组合;
(2)涉及将在生物之外制备的可遗传物质直接引入到生物中的技术,包括显微注射法、宏观注射法和微囊法;或
(3)细胞融合(包括原生质体融合)或杂交技术,其中使用并非天然存在的方法,通过融合两个或两个以上细胞形成具有可遗传的遗传物质的新组合的活细胞。
如果菌株具有至少99.95%,例如至少99.99%,优选地至少99.995%的核酸同一性,那么认为菌株是约氏乳杆菌菌株CNCMI-1225的“衍生物”。例如,如果与约氏乳杆菌CNCMI-1225的核酸序列比较,菌株具有500个以下,例如100个以下,优选地50个以下的核酸改变,那么认为该菌株是约氏乳杆菌菌株CNCMI-1225的衍生物。
如果细菌菌株在8℃和/或15℃储存45天后,在Lc1饮用产品中显示出增加的存活,那么认为该细菌菌株“与约氏乳杆菌菌株CNCMI-1225比较,显示出增加的贮藏期限”。因此,在产品中使用本发明约氏乳杆菌菌株CNCMI-1225的天然衍生物在贮藏期限的最后会导致增加的活细菌数量。
本发明人发现,本发明约氏乳杆菌菌株CNCMI-1225的天然衍生物在负责D-乳酸缺失表型的d-ldh基因中显示出突变。
因此,约氏乳杆菌菌株CNCMI-1225的天然衍生物具有改变的D-乳酸脱氢酶序列。例如,约氏乳杆菌菌株CNCMI-1225的天然衍生物可以在D-乳酸脱氢酶序列的氨基酸位置77处包含精氨酸至组氨酸的氨基酸改变。
这种改变明显显著改变了D-乳酸脱氢酶的功能,从而产生了极少的或者不会产生D-乳酸。
D-乳酸脱氢酶蛋白质序列的这种改变基于D-乳酸脱氢酶基因的核酸序列的改变。
失活所得的酶的D-乳酸脱氢酶基因的核酸序列的任何天然改变都可以实现本发明的主题。此外,野生型序列或者一部分或整个D-乳酸脱氢酶基因内一个或多个后续(subsequent)核苷酸的至少一个缺失都可以实现本发明的主题。
本发明人分析了本发明约氏乳杆菌菌株CNCMI-1225的天然衍生物中D-乳酸脱氢酶基因的核酸序列。
因此,本发明还涉及约氏乳杆菌菌株CNCMI-1225的天然衍生物,其中在约氏乳杆菌菌株CNCMI-1225的天然衍生物中D-乳酸脱氢酶基因的核酸序列包含在核酸位置270处G至A的转换。
尽管导致D-乳酸脱氢酶失活的此种改变在大自然中可以以低频率自发地发生,但其是随机的,并且可以以相同的频率修复回亲本序列(野生型)。
如果失活的基因赋予了变体生长缺陷,那么这种修复频率可以甚至更高。考虑到从培养物保藏到最终产品之间的大量传代,重要的是此种改变是稳定的,特别是因为其是单碱基对改变,其在遗传上比缺失更不稳定。
有利地,还发现该改变是稳定的。
实现本发明目的的约氏乳杆菌菌株CNCMI-1225的天然衍生物的一个实例是分离的、纯化的,并且详细表征的。
因此,本发明涉及约氏乳杆菌菌株CNCMI-1225的天然衍生物,其中约氏乳杆菌菌株CNCMI-1225的天然衍生物可以是约氏乳杆菌CNCMI-4437。
约氏乳杆菌CNCMI-4437根据布达佩斯条约于2011年2月8日保藏于国立微生物保藏中心(CollectionNationaledeCulturesdeMicroorganismes(CNCM)),InstitutPasteur,25RueduDocteurRoux,F-75724ParisCedex15,法国。
菌株是已经完全测序的。
约氏乳杆菌CNCMI-1225在布达佩斯条约下于1992年6月30日保藏于CNCM。
约氏乳杆菌CNCMI-4437含有总共23个改变,包括该菌株中负责缺失D-乳酸产生的D-乳酸脱氢酶基因的期望改变。在预测对生长起重要作用的两种基因中存在改变,即LJ0874-磷酸丙糖异构酶和LJ0515–磷酸甲基嘧啶激酶。在MRS培养基中,与La1比较,约氏乳杆菌CNCMI-4437以相似的速率生长,因此上述改变没有显示出不利影响。值得注意的是LJ1658–双组分调节子的组氨酸激酶中的第二个改变。该菌株的初步表达谱显示出构成可能对甘露糖特异的PTS操纵子的五种基因,LJ1652至LJ1656的显著下调。与La1比较,这不会导致糖发酵模式的任何改变,但这可能归因于La1中几种预测的甘露糖PTS系统的存在。
本发明还延伸至根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物,其中约氏乳杆菌菌株CNCMI-1225的天然衍生物以在生物学上纯的培养物存在。
根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物可以根据任何合适的方法培养,并可以通过例如冷冻干燥或者喷雾干燥制备用于添加到本发明的组合物。
益生菌菌株约氏乳杆菌CNCMI-1225提供了许多明确记录的健康益处,其中一些在上文中进行了详述。
“益生菌”意为对宿主的健康或保健具有有益作用的微生物细胞制备物或者微生物细胞的组分(SalminenS,等人“Probiotics:howshouldtheybedefined”TrendsFoodSci.Technol.1999:10107-10)。
就提供的健康益处而言,基本上可以把根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物看作生物等同物(bioequivalent)。
科学工作显示,分离自在生物学上纯的约氏乳杆菌菌株CNCMI-1225的培养物的无细胞培养上清液也具有几种健康益处(参见例如,Bernet-Camard,M.-F.,等人,1997,APPLIEDANDENVIRONMENTALMICROBIOLOGY,第2747–2753页)。
因此,本发明还延伸至分离自在生物学上纯的根据本发明约氏乳杆菌菌株CNCMI-1225的天然衍生物的培养物的无细胞培养上清液。
由于本发明约氏乳杆菌菌株CNCMI-1225的天然衍生物和无细胞培养上清液具有大量健康益处,因此可以将它们用于治疗或预防人类或动物体的疾病。
因此,本发明涉及包含根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或无细胞培养上清液的组合物,用于制备在通过疗法治疗人类或动物体的方法中的组合物。
本发明还涉及包含根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或无细胞培养上清液的组合物的用途,用于制备药物组合物或者药物。
已经详细研究了约氏乳杆菌CNCMI-1225的益生菌相关活性,包括免疫调节(Haller,D.,等人,2000,Infect.Immun.68,752-759;Haller,D.,等人,2000,Gut47,79-87;Ibnou-Zekri,N.,等人,2003,Infect.Immun.71,428-436)、病原体抑制(Bernet,M.F.,等人,1994,Gut35,483-489)和上皮细胞附着(Neeser,J.R.,等人,2000,Glycobiology10,1193-1199;Granato,D.,等人,1999,Appl.Environ.Microbiol.65,1071-1077)。
由于生物等同性(bioequivalence),根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或其无细胞培养上清液提供了相同的健康益处。
因此,本发明涉及包含根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或无细胞培养上清液的组合物,用于治疗或预防与减弱的免疫系统相关的病症。
本发明还涉及根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或无细胞培养上清液的用途,用于制备治疗或预防与减弱的免疫系统相关的病症的组合物。
与减弱的免疫系统相关的病症是本领域熟知的,并且本领域技术人员理解,哪些病症与减弱的免疫系统相关。
与减弱的免疫系统相关的病症的一般实例可以选自流感、鼻炎、普通感冒及其组合。
还可以将包含根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或无细胞培养上清液的组合物用于治疗或预防与由于肠毒性细菌或病毒的细胞附着和细胞侵入相关的病症。
本发明还延伸至根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物和/或无细胞培养上清液的用途,用于制备治疗或预防与由于肠毒性细菌或病毒的细胞附着和细胞侵入相关的疾病的组合物。
肠毒性细菌或病毒是本领域熟知的。欧洲食品安全局(EuropeanFoodSafetyAuthority)(EFSA)已经在2010年11月公布了病原体的列表。
例如,肠毒性细菌或病毒可以选自沙门氏菌属(Salmonella);弯曲杆菌属(Campylobacter);利斯特氏菌属(Listeria);大肠杆菌菌株,例如如ETEC、EHEC、EPEC或EIEC菌株;耶尔森菌属(Yersinia);志贺氏菌属(Shigella);产毒素细菌,如金黄色葡萄球菌(Staphylococcusaureus)、肉毒梭菌(Clostridiumbotulinum)或蜡状芽孢杆菌(Bacilluscereus);Vibriovulnifucus/parahaemolyticus(副溶血弧菌);轮状病毒(rotavirus);类诺瓦克病毒(norovirus);产vero细胞毒素大肠杆菌(verotoxigenicE.coli);阪崎肠杆菌(Enterobactersakazakii);产毒的产气荚膜梭菌(toxigenicC.perfringens)(A型和B型);食源性寄生虫,如棘球属(Echinococcus)、弓形体属(Toxoplasma)或贾第鞭毛虫属(Giardia);幽门螺杆菌(Helicobacterpylori);艰难梭菌(Clostridiumdifficile);破伤风梭菌(Clostridiumtetani)及其组合。
本领域技术人员明确,哪种病症与哪种肠毒性细菌或病毒物种相关。
例如,与由于肠毒性细菌或病毒物种的细胞附着和细胞侵入相关的病症可以选自下呼吸道感染、胃肠道感染、中耳炎及其组合。
本发明组合物可以是任何类型的组合物,只要其适合于施用给人类或动物。
特别地,本发明组合物可以口服地、肠内地、肠胃外地或者局部地施用。可以以通常对于选定的施用模式可用的任何盖伦形式提供组合物。
可以将本发明组合物施用给任何年龄组。
优选地,可以在冷季,例如从秋天至春天期间,施用本发明组合物。
还可以在任何时间消费本发明组合物。优选地,在早晨消费本发明组合物,例如以增强一天的免疫系统。
组合物可以例如选自食物组合物、宠物食物组合物、饮料、乳制品、营养配方、婴儿配方奶粉、食物添加剂、营养治疗品、药物组合物、食物成分和/或美容组合物。
例如,组合物可以选自酸化的乳产品,例如酸奶或酸奶饮料;或者基于乳的粉末。
如果以贮藏稳定粉末的形式提供组合物,那么特别优选的是粉末状产品。为了获得贮藏稳定性,并且确保益生菌的存活,可以以0.2以下,例如0.19-0.05,优选地0.15以下的水活度提供组合物。水活度或aw是系统中水的能量状态的测量。其定义为来源于粉末/产品的水的蒸汽压除以在相同温度下纯水的蒸汽压;因此,纯蒸馏水具有恰好为1的水活度。
本发明组合物可以是清洁剂、保护剂、治疗或护理乳膏、护肤洗剂、凝胶剂或泡沫剂,如清洁或消毒洗剂、洗浴组合物或除臭组合物。
更特别地,对于用于外部局部施用的组合物,它们可以是水溶液、水-醇溶液或油性溶液、洗剂或浆液类型的溶液或分散剂、通过分散水相包脂肪相(O/W)或反过来(W/O)获得的液体或半液体稠度的乳型乳剂或者软的、半固体或固体稠度的乳膏型混悬剂或乳剂、水凝胶或无水凝胶剂、微乳剂、微胶囊剂、微粒或者离子和/或非离子型的多孔分散剂。
根据本发明的局部组合物可以有利地以适合于护发的任何盖伦形式,特别地以洗发液、香波,特别地去屑香波、护发素、护发素、发乳或凝胶、stylinglacquer、hairsettinglotion、治疗洗剂、染色组合物(特别地氧化染色)的形式,任选地以染色香波、头发重构洗剂、永久波动组合物(apermanent-wavingcomposition)、抗脱发洗剂或凝胶剂、抗寄生虫香波或药用香波,特别低抗-皮脂溢香波、头皮护理产品(其特别地抗刺激、抗老化或者重建,或者其活化血液循环)的形式配制。
当本发明组合物是乳剂时,相对于组合物的总重量,脂肪相的比率按重量计可以为5%至80%,以及优选地按重量计10%至50%。在乳剂形式的组合物中使用的油、乳化剂和辅助乳化剂可以选自那些在化妆品和/或皮肤病领域中常规使用的那些油、乳化剂和辅助乳化剂。相对于组合物的总重量,在组合物中,乳化剂和辅助乳化剂按重量计可以以0.3%至30%,以及优选地按重量计0.5%至20%的比率存在。
当本发明的组合物是油性凝胶剂或溶液剂时,脂肪相可以占组合物的总重量的90%以上。
用于局部施用的盖伦形式还可以含有在化妆品、药物和/或皮肤病领域中常用的佐剂,例如亲水性或亲脂性胶凝剂、亲水性或亲脂性活性剂、防腐剂、抗氧化剂、溶剂、香味剂、填充剂、掩蔽物(screens)、吸味剂和染料。这些多种佐剂的量为那些常规用于所考虑领域的量,并且例如为组合物的总重量的0.01%至20%。取决于其性质,可以将这些佐剂引入到脂肪相中和/或水相中。
对于可以用于本发明的脂肪物质,提及的包括矿物油例如如氢化聚异丁烯和液体石油凝胶、植物油例如如牛油树脂的液体级分、向日葵油和杏仁油、动物油例如如全氢角鲨烷、合成油,特别地Purcellinoil、肉豆蔻酸异丙酯和棕榈酸乙基己酯、不饱和脂肪酸和氟代油例如如全氟聚醚。还可以使用脂肪醇、脂肪酸例如硬脂酸和例如如蜡,特别地石蜡、腊棕榈蜡和蜂蜡。还可以使用硅酮化合物例如硅酮油和例如环甲基硅酮和二甲硅油,以及硅酮蜡、树脂和树胶。
对于可以用于本发明的乳化剂,提及的例如包括硬脂酸甘油酯、聚山梨酯60、由Henkel公司以名称Sinnowax销售的包含33摩尔环氧乙烷的鲸蜡基硬脂醇/氧乙烯化鲸蜡基硬脂醇的混合物、由Gattefosse公司以名称63销售的PEG-6/PEG-32/甘油硬脂酸酯的混合物、PPG-3肉豆寇基醚、硅酮乳化剂例如cetyldimethiconecopolyol和山梨醇酐单硬脂酸酯或三硬脂酸酯、PEG-40硬脂酸酯或者氧乙烯化山梨聚糖单硬脂酸酯(20EO)。
对于可以用于本发明的溶剂,提及的包括低级醇,特别地乙醇和异丙醇,以及丙二醇。
本发明组合物还可以有利地含有泉水和/或矿质水,特别地选自Vittel水、来自Vichy盆地的水和laRochePosay水。
对于亲水性胶凝剂,提及的可以包括羧基聚合物如卡波姆、丙烯酸共聚物如丙烯酸酯/烷基丙烯酸酯共聚物、聚丙烯酰胺,以及特别地由SEPPIC公司以名称Sepigel销售的聚丙烯酰胺、C13-14异构烷烃和Laureth-7的混合物、多糖例如衍生物如羟基烷基纤维素,和特别地羟基丙基纤维素和羟基乙基纤维素、天然树胶如瓜尔豆胶、豆角胶、角豆胶和黄原胶,以及粘土。
对于亲脂性胶凝剂,提及的可以包括经修饰的粘土如有机皂土、脂肪酸的金属盐如硬脂酸铝和疏水性二氧化硅或者乙基纤维素和聚乙烯。
根据本发明的组合物还可以构成清洁皂或条的固体制剂。
还可以将它们以溶液剂、乳膏、凝胶剂、乳剂或慕斯形式或者备选地以还含有压力下的推进剂的气雾剂组合物形式用于头皮。
在用于口服施用的根据本发明的口服用途的情况下,优选的是使用可消化的支持体或载体。取决于所考虑的组合物的类型,可消化的支持体或载体可以具有不同的性质。
乳、酸奶、奶酪、发酵乳、基于乳的发酵产品、冰淇淋、基于谷物的产品或者基于发酵的谷物的产品、早餐谷物、谷物或格兰诺拉麦片棒、基于乳的粉末、幼儿和婴儿配方、糖食的食物产品、巧克力或谷物类型、特别地用于家畜的动物饲料、片剂、凝胶胶囊或锭剂、液体细菌混悬剂、干燥形式的口服补充剂或液体形式的口服补充剂都特别适合作为可消化的支持体或载体。
待口服施用的根据本发明的组合物可以例如以包衣的片剂、凝胶胶囊、凝胶剂、乳剂、片剂、胶囊剂、水凝胶、食物棒、压缩的或者松散的粉末、液体混悬剂或溶液剂、甜食产品、发酵乳、发酵乳酪、口香糖、牙膏或喷雾溶液或者食物载体的形式配制。
干燥形式的片剂或锭剂、口服补充剂,以及以液体形式的口服补充剂适合用作为饮食或药物支持体或者食物载体。
具体而言,可以通过产生糖-包衣片剂、凝胶胶囊、凝胶剂、乳剂、片剂、胶囊剂和允许控释的水凝胶的常用方法配制组合物,例如食物补充剂。
具体而言,可以将根据本发明的微生物掺入到所有形式的食物补充剂或强化食物,例如食物棒或者压缩的或非压缩的粉末中。可以将粉末稀释于水、苏打水、乳制品或大豆衍生物中,或者可以掺入到食物棒中。
此外,可以使用用于此类口服组合物或食物补充剂的常用的赋形剂和组分,具体而言即脂肪和/或含水组分、润湿剂、增稠剂、防腐剂、质地改进剂、风味增强剂和/或包衣剂、抗氧化剂、防腐剂和常规用于食品工业的染料,配制本发明的微生物、其部分和/或其代谢物。
用于口服组合物,以及具体而言用于食物补充剂的配制剂和赋形剂是本领域已知的,并不是本文中详细描述的主题。
在治疗性应用中,以足以至少部分治愈或者抑制疾病及其并发症的症状的量施用组合物。将足够实现该目的的量定义为“治疗有效剂量”。对于该目的有效的量将取决于本领域技术人员已知的许多因素,例如疾病的严重程度和患者的体重和一般状态。
在预防性应用中,以足以至少部分降低发展中的疾病的风险的量将根据本发明的组合物施用给对特定疾病敏感或者另外地处于特定疾病风险的患者。将该量定义为“预防有效剂量”。此外,精确的量取决于许多的患者特定因素,例如患者的健康状态和体重。
本发明组合物以治疗或预防有效剂量包含本发明的约氏乳杆菌菌株CNCMI-1225的至少一种天然衍生物和/或无细胞培养上清液。
本领域技术人员能相应地调整剂量。
例如,组合物可以以每天剂量106-1012cfu,例如108-1010cfu的量包含根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物。
以前的工作还显示,可以将非复制性约氏乳杆菌CNCMI-1225用于治疗或预防与免疫系统相关的病症包括感染,参见WO2010/133475,所述文献全部并入本文作为参考。发现约氏乳杆菌CNCMI-1225强烈地诱导了组成型hBD1表达,并且经热处理的约氏乳杆菌CNCMI-1225比其活的对应物更强烈上调hBD1。
因此,根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物还可以以非复制性形式存在。
约氏乳杆菌菌株CNCMI-1225的“非复制性”天然衍生物包括已经经热处理的衍生物。这包括灭活的、死的、不存活的和/或表现为片段如DNA、代谢物、胞质化合物和/或细胞壁物质的约氏乳杆菌菌株CNCMI-1225的天然衍生物。
“非复制性”意为通过经典的平板方法没有检测到活细胞和/或菌落形成单位。此类经典的平板方法总结于微生物教科书:JamesMonroeJay,MartinJ.Loessner,avidA.Golden。2005。Modernfoodmicrobiology。第7版,SpringerScience,NewYork,N.Y.第790页中。通常,不存在活细胞可以表现如下:在琼脂板上没有可见的菌落或者在用不同浓度的细菌制备物(‘非复制性’样品)接种,并在合适的条件(有氧和/或厌氧气氛至少24个小时)下温育后,在液体生长培养基中混浊度没有增加。
显而易见,非复制性微生物不会形成菌落,因此,应将该术语理解为获自106-1012cfu/g可复制细菌的非复制性微生物的量。
组合物还可以以每天剂量0,005mg–5000mg,例如0.5mg-50mg的量包含根据本发明的约氏乳杆菌菌株CNCMI-1225的天然衍生物。
本领域技术人员应当理解,在不背离所公开的本发明范围的情况下,他们可以自由地组合本文中所述的本发明的全部特征。具体而言,可以将用于本发明的用途所述的特征应用于本发明的食物,并且反之亦然。
本发明的其他优点和特征从以下实施例和附图中是显而易见的。
图1显示了用甲磺酸乙酯处理的约氏乳杆菌CNCMI-1225的‘存活曲线’。
图2显示了从G碱基的氧化到DNA链的分离的天然突变的分子过程,其产生了亲本和经修饰的DNA类型的混合物,以及混合的亲本和经修饰的菌落。
图3显示约氏乳杆菌CNCMI-1225D-乳酸脱氢酶基因的基因序列(SEQIDNO:1),并圈出了在约氏乳杆菌CNCMI-4437中的相应基因中鉴定的改变。还用约氏乳杆菌CNCMI-4437的相应改变显示了翻译的D-乳酸脱氢酶(SEQIDNO:2)。
图4A显示与亲本菌株约氏乳杆菌CNCMI-1225(G)比较,具有在位置230处加框和改变的碱基的约氏乳杆菌CNCMI-4437D-乳酸脱氢酶基因的基因序列(SEQIDNO:6)。
图4B显示与亲本菌株约氏乳杆菌CNCMI-1225(R)比较,具有在位置77处加框和改变的氨基酸的约氏乳杆菌CNCMI-4437D-乳酸脱氢酶基因的蛋白质序列(SEQIDNO:7)。
实施例1.甲磺酸乙酯处理约氏乳杆菌CNCMI-1225培养物。用Dulbecco’s磷酸盐缓冲盐水洗涤100μl含大约108个菌落形成单位的16个小时的约氏乳杆菌CNCMI-1225培养物的样品3次。最后,将细胞悬浮在1mlPBS中,并加入0或10μl甲磺酸乙酯并在37℃,在没有振荡的情况下温育。在PBS中洗涤经处理的细胞2次,测定经处理的和未处理培养物的CFU,并作为存活者作图,以产生图1中所述的‘存活曲线’。最初靶向了产生1%存活者的条件,并用之前和之后的时间点归类,稀释细胞,并在MRS平板上接种为单个菌落用于计数。然后,将剩余的经处理的细胞用于接种10mlMRS肉汤,并在37℃温育16小时。然后,稀释培养物,并铺展在MRS平板上,以产生用于筛选的单个菌落。
如图2中所示,当通过DNA复制解析时,在细菌中主要通过双链DNA中鸟苷(G)残基的氧化,天然地发生突变,并通常产生单碱基对改变。取决于所测序的DNA链,这导致G至腺苷(A)和胞嘧啶(C)至胸腺嘧啶(T)改变的天然突变谱。甲磺酸乙酯起着化学氧化G碱基的作用,并导致与天然突变相同的突变谱,即取决于所测序的DNA链,G至A和C至T。这随后通过D-乳酸脱氢酶基因和基因组序列的DNA测序进行了确证,其中大多数观察到的改变都是G至A或C至T。
实施例2.筛选缺失D-乳酸产生菌株的各个菌落。将各个经甲磺酸乙酯处理的菌落挑取到含200μlMRS肉汤的96孔板中,并在37℃温育24小时,以形成最小的培养物。使用Tecansunrise微孔板读取器,通过620nm处的吸光度估计培养物的生长。用190μl含100mMTrisHClpH9,2.5mMEDTApH8.0,20U/mlD-乳酸脱氢酶(来自Leuconostocmesenteroides),1mg/mlNAD(β-烟酰胺腺嘌呤二核苷酸)加上3%水合肼的反应混合物混合10μl的培养上清液,并在室温温育1小时。然后使用Tecansunrise微孔板读取器测量320nm(形成β-NADH)的吸光度,并将数据输出至excel用于分析。在每一平板上,将含仅MRS和5%、25%、50%或100%浓度的约氏乳杆菌CNCMI-1225培养上清液包括为用于归一化数据的标准品。
这是分离的菌落的表型分析,以确定培养后培养基中D-乳酸的存在或者不存在。当筛选各个菌落D-乳酸的存在时,还显而易见的是含有50%的D-乳酸产生者和50%的D-乳酸非产生者的‘混合’培养物将导致为D-乳酸存在‘阳性’的培养物。因此,认为我们靶向的D-乳酸缺失表型为D-乳酸产生表型‘隐性的’。当我们考虑图2中所示的诱变的示意图时这是重要的,因为单个氧化的G碱基会导致最终的菌落,其为亲本和改变的个体二者的混合物,并因此在表型上为D-乳酸阳性的。在这种情况下,对于分离缺失D-乳酸产生的菌株,纳入甲磺酸乙酯处理后在MRS肉汤中的生长是重要的。筛选了大约21000个各个经甲磺酸乙酯处理的菌落后,分离了15个缺失D-乳酸产生的菌落,并进行了进一步分析。
实施例3.测定培养基中D-乳酸水平。在37℃,在MRS肉汤中培养培养物16个小时,并通过离心去除细菌。为了测定D-乳酸浓度,将无细胞培养上清液稀释于水中,如上所述进行分析,并与用D-乳酸钠稀释液制备的标准曲线比较。通过用兔肌肉L-乳酸脱氢酶交换酶D-乳酸脱氢酶,并使用L-乳酸钠作为标准,以相同的方式测定L-乳酸浓度。对CNCMI-4437的这种分析的结果显示于表1中,并且包括对照:约氏乳杆菌CNCMI-1225、具有GMO失活的D-乳酸脱氢酶基因的约氏乳杆菌NCC9006,以及均被认为是L-乳酸产生菌株的副干酪乳杆菌(Lactobacillusparacasei)NCC2461和鼠李糖乳杆菌(Lactobacillusrhamnosus)NCC4007。
副干酪乳杆菌NCC2461(检索号:CNCMI-2116)于1999年1月12日根据布达佩斯条约保藏于CNCM(地址已经提及)。鼠李糖乳杆菌NCC4007(检索号:CGMCC1.3724)于2004年10月根据布达佩斯条约保藏于中国普通微生物菌种保藏管理中心(ChinaGeneralMicrobiologicalCultureCollectionCenter)(CGMCC),InstituteofMicrobiology,ChineseAcademyofSciences,北辰西路1号,朝阳区,北京100101,中国)。如在Lapierre等人,题为“D-LactateDehydrogenaseGene(ldhD)InactivationandResultingMetabolicEffectsintheLactobacillusjohnsoniiStrainsLa1andN312:(Appl.Environ.Microbiol.1999,65(9):4002)的文章中所述,约氏乳杆菌NCC9006是来源于约氏乳杆菌La1的菌株。
表1.从无细胞培养物中测定的CNCMI-1225、CNCMI-4437的D-和L-乳酸的值。将菌株NCC9006(具有D-乳酸脱氢酶基因的GMO失活的约氏乳杆菌菌株)、均被认为是L-乳酸产生菌株的副干酪乳杆菌NCC2461和鼠李糖乳杆菌NCC4007包括为对照。实验一式三份地进行,并表示为平均值加上括号内的标准差。
*,以g/l表示的乳酸值。
**,仅一个样品给出高于定量下限的值。
***,低于定量下限。
****,使用定量下限值计算的%。
结果显示,约氏乳杆菌CNCMI-1225的D-乳酸产生值为总乳酸的大约65%,并且约氏乳杆菌CNCMI-4437的D-乳酸产生值是极大降低的。约氏乳杆菌CNCMI-4437的D-乳酸产生水平非常低,并且低于总乳酸的1%。该结果与用含使用基因技术方法失活的D-乳酸脱氢酶基因的约氏乳杆菌NCC9006获得的结果相似。此外,值得注意的是,均被认为是L-乳酸产生菌株的副干酪乳杆菌NCC2461和鼠李糖乳杆菌NCC4007的对照菌株,它们在这些条件下分别产生了3和3.5%的D-乳酸。因为这些数据,可以认为菌株约氏乳杆菌CNCMI-4437是L-乳酸产生者,并在表型上与约氏乳杆菌CNCMI-1225不同。
实施例4.鉴定D-乳酸脱氢酶基因的改变。为了鉴定负责D-乳酸缺失表型的D-乳酸脱氢酶基因的改变,我们PCR扩增了用于DNA序列分析的区域。使用引物P1TCAGCACATAACCAGCAGCT(SEQIDNO:3)和P2GCAATAATACTGTCGCCGGT(SEQIDNO:4),从1μl的细菌培养物中扩增了该区域。纯化扩增子,并用引物P1、P3GTGTATAATAAAAGACGGTC(SEQIDNO:5)和P2测序,在DNASTAR程序套件中编辑并分析。结果显示于图3中。如图3中所示,在图3和图4A的碱基对270处,菌株约氏乳杆菌CNCMI-4437含有G至A的改变,并且这导致位于D-乳酸脱氢酶序列的保守标记结构域之外的位置77处精氨酸至组氨酸的氨基酸改变(R77H)(图4B)。基因测序数据显示,约氏乳杆菌CNCMI-4437中D-乳酸缺失产生表型伴随着D-乳酸脱氢酶基因和酶序列的相应改变。
实施例5.测定约氏乳杆菌CNCMI-4437的表型稳定性。考虑到从培养物保藏到最终产品之间的大量传代,重要的是D-乳酸缺失表型是稳定的,并且回复突变成产生D-乳酸非常罕见。为了研究这个,我们在MRS肉汤中培养了总共100代的约氏乳杆菌CNCMI-4437,并然后测试了300个单独菌落的D-乳酸产生。结果是测试的菌落都没有显示出比菌株约氏乳杆菌CNCMI-4437测定的D-乳酸水平高的D-乳酸水平。在中试规模产生喷雾干燥的粉末和Lc1饮料产品后,也进行了该分析,得到相同的结果。
在乳酸菌中,单拷贝的D-或L-乳酸脱氢酶对于乳酸的产生和NADH(该反应中的辅因子)至NAD的再生是必需的。在乳酸菌中,这是厌氧条件下再生NAD的唯一途径,并且对于生长是必需的。在D-乳酸缺失菌株的情况下,对于回复突变成D-乳酸产生,不存在选择压力,因为L-乳酸脱氢酶足以掩盖D-乳酸脱氢酶活性的缺失。
实施例6.Lc1饮料产品中约氏乳杆菌CNCMI-1225和约氏乳杆菌CNCMI-4437的存活。对于储存45天后,确保期望的107个菌落形成单位,产品储存期间益生菌的存活是重要的因素。活力丧失原因还未完全理解,但一些证据显示与氧气有关。为了测试在模拟的储存条件下,约氏乳杆菌CNCMI-4437是否和约氏乳杆菌CNCMI-1225存活的一样好,我们取用新鲜的Lc1饮料产品,并且通过加热至85℃10分钟,去除活细菌。然后,将约氏乳杆菌CNCMI-1225或约氏乳杆菌CNCMI-4437接种到无菌产品中,密封瓶子并在8℃或15℃,在标准测试条件下储存45天。来自三次独立试验的结果显示如下。令我们惊奇的是,约氏乳杆菌CNCMI-4437在两种温度和在全部三个试验中都比约氏乳杆菌CNCMI-1225显示出一贯更好的存活,具有0.42logs至2logs以上的改善。
试验1.
试验2.
试验3.
SEQID的列表:
Claims (27)
1.约氏乳杆菌CNCMI-4437。
2.权利要求1的约氏乳杆菌CNCMI-4437,其中所述约氏乳杆菌CNCMI-4437与约氏乳杆菌菌株CNCMI-1225相比是缺失D-乳酸产生的,并且表现出增加的贮藏期限。
3.权利要求1的约氏乳杆菌CNCMI-4437,其中所述约氏乳杆菌CNCMI-4437的D-乳酸脱氢酶序列在氨基酸位置77处包含精氨酸至组氨酸的氨基酸改变。
4.权利要求1的约氏乳杆菌CNCMI-4437,其中所述约氏乳杆菌CNCMI-4437中所述D-乳酸脱氢酶基因的核酸序列在核酸位置270处包含G至A的转换。
5.权利要求1-4中任一项的约氏乳杆菌CNCMI-4437,其中所述约氏乳杆菌CNCMI-4437作为在生物学上纯的培养物存在。
6.无细胞培养上清液,其分离自权利要求1-4的任一项中要求保护的所述约氏乳杆菌CNCMI-4437的在生物学上纯的培养物。
7.权利要求1-5的任一项中要求保护的约氏乳杆菌CNCMI-4437和/或权利要求6中要求保护的无细胞培养上清液在制备用于通过疗法治疗人类或动物体的组合物中的用途。
8.权利要求1-5的任一项中要求保护的所述约氏乳杆菌CNCMI-4437和/或权利要求6中要求保护的所述无细胞培养上清液在制备用于治疗或预防与减弱的免疫系统相关的病症的组合物中的用途。
9.如权利要求8中要求保护的用途,其中所述与减弱的免疫系统相关的病症选自流感、鼻炎、普通感冒及其组合。
10.权利要求1-5的任一项中要求保护的所述约氏乳杆菌CNCMI-4437和/或权利要求6中要求保护的所述无细胞培养上清液在制备用于治疗或预防与由于肠毒性细菌或病毒的细胞附着和细胞侵入相关的病症的组合物中的用途。
11.权利要求10中要求保护的用途,其中所述与由于肠毒性细菌或病毒物种的细胞附着和细胞侵入相关的病症选自下呼吸道感染、胃肠道感染、中耳炎及其组合。
12.权利要求10中要求保护的用途,其中所述肠毒性物种选自沙门氏菌属(Salmonella);弯曲杆菌属(Campylobacter);利斯特氏菌属(Listeria);大肠杆菌菌株;耶尔森菌属(Yersinia);志贺氏菌属(Shigella);产毒素细菌;Vibriovulnifucus/parahaemolyticus;轮状病毒(rotavirus);类诺瓦克病毒(norovirus);产vero细胞毒素大肠杆菌(verotoxigenicE.coli);阪崎肠杆菌(Enterobactersakazakii);产毒的产气荚膜梭菌(toxigenicC.perfringens);食源性寄生虫;幽门螺杆菌(Helicobacterpylori);艰难梭菌(Clostridiumdifficile);破伤风梭菌(Clostridiumtetani)及其组合。
13.权利要求12中要求保护的用途,其中所述大肠杆菌菌株选自ETEC、EHEC、EPEC或EIEC菌株。
14.权利要求12要求保护的用途,其中所述产毒素细菌选自金黄色葡萄球菌(Staphylococcusaureus)、肉毒梭菌(Clostridiumbotulinum)或蜡状芽孢杆菌(Bacilluscereus)。
15.权利要求12中要求保护的用途,其中产毒的产气荚膜梭菌(toxigenicC.perfringens)选自A型产毒的产气荚膜梭菌和B型产毒的产气荚膜梭菌。
16.权利要求12中要求保护的用途,其中所述食源性寄生虫选自棘球属(Echinococcus)、弓形体属(Toxoplasma)或贾第鞭毛虫属(Giardia)。
17.权利要求7-16的任一项中要求保护的用途,其中所述组合物选自食物组合物、营养制剂、营养治疗品、药物组合物和/或美容组合物。
18.权利要求17的用途,其中所述食物组合物为饮料。
19.权利要求17的用途,其中所述食物组合物为乳制品。
20.权利要求17的用途,其中所述食物组合物为宠物食物组合物。
21.权利要求7-16的任一项中要求保护的用途,其中所述组合物是食物成分。
22.权利要求7-16的任一项中要求保护的用途,其中所述组合物是食物添加剂。
23.权利要求7-22的任一项中要求保护的用途,其中所述组合物选自酸化的乳制品;或者基于乳的粉末。
24.权利要求23的用途,其中所述酸化的乳制品是酸奶。
25.权利要求23的用途,其中所述酸化的乳制品是酸奶饮料。
26.权利要求7-25的任一项中要求保护的用途,其中所述组合物包含权利要求1-5的任一项中要求保护的所述约氏乳杆菌CNCMI-4437,其中所述组合物中CNCMI-4437的量为每天剂量106-1012cfu。
27.权利要求7-25的任一项中要求保护的用途,其含有权利要求1-5的任一项中要求保护的所述约氏乳杆菌CNCMI-4437,其中所述组合物中CNCMI-4437的量为每天剂量约0.005mg–5000mg。
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