CN1738595B - 用于减弱炎症过程和非特异性免疫参数的异常激活的含有益生素的组合物 - Google Patents
用于减弱炎症过程和非特异性免疫参数的异常激活的含有益生素的组合物 Download PDFInfo
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- CN1738595B CN1738595B CN02807078XA CN02807078A CN1738595B CN 1738595 B CN1738595 B CN 1738595B CN 02807078X A CN02807078X A CN 02807078XA CN 02807078 A CN02807078 A CN 02807078A CN 1738595 B CN1738595 B CN 1738595B
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Abstract
本发明涉及能通过改善非特异性免疫参数和淋巴细胞亚群的体内稳态减弱炎症过程的含有益生素(益生佐剂)的组合物。本发明还涉及将益生制剂用于生产用来减弱炎症过程和/或诸如吞噬细胞的非特异性免疫参数的异常激活的药物或食品或宠物食品组合物的用途。
Description
发明领域
本发明涉及能通过改善非特异性免疫防御参数和淋巴细胞亚群体的体内稳态减弱炎症过程的含有益生素(益生佐剂)的组合物。本发明还涉及将益生制剂用于生产用来减弱炎症过程和/或诸如吞噬细胞的非特异性免疫参数的异常激活的药物或食品或宠物食品组合物的用途。
发明背景
众所周知,益生素包括糖类,更具体地讲,包括寡糖。另外,已知所述糖类业已被广泛用作功能性食品成分。它们能抗人类消化道中的酶的水解作用,能够不被降解地到达结肠,并且提供特别适合双歧杆菌属生长的糖类物质。寡糖可以由葡萄糖、半乳糖、木糖、麦芽糖、蔗糖、乳糖、淀粉、木聚糖、半纤维素、菊糖、树胶或其混合物产生。纯化的市售产品,如寡聚果糖含有超过大约95%的寡糖形式的固体。
主要针对与矿物质的生物可利用性、脂类代谢和胃肠习性的调控的功能作用,业已在人类中研究了寡聚果糖(Roberfroid,M.B.Delzenne,N.M.Annu Rev Nutr 1998;18:117-143。对于它们对免疫学功能的影响的关注较少,同时,通过动物研究了解了它们对与肠道相关的淋巴组织的致癌作用和刺激的修饰作用(Pierre,F.,等.Cancer Res 1997;57:225-228)。
实际上,寡聚果糖、长链(菊糖)和短链(寡聚果糖)是能够逃脱在上胃肠道中被消化的糖类。然后它们在结肠中发酵,并且选择地刺激双歧杆菌属生长。
人类肠道菌群及其重要的代谢活性可能与多种和健康相关的功能相关,如维持肠道稳态,生物异源物质的代谢和肠道免疫的刺激。它们受疾病、饮食、紧张的影响,并且有可能受衰老的影响。大肠中的每克粪便含有高达1012个细菌,具有来自细菌的大约40-50个属的大约103个不同的种。其中的大部分是专性厌氧菌,不过具有一个庞大的兼性厌氧菌群。主要的厌氧菌物种是拟杆菌、双歧杆菌、真杆菌,它们占整个粪便菌群的99%,其后是梭菌、乳杆菌和格兰氏阳性球菌、肠球菌、大肠菌类、产甲烷菌,以及含量低得多的硫酸还原细菌(Hill,M.J.Normal gut bacterial flora.1995;3-17)。
成年人的微菌群特征是在大约两岁的时候出现的。成年人肠道微菌群似乎相当稳定;不过,业已报导过随着衰老会发生某些改变,主要是低水平的双歧杆菌属和拟杆菌属(Hopkins,M.J.,等.Gut2001;48:198-205)。肠道菌群可以划分成具有有益作用的菌种,如双歧杆菌,或具有有害作用的菌种,如铜绿甲单胞菌、变形菌、葡萄球菌、某些梭菌和韦荣氏球菌,和具有中间作用的菌种,如肠球菌、大肠杆菌、肠球菌和拟杆菌。业已报导过双歧杆菌属和乳杆菌属对特异的免疫功能有有益作用(Schiffrin,E.J.,等.,J Dairy Sci1995;78:491-497)。
据报导,随着衰老,双歧杆菌属通常会减少,同时,产气荚膜梭菌、肠球菌和肠杆菌科增加(Mitsuoka,T.Hayakawa,K.ZentralblBakteriol[Orig A]1973;223:333-342)。由于较高的萎缩性胃炎和胃酸过少的倾向,在老年人群中细菌过度生长更为常见。在健康的老年人群中,细菌的过度生长似乎没有临床症状,它可能在75岁以上的虚弱的老年人群中具有某些重要作用,并且在与抗生素治疗相关的急性护理或长期护理的老年人群中,与艰难梭菌相关的腹泻更为常见,并且有可能减弱免疫反应。业已认识到衰老与免疫功能的丧失相关,并且在营养和免疫功能之间存在相关性(Meydani,S.N.营养免疫学研究现状:J Nutr Immunol 1994;2:93-97)。
免疫反应的改变(细胞因子生产的重建和免疫功能失调)与感染和与感染相关的死亡的发病率增加相关。营养干预,主要是维生素和矿物质补充,可以改善虚弱老年人的免疫反应[Lesourd,B.M.Am JClin Nutr 1997;66:478S-484S 41]。
本发明的目的是提供能够限制免疫功能失调,尤其是诸如吞噬细胞和单核细胞巨噬细胞系统的非特异性免疫反应的异常激活,以及将淋巴细胞亚群保持在正常的激活水平上的另一种组合物。
发明概述
因此,在第一方面,本发明提供了一种含有至少一种益生素的,用于减弱炎症过程和/或非特异性免疫参数的异常激活的组合物。
例如,该组合物特别适用于减弱吞噬细胞的异常激活。
业已惊奇地发现,益生添加剂可以诱导炎症过程的减弱,特别是能诱导非特异性免疫的改变,如减弱了的吞噬细胞活性,以及白介素-6mRNA在外周血单核细胞中的减弱了的表达。
在第二方面,本发明提供了至少一种益生素用于生产用来减弱哺乳动物的炎症过程的药物或食品或宠物食品组合物的用途。
在第三方面,本发明提供了至少一种益生素用于生产用来减弱哺乳动物的非特异性免疫参数的异常激活的药物或食品或宠物食品组合物的用途。
在第四方面,本发明提供了一种减弱哺乳动物体内炎症过程的方法,该方法包括给予有效量的益生素或含有至少一种益生素的组合物。
在第五方面,本发明提供了一种减弱哺乳动物体内非特异性免疫参数的异常激活的方法,该方法包括给予有效量的益生素或含有至少一种益生素的组合物。
本发明的一个优点是,它能够减弱炎症过程,特别是减弱白介素-6mRNA在外周血单核细胞中的表达。
本发明的另一个优点是它能减弱粒细胞和单核细胞的吞噬细胞活性,特别是在患有慢性炎症状况的虚弱患者体内的活性。
本发明的再一个优点是,通过简单地消耗本发明的食品组合物,它可用于改善哺乳动物的炎症状况,并且因此降低发生有害感染的危险。可以理解的是,静脉内或皮下注射药物需要专业知识,并且与口服相比,注射不够安全、方便或者不容易被患者接受。考虑到这些因素,本发明提供了具有可以口服的营养性和/或治疗性产品的明确优点。
详细说明
根据第一方面,所述组合物优选含有至少一种益生素或益生混合物。
所述益生素优选包括由葡萄糖、半乳糖、木糖、麦芽糖、蔗糖、乳糖、淀粉、木聚糖、半纤维素、菊糖、树胶(例如阿拉伯树胶)或其混合物产生的寡糖。所述寡糖更优选包括寡聚果糖(FOS),所述益生素最优选是寡聚果糖和菊糖的混合物。该混合物优选包括或市售和的混合物。
所述益生素优选包括大约50%-95%FOS。更优选包括大约60%-80%FOS。最优选包括大约70%FOS。
所述益生素优选包括大约10%-50%菊糖。更优选包括大约20%-40%菊糖。最优选包括大约30%菊糖。
所述益生素可以包括寡聚果糖和菊糖的混合物,其重量百分比为寡聚果糖70%,菊糖30%。
所述组合物除了所述益生素之外,优选含有一种益生菌。例如,所述益生菌可以是双歧双歧杆菌或嗜热链球菌。所述双歧杆菌优选是乳酸双歧杆菌。
在一种实施方案中,所述组合物可以是完全的和营养平衡的食品或宠物食品。例如,它还可以是食品添加剂。所述组合物优选是针对老年人或老年宠物或患有慢性炎症的危重疾病患者。
因此,可以制备一种营养全面的宠物食品。所述营养全面的宠物食品可以是任何合适形式的;例如干燥形式、半潮湿形式或潮湿形式;它可以是冷藏或货架储存稳定的宠物食品。所述宠物食品可以按常规方法生产。所述益生素优选是以含有所述益生素的植物材料形式提供的。合适的植物材料包括芦笋、洋蓟、洋葱、小麦、yacon或菊苣,或所述植物材料的残余物。另外,所述益生素能够以菊糖提取物或其水解产物形式提供,通常被称为寡聚果糖、寡聚半乳糖、寡聚木糖或淀粉的寡聚衍生物。来自菊苣的提取物是特别适合的。益生素在宠物食品中的最大含量优选占重量的大约20%;特别是占重量的大约10%。例如,所述益生素可以占宠物食品重量的大约0.1%-大约5%。对于用菊苣作为益生素的宠物食品来说,所添加的菊苣可以占该食品混合物重量的大约0.5%-大约10%;更优选占重量的大约1%-大约5%。
除了本发明的益生素之外,所述宠物食品还可以包括任何一种或多种糖类来源、蛋白来源或脂类来源。
可以使用任何合适的糖类来源。所述糖类来源优选是以谷物、面粉和淀粉形式提供的。例如,所述糖类来源可以是大米、大麦、高粱、小米、燕麦、玉米粉或小麦面。还可以使用诸如蔗糖、葡萄糖和玉米浆的简单糖类。可以根据需要选择由所述糖类来源所提供的糖类数量。例如,宠物食品可以含有高达占重量的大约60%的糖类。
合适的蛋白来源可以从任何合适的动物和植物蛋白来源中选择;例如肌肉或骨骼肉,肉和骨粉、禽肉粉、鱼肉粉、乳蛋白、玉米面筋、小麦面筋、大豆粉、大豆蛋白浓缩物、大豆蛋白分离物、卵蛋白、乳清、酪蛋白、和面筋等。对于老年动物来说,所述蛋白来源优选含有优质动物蛋白。可以根据需要选择由所述蛋白来源所提供的蛋白数量。例如,以干重量为基础计算,宠物食品可以含有大约12%-70%的蛋白。
所述宠物食品可以含有脂肪来源。任何合适的脂肪来源都可以使用,包括动物脂肪和植物脂肪。优选的脂肪来源是动物脂肪来源,如牛脂。还可以使用植物油类,如玉米油、向日葵油、红花油、油菜籽油、大豆油、橄榄油和富含单不饱和和多不饱和脂肪酸的其他油类。除了必须脂肪酸(亚油酸和α亚油酸)之外,所述脂肪来源还可以包括长链脂肪酸。合适的长链脂肪酸包括γ亚油酸、硬脂烯酸、花生四烯酸、十二碳五烯酸、和二十二碳六烯酸。鱼油是十二碳五烯酸和二十二碳六烯酸的合适来源。玻璃苣油、黑醋栗籽油和月见草油是γ亚油酸的合适来源。油菜籽油、大豆油、亚麻油和胡桃油是α亚油酸的合适来源。红花油、向日葵油、玉米油和大豆油是亚油酸的合适来源。橄榄油、油菜籽油(canola)、高油向日葵和红花、花生油、稻米糠油是单不饱和脂肪酸的合适来源。可以根据需要选择由所述脂肪来源所提供的脂肪的数量。例如,以干重量计,所述宠物食品可以含有重量百分比为大约5%-大约40%的脂肪。所述宠物食品优选具有较少数量的脂肪。
所述宠物食品可以含有其他活性剂,如长链脂肪酸。合适的长链脂肪酸包括α亚油酸、γ亚油酸、亚油酸、十二碳五烯酸、和二十二碳六烯酸。鱼油是十二碳五烯酸和二十二碳六烯酸的合适来源。玻璃苣油、黑醋栗籽油和月见草油是γ亚油酸的合适来源。红花油、向日葵油、玉米油和大豆油是亚油酸的合适来源。
糖类、蛋白和脂类来源的选择并不是关键的,并且可以根据动物的营养需要、适口性因素、以及所生产的产品类型选择。另外,根据需要,还可以将各种其他成分掺入所述宠物食品,例如糖、盐、香料、调料、维生素、矿物质、芳香剂、树胶、和益生性微生物。
还可以添加益生性微生物。所述益生性微生物可以选自适合动物消耗并且能够改善肠道中微生物平衡的一种或多种微生物。合适的益生性微生物的例子包括酵母,如啤酒糖酵母属、德巴利酵母属、念珠菌属、毕赤酵母属和球拟酵母属;霉菌,如曲霉属、根霉属、毛霉属、和青霉属以及球拟酵母属;以及细菌,如双歧杆菌属、拟杆菌属、梭菌属、梭杆菌属、蜜蜂球菌属、原粘杆菌属、链球菌属、肠球菌属、乳球菌属、葡萄球菌属、消化链球菌属、芽孢杆菌属、片球菌属、微球菌属、明串珠菌属、魏斯氏菌属、气球菌属、酒球菌属和乳杆菌属。所述益生性微生物可以是粉状的干燥形式的;特别是对于能产生孢子的微生物来说是孢子形式的。另外,如果需要的话,可以将所述益生性微生物胶囊化,以便进一步提高存活的可能性;例如,胶囊化于糖基质、脂肪基质或多糖基质中。如果使用益生性微生物的话,所述宠物食品优选在每克宠物食品中含有大约104-1010细胞的所述益生性微生物;更优选每克食品含有大约106-大约108细胞的所述益生性微生物。所述宠物食品含有重量百分比为大约0.5%-大约20%的所述益生性微生物的混合物;优选大约1%-大约6%的重量百分比;例如,大约3%-大约6%的重量百分比。
对于老年宠物来说,所述宠物食品优选含有与用于年轻宠物的食品相比成比例地减少的脂肪。另外,所述淀粉来源可以包括燕麦、大米、大麦、小麦和玉米中的一种或多种。
对于干燥的宠物食品来说合适的工艺是挤压蒸煮,不过,也可以使用烘烤和其他合适的工艺。在进行挤压蒸煮时,所述干燥的宠物食品通常是以粗粒形式提供的。如果使用益生素的话,可以在加工之前将所述益生素与所述干燥宠物食品的其他成分混合。在欧洲专利申请号0850569中披露了一种合适的工艺。如果使用益生性微生物的话,最好将所述微生物包衣在干燥的宠物食品上或填充到宠物食品内。在欧洲专利申请号0862863中披露了一种合适的工艺。
对于潮湿的宠物食品来说,可以用披露于美国专利4,781,939和5,132,137中的方法生产模拟的肉类食品。还可以使用用于生产大块型产品的其他方法;例如,在蒸汽烤炉中蒸煮。另外,可以生产长方形食品,包括乳化合适的肉类材料以便生产肉类乳液,添加合适的胶凝剂,并且在填充到包装罐或其他容器中之前,对所述肉类乳液进行加热。
在另一种实施方案中,制备了一种供人类消耗的食品组合物。该组合物可以是营养全面的配方、乳制品、冷藏的或货架储存稳定的饮料、汤、食品添加剂、谷粉替代品、和营养棒或糖果。
除了本发明的益生素之外,所述营养配方还可以包括蛋白来源。优选将饮食蛋白用作蛋白来源。饮食蛋白可以是任何合适的饮食蛋白;例如动物蛋白(如乳蛋白、肉蛋白和卵蛋白);植物蛋白(如大豆蛋白、小麦蛋白、大米蛋白和豌豆蛋白);游离氨基酸的混合物;或其组合。诸如酪蛋白的乳蛋白、乳清蛋白和大豆蛋白是特别优选的。所述组合物还可以含有糖类来源和脂肪来源。
如果所述营养配方包括脂肪来源的话,所述脂肪来源优选能提供所述营养配方的大约5%-大约55%的能量;例如大约20%-大约50%的能量。组成所述脂肪来源的脂类可以是任何合适的脂肪或脂肪混合物。植物脂肪是特别适合的;例如大豆油、棕榈油、椰子果油、红花油、向日葵油、玉米油、canola油、和卵磷脂等。如果需要的话,还可以添加动物脂肪,如乳类脂肪。
可以在所述营养配方中添加糖类来源。糖类来源优选提供所述营养组合物的大约40%-大约80%的能量。可以使用任何合适的糖类,例如蔗糖、乳糖、葡萄糖、果糖、玉米糖浆固体和麦芽糖糊精及其混合物。如果需要的话,还可以添加食用纤维。如果使用的话,它优选占所述营养配方能量的大约5%。所述食用纤维可以来自任何合适的来源,例如,包括大豆、豌豆、燕麦、果胶、瓜耳胶、阿拉伯树胶、和寡聚果糖。在所述营养配方中还可以添加满足适应的指导数量的合适的维生素和矿物质。
如果需要的话,可以将一种或多种食品级乳化剂添加到所述营养配方中;例如二乙酰酒石酸单-和双-甘油酯,卵磷脂和单-和双-甘油酯。类似地,可以添加合适的盐和稳定剂。
所述营养配方优选是可以肠道给药的;例如以粉末、片剂、胶囊、液体浓缩物、固体产品或便于饮用的饮料形式提供。如果需要生产粉末状的营养配方的话,可以将均化的混合物转移到一个诸如喷雾干燥器或冷冻干燥器的合适的干燥装置中,并且转化成粉末。
在另一种实施方案中,一种营养组合物包括与益生制剂混合在一起的乳基谷物。所述乳基谷物优选是婴儿谷物,它起着所述益生制剂的载体的作用。
在另一种实施方案中,可以用本发明的至少一种益生素强化常规的食品。例如,发酵乳、酸奶、鲜奶酪、凝乳、糖果食品,例如甜味或增甜饮料、糖果棒、早餐谷片或条、饮料、奶粉、大豆基产品,非乳发酵产品或用于临床营养的营养添加剂。所述组合物的添加量优选至少占重量的大约0.01%。
下面所提供的实施例仅仅是说明性的,并且不能以任何形式理解成是对本发明主题的限定。百分比和份数是以重量为基础的,除非另有说明。在实施例之前是对附图的扼要说明。
图1:食用8克短链寡聚果糖(FOS)对疗养院的老人的新鲜粪便样品中双歧杆菌的活菌数量的影响。所述时间点是在为期3周FOS摄取之前(2)、期间(3)、和之后(4)。(A)各个值。(B)方框图。方框表示25th和75th百分位数,在方框内的实线条表示中间值,和t型5th和95th百分位数;实心点表示外侧值。
图2:寡聚果糖(FOS)对成年人和老年人体内双歧杆菌的影响。编号24(Bouhnik Y等,J Nutr 1999;129:113)在18-47岁的成年人中来自2.5-20克FOS/天的剂量反应;编号25(Menne E等,J Nutr2000;130:1197)在20-50岁的成年人体内对8克FOS/天的反应;编号26(Gibson GR J Nutr 1999;129:1438S)在年轻成年人体内对15克FOS/天的反应;编号27(Kruse HP等,Brit J Nutr 1999;82:375)在26-53岁成年人体内对高达34克/天的菊糖的反应;编号28(Kleessen B等,Am J Clin Nutr 1997;65:1397)在年龄为68-89岁的老年便秘受试者中对20克FOS/天的反应。本研究:在年龄为77-91岁的疗养院的受试者对8克FOS/天的反应。
图3:在补充了FOS的老年人的外周血单核细胞(PBMC)中IL-6mRNA表达的差异。通过逆转录-聚合酶链式反应(RT-PCR)测定IL-6mRNA在PBMC中的表达。通过用NIH-图象程序扫描带强度,估算定量改变,并且以IL-6 mRNA与β-肌动蛋白mRNA的百分比的密度比例形式计算。时间点为在为期3周的FOS摄取之前(2)、期间(3)、和之后(4)。(A)来自溴化乙锭染色凝胶的各个改变的典型图象。(B)IL-6mRNA表达的定量评估:在FOS摄取期间,IL-6mRNA的改变明显不同于在摄取FOS之前的改变(p=0.018)。
实施例1:寡糖对老年人粪便菌群和非特异性免疫系统的影响
材料和方法
研究设计
本研究是对19位疗养院的老年患者的试验前/试验后研究(参见研究方案)。
在时间点1(试验前或在洗脱期开始的时候),2(在摄取益生素之前),3(在摄取益生素期间),和4(随访期后)测定体重并且采集大便、血液和尿液样品。
在整个研究期间,限制发酵乳制品的摄取,并且限制含有寡聚果糖的食品(洋葱、韭菜、菊苣根)。
受试者
征集19位疗养院的老年受试者用于研究。在本研究中排除满足下列标准中的一项或多项的受试者:
在上个月做过抗生素治疗
慢性肠道疾病
特殊饮食方案(即素食者)
诊断出胃肠癌
出现肠胃气胀
获得了研究伦理委员会的批准。获得了来自所有受试者的知情同意书。本研究是在一家当地疗养院进行的,Le Mont-P lerin,Switzerland。
营养状态
在开始研究时,通过最低营养评估(MNA)测试评估营养状态,该测试包括以下项目:人体测量(小腿围和臂围、身高、体重、和体重减少),一般性评估(生活方式、医疗、运动能力),饮食询问(用餐次数、摄取的液体和食物、进食的自主性),和主观评估(对健康和营养的自我感觉)[Guigoz,Y.,等.Nutr Rev 1996;54:S59-S65]。MNA使用30分的标准对老年人营养状态进行分类:MNA≥24=营养良好,MNA 17-23.5=有营养不良的危险,而MNA<17=营养不良。
益生素补充
按以下方式每天给予8克短链寡聚果糖(FOS):每天2次,4克FOS粉末(Actilight 950P,Béghin-Meiji industries,Neuilly-sur-Seine,France),由护理人员在用餐时掺入饭菜中。为了评估并发症,由护士在每天的记录表上记录每天对添加剂的消耗量。
微生物学研究
统计了乳杆菌属、拟杆菌属、肠杆菌科、肠球菌属、双歧杆菌属和产气荚膜梭菌的内源群体。
在第0、21、42和63天收集每一位受试者的大便样品。将所述大便样品立即(在30分钟之内)放入一个厌氧罐中,并且在4℃下保存直到分析(最长6小时)。用含有0.5%半胱氨酸的预先还原的Ringer溶液进行100倍的系列稀释,从-2到-8。接种各种培养基的培养皿,并且使用Anaerocult A(Merck,Darmstadt,Germany)在37℃下,在厌氧环境中温育48小时,所不同的是,肠球菌和肠杆菌是在37℃下,在需氧环境下温育24小时。按以下方法在选择性或半选择性培养基上检测细菌:在Drigalski培养基上检测肠杆菌科(SanofiDiagnostics Pasteur,France),在Eugon Tomato培养基上检测双歧杆菌属(Wadsworth Anaerobic Bacteriology Manual,V.Suter,D.Citron and S.Finegold Third edition),在含有抗生素(Phosphomycine(79.5mg/l)+磺甲恶唑(0.93mg/l)+甲氧苄啶(5mg/l))的MRS(Difco,MI.USA)中检测乳杆菌属,在NN琼脂(Lowbury and Lilly,1995)检测产气荚膜梭菌,在Neo-Vanco培养基(BioMrieux,Marcy-1′Etoile,France)上培养拟杆菌属,并且在Azide琼脂上检测肠球菌(Difco)。
在温育之后,统计菌落数量,并且在必要时进一步鉴定。通过显微技术鉴定乳杆菌和双歧杆菌菌株,并且使用API观察系统(BioMrieux)进行生物化学鉴定,分别将API 50 CHL观察系统用于乳杆菌,将API ID 32A观察系统用于双歧杆菌属。细菌数量是以每克新鲜粪便样品中的log10菌落形成单位(CFUs)形式表示的,检测限为3.30cfu/g。
粪便pH
在排便后不久由护士用微电极(Orion)在粗粪便上的三个不同点上测定粪便pH。计算每一个时间点上的平均pH。
血液取样
在研究开始之前(4ml)以及在第0、21、42和63天(23ml)从每一位禁食的受试者体内抽取血液样品。血液是在禁食一夜之后在上午(在上午10点之前)采集的。将样品收集在真空容器管中。将13ml样品收集在肝素化试管中,并且让10ml样品在室温下凝固30-60分钟。血清样品在-70℃下冷冻保存直到分析。将肝素化的血液用于功能性免疫参数(吞噬作用,参见下文)和血液学。
免疫学分析
通过Simulset和Pagotest(Becton Dickinson,Basel,Switzerland)在新鲜肝素化血液样品中分析外周血单核细胞群及其吞噬活性。
通过PCR分析白介素-6mRNA表达:在外周血单核细胞中mRNA表达的分析是按照Delneste,Y.,等(Nutr Rev 1998;56:S93-S98)所披露的方法通过PCR评估的。引物序列为:
5′-CTGCAGGAACTGGATCAGGACTTTTGTACT-3′和
5′-GCCTTCGGTCCAGTTGCCTTCTCCCTGGGG-3′用于白介素-6(IL-6)和
5′CGTTTCCCGCTCGGCCGTGGTGGTGAAGC-3′和
5′-GGCGACGAGGCCCAGAGCAAGAGAGGCATC-3′用于β-肌动蛋白。
生物学测定
使用Behring浊度计,通过免疫-浊度法分析血清白蛋白、运甲状腺素蛋白(前白蛋白)、C-反应蛋白、和α1-酸性糖蛋白浓度(方法和试剂由Behring,Marburg,Germany提供)。通过放射免疫测定分析叶酸和维生素B12(钴胺素)浓度(Dual Count,Diagnostic ProductCorporation,Los Angeles,California,USA)。
统计学分析
根据平均差计算单样品t-检验或Wilcoxon signed-rank检验,以便评估主要假设的在0.05%水平上的统计学显著性:益生素对所测定的不同参数是否有作用?根据所述平均差计算另一个单样品t-检验,以便评估与本研究的设计相关的两个问题(包括试验前和试验后期)在0.025%水平上的统计学显著性(对自由度损失进行校正:终止发酵乳(=洗脱期)是否对测定的不同参数有影响?终止益生素(=随访期)是否对测定的不同参数有影响?所有统计学分析都是用NCSS 6.0.22统计学软件完成的。
结果
受试者
所述19位受试者(4个男性和15个女性)的平均年龄为85±6.0(77-97岁)。女性体重明显比男性轻。在表1中提供了在研究开始时受试者的特征。
表1:受试者特征
只有一位受试者患有营养不良,7位具有患营养不良危险的女性处在21.5-23的较高得分范围内,而营养良好的平均MNA得分范围为24.6±3.0分。
血清白蛋白(正常范围35-55g/L)和运甲状腺素蛋白(前白蛋白;正常范围0.16-0.40)在较低的正常范围内,而急性期蛋白、α1-酸性糖蛋白(正常范围0.5-1.3)和C-反应蛋白(正常值<10mg/L,而老年人的异常值>20mg/L)表明不存在炎症过程,只有2位男性(具有较高的C-反应蛋白水平)和2位女性(低白蛋白水平,具有正常的运甲状腺素蛋白和临界C-反应蛋白)。以上结果表明,有一类老年人仍然营养良好,但是相当虚弱。
细菌学分析和粪便pH
在补充FOS的3周时间内,双歧杆菌属的细菌数量的log10CFU平均值提高了2.8±0.57(p<0.001),但是拟杆菌属数量也增加了(p<0.032)(参见表2a和2b以及图1)。
表2a:给予FOS对粪便菌群和pH的影响
95%CI=±95%置信区间
肠杆菌科、肠球菌属和乳杆菌属的细菌数量没有受到抑制乳制品发酵和/或摄取寡聚果糖(FOS)的显著影响。不过,对于双歧杆菌属来说,所述影响似乎是对摄取FOS的一种特异性反应,它主要导致了在开始使用FOS之前表现出log10CFU低于7的受试者的双歧杆菌属数量的增加。抑制FOS补充,显著降低了双歧杆菌属的数量,log10CFU降低了1.1±0.39,不过,没有达到起始水平(表2a和2b以及图1)。在图2中,比较了在本研究中获得的双歧杆菌属的变化和以前研究中的变化,表明老年人对FOS作用的敏感程度至少与年轻的成年人一样。抑制发酵的乳制品或摄取FOS,都没有改变粪便pH(表2a)。
非特异性免疫
摄取FOS导致了外周T淋巴细胞以及淋巴细胞亚型CD4+,CD8+T细胞百分比显著增加(表3a和3b)。白细胞、激活的T淋巴细胞和天然杀伤(NK)细胞的总数量没有受到摄取FOS的影响(表3a和3b)。
表3a:外周免疫学参数:淋巴细胞亚群
95%CI=+/-95%置信区间
表3b-c-d:外周免疫学参数的改变
所提供的数据是在每个3周时间之后的平均差±平均值的标准误差(sem)
1FOS=4g/每日两次,2终止发酵乳制品的作用=在试验前取样和开始补充益生素之间的平均变化(3周洗脱期),3补充FOS的作用(4克/每日两次)=在开始补充益生素和补充结束时之间的平均变化(3周益生素摄取期),4终止补充FOS的作用=补充益生素结束和本研究结束之间的平均变化(3周随访期),5对平均差的单样品t-检验。
通过摄取FOS显著降低了粒细胞和单核细胞的吞噬活性:吞噬活性以平均荧光强度改变形式表示,对于粒细胞来说从130±10降低到52±2(p<0.001),而对于单核细胞来说从75±5降低到26±2(p<0.001)。
在摄取FOS之后外周血单核细胞中白介素-6mRNA的含量的显著降低,同样表明了炎症过程的可能的减轻(图3)。
维生素B12和叶酸状态
补充FOS没有影响维生素B12或叶酸血清含量:在补充之前血清维生素B12含量为271±143ng/L,而在补充期间为289±160ng/L。不过有3位受试者缺乏维生素B12。有2位受试者在研究期间恢复到正常状态,而其他的受试者在整个研究期间保持缺乏。在补充之前血清叶酸含量为5.9±2.0ug/L,而在补充期间为5.7±1.9ug/L。
我们的结果有力支持了寡聚果糖在老年受试者中的产双歧杆菌作用,双歧杆菌属数量增加了2个对数值,因为我们的虚弱的老年受试者在研究开始时表现出低的双歧杆菌属数量。外周血单核细胞中IL-6mRNA表达的减弱表明了炎症过程的减弱。
实际上,本研究证明了补充FOS对在成年人和老年人中所观察到的双歧杆菌属的正影响(图2),表明与年轻的成年人一样,老年人对益生素(FOS)的反应是双歧杆菌属的增加,或者如果双歧杆菌属的数量很低的话结果会更好。另外,8克或更少的FOS似乎就足以获得对双歧杆菌属数量的最大影响。尽管不同的研究似乎存在一种剂量反应,单一的研究表明,超过4-5克/天的阈值获得了最大反应,并且双歧杆菌属的增加似乎在更大程度上取决于起始数量(图1和2)。通常添加到饮食中的FOS可以增加双歧杆菌属的含量,其代价是减少了潜在的有害细菌,主要是梭菌属和拟杆菌属。不过,在本研究中我们观察到了拟杆菌属的显著增加(表2a和2b)。
我们发现了吞噬活性的显著减弱。这种吞噬活性的减弱病可能是与致病细菌的可能减少相关的巨噬细胞激活减弱的一种反映,并因此暗示了由于具有较低的内毒素负担而减轻了炎症。不过,令人吃惊的是,炎症过程的这种可能的减轻,是通过外周血单核细胞中白介素-6mRNA含量的降低表明的(图3)。
例2:食品添加剂
通过以大约70%的寡聚果糖与大约30%的菊糖的重量比例混合或掺合寡聚果糖和菊糖制备了一种食品添加剂。所得到的益生混合物可以添加或掺合任何合适的载体,例如,发酵乳、酸乳、鲜奶酪、凝乳、糖果条、早餐谷物片或条、饮料、奶粉、大豆基食品、非乳发酵制品或用于临床营养的营养添加剂。
例3:干燥的宠物食品
一种饲料混合物是由重量百分比为大约58%的玉米、重量百分比为大约5.5%的玉米面筋、重量百分比为大约22%的鸡肉粉、2.5%的干燥菊苣、1%的肉碱、组成其余部分的盐、维生素和矿物质组成。
将所述饲料混合物添加到一个预调节器中,并且湿化。然后将所述湿化的饲料添加到一个挤出器-蒸煮器中,并且明胶化。离开所述挤出器的明胶化基质强制通过一个模具并且挤出。将挤出物切割成适合饲养狗的小块,在大约110℃下干燥大约20分钟,并且冷却以便形成团。
这种干燥的狗食特别适用于减轻炎症过程和/或诸如吞噬细胞的非特异性免疫参数的异常结果。
例4:湿的罐装宠物食品
用56%的禽骨架,猪肺和猪肝(研磨物),3%的鱼,16%的小麦粉,8%的血浆,10.8%的水,2.2%的染料、1%的半精炼κ角叉菜胶,无机盐和9%的富含单不饱和脂肪酸的油(橄榄油)和3%的菊苣制备一种混合物。在12℃下乳化该混合物,并且以布丁形式挤出,然后在90℃下蒸煮。将其冷却到30℃,并且切割成厚块。
将30%的这种厚块(含水量为58%)掺入由23%的禽骨架,1%的瓜耳树胶,1%的染料和香料以及75%的水制备的基料中。然后填充到镀锡铁皮罐中,并且在127℃下消毒60分钟。
Claims (7)
1.至少一种益生素用于生产用来减轻老年人或老年宠物的炎症过程的药物或食品或宠物食品组合物的用途,其中所述益生素是寡糖。
2.权利要求1的用途,其中所述药物或食品或宠物食品组合物减轻老年人或老年宠物的非特异性免疫参数的异常激活。
3.如权利要求1或2的用途,其中,所述益生素包括由葡萄糖、半乳糖、木糖、麦芽糖、蔗糖、乳糖、淀粉、木聚糖、半纤维素、菊糖、树胶或其混合物产生的寡糖。
4.如权利要求1或2的用途,其中,所述益生素包括寡聚果糖或寡聚果糖和菊糖的混合物。
5.如权利要求1或2的用途,其中,所述益生素包括重量百分比为60%-80%的寡聚果糖,和20%-40%的菊糖。
6.如权利要求1或2的用途,其中,所述益生素是以与益生菌的混合物形式提供的。
7.权利要求6的用途,其中所述益生菌是双歧杆菌或嗜热链球菌。
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EP01201091A EP1243273A1 (en) | 2001-03-22 | 2001-03-22 | Composition comprising a prebiotic for decreasing infammatory process and abnormal activation of non-specific immune parameters |
EP01201091.4 | 2001-03-22 | ||
PCT/EP2002/002905 WO2002076471A1 (en) | 2001-03-22 | 2002-03-15 | Composition comprising a prebiotic for decreasing inflammatory process and abnormal activation of non-specific immune parameters |
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CN1738595A CN1738595A (zh) | 2006-02-22 |
CN1738595B true CN1738595B (zh) | 2011-01-12 |
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US (1) | US8092608B2 (zh) |
EP (2) | EP1243273A1 (zh) |
JP (1) | JP4456329B2 (zh) |
CN (1) | CN1738595B (zh) |
AR (1) | AR033439A1 (zh) |
AT (1) | ATE350045T1 (zh) |
CA (1) | CA2442053C (zh) |
DE (1) | DE60217319T3 (zh) |
EA (1) | EA006441B1 (zh) |
ES (1) | ES2278021T5 (zh) |
PE (1) | PE20020986A1 (zh) |
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WO2002076471A1 (en) | 2002-10-03 |
DE60217319D1 (de) | 2007-02-15 |
EP1383514B1 (en) | 2007-01-03 |
DE60217319T3 (de) | 2019-04-18 |
ES2278021T3 (es) | 2007-08-01 |
CN1738595A (zh) | 2006-02-22 |
DE60217319T2 (de) | 2007-10-04 |
UY27221A1 (es) | 2002-08-30 |
EA200301044A1 (ru) | 2004-04-29 |
EP1243273A1 (en) | 2002-09-25 |
JP4456329B2 (ja) | 2010-04-28 |
CA2442053A1 (en) | 2002-10-03 |
EP1383514B2 (en) | 2018-12-19 |
ES2278021T5 (es) | 2019-05-13 |
ATE350045T1 (de) | 2007-01-15 |
JP2004529910A (ja) | 2004-09-30 |
ZA200308189B (en) | 2005-03-30 |
PE20020986A1 (es) | 2003-01-02 |
AR033439A1 (es) | 2003-12-17 |
EA006441B1 (ru) | 2005-12-29 |
EP1383514A1 (en) | 2004-01-28 |
US8092608B2 (en) | 2012-01-10 |
US20040219157A1 (en) | 2004-11-04 |
CA2442053C (en) | 2012-07-10 |
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