CN103347405A - 使用人乳中发现的寡糖的组合调节双歧杆菌属的生长 - Google Patents
使用人乳中发现的寡糖的组合调节双歧杆菌属的生长 Download PDFInfo
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- CN103347405A CN103347405A CN2012800083041A CN201280008304A CN103347405A CN 103347405 A CN103347405 A CN 103347405A CN 2012800083041 A CN2012800083041 A CN 2012800083041A CN 201280008304 A CN201280008304 A CN 201280008304A CN 103347405 A CN103347405 A CN 103347405A
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Abstract
用于口服施用于人类的益生元制剂,其包含2'-岩藻糖基乳糖、3'-唾液酰乳糖和6'-唾液酰乳糖,以及用于调节或刺激双歧杆菌属在肠中生长的方法。制剂可以是婴儿配方奶。
Description
发明背景
本发明涉及含有用于人类营养,例如婴儿营养的寡糖的益生元制剂,其调节双岐杆菌属(Bifidobacteria)的生长。
益生元制剂为有益菌,例如定居在宿主动物如哺乳动物,例如人类的肠中的双歧杆菌属的多种物种提供营养。这些细菌群体帮助排斥肠中的有害细菌,并且还能消化宿主不能消化的一些化合物。因此,益生元制剂通过帮助双歧杆菌属活跃于宿主的肠中来为宿主提供益处。
根据Wang,等人,Am.J.Clin.Nutr.,74(4),510(2001),常规婴儿配方奶比人乳含有更少量的结合寡糖的唾液酸。此外,根据Wang,等人,J.Pediatr.,138,914-6(2001),喂养人乳的早产婴儿的唾液含有的唾液酸水平是喂养市售婴儿配方奶的婴儿的两倍。
EP1255449描述了含寡糖的营养制剂。根据EP1255449,寡糖(特别是低聚果糖和唾液酰乳糖能通过人类的小肠,而不会被消化,并且通过数量有限的微生物,包括双歧杆菌属和乳杆菌属(Lactobacilli)发酵。认为唾液酰乳糖,特别是天然地存在于人乳中的3'-和6'-唾液酰乳糖可以抑制病原菌在人类中的结合。本专利描述了低聚果糖和3'-和6'-唾液酰乳糖的组合在营养制剂中的用途。
WO2004/093557描述了含唾液酸来源,如唾液酰乳糖的婴儿配方奶。
WO2004/112509描述了诱导新生婴儿中肠屏障成熟的营养组合物,其中该组合物包含微生物和不可消化的寡糖,其可以是唾液酸寡糖。
WO2007/114696描述了绵羊寡糖,例如3-或6-唾液酰乳糖的用途,所述绵羊寡糖用于刺激免疫系统。
WO2009/059996描述了含有3'-或6'-唾液酰乳糖的营养组合物,其用于预防二次感染。
美国专利号5,260,280描述了通过给人类患者施用有效量的分离的唾液酰乳糖,来中和细菌肠毒素的方法。
发明概述
本发明提供了用于给人类口服施用的益生元制剂,其包含2'-岩藻糖基乳糖、3'-唾液酰乳糖和6'-唾液酰乳糖。该制剂调节双歧杆菌属在人类肠中的生长。所述制剂可以是婴儿配方奶。
本发明还提供了用于调节或刺激双歧杆菌属,例如两歧双岐杆菌(Bifidobacterium bifidum)、短双岐杆菌(Bifidobacterium breve)和婴儿双岐杆菌(Bifidobacterium infantis)的生长的方法。
附图简述
图1显示两歧双岐杆菌在对照培养基和在含2'-岩藻糖基乳糖的培养基中的生长曲线。
图2显示图1中的曲线的AUC。
图3显示婴儿双岐杆菌在对照培养基和在含2'-岩藻糖基乳糖的培养基中的生长曲线。
图4显示图3中的曲线的AUC。
图5显示婴儿双岐杆菌在对照培养基和在含6'-唾液酰乳糖的培养基中的生长曲线。
图6显示短双岐杆菌在对照培养基和在含3'-唾液酰乳糖的培养基中的生长曲线。
图7显示婴儿双岐杆菌在对照培养基和在含3'-唾液酰乳糖的培养基中的生长曲线。
发明详述
本发明提供了包含寡糖组合的营养制剂,所述寡糖组合比单一寡糖刺激更多的双歧杆菌属的物种的生长。本发明的寡糖组合帮助了更广谱的有益肠细菌活跃,在摄取了该制剂的人类受试者的肠中提供了更多样的和更自然平衡的双歧杆菌属群体。
本发明制剂通常是口服,例如通过饮用液体制剂摄取的食品或食品补充剂。在一个实施方案中,所述制剂是婴儿可以以常规方式饮用的婴儿配方奶或者乳替代物。
在本发明的一个实施方案中,制剂是包含至少约0.1mg/ml的2'-岩藻糖基乳糖、至少约0.1mg/ml的6'-唾液酰乳糖和至少约0.1mg/ml的3'-唾液酰乳糖的液体。在本发明的一个实施方案中,制剂是包含约0.1-10mg/ml的2'-岩藻糖基乳糖、约0.1-10mg/ml的6'-唾液酰乳糖和约0.1-20mg/ml的3'-唾液酰乳糖的液体。在另一个实施方案中,制剂是包含约0.5-2mg/ml的2'-岩藻糖基乳糖、约0.5-2mg/ml的6'-唾液酰乳糖和约1-4mg/ml的3'-唾液酰乳糖的液体。在另一个实施方案中,制剂是包含约1mg/ml的2'-岩藻糖基乳糖、约1mg/ml的6'-唾液酰乳糖和约2mg/ml的3'-唾液酰乳糖的液体。
在本发明的一个实施方案中,制剂分别以约0.5-2.0比0.5-2.0比1.0-4.0的重量比包含2'-岩藻糖基乳糖、6'-唾液酰乳糖和3'-唾液酰乳糖。在一个实施方案中,以重量计,3'-唾液酰乳糖的量与2'-岩藻糖基乳糖和/或6'-唾液酰乳糖的量至少一样多。在本发明的一个实施方案中,制剂分别以约0.9-1.1比0.9-1.1比1.8-2.2的重量比包含2'-岩藻糖基乳糖、6'-唾液酰乳糖和3'-唾液酰乳糖。
在本发明的一个实施方案中,制剂是干粉。除已经干燥去除了水以外,本发明的干粉制剂与本发明的液体制剂是相同的。可以通过添加水来重构干粉制剂,以形成本发明的液体制剂。在本发明的一个实施方案中,干粉制剂包含干粉制剂总重量的至少约0.4wt%的2'-岩藻糖基乳糖、至少约0.4wt%的6'-唾液酰乳糖和至少约0.8wt%的3'-唾液酰乳糖。在本发明的一个实施方案中,干粉制剂包含干粉制剂总重量的约0.4-1.5wt%的2'-岩藻糖基乳糖、约0.4-1.5wt%的6'-唾液酰乳糖和约0.8-3.0wt%的3'-唾液酰乳糖。在本发明的一个实施方案中,干粉制剂包含干粉制剂总重量的约0.7-0.9wt%的2'-岩藻糖基乳糖、约0.7-0.9wt%的6'-唾液酰乳糖和约1.4-1.7wt%的3'-唾液酰乳糖。因此,这三种寡糖包含干粉重量的约1.6-6.0%。本段中的重量百分数与用于本发明的任何液体制剂的重量百分数相同,条件是重量百分数是基于除水外的全部成分计算(在下文中称为“干重”)。
本发明制剂还可以是液体浓缩物的形式,所述浓缩物以与前述液体制剂相同的比率包含相同的成分,但含有较少的水,因此含有较高浓度的其他成分。基于干重的寡糖的百分数将与本发明的任何其他制剂相同。可以将此类液体浓缩物与合适量的水混合,以产生前述液体制剂。本领域技术人员将容易地理解,如何制备此类液体浓缩物,以及在不过度实验的情况下如何确定与所述浓缩物混合的水的量。
如本文中所用,术语“婴儿配方奶”指营养制剂(或者是液体形式,或者是通过添加水可以重构形成液态的婴儿配方奶的干粉形式),所述营养制剂为婴儿提供完全营养,并且在喂养婴儿中可被用作人乳的替代物。此类制剂是本领域熟知的。
通常,婴儿配方奶以方便的液体形式,每100ml提供60-70Kcal。婴儿配方奶通常每100Kcal包含:1.8-4.5g蛋白质;3.3-6.0g脂肪(脂类);300-1200mg亚油酸;9-14g糖,其选自乳糖、蔗糖、葡萄糖、葡萄糖浆、淀粉、麦芽糖糊精和麦芽糖及其组合;以及必需的维生素和矿物质。乳糖可以是婴儿配方奶中主要的糖。每100ml液态的婴儿配方奶可以含有约67Kcal。每100Kcal的婴儿配方奶可以包含约1.8-3.3g蛋白质,例如每100Kcal包含约1.8-1.1g蛋白质。
婴儿配方奶还可以包含选自核苷酸,其选自胞苷5'-一磷酸(CMP)、尿苷5'-一磷酸(UMP)、腺苷5'-一磷酸(AMP)、鸟苷5'-一磷酸(GMP)和肌苷5'-一磷酸(IMP)及其混合物。婴儿配方奶还可以包含黄体素、玉米黄质、低聚果糖、低聚半乳糖、唾液酰乳糖和/或岩藻糖基乳糖。婴儿配方奶中可以包含长链多不饱和脂肪酸,例如二十二碳六烯酸(DHA)和花生四烯酸。婴儿配方奶还可以包含本领域熟知的其他成分。
婴儿配方奶的目的是为婴儿提供婴儿所需的全部能量和营养,并且婴儿可以另外地从其母亲的人乳中接受所述能量和营养。然而,人乳是非常复杂的组合物,并且婴儿配方奶没有准确地模拟该组合物。多年来,本领域技术人员致力于制备更类似于人乳的婴儿配方奶,并使得婴儿配方奶将给婴儿提供尽可能多的人乳的益处。
常规实践是从牛乳中制备婴儿配方奶,因为婴儿所需的大多数或者全部营养物都存在于牛乳中。然而,牛乳具有的组合物与人乳明显不同。例如,牛乳包含的蛋白质含量具有约20:80的乳清-酪蛋白比,而人乳具有约60:40的比例。牛乳具有的α乳清蛋白(一种乳清蛋白)还比人乳少,并且许多牛酪蛋白含量是β-乳球蛋白,其并不以有效的量存在于人乳中。由于这些以及其他差异,本领域技术人员已经研发了修饰牛乳的方法,例如通过制备可用于婴儿配方奶中的富集α-乳清蛋白的牛乳乳清级分以提供具有更类似于人乳的蛋白质谱的制剂。这些方法是本领域熟知的。例如,美国专利号6,913,778描述了通过将富集α-乳清蛋白的乳清级分与脱脂牛乳级分混合来制备婴儿配方奶。该专利还描述了具有降低的蛋白质含量的婴儿配方奶。牛乳含有约15-16g/L的蛋白质,而人乳含有约11g/L。
除婴儿配方奶之外的营养制剂也是本领域熟知的。例如,有设计用于儿童或成人的制剂,其旨在提供完全的营养或为儿童或成人消费者的饮食提供补充。本领域技术人员致力于为消费者制备尽可能有益的这些制剂。
寡糖2'-岩藻糖基乳糖、6'-唾液酰乳糖和3'-唾液酰乳糖是商业上可得到的熟知化合物。
已经发现,2'-岩藻糖基乳糖刺激两歧双岐杆菌和婴儿双歧杆菌的生长,但可以抑制短双岐杆菌的生长,6'-唾液酰乳糖刺激婴儿双歧杆菌的生长,而3'-唾液酰乳糖刺激短双歧杆菌和婴儿双歧杆菌的生长。已经发现,以本文中所述的量和比例,组合使用这三种寡糖有益地影响了这三种双歧杆菌属在人类肠中的生长。因此,本发明促进了细菌在消费者的肠中的健康平衡。
可以使用用于组合制剂的成分(所述成分都是本领域已知的)的常规方法制备液体、液体浓缩物形式或是干粉形式的本发明制剂。本领域技术人员将容易地理解如何制备制剂,或者能确定如何在不过度实验的情况下制备制剂。
在本发明方法中,将本发明的液体营养制剂口服施用于人类消费者,以调节双歧杆菌属,例如两歧双岐杆菌、短双岐杆菌和婴儿双岐杆菌的生长。
提出以下实施例以说明本发明的某些实施方案,不应认为所述实施例限制了本发明的范围。
实施例1:2'-岩藻糖基乳糖、3'-唾液酰乳糖和6'-唾液酰乳糖对婴儿双岐杆菌ATCC15697、短双岐杆菌ATCC15700和两歧双岐杆菌ATCC29521的生长的影响
将梭菌加强培养基(Reinforced Clostridia Medium)(RCM)用于培养双歧杆菌属。将不具有葡萄糖的RCM用作对照培养基。RCM是用于培养和计数厌氧菌的熟知培养基,其由Hirsch和Grinstead,J.Dairy Res.,21:101(1954)首次配制。
将测试成分(2'-岩藻糖基乳糖、6'-唾液酰乳糖和3'-唾液酰乳糖)以以下浓度:1mg/ml2'-岩藻糖基乳糖;1mg/ml6'-唾液酰乳糖;和2mg/ml、3'-唾液酰乳糖溶解在对照培养基中。
双歧杆菌属分别在RCM中厌氧生长过夜,然后测量所述双歧杆菌属并根据在595nm的吸收光谱所测量的,将所述双歧杆菌属稀释至0.1的光密度,然后进一步稀释10倍,以制备工作原种。对于每次测试,将50μl的工作原种加入到含200μl对照培养基(48孔)或者200μl测试成分溶液(48孔)的96孔板的每一孔中。将平板厌氧培养30个小时,并且每15分钟读取595nm处的光密度。平均类似孔的值,并随时间作图(图1、3和5-7),计算30个小时的时期中的曲线下面积(AUC)(图2和4)。
A.2'-岩藻糖基乳糖和两歧双岐杆菌
平均两次实验的值。根据上述方法培养细菌8个小时后,如图1和2所示,在含测试成分的孔中,生长明显更快,并且测试成分的AUC大约多25.1%。
B.2'-岩藻糖基乳糖和婴儿双歧杆菌
根据上述方法培养细菌8个小时后,如图3和4所示,在含测试成分的孔中,生长明显更快,并且测试成分的AUC大约多30.5%。
C.6'-唾液酰乳糖和婴儿双歧杆菌
根据上述方法培养细菌12个小时后,如图5所示,在含测试成分的孔中,生长明显更快。
D.3'-唾液酰乳糖和短双歧杆菌
根据上述方法培养细菌6个小时后,如图6所示,在含测试成分的孔中,生长明显更快。
E.3'-唾液酰乳糖和婴儿双歧杆菌
根据上述方法培养细菌8个小时后,如图7所示,在含测试成分的孔中,生长明显更快。
实施例2:
适合于喂养婴儿的本发明液体益生元制剂描述如下:
该示例性制剂中所使用的蛋白质来自牛乳和乳清,其中60-65%是乳清蛋白且35-40%是酪蛋白。在该制剂中,将具有提高的α-乳清蛋白含量的乳清级分用于提供每100ml制剂约0.2gα-乳清蛋白的蛋白质含量。
实施例3:
适合于喂养婴儿的本发明液体益生元制剂描述如下:
该示例性制剂中所使用的蛋白质来自牛乳和乳清,其中60-65%是乳清蛋白且35-40%是酪蛋白。在该制剂中,将具有提高的α-乳清蛋白含量的乳清级分用于提供每100ml制剂约0.2gα-乳清蛋白的蛋白质含量。
对于本领域技术人员,将出现本文中并未说明的本发明的许多变化。本发明并不限于本文中所说明和描述的实施方案,还包括所附权利要求范围内的全部主题。
Claims (11)
1.用于口服施用于人类的益生元制剂,其包含2'-岩藻糖基乳糖、3'-唾液酰乳糖和6'-唾液酰乳糖,其中以重量计,所述3'-唾液酰乳糖的量与2'-岩藻糖基乳糖的量至少一样多。
2.权利要求1的制剂,其中所述2'-岩藻糖基乳糖、3'-唾液酰乳糖和6'-唾液酰乳糖占所述制剂干重的约1.6-6.0%。
3.权利要求1或权利要求2的制剂,其中所述制剂分别以约0.5-2.0比0.5-2.0比1.0-4.0的重量比包含2'-岩藻糖基乳糖、6'-唾液酰乳糖和3'-唾液酰乳糖。
4.权利要求3的制剂,其中所述重量比分别为约0.9-1.1比0.9-1.1比1.8-2.2。
5.权利要求1的制剂,其中所述制剂包含婴儿配方奶。
6.权利要求5的婴儿配方奶,其中所述婴儿配方奶是液体,其包含约0.1-10mg/ml的2'-岩藻糖基乳糖、约0.1-10mg/ml的6'-唾液酰乳糖和约0.1-20mg/ml的3'-唾液酰乳糖。
7.权利要求5的婴儿配方奶,其中所述婴儿配方奶是液体,其包含约0.5-2mg/ml的2'-岩藻糖基乳糖、约0.5-2mg/ml的6'-唾液酰乳糖和约1-4mg/ml的3'-唾液酰乳糖。
8.权利要求5的婴儿配方奶,其中所述婴儿配方奶是液体,其包含约1mg/ml的2'-岩藻糖基乳糖、约1mg/ml的6'-唾液酰乳糖和约2mg/ml的3'-唾液酰乳糖。
9.权利要求5的婴儿配方奶,其中所述婴儿配方奶是干粉,其包含约0.4-1.5wt%的2'-岩藻糖基乳糖、约0.4-1.5wt%的6'-唾液酰乳糖和约0.8-3.0wt%的3'-唾液酰乳糖。
10.权利要求9的婴儿配方奶,其包含约0.7-0.9wt%的2'-岩藻糖基乳糖、约0.7-0.9wt%的6'-唾液酰乳糖和约1.4-1.7wt%的3'-唾液酰乳糖。
11.用于调节或刺激双歧杆菌属在人类肠中生长的方法,其包括将权利要求1-10的任一项的制剂口服施用于人类。
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