CN105025907B - 动物双歧杆菌动物亚种菌株 - Google Patents
动物双歧杆菌动物亚种菌株 Download PDFInfo
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Abstract
本发明涉及一种新的动物双歧杆菌动物亚种菌株，其尤其能够改善肠神经系统的功能，以及包括所述新的菌株的组合物。
Description
技术领域
本发明涉及一种新的动物双歧杆菌动物亚种(Bifidobacterium animalisssp.animalis)菌株。此菌株尤其能够通过增加肠神经系统(ENS)的血管活性肠肽(VIP)水平来改善肠神经系统的功能，以例如促进肠道运动性或屏障完整性的功能。因此，此菌株可用于预防和/或减轻胃肠不适或胃肠疾病。
背景技术
肠神经系统(ENS)是整合的神经系统，其沿肠道形成2个主要的具有功能特异性作用的神经丛(Furness,J.Auton.Nerv.Syst.,81(1-3):87-96,2000)。位于纵肌和环状肌之间的肠肌丛包含主要参与控制胃肠(GI)运动性的神经元。位于环状肌和粘膜之间的黏膜下神经丛包含主要参与控制肠上皮屏障(IEB)功能的神经元。肠神经元通过释放作用于肠神经元支配的靶细胞的神经介质来控制GI功能。
对于消化系统，神经介质VIP诱导平滑肌松弛(低级食管括约肌、胃和胆囊)、刺激水分泌至胰液和胆汁中，并使得抑制胃酸分泌和自肠腔中的吸收。其在肠道中的作用是极大地刺激了水和电解质的分泌，以及扩张肠平滑肌、扩张周边血管、刺激胰碳酸氢盐的分泌并抑制由胃泌素刺激的胃酸分泌。这些影响共同作用以增强运动性。因此，增加VIP有益地改善了胃肠道肌肉的松弛。
肠上皮屏障由极化的特异化细胞的连续单层构成，特异化细胞通过控制屏障渗透性以及上皮细胞极性的紧密的连接相关蛋白如紧密连接蛋白-1(ZO-1)而聚在一起。体外和体内的研究表明，益生菌可以通过调节ZO-1来影响上皮抵抗力并降低渗透性(Eun等人,APMIS,119(1):49-56,2011；Miyauchi等人,J.Dairy Sci.,92(6):2400-8,2009)。由ENS合成的神经介质也参与了肠道渗透性调节(等人,Am.J.Physiol.Gastrointest.Liver Physiol.,275:G95-103,1998；Hayden等人,Am.J.Physiol.Regul.Integr.Comp.Physiol.,278:R1589-94,2000；Neunlist等人,Am.J.Physiol.Gastrointest.Liver Physiol.,285(5):G1028-36,2003)。例如，在由新生儿母体分离所诱导的结肠中的增强的乙酰胆碱的神经元合成导致了大鼠中上皮细胞旁渗透性的增加(Gareau等人,Am.J.Physiol.Gastrointest.Liver Physiol.,293:G198-203,2007)。相比之下，由粘膜下神经元所释放的血管活性肠肽(VIP)通过调节紧密连接蛋白ZO-1在肠上皮细胞中的表达增加了IEB的抵抗力(Neunlist等人,J.Intern.Med.,263(6):577-83,2008)进一步增强VIP在调节屏障功能中的作用的最近研究表明VIP增加了在小鼠的由鼠类柠檬酸杆菌(Citrobacter rodentium)诱导的结肠炎中的屏障抵抗力(Conlin等人,Am.J.Physiol.Gastrointest.Liver Physiol.,297(4):G735-50,2009)。
数项研究测量了一些益生菌对ENS神经介质(包括VIP)的作用。Kamm等人(Neurogastrointest.Motil.,16:53-60,2004)公开了布拉氏酵母菌(Saccharomycesboulardii)对减少钙结合蛋白-28k(CALB)的作用，而对猪空肠的其它神经元标记物(ChAT、VIP、SP、NF-160等)没有作用。Metugriachuk等人(Rejuvenation Res.,9:342-345,2006)公开了在年老的Wistar大鼠的小肠和大肠的运动性上的共生制备显著地增加了小肠和结肠的肌电活性，并导致VIP的mRNA表达增加，但对VIP浓度没有显著的作用。
已知细菌菌株可提供与健康相关的益处。这样的菌株通常被称为益生菌，并用于食品。需要新的可以引入到食品中且具有改良的与健康相关的益处和/或其它益处如感官性质和/或可加工性的菌株。
在国际申请WO 2011/148219中，发现包括乳酸菌的组合物在ENS中增加了VIP水平，从而提高了ENS功能。这些乳酸菌尤其是植物乳杆菌(Lactobacillus plantarum)CNCMI-4318、短双歧杆菌(Bifidobacterium breve)CNCM I-2219、动物双歧杆菌乳酸菌亚种(Bifidobacterium animalis ssp.lactis)CNCM I-2494和短双歧杆菌(Bifidobacteriumbreve)CNCM I-4321。
发明内容
在本文中，本发明人鉴定了一种新的细菌菌株，特别地，其尤其可以调节肠神经系统以促进肠道运动性和屏障完整性功能，并且特别地，其所增加的VIP水平多于WO 2011/148219中所公开的菌株。此菌株被归类为动物双歧杆菌动物亚种菌株，并根据布达佩斯条约在2012年3月6日保藏于作为国际保藏机构的CNCM(Collection Nationale de Culturesde Microorganismes，法国国家微生物保藏中心)，编号I-4602。
因此，本发明涉及动物双歧杆菌动物亚种CNCM I-4602菌株。
此菌株的特征如下：
-菌落形态(固体MRS+半胱氨酸，于37℃72h，厌氧条件)：小的、圆形的且白的、规则的且半透明的菌落；
-微观形态(液体MRS+半胱氨酸，于37℃16h，厌氧条件)：厚的、大的、两极的、葫芦形杆菌；
-下列底物的发酵(API 50CH条上，API MRS培养基中，于37℃48h)：L-阿拉伯糖、D-木糖、D-半乳糖、D-葡萄糖、D-麦芽糖、D-蜜二糖、D-蔗糖和D-棉子糖；
-生化鉴定(API 20A条上，API 20A培养基中于36℃24h)：无芽孢，革兰氏染色阳性，棒状，在存在D-葡萄糖、D-蔗糖、D-麦芽糖、水杨苷、D-木糖、L-阿拉伯糖和D-棉子糖、明胶和七叶苷水解作用的情况下酸化；
-所检测到的酶活性(API ZYM条上，于37℃4h)：酯酶(C4)、亮氨酸芳基酰胺酶、胱氨酸芳基酰胺酶、α-半乳糖苷酶、β-半乳糖苷酶和α-葡糖苷酶。
本发明还包括衍生自本发明的菌株的仍对VIP有活性的突变菌株或遗传转化菌株。这些突变体或遗传转化菌株可以是其中一种或多种亲本菌株的内源基因被突变的菌株，例如以修改其一些代谢性质(例如其发酵糖类的能力、其对酸度的抗性、其运送至胃肠道的生存力、其后酸化或其代谢物生产)。它们还可以是用一个或多个目标基因遗传转化亲本菌株所获得的菌株，例如以赋予所述菌株另外的生理特征，或以使其表达希望通过所述菌株施用的具有治疗或疫苗兴趣的蛋白。
本发明的菌株以及如上所定义的突变体可用作益生菌。其可用于预防和/或减轻胃肠不适或胃肠疾病。菌株增加了受试者中肠神经系统的血管活性肠肽(VIP)水平。其可以在施用后抑制食品和/或受试者中的病原体的生长。其可以例如通过增加肠上皮细胞的跨上皮电阻(TEER)来提供改善的肠道屏障性质。其可以借由发酵和/或消化提供目标短链脂肪酸的生产，例如增加的产量或改善的脂肪酸组成。其可以借由发酵和/或消化提供降低的气体产生。其可以对胃应激具有感兴趣的抵抗力。其可以例如为新生哺乳动物尤其是新生人类提供良好的胃肠运动行性质。其可以促进肠神经系统和肠道功能以及胃肠运动性的成熟，从而尤其是降低胃肠转运时间、增加胃肠肌肉收缩力、改善胃肠蠕动、预防延迟的胃排空、增加胃肠系统的协调收缩、降低胎粪排泄时间、降低出生后胃肠运动开始的时间并允许在新生哺乳动物例如新生人类出生后的早期肠道喂养。此菌株可以例如在存在维生素K的情况下在发酵乳制品中呈现出良好的生长和/或存活。
本发明还涉及包含一种或多种其它的(益生的或非益生的)细菌菌株的组合物，例如一种或多种选自乳杆菌(Lactobacillus)、乳球菌(Lactococcus)、链球菌(Streptococcus)和双歧杆菌(Bifidobacteria)属的细菌菌株。特别地，这类其它菌株可包括一种或多种嗜热链球菌(Streptococcus thermophilus)的菌株和/或一种或多种保加利亚乳杆菌(Lactobacillus bulgaricus)的菌株。
根据本发明的这一组合物可有利地包括选自短双歧杆菌CNCM I-4321、短双歧杆菌CNCM I-2219、动物双歧杆菌乳酸菌亚种CNCM I-2494、植物乳杆菌CNCM I-4318、两歧双歧杆菌CNCM I-4319和鼠李糖乳杆菌CNCM I-4316中的至少一种细菌菌株。两歧双歧杆菌CNCM I-4319和鼠李糖乳杆菌CNCM I-4316公开于国际申请WO 2011/148220。
本发明的组合物可以是任何适于给药的形式，尤其是口服给药。这包括例如固体、半固体、液体和粉剂。通常优选液体组合物以易于给药，例如作为饮料。
本发明的组合物的实例是包括食品的营养组合物。组合物可以是例如乳制品，优选发酵乳制品。任选地，可以存在其它的乳酸菌菌株。在一个优选的实施方式中，存在嗜热链球菌和保加利亚乳杆菌菌株。发酵产品可以是液体形式或由干燥发酵液所获得的干燥粉末形式。优选地，发酵产品是新鲜产品。新鲜产品(其没有经历剧烈的热处理步骤)具有细菌菌株以活的形式存在的优点。发酵产品可以尤其是乳制品，更优选的是发酵乳和/或发酵乳清。例如，营养组合物是凝固的、搅拌的或可饮用形式的酸奶或发酵乳。发酵产品也可以是干酪或发酵植物，如凝固的、搅拌的或可饮用形式的发酵大豆、谷物和/或水果。本发明的营养组合物可以是婴儿食品、婴儿配方奶粉或后续婴儿配方奶粉。
本发明的组合物还可以是营养产品或药物产品、营养补充剂或医疗食品。
本发明的营养组合物还包括食品补充剂和功能食品。“食品补充剂”是指由通常在食品中使用的化合物所制造的产品，但所述化合物通常为与养料(aliment)无关的片剂、粉剂、胶囊剂、饮剂或任何的其它形式，并且对人的健康具有有益作用。“功能性食品”是也对人的健康具有有益作用的滋养品。特别地，食品补充剂和功能性食品可以对疾病(例如对慢性疾病)具有生理作用(防护或治疗)。
根据本发明的组合物可例如在每g干重中包括至少105个菌落形成单位(cfu)，优选至少106cfu的本发明的菌株(动物双歧杆菌动物亚种CNCM I-4602)。
在本发明的组合物中，细菌菌株尤其是动物双歧杆菌动物亚种CNCM I-4602可以以完整细菌(可以是活的或死的)的形式使用。或者，其可以以细菌裂解物的形式或以细菌部分的形式使用。可以选择适合于这一用途的细菌部分，例如，通过测试其缓解对本申请所述的共培养模型的VIP水平的作用的性质。优选地，细菌细胞以活的、有活性的细胞的形式存在。
当细菌是活细菌的形式时，组合物典型地可以在每g干重的组合物中包括105至1013cfu，优选至少106cfu，更优选至少107cfu，还更优选至少108cfu，和最优选至少109cfu。在液体组合物的情况下，这通常对应于104至1012cfu/ml，优选至少105cfu/ml，更优选至少106cfu/ml，还更优选至少107cfu/ml和最优选至少109cfu/ml。
本发明的菌株或组合物特别适用于预防和/或减轻胃肠不适或胃肠疾病。其可以施用于患有肠道不适或肠道疾病的受试者。因此，有利地用于预防或解决肠道不适或肠道疾病。此类不适或疾病包括例如与气体相关的不适或疾病、胃气胀、腹胀、腹痛、便秘，以及在一些严重的方面，肠易激综合征(IBS)和炎性肠疾病(IBD)。
根据一个具体的方面，菌株CNCM I-4602或根据本发明的组合物用于增加受试者中肠神经系统的血管活性肠肽(VIP)水平。更具体地，这种VIP水平的增加用于改善肠道运动性、改善肠道蠕动和/或降低肠渗透性，尤其是在患有选自如下疾病的受试者中：肠道运动性和/或渗透性疾病、肠易激综合征(IBS)如IBS-C、IBS-D和IBS-PI、炎性肠疾病(IBD)、便秘和肠道感染，或者是在选自老人、婴幼儿和肥胖者的受试者(其肠道功能总体上得到了改善)中。IBS(或痉挛性结肠)是特征为慢性腹痛、不适、鼓胀和在缺少任何可检测的器官原因的情况下改变排便习惯的功能性肠疾病。腹泻或便秘可能占主导地位，因此IBS分别被分为IBS-D或IBS-C，或可能两者交替。IBS可以开始于感染后(此时被称为IBS-PI(指感染后))，应激性生活事件后或没有任何其它医学指标的成熟开始后。
因此，本发明的组合物可用于治疗和/或预防选自肠道运动性疾病、肠道渗透性疾病、IBS(包括IBS-D、IBS-C和IBS-PI)、IBD、便秘和肠道感染的疾病，尤其是在老人或肥胖者或婴幼儿中。
具体实施方式
可以在下列非限制性实施例中找到本发明的详细说明或优点。
实施例1：在ENS原代培养物和T84细胞的共培养模型中乳酸菌对神经元VIP生产的作用
研究了与107种益生菌菌株相互作用后原代肠神经元对VIP生产的不同响应，其中益生菌菌株包括乳酸菌，尤其是双歧杆菌。
细菌菌株和生长条件
乳酸杆菌生长于Man、Rogosa和Sharpe(MRS)培养基(Biokar Diagnostics,Beauvais，法国)中，于37℃需氧条件下培养24小时。
双歧杆菌培养于确定的培养基中，此培养基由补充有葡萄糖(Sigma，法国)、酵母提取物(Becton Dickinson，美国)和氯化血红素(Calbiochem，法国)的胰蛋白胨(BectonDickinson，美国)组成，并于37℃在厌氧条件下生长24小时。动物双歧杆菌乳酸菌亚种培养于补充有半胱氨酸(Sigma，法国)的MRS培养基(Biokar Diagnostics)中，并于37℃在厌氧条件下生长24小时。
获得ENS原代培养物和T84细胞的共培养模型
共培养模型，其改编自Moriez等人(Biochem.Biophys.Res.Commun.,382(3):577-82,2009)开发的模型，其具有与肠上皮细胞T84细胞培养的肠神经元细胞，用于细菌的筛选。
如前所述(Chevalier等人,J.Physiol.,586(7):1963-75,2008)，获得大鼠ENS的原代培养物。
T84细胞系培养于补充有10％热灭活的FBS和50IU/ml青霉素和50μg/ml链霉素的DMEM-F12(1：1，GIBCO)中。以2×105个细胞/插入物的密度将细胞接种于12孔过滤器(Corning,NY，美国)并培养，以获得汇合。建立共培养前24小时，T84细胞的培养基被原代ENS培养基替代。
上皮细胞达到汇合后1天，将过滤器转移至底部接种有肠神经细胞的12孔板中。上皮细胞和神经元细胞共培养于上皮细胞的培养基中。
将细菌培养物在PBS中洗涤，并再悬浮于Dulbecco’s Modified Eagle Medium(DMEM)：Nutrient Mixture F-12(Ham’s)(1:1)培养基中，其包含1％的N-2补充剂(LifeTechnologies,Cergy-Pontoise，法国)。细菌的每种菌株至少一式三份进行测试以进行方差分析。以40个细菌/上皮细胞的MOI将菌株添加到过滤器隔室中。对照不加入细菌。共孵育8小时后，移除含有上皮细胞和细菌的过滤器隔室并将原代ENS培养物在恒温箱(95％空气，5％CO2)中于37℃保持22小时。在对照孔中，进行VIP测量时，用40mM KCl刺激神经元细胞。
VIP测量
然后，从12孔板中收集ENS细胞并用于制备蛋白提取物，以定量血管活性肠肽(VIP)。为了确定细胞内VIP，用含有蛋白酶抑制剂混合物(Roche Diagnostics,Meylan，法国)的RIPA裂解缓冲液(Millipore,Billerica,MA，美国)提取细胞内蛋白。用购买的酶免疫分析(EIA)试剂盒(Peninsula laboratories,Bachem,SA)定量VIP。
ENS模型的统计方法
根据下式，用阴性对照(T84)将所有的菌株VIP标记值归一化：(VIP菌株-VIPT84)/VIPT84。
根据其所估计的归一化值将菌株排序，其中所估计的归一化值来自具有作为固定影响的菌株的混合模型。模型给出了与每种菌株所估计的归一化值相关的P值。如果P值小于5％(<0.05)，则菌株作用是显著的。
结果
结果示于下述表1中。
表1
KCl，用作阳性对照，平均增加15.7％的VIP水平。
9个菌株能显著地增加VIP水平，从0.18％至62.5％。表1给出了其中7个所得到的结果。在9个菌株中，动物双歧杆菌动物亚种CNCM I-4602示出了最高水平的VIP增加。其与不存在任何菌株的水平相比，具有62.5％的增加。
在另一方面，观察到15个菌株显著地(P<0.05)降低了VIP水平。表1中示出了这15个菌株中的10个的结果。
所测试的其它菌株对VIP没有显著(P>0.05)的作用，其包括例如长双歧杆菌NCC2705(CNCM I-2618)、鼠李糖乳杆菌GG(ATCC 53103)、婴儿双歧杆菌(Bifidobacteriuminfantis)UCC 3564、长双歧杆菌Bb536、动物双歧杆菌乳酸菌亚种Bi-07(ATCC SD 5220)、约氏乳杆菌La1(CNCM I-1225)、植物乳杆菌299v(DSM 9843)和罗伊氏乳杆菌(Lactobacillus reuteri)SD 2112(ATCC 55730)。
Claims (18)
1.一种动物双歧杆菌动物亚种(Bifidobacterium animalis subspecies animalis)菌株，其于2012年3月6日保藏于CNCM(Collection Nationale de Cultures deMicroorganismes，法国国家微生物保藏中心)，保藏号为I-4602。
2.一种组合物，其包含权利要求1所述的菌株和至少一种其它的选自短双歧杆菌(Bifidobacterium breve)CNCM I-4321、短双歧杆菌CNCM I-2219、动物双歧杆菌乳酸菌亚种(Bifidobacterium animalis ssp.lactis)CNCM I-2494、植物乳杆菌(Lactobacillusplantarum)CNCM I-4318、两歧双歧杆菌(Bifidobacterium bifidum)CNCM I-4319和鼠李糖乳杆菌(Lactobacillus rhamnosus)CNCM I-4316的菌株。
3.如权利要求2所述的组合物，其是乳制品。
4.如权利要求2所述的组合物，其是发酵乳制品。
5.如权利要求2至4中任一项所述的组合物，其每g干重包括至少105cfu的动物双歧杆菌动物亚种CNCM I-4602。
6.如权利要求2至4中任一项所述的组合物，其每g干重包括至少106cfu的动物双歧杆菌动物亚种CNCM I-4602。
7.如权利要求1所述的菌株在制备用于预防和/或减轻胃肠不适或胃肠疾病的食品产品中的用途。
8.如权利要求1所述的菌株在制备用于增加受试者的肠神经系统的血管活性肠肽水平的食品产品中的用途。
9.如权利要求1所述的菌株在制备用于改善胃肠运动性、改善肠道蠕动和/或降低肠道渗透性的食品产品中的用途。
10.如权利要求7所述的用途，其中所述菌株被施与患有选自肠道运动性疾病、肠道渗透性疾病、便秘和肠道感染的疾病的受试者。
11.如权利要求7所述的用途，其中所述菌株被施与患有肠易激综合征的受试者。
12.如权利要求7所述的用途，其中所述菌株被施与患有炎性肠疾病的受试者。
13.如权利要求2至6中任一项所述的组合物在制备用于预防和/或减轻胃肠不适或胃肠疾病的食品产品中的用途。
14.如权利要求2至6中任一项所述的组合物在制备用于增加受试者的肠神经系统的血管活性肠肽水平的食品产品中的用途。
15.如权利要求2至6中任一项所述的组合物在制备用于改善胃肠运动性、改善肠道蠕动和/或降低肠道渗透性的食品产品中的用途。
16.如权利要求13所述的用途，其中所述组合物被施与患有选自肠道运动性疾病、肠道渗透性疾病、便秘和肠道感染的疾病的受试者。
17.如权利要求13所述的用途，其中所述组合物被施与患有肠易激综合征的受试者。
18.如权利要求13所述的用途，其中所述组合物被施与患有炎性肠疾病的受试者。
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