CN103230005B - 刺激肠内菌群的方法 - Google Patents

刺激肠内菌群的方法 Download PDF

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CN103230005B
CN103230005B CN201310148476.5A CN201310148476A CN103230005B CN 103230005 B CN103230005 B CN 103230005B CN 201310148476 A CN201310148476 A CN 201310148476A CN 103230005 B CN103230005 B CN 103230005B
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bifidobacterium
composition
baby
caesarean section
purposes
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CN103230005A (zh
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G·伯姆
B·斯塔尔
J·诺尔
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Nutricia NV
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Nutricia NV
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Abstract

本发明涉及喂养的方法及对经剖腹生产术分娩的婴儿施用的组合物及尤其是a）至少两种不同的微生物；或b）至少一种微生物和至少一种不消化的寡糖；或c）至少两种不同的双歧杆菌种、亚种或菌株在制备对经剖腹生产术分娩的婴儿经肠施用的组合物中的用途。因此可以刺激所述婴儿肠内菌群的健康发展。

Description

刺激肠内菌群的方法
本申请是申请日为2006年10月20日、发明名称为“刺激肠内菌群的方法”的中国专利申请200680035474.3(国际申请号PCT/EP2006/010159)的分案申请。
发明领域
本发明涉及喂养的方法及对经剖腹生产术分娩的婴儿施用的组合物。
发明背景
当为婴儿配制营养品时，使用母乳作为金标准。通常组合成分尤其是非人类来源的成分来模拟人母乳的组成特征和生理效应。
人乳的一个重要方面是它向婴儿提供能量和液体。除了提供能量，人母乳包含许多额外的组分，其旨在维持所述婴儿的健康。人母乳例如含有益生纤维，该益生纤维刺激健康的肠内菌群的发展。健康的肠内菌群对所述婴儿有许多积极影响，例如减少受感染的几率并增强免疫***。
几种市售婴儿奶粉配方含有旨在刺激肠内菌群发展的成分。婴儿配方例如可含有益生纤维或活益生生物。所述益生纤维通过上部胃肠道时通常未经消化并选择性刺激结肠内有益细菌的生长。活益生生物增加肠内特定细菌的数目。
EP1105002描述了含有一种或多种可溶性寡糖和一种或多种可溶性多糖的益生碳水化合物混合物，其中每种的至少80wt.%为益生的。
WO2005039319描述了为非母乳喂养或部分母乳喂养的婴儿制备的含有短双歧杆菌（Bifidobacteriumbreve）和不可消化的碳水化合物混合物的制剂及其在非母乳喂养或部分母乳喂养的婴儿中治疗或预防免疫紊乱的用途。
发明概述
通常设计婴儿配方来模拟接受人母乳的婴儿肠内菌群的发展，这暗示所有婴儿对人母乳和婴儿配方作出相似的反应。然而，本发明人已发现婴儿的一个亚群体，即那些经剖腹生产术分娩的婴儿，因为他们出生时肠内菌群完全不同于经***出生的婴儿的肠内菌群，所以将会作出不同反应。尤其是，经剖腹生产术分娩的婴儿的双歧杆菌种的分布型和含量不同于经***途径分娩的婴儿的双歧杆菌种的肠内分布型和含量。
本发明人已分析了剖腹产分娩后的新生儿和***分娩后的新生儿的肠内菌群。惊奇地发现两组新生儿在所述肠内菌群组成上存在巨大差异。尤其是经***途径出生的婴儿含有至少3种不同的双歧杆菌种，而经剖腹生产术出生的婴儿缺少最重要的双歧杆菌种。
也发现经剖腹生产术出生的婴儿尤其缺少短双歧杆菌、婴儿双歧杆菌（Bifidobacteriuminfantis）、两歧双歧杆菌（Bifidobacteriumbifidum）、链状双歧杆菌（Bifidobacteriumcatenulatum）、青春双歧杆菌（Bifidobacteriumadolescentis）和长双歧杆菌（Bifidobacteriumlongum）。这些种存在于经***途径出生的绝大多数婴儿的菌群中。
因为所述肠内菌群在婴儿发育过程中，尤其是在刺激免疫***和抵御感染中起至关重要的作用，所以刺激经剖腹生产术出生的婴儿肠内菌群的健康发展是最重要的。
此外，经剖腹生产术分娩的新生儿肠内菌群缺少生物多样性。在经剖腹生产术出生的婴儿中只检测到一种或两种不同种的双歧杆菌，而经***途径分娩的新生儿其肠内菌群通常含有若干不同种的双歧杆菌。本发明人相信这些观察结果表明经剖腹生产术分娩的婴儿在肠道中普遍缺少物种多样性。所述生物多样性对实现预期的生理效应是非常重要的，并最适宜于刺激所述婴儿的健康。
生产专为婴儿设计的益生产品Life并适用于经剖腹生产术分娩的婴儿。Life由婴儿双歧杆菌制成。因为所述Life产品含有仅一种双歧杆菌种，所以它对婴儿的益处将非常有限。
尤其预料不到的还有即使当母乳喂养婴儿时也会改善婴儿的健康的观察结果。在经剖腹生产术分娩婴儿的情况下，母乳（绝大多数情况下）是对该婴儿最好的营养品。然而，所述母乳同样不能同时导致如在经***分娩出生的婴儿中获得的相似的菌群。因此当婴儿接受人母乳时也可有利地使用本方法。
因此，本发明尤其旨在：a）增加经剖腹生产术出生的婴儿肠内菌群的特定物种的发生；b）增加肠内菌群的微生物的生物多样性；和/或c）刺激有益微生物，尤其是双歧杆菌的生长。
因此，一方面本发明提供通过施用组合物刺激经剖腹生产术出生的婴儿肠内菌群健康发展的方法，其中所施用的组合物含有：
－至少两种不同的微生物；或
－至少一种微生物和至少一种不消化的寡糖；或
－至少两种不同的双歧杆菌种、亚种或菌株。
所述微生物将增加婴儿菌群的生物多样性，而所述不消化的寡糖刺激固有的和所施用的微生物的发展和生长。
本方法中所施用的组合物优选包含多种不同的细菌种类，优选多种双歧杆菌种。这导致对剖腹生产术分娩的婴儿肠内菌群的最佳刺激。
优选实施方案详述
本发明提供喂养婴儿的方法，所述方法包含对经剖腹生产术分娩的婴儿经肠施用组合物，所述组合物包含：a）至少两种不同的微生物；或b）至少一种微生物和至少一种不消化的寡糖；或c）至少两种不同的双歧杆菌种、亚种或菌株。
另一方面，本发明提供刺激经剖腹生产术分娩的婴儿的健康的方法，该方法包含对婴儿施用并在其出生后100小时内施用含有微生物和/或不消化的寡糖的组合物。
另一方面，本发明提供了制备适合于经剖腹生产术出生的婴儿的婴儿营养品的方法，该方法包含混合：A）人母乳；和B）含有：ⅰ）至少两种不同的微生物；或ⅱ）至少一种微生物和至少一种不消化的寡糖；或ⅲ）至少两种不同的双歧杆菌种、亚种或菌株的组合物。
另一方面，本发明提供组合物，该组合物包含至少四种双歧杆菌种、亚种和/或菌株，其选自短双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌、链状双歧杆菌、青春双歧杆菌、嗜热双歧杆菌（Bifidobacteriumthermophilum）、高卢双歧杆菌（Bifidobacteriumgallicum）、动物双歧杆菌（Bifidobacteriumanimalis）、角双歧杆菌（Bifidobacteriumangulatum）、假小链双歧杆菌（Bifidobacteriumpseudocatenulatum）、嗜热嗜酸双歧杆菌（Bifidobacteriumthermacidophilum）和长双歧杆菌。
本发明也提供营养组合物，其包含5到25en％的蛋白质；25到60en％的脂肪；30到70en％的碳水化合物；至少两种不同种的双歧杆菌和至少一种乳杆菌。
本发明也提供液体体积为0.5到50ml的容器，该容器含有对婴儿施用的组合物，该组合物包含至少两种不同的微生物；或至少一种微生物和至少一种不消化的寡糖；或至少两种不同的双歧杆菌种、亚种或菌株。
另一方面，本发明提供刺激婴儿健康的肠内菌群发展的方法，该方法包括步骤：A）混合I）营养或药学上可接受的液体；和II）干燥组合物，其中所述干燥组合物II包含至少两种不同的微生物；或至少一种微生物和至少一种不消化的寡糖；或至少两种不同的双歧杆菌种、亚种或菌株；及B）对经剖腹生产术出生的婴儿施用步骤a）中获得的组合物。
本发明中使用的组合物优选为营养和/或药物组合物并适合对婴儿施用。
剖腹生产术
本发明涉及对经剖腹生产术分娩的婴儿经肠施用含有微生物的组合物。剖腹生产术是手术操作，其中婴儿通过母亲腹壁上的切口，然后通过子宫壁分娩。当剖腹生产术比***分娩对母亲或婴儿更安全时通常实施剖腹生产术。其他时间，女人也可选择剖腹生产术而不是***分娩她的婴儿。
多样性
本组合物含有至少两种不同的微生物；或至少一种微生物和至少一种不消化的寡糖；或至少两种不同的双歧杆菌种、亚种或菌株。所述微生物优选为细菌和/或酵母。本发明中使用的微生物优选为益生的，即当将其应用到人或动物时，它可通过改善土著菌群的性质来对宿主产生有益影响。上述组合通常旨在增加经剖腹生产术分娩的婴儿肠内的微生物的多样性和/或数量。这对所述婴儿具有有益影响，证明具有大量健康益处。
本发明中使用的术语“不同的微生物”指属于不同属和/或种的微生物。所述不同的微生物优选为不同的细菌。优选地，所述不同的微生物为不同种。例如并优选地，本组合物包含或由短双歧杆菌和链状双歧杆菌组成。此处将短双歧杆菌和链状双歧杆菌视为两种不同的细菌种类。如果细菌为不同的亚种，则不认为它们是不同的。例如，不认为德氏乳杆菌德氏亚种和德氏乳杆菌保加利亚亚种是两种不同的物种。本组合物含有至少两种不同的微生物，优选至少两种不同的细菌种。本组合物优选包含至少三种不同的种，更优选至少四种不同的种。优选地，所用的微生物或细菌种为益生的。
在一个实施方案中，本组合物含有至少两种不同的双歧杆菌种、亚种或菌株。本组合物优选包含至少一种，更优选至少两种，甚至更优选至少三种，最优选至少四种不同的双歧杆菌菌株。本组合物优选包含至少一种，更优选至少两种，甚至更优选至少三种，最优选至少四种不同的双歧杆菌亚种。例如动物双歧杆菌动物亚种和动物双歧杆菌亚乳糖亚种为不同的亚种。短双歧杆菌M16V与短双歧杆菌R0070为不同的菌株。
微生物
本组合物优选含有至少一种选自乳酸细菌、芽孢杆菌和酵母的微生物，优选至少一种选自肉杆菌属（Carnobacterium）、肠球菌属（Enterococcus）、乳杆菌属（Lactobacillus）、乳球菌属（Lactococcus）、明串珠菌属（Leuconostoc）、酒球菌属（Oenococcus）、片球菌属（Pediococcus）、链球菌属（Streptococcus）、四联球菌属（Tetragenococcus）、漫游球菌属（Vagococcus）和魏斯氏菌属（Weissella）和双歧杆菌属（Bifidobacteria）的乳酸细菌。本组合物优选含有至少两种，更优选至少三种，最优选至少四种不同的微生物。
本组合物优选含有至少一种选自双歧杆菌属的种，更优选至少两种，甚至更优选至少三种，更优选至少四种，最优选至少五种选自双歧杆菌属的种。双歧杆菌为革兰氏阳性、厌氧型棒状细菌。当与各个双歧杆菌属的模式株比较时，本双歧杆菌属优选具有与16SrRNA序列至少95％的同一性，更优选具有至少97％的同一性，如在关于本主题的手册例如Sambrook,J.，Fritsch,E.F.和Maniatis,T.(1989)，分子克隆，实验室手册，第二版，冷泉港（N.Y.）实验室出版中定义。优选使用的双歧杆菌在Scardovi,V.GenusBifidobacterium,p.1418-p.1434.In:Bergey'smanualofsystematicBacteriology.Vol.2.Sneath,P.H.A.,N.S.Mair,M.E.SharpeandJ.G.Holt(ed.).Baltimore:Williams&Wilkins.1986.635p中也有描述。
本组合物优选包含至少一种，更优选至少两种，甚至更优选至少三种，最优选至少四种优选选自短双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌、链状双歧杆菌、青春双歧杆菌、嗜热双歧杆菌、高卢双歧杆菌、动物双歧杆菌、角双歧杆菌、假小链双歧杆菌、嗜热嗜酸双歧杆菌和长双歧杆菌的双歧杆菌种。更优选地本组合物优选包含至少一种，更优选至少两种，甚至更优选至少三种，最优选至少四种优选选自短双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌、链状双歧杆菌、青春双歧杆菌和长双歧杆菌的双歧杆菌种。最优选地本组合物含有短双歧杆菌和/或链状双歧杆菌。
在进一步优选的实施方案中，本组合物包含至少一种，更优选至少两种，甚至更优选至少三种，最优选至少四种双歧杆菌亚种。在更进一步优选的实施方案中，本组合物包含至少一种，更优选至少两种，甚至更优选至少三种，最优选至少四种双歧杆菌菌株。
在进一步优选的实施方案中，本组合物包含乳酸细菌，优选至少选自乳杆菌、乳球菌和链球菌的细菌。更优选地，本组合物包含至少一种选自植物乳杆菌（Lactobacillusplantarum）、路氏乳杆菌（Lactobacillusreuteri）、约氏乳杆菌（Lactobacillusjohnsonii）、干酪乳杆菌（Lactobacilluscasei）、鼠李糖乳杆菌（Lactobacillusrhamnosus）、嗜酸乳杆菌（Lactobacillusacidophilus）、发酵乳杆菌（Lactobacillusfermentum）、乳乳杆菌（Lactobacilluslactis）、嗜热链球菌（Streptococcusthermophilus）和类干酪乳杆菌（Lactobacillusparacasei）的细菌。进一步增加的生物多样性将对新生儿的健康有刺激作用。
本组合物优选含有每克本组合物干重101至1013菌落形成单位（cfu）的微生物，优选102至1012，更优选103至1010。优选地，本组合物含有每克本组合物干重101至1013菌落形成单位（cfu）的双歧杆菌，更优选102至1012，最优选103至1012。
本方法优选包含施用的每份含有101至1013cfu的微生物，更优选102至1011，最优选103至1010。本方法优选包含施用的每份含有101至1013cfu的双歧杆菌，更优选102至1012，最优选103至1011。
寡糖
本发明中使用的术语“不消化的寡糖”指寡糖/碳水化合物，其在肠中不能或只能部分通过人类上消化道（小肠和胃）中存在的酸或消化酶被消化，但可通过人类肠内菌群发酵。优选地不消化的寡糖具有2到100的聚合度（DP），优选2到50的聚合度。
该不消化的寡糖优选为益生纤维。术语“益生纤维”指不消化的纤维，其通过选择性刺激结肠内一种或有限数目的细菌的生长和/或活性对所述宿主产生有益影响。
该不消化的寡糖优选为可溶性的。当提及多糖、纤维或寡糖时，此处所用的术语“可溶性的”指根据L.Prosky等人,J.Assoc.Off.Anal.Chem.71,1017-1023(1988)描述的方法，该物质至少50％为可溶的。
本组合物优选包含至少一种选自半乳寡糖、不消化糊精、木寡糖、***寡糖、葡寡糖、甘露寡糖、异麦芽寡糖和果多糖的寡糖。
此处所用的术语“果多糖”涉及多糖碳水化合物，其包含至少3个β-连接果糖的单元链，其DP在3到300之间，优选在20到150之间。优选使用菊粉。菊粉可以商标名，(Orafti)获得。所述果多糖的平均DP优选至少为15，更优选至少为20或更高，高达300。菊粉中所述果糖单元通过β(2→l)键连接。
不消化的多聚糊精指抗消化（麦芽）糊精或抗消化聚葡萄糖，该抗消化（麦芽）糊精或抗消化聚葡萄糖具有10到50，优选10到20之间的DP。所述不消化的多聚糊精包含α(l→4)、α(l→6)糖苷键及1→2和l→3键。不消化的多聚糊精例如在可从MatsutamiInductries商标名或Danisco商标名下获得。
本发明人发现半乳寡糖可有利地用于本组合物，因为这些寡糖在刺激双歧杆菌生长上尤其有效。因此，在优选的实施方案中，本组合物含有半乳寡糖。此处所用的术语“半乳寡糖”指不消化的的糖类，其中至少30％的糖单元为半乳糖单元，优选至少50％，更优选至少60％。优选地，所述半乳寡糖的糖为β-连接，与在人乳中的情况一样。
本组合物优选含有选自反式半乳寡糖、乳-N-四糖(LNT)和乳-N-新四糖(neo-LNT)的半乳寡糖。在尤其优选的实施方案中，本方法包含施用反式半乳寡糖（[半乳糖]n-葡萄糖；其中n为1到60之间的整数，即2、3、4、5、6…、59、60；优选地n选自2、3、4、5、6、7、8、9或10）。例如在商标VivinalTM（BorculoDomoIngredients，荷兰）下出售反式半乳寡糖（TOS）。优选地，所述反式半乳寡糖的糖为β-连接。
本组合物每100克干重优选含有0.5至75克的不消化的可溶寡糖，优选0.5到50克。本组合物每100克干重优选包含0.1至95克半乳寡糖，优选0.1到50克。
本方法优选包括施用含有0.05到25克的不消化的寡糖，优选0.1到5克的一份。本方法优选包括施用含有0.05到25克的半乳寡糖，优选0.1到5克半乳寡糖的一份。
本发明人也已发现长链的不消化的寡糖和短链的不消化的寡糖的混合物协同刺激健康的肠内菌群，尤其是双歧杆菌的生长。因此本组合物优选含有至少两种具有不同平均聚合度（DP）的寡糖。优选地，所述重量比值：
a.（DP为2到5的不消化的寡糖）：（DP为6、7、8和/或9的不消化的寡糖）＞1；并且
b.（DP为10到60的不消化的寡糖）：（DP为6、7、8和/或9的不消化的寡糖）＞1
优选地，两个重量比值都大于2，甚至更优选大于5。
为进一步改进，所述寡糖优选含有相对高含量的短链寡糖，因为这些短链寡糖可强烈刺激双歧杆菌的生长。因此，在本组合物中优选至少10wt.％的寡糖具有2至5的DP（即2、3、4和/或5）并且至少5wt.％具有10到60的DP。优选至少50wt.％，更优选至少75wt.％的所述寡糖具有2到9的DP（即2、3、4、5、6、7、8和/或9）。
为提高多数肠内生物的生物多样性并刺激其生长，本组合物优选包含两种具有不同结构的寡糖。本组合物包含至少两种不同的寡糖，其中所述寡糖的糖单元的同源性低于约90％，优选低于50％，甚至更优选低于25％，甚至更优选低于5％。本发明中所用的术语“同源性”指在不同寡糖中相同糖单元的百分率的累加。例如，寡糖1（OL1）具有果糖-果糖-葡萄糖-半乳糖的结构，因此含有50％的果糖，25％的半乳糖和25％的葡萄糖。寡糖2（OL2）具有果糖-果糖-葡萄糖的结构，因此含有66％的果糖，33％的葡萄糖。所述不同的寡糖因此具有75％的同源性（50％果糖＋25％的葡萄糖）。
本组合物优选含有半乳寡糖和果多糖。
应用
本组合物优选经肠施用，更优选口服施用。本组合物因此优选为液体。此处所用的术语“经肠的”也包括直肠或***给药。
当婴儿接受婴儿奶粉配方时，在营养配方中优选包括本组合物，其含有微生物及任选地含有至少一种不消化的寡糖。可分别将益生菌和/或益生素加入该婴儿配方中。当新生儿通过导管接受营养品时，经导管施用的营养品中可适当包含所述微生物。刺激多样性是很重要的，因此本发明也提供了营养组合物，该营养组合物包含5到25en％的蛋白质；25到60en％的脂肪；30到70en％的碳水化合物；至少两种不同种的双歧杆菌和至少一种乳杆菌。
当婴儿接受人母乳时，本组合物可适当地与人母乳混合。本发明因而也提供制备适合经剖腹生产术出生的婴儿的婴儿营养品的方法，该方法包括混合：A）人母乳；和B）包含（ⅰ）至少两种不同的微生物；或（ⅱ）至少一种微生物和至少一种不消化的的寡糖；或（ⅲ）至少两种不同的双歧杆菌种、亚种或菌株的组合物。组合物B）的优选特征如上文提供。
在进一步优选的实施方案中，本组合物以非常小的体积对所述婴儿施用，例如通过“接种”所述婴儿。优选地，用注射器、移液器或导管对所述婴儿施用本组合物，优选在其出生后直接施用。在进一步优选的实施方案中，经直肠或***对经剖腹生产术分娩的所述婴儿施用本组合物，该组合物优选为栓剂、丸剂或片剂的形式。因此，本发明也提供适于对年龄低于1岁的婴儿直肠施用的栓剂、丸剂或片剂，其中所述栓剂、丸剂或片剂含有微生物和/或不消化的寡糖，优选地如上文所述的本组合物。
在进一步优选的实施方案中，本发明提供了刺激婴儿肠内菌群的方法，该方法包括对年龄低于3岁的婴儿直肠施用包含微生物和/或不消化的寡糖的组合物。优选地，所述婴儿年龄低于1岁，更优选地低于2周。优选地所述婴儿经剖腹生产术分娩。优选地直肠施用的该组合物包含上述组合物。
该方法具有不干扰正常的人乳喂养活动的优点并与出生过程中发生的***接种有很高的相似性。本发明也提供增强经剖腹生产术分娩的婴儿的健康的方法，该方法包括对出生后100小时内，优选出生后72小时内，最优选出生后48小时内的婴儿施用含有微生物和/或不消化的寡糖的组合物。
所述组合物优选适于出生后直接施用。因此，在进一步优选的实施方案中，本发明提供含有体积为0.5至50ml的液体组合物的容器，其含有本组合物。所述液体与本微生物，任选地与不消化的寡糖组合，可适用于本方法。优选地所述液体具有0.5到25ml的体积。该体积优选为小体积，因为否则它将干扰所述婴儿的食欲和饮水行为。
本发明类似地包含具有可重构的干燥组合物的容器，所述组合物含有本组合物，，其中所述容器具有0.5到50ml的容积。该容器优选附带说明书以用小体积液体，例如水重构所述粉末。
本发明因此也提供刺激婴儿健康的肠内菌群发展的方法，该方法包括步骤A：混合I）尤其是营养或药学上可接受的液体；和II）干燥组合物，其中所述干燥组合物II中包含至少两种不同的微生物；或至少一种微生物和至少一种不消化的的寡糖；或至少两种不同的双歧杆菌种、亚种或菌株；及步骤B）对经剖腹生产术出生的婴儿施用步骤a）中得到的组合物。
本组合物优选对出生后1年内的婴儿施用，优选在其出生后两周内，甚至更优选在其出生后一周内，最优选在其出生后48小时内施用。
实施例
实施例1：***分娩出生的婴儿对比剖腹生产术分娩出生的婴儿肠内 小型生物群的分子表征
在本研究中，通过使用PCR变性梯度凝胶电泳（DGGE）和PCR温度梯度凝胶电泳（TGGE）研究分娩方式（剖腹生产术分娩对***分娩）在出生后第三天对肠内微生物组成的影响。使用DGGE和TGGE分析以及10种双歧杆菌种特异性扩增。
在获得父母的书面知情同意书后，剖腹生产术分娩和***分娩后的23个新生儿参与了此项研究。在他们出生后第三天取得其粪便样品。
提取微生物DNA并根据Favier等人，EnvironMicrobiol2002;68:219-226和Satokari等人，ApplEnvironMicrobiol2001;67:504-513;Satorkari等人SystemApplMicrobiol2003;26:572-584的方法分析。
表1给出的是对剖腹生产术分娩的新生儿出生后第三天的粪便样品中双歧杆菌的检测结果。表2给出的是在经***分娩的新生儿出生后第三天的粪便样品中检测的双歧杆菌的结果。
可以看出经剖腹生产术出生的婴儿的菌群与经***途径出生的婴儿的菌群存在巨大差异。认识这些物种水平上的差异使本发明人能够设计本组合物和方法。
这些结果表明根据本发明的组合物和方法的有利用途，例如喂养经剖腹生产术出生的婴儿、刺激健康的肠内菌群并因而预防感染及刺激健康的免疫***的方法。
表1：剖腹生产术
(-)=无扩增
(+/-)=弱扩增
(+)=阳性扩增
(++)=强扩增
表2：正常生产
(-)=无扩增
(+/-)=弱扩增
(+)=阳性扩增
(++)=强扩增
实施例2：通过婴儿粪便进行半乳寡糖的体外发酵
目标：通过体外半动力分批发酵***使用婴儿粪便来评价半乳寡糖（GOS）及GOS和菊粉的组合提高乳酸细菌活性的能力。测定乳酸盐的量，因为它是乳酸细菌包括双歧杆菌的发酵产物。
方法：从健康的人工喂养的婴儿中得到新鲜粪便。将从1至4个月大的婴儿得到的新鲜粪便材料在1小时内合并并放进防腐培养基中（缓冲蛋白胨20.0g/l，L-半胱氨酸－盐酸0.5g/l，巯基乙酸钠0.5g/l，刃天青片剂1/1，pH6.7）并且在发酵实验开始前于4℃最多保存2小时。
将保存的粪便溶液在13,000rpm（转/分钟）下离心15分钟。去除所述上清液并将所述粪便与McBain&MacFarlane培养基（缓冲蛋白胨水3.0g/l，酵母提取物2.5g/1，粘蛋白（刷状缘）0.8g/1，胰蛋白胨3.0g/1，L-半胱氨酸盐酸0.4g/l，胆盐0.05g/1，K2HPO4.3H2O2.6g/1，NaHCO30.2g/1，NaCl4.5g/1，MgSO4.7H2O0.5g/1，CaCl20.228g/1，FeSO40.005g/1）以1：5的重量比混合，该培养基代表肠内环境。
在t=0时，将15ml所述粪便悬浮液与500mg益生菌在瓶中组合并彻底混合。将不加入益生菌的作为对照。加入的益生菌如下：
培养基：123
GOS：0500500
菊粉：0050
作为GOS来源，使用VivinalGOS（BorculoDomo）。作为菊粉来源，使用RaftilinHP（Orafti）。
将15ml转移到透析管中，该透析管在一个充满250ml缓冲培养基（K2HPO4.3H2O2.6g/1，NaHCO30.2g/1，NaCl4.5g/1，MgSO4.7H2O0.5g/1，CaCl20.228g/1，FeSO4.7H2O0.005g/1，pH6.3(?)）的250ml瓶中。封闭所述瓶子并在37℃下温育。3小时后用皮下注射器从透析管并从透析缓冲液取出1ml样品并在-18℃下保存。以一式两份（duplo）进行实验。
使用L-乳酸检测试剂盒及D-和L-乳酸脱氢酶（BoehringerMannheim,Mannheim,德国）通过酶法测定乳酸。
结果与结论：所述结果用每g加入的益生菌形成的乳酸的量来表示。所述结果表明当婴儿的菌群使GOS或GOS和菊粉的混合物发酵时，乳酸的量会增加。当不加入益生菌时，观察不到乳酸的产生。只有GOS时会产生每g益生菌0.1801毫mol乳酸。当加入GOS与菊粉的组合时产生每g益生菌0.2119毫mol乳酸。
所述结果表明所述寡糖刺激婴儿乳酸细菌，包括双歧杆菌的生长和/或活性。这些结果表明了所述不消化的寡糖，尤其是半乳寡糖在本组合物和根据本发明的方法即喂养经剖腹生产术出生的婴儿的方法中的有利用途。
实施例3：喂养经剖腹生产术出生的婴儿的方法
在所述婴儿经剖腹生产术出生后的两天内，对其施用营养组合物，该组合物每100ml含有现成喂养配方：1.6克蛋白质，3.6克脂肪，6.4克可消化性碳水化合物（主要是乳糖），0.8克不消化的碳水化合物（其中有0.60克反式半乳寡糖，0.07克菊粉，1×109cfu短双歧杆菌，1×109cfu链状双歧杆菌，1×109cfu长双歧杆菌和1×109cfu类干酪乳杆菌）。
实施例4：包装组合物
包装组合物中所述包装指出产品尤其适于口服喂养经剖腹生产术出生的婴儿，所述组合物包含短双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌、链状双歧杆菌、青春双歧杆菌、长双歧杆菌、半乳寡糖和菊粉。
实施例5：包装组合物
如实施例4中的组合物，其中所述组合物具有10ml的体积并且包装于注射器中。
实施例6：直肠给药
适合对出生后两周内的婴儿直肠给药的PII,其包含半乳寡糖和短双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌。

Claims (14)

1.至少一种微生物和至少一种不消化的寡糖在制备对经剖腹生产术分娩的婴儿经肠施用的组合物中的用途，其中所述组合物含有至少一种双歧杆菌物种，所述双歧杆菌物种包含长双歧杆菌和短双歧杆菌。
2.权利要求1所述的用途，用于通过经肠施用所述组合物刺激经剖腹生产术分娩的婴儿中健康肠内菌群的发展。
3.权利要求1或2所述的用途，其中所述组合物经口施用并且其中所述组合物为婴儿奶粉配方或可重构的干燥组合物的形式；或其中所述组合物经直肠施用并且其中所述组合物为栓剂、丸剂或片剂的形式。
4.权利要求1或2所述的用途，其中所述组合物包含5到25en％的蛋白质，25到60en％的脂肪和30到70en％的碳水化合物。
5.权利要求1或2所述的用途，其中对接受人母乳的婴儿施用所述组合物。
6.权利要求1或2所述的用途，其中在婴儿出生后一周内对其施用所述组合物。
7.制备适合经剖腹生产术出生的婴儿的婴儿营养品的方法，该方法包括混合：
a.人母乳；和
b.包含长双歧杆菌和/或短双歧杆菌和至少一种不消化的寡糖的组合物。
8.至少一种微生物和至少一种不消化的寡糖在生产用于刺激经剖腹生产术分娩的婴儿的健康的肠内菌群发展的方法中的组合物中的用途，所述方法包括步骤：
A)混合I)营养或药学上可接受的液体；和II)干燥组合物，其中所述干燥组合物II包含长双歧杆菌和/或短双歧杆菌和至少一种不消化的寡糖；
B)对婴儿施用步骤a)中获得的组合物。
9.包含微生物和不消化的寡糖的组合物在生产组合物中的用途，通过对婴儿并且在其出生后100小时内施用所述生产的组合物来刺激经剖腹生产术分娩的婴儿肠内菌群，所述组合物包含长双歧杆菌和/或短双歧杆菌和至少一种不消化的寡糖。
10.包含微生物和不消化的寡糖的组合物在生产组合物中的用途，通过对3岁以下的婴儿经直肠施用所述生产的组合物来刺激经剖腹生产术分娩的婴儿的肠内菌群，所述组合物包含长双歧杆菌和/或短双歧杆菌和至少一种不消化的寡糖。
11.根据权利要求1、2、7、8、9或10的用途，其中所述组合物包含选自半乳寡糖、不消化的糊精、木寡糖、***寡糖、葡寡糖、甘露寡糖、异麦芽寡糖和果多糖的不消化的寡糖。
12.根据权利要求1、2、7、8、9或10的用途，其中所述组合物包含半乳寡糖和/或果寡糖。
13.根据权利要求12的用途，其中所述组合物包含半乳寡糖。
14.待施用于经剖腹生产术分娩的婴儿的组合物，其包含至少一种不消化的寡糖和至少两种选自短双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌、链状双歧杆菌、青春双歧杆菌、嗜热双歧杆菌、高卢双歧杆菌、动物双歧杆菌、角双歧杆菌、假小链双歧杆菌、嗜热嗜酸双歧杆菌和长双歧杆菌的双歧杆菌种，其中，所述至少一种不消化的寡糖包含不消化的半乳寡糖，并且其中所述至少两种不同的双歧杆菌种包括长双歧杆菌和/或短双歧杆菌。
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