CN102858354B - 包含用于婴儿和幼儿的益生菌的婴儿和幼儿喂养配方 - Google Patents
包含用于婴儿和幼儿的益生菌的婴儿和幼儿喂养配方 Download PDFInfo
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- CN102858354B CN102858354B CN201080031177.8A CN201080031177A CN102858354B CN 102858354 B CN102858354 B CN 102858354B CN 201080031177 A CN201080031177 A CN 201080031177A CN 102858354 B CN102858354 B CN 102858354B
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Abstract
本发明涉及婴儿和幼儿营养领域。特别的是,本发明涉及施用于大于6个月的婴儿和幼儿的包含益生微生物的婴儿和幼儿喂养配方。这些益生微生物可以是非复制的益生微生物,例如生物活性热处理的益生微生物。
Description
本发明涉及婴儿和幼儿营养领域。特别的是,本发明涉及施用于大于6个月的婴儿和幼儿的包含益生微生物的婴儿和幼儿喂养配方。这些益生微生物可以是非复制的益生微生物,例如生物活性热处理的益生微生物。
母乳是婴儿健康成长和发育的理想食物。在2001年世界卫生组织(WHO)改变了它推荐的单独母乳喂养持续时间,从4至6个月变为6个月,因此,母乳喂养应该得到鼓励和相应地促进。
从第6个月开始婴儿的行为变化。他们第一次坐起来或仅用一只手抓起玩具。婴儿的营养需求随着婴儿发育而变化。营养物如铁和钙变得甚至更加重要。
为了通过合适的营养来满足如此快速的发育,开发了婴儿喂养配方,在从母乳喂养转变成食用固体食物时期该配方提供了最佳营养。
这种婴儿和幼儿喂养配方是例如后继配方(follow-up-formula)。后继配方表示旨在用作第6个月后的婴儿和幼儿的断乳膳食的液体部分的食物。
典型的婴儿和幼儿喂养配方是营养完整的,包含完整蛋白质,并且是以牛乳为基础。它们设计用于补充更大婴儿改变的膳食。
婴儿早期营养的一个重要功能是产生健康的肠道菌群和产生强大的免疫系统。
健康的肠道菌群将有助于功能的胃肠道,反过来其将帮助适当地消化摄入的食物并且减轻婴儿和幼儿胃痛。
婴儿和幼儿典型地每年感冒约10次。由于婴儿在奶瓶喂养时通过鼻子呼吸,感冒会引起问题。
因此,需要进一步改善婴儿和幼儿喂养配方的免疫增强作用。
还需要进一步改善婴儿和幼儿喂养配方的抗炎作用。
因此,本领域需要给婴儿提供尽可能接近母乳的营养的婴儿和幼儿喂养配方。这种婴儿喂养配方应当具有改善的免疫增强作用、抗炎作用和/ 或应当易于消化。优选使用天然成分获得这种配方,所述的天然成分对于施用者是安全的,无副作用并且易于使用本领域的工业技术掺入到婴儿喂养配方中。
本发明人解决了该需求。因此,本发明的目的是改善现有技术状态并且提供能满足上述需求的婴儿和幼儿喂养配方。
本发明人令人惊讶地发现他们通过独立权利要求的主题能够实现该目的。从属权利要求进一步发展了本发明的想法。
因此,本发明人计划提供一种包含益生菌的婴儿和幼儿喂养配方。
FDA规定定义婴儿为不超过12个月的人(标题21,Code of Federal Regulations 21 CFR 105.3(e))。
发现益生菌能够在婴儿和幼儿喂养配方的框架中提供它们的健康益处。因此,例如双歧杆菌属存在于母乳中并且是给予母乳天然保护性质的部分。
因此,向婴儿和幼儿喂养配方中添加益生微生物将使得它们更接近母乳。
然而,特别是由于用水重构的粉末喂养配方的贮存期限通常超过例如酸奶饮料(yoghurt drinks)(包含益生菌)的贮存期限,因此通常不向这类喂养配方中添加益生菌,因为例如在延长的贮存期限内确保益生菌存活的不确定性。
本发明人目前能证明,甚至非复制的益生菌能提供益生菌的健康益处并且甚至可能具有改善的益处。
因此,本发明的一个实施方案是施用于从6个月开始的婴儿或幼儿的婴儿或幼儿喂养配方,所述的配方向婴儿或幼儿提供完整的营养并且包含益生微生物。
本发明的喂养配方可施用于6-36个月、例如6-18个月的婴儿或幼儿。
术语“婴儿”表示不超过12个月的人。
术语“幼儿”表示超过12个月至36个月的人。
喂养配方可以以易于施用的液体组合物或者以在使用前用水重构的干燥组合物提供。
如果以干燥组合物提供,优选该组合物具有低于0.2、优选低于0.15的水活度,以进一步增加贮存稳定性。例如大多数细菌在低于0.91的水活度时不能生长,而且大多数霉菌在低于0.80的水活度时停止生长。
水活度(aw)是体系中水的能量状态的度量。其定义为水的蒸气压除以纯水的蒸气压。因此,蒸馏水的水压为1。
典型的后继配方提供了每100mL不小于60kcal(或250kJ)并且不大于85kcal(或355kJ)。后继配方还包含每100卡不小于3g并且不大于6g的脂肪。
本发明的喂养配方可以具有热量密度范围为62-68kcal/100mL,并且可以包含蛋白质源的量为1.9-3.5g/100kcal、碳水化合物源的量为12-13g/100kcal、脂质源的量为4-5g/100kcal。
蛋白质源可以包括乳清蛋白和酪蛋白。例如可以使用乳清与酪蛋白的比例范围为约35∶65至55∶45。为了增加的蛋白质需要,增加酪蛋白组分。例如对于2g蛋白质/100kcal,可以使用乳清/酪蛋白的重量比为1∶1。对于2.5g蛋白质/100kcal,可以使用乳清/酪蛋白的重量比为2∶3。
碳水化合物源可以基本上包括乳糖。然而,还可以使用例如乳糖和麦芽糊精比例范围为3∶1至1∶1。
本发明的喂养配方可以包含0.2-0.3g LC-PUFA/100g脂肪酸。LC-PUFA可以选自ARA、DHA或其组合。例如,LC-PUFA可以包含ARA和DHA的组合。含有DHA和ARA的配方已经显示出提供与母乳喂养的婴儿类似的视觉和智力发育。
本发明的喂养配方还可以包含每100mL配方1.5-2.5mg核苷酸。并不认为核苷酸及其碱基是“必需的”,因为它们能够通过婴儿机体由简单的化合物合成。然而,在某些时候,合成过程可能不能满足需求,例如,快速细胞更新期,如正常生长或肠疾病。在这些时候,机体更多地依赖核苷酸的饮食来源。
喂养配方可以包含部分或仅非复制的益生微生物。
本发明人令人惊讶地发现,例如就免疫增强作用和/或抗炎作用而言,非复制的益生微生物可能甚至比复制的益生微生物更有效。
令人惊讶的是,因为益生菌通常被定义为“当足量施用时,能向宿主提供健康益处的活的微生物”(FAO/WHO准则)。大多数公开的文献涉及活的益生菌。此外,某些研究调查非复制细菌产生的健康益处,并且它们大多数表明灭活的益生菌(例如通过热处理)导致它们声称的健康益处的丧失(Rachmilewitz,D.等人,2004,Gastroenterology 126:520-528;Castagliuolo等人,2005,FEMS Immunol.Med.Microbiol.43:197-204;Gill,H.S.和K.J.Rutherfurd,2001,Br.J.Nutr.86:285-289;Kaila,M.等人,1995,Arch.Dis.Child 72:51-53)。某些研究表明死的益生菌可能保留某些健康作用(Rachmilewitz,D.等人,2004,Gastroenterology 126:520-528;Gill,H.S.和K.J.Rutherfurd,2001,Br.J.Nutr.86:285-289),但是显然,目前在本领域中活的益生菌被认为更有效。
本发明的喂养配方可以包含任何有效量、例如相应于约106至1012cfu/g干重的量的益生微生物。
益生微生物可以是非复制的益生微生物。
“非复制的”益生微生物包括已经热处理的益生菌。这包括灭活的、死的、不存活的和/或以碎片(例如DNA、代谢物、细胞质化合物和/或细胞壁物质)存在的微生物。
“非复制的”表示没有可以通过常规平板培养法检测到的存活的细胞和/或集落形成单元。这些常规的平板培养法在微生物学书中有概述:James Monroe Jay,Martin J.Loessner,David A.Golden.2005.Modern food microbiology.第7版,Springer Science,New York,N.Y.790p。典型地,没有存活细胞具有如下表现:在接种不同浓度的细菌制品(“非复制的”样品)并且在合适的条件下温育(需氧和/或缺氧气氛下至少24小时)后琼脂平板上无可见的集落或液体生长培养基中浊度不增加。
对于本发明的目的,益生菌被定义为“对宿主的健康或幸福(well-being)具有有益作用的微生物细胞制品或微生物细胞组分”(Salminen S,Ouwehand A.Benno Y.等人“Probiotics:how should they be defined(益生菌:如何定义它们)”Trends Food Sci.Technol.1999:10107-10)。
使用非复制的益生微生物提供某些优势是可能的。在严重的免疫缺乏 婴儿中,在罕见的病例中由于存在发生菌血症的潜在风险而可能限制使用活的益生菌。可以使用非复制的益生菌而不存在任何问题。
此外,供应非复制的益生微生物允许热的重构,同时保留健康益处。
本发明的喂养配方包含足以至少部分产生健康益处量的益生微生物和/或非复制的益生微生物。足以实现该目的的量定义为“治疗有效剂量”。有效用于该目的的量取决于本领域技术人员已知的多种因素(例如婴儿的体重和一般健康状态)和食物基质的作用。
在预防应用中,本发明的喂养配方以足以至少部分降低发生该障碍的风险的量施用于易于发生该障碍或处于该障碍风险中的食用者。该量被定义为“预防有效剂量”。而且,准确的量取决于多种因素(例如婴儿的健康状态和体重)和食物基质的作用。
本领域的那些技术人员将能够适当地调整治疗有效剂量和/或预防有效剂量。
通常,本发明的喂养配方包含治疗有效剂量和/或预防有效剂量的益生微生物和/或非复制的益生微生物。
典型地,治疗有效剂量和/或预防有效剂量的范围为每日剂量约0.005mg-1000mg益生微生物和/或非复制的益生微生物。
就数值量而言,“短时高温”处理的非复制的微生物可以以相应于104至1012当量cfu/g干燥的组合物的量存在于喂养配方中。显然,非复制的微生物不形成集落,因此,该术语应当被理解为非复制的微生物的量,该量是由104至1012cfu/g复制的细菌获得的。这包括灭活的、无存活的或死的或以碎片(例如DNA或细胞壁或细胞质化合物)存在的微生物。换言之,喂养配方包含的微生物的量是以该量的微生物的集落形成能力(cfu)方式表示的,如同所有微生物是活的,无论它们事实上是否是非复制的,例如灭活的或死的、碎片或者任何或所有这些状态的混合物。
优选地,非复制的微生物是以等价于104至109cfu/g干燥的喂养配方的量存在的,甚至更优选是以等价于105至109cfu/g干燥的喂养配方的量存在的。
可以通过本领域已知的任何方法使得益生菌非复制。
目前可用于使得益生菌株非复制的技术通常是热处理、γ-辐射、UV光或使用化学试剂(福尔马林、低聚甲醛)。
优选使用食品工业中在工业环境下相对易于使用的技术,以使得益生菌非复制。
目前市售包含益生菌的大多数产品是在它们制备过程中热处理的。因此其便于能够与制备的产品一起或至少以类似的方式热处理益生菌,而益生菌保留或改善了它们的有益性质或甚至获得对食用者新的有益性质。
然而,文献中通过热处理灭活益生微生物通常与至少部分有益活性丧失有关。
目前本发明人令人惊讶地发现,例如通过热处理使得益生微生物非复制不会导致益生健康益处的丧失,相反,可能增强现存的健康益处,并且甚至产生新的健康益处。
因此,本发明的一个实施方案是喂养配方,其中非复制的益生微生物是通过热处理使得其非复制的。
这种热处理可以在至少71.5℃下进行至少1秒。
可以使用长期热处理或短期热处理。
目前在工业规模中通常优选短期热处理,例如UHT-样的热处理。这种方式的热处理减少了细菌载量,并且缩短了处理时间,从而降低了营养物的破坏。
本发明人首次证实了高温短时热处理的益生微生物表现出抗炎免疫特性,无论它们最初的性质。特别是通过这种热处理产生了新的抗炎特性或增强了现存的抗炎特性。
因此,目前通过使用特定的热处理参数产生具有抗炎免疫特性的非复制益生微生物是可能的,所述的参数相应于典型的工业上可用的热处理,即使活的对应物(counterpart)不是抗炎菌株。
因此,例如,热处理可以是在约71.5-150℃下高温处理约1-120秒。高温处理可以是高温/短时(HTST)处理或超高温(UHT)处理。
益生微生物可以在约71.5-150℃下高温处理约1-120秒的短期。
更优选的是益生微生物可以在约90-140℃、例如90-120℃下进行高温 处理约1-30秒的短期。
高温处理使得微生物至少部分非复制。
高温处理可以在正常大气压下进行,但是还可以在高压下进行。典型的压力范围是1至50巴、优选1-10巴、甚至更优选2-5巴。显然,当应用加热时,优选益生菌是在液体或固体培养基中热处理的。因此,应用的理想压力取决于其中提供微生物的组合物的性质以及所用的温度。
高温处理可以在温度范围为约71.5-150℃、优选约90-120℃、甚至更优选约120-140℃下进行。
高温处理可以进行约1-120秒、优选约1-30秒、甚至更优选约5-15秒的短期。
该给定的时间范围表示益生微生物经历给定温度的时间。值得注意的是,取决于其中提供微生物的组合物的性质和量以及取决于所用的加热装置的构造,热应用的时间可以不同。
然而,典型的是,本发明的喂养配方和/或微生物是通过高温短时(HTST)处理、快速巴氏消毒法或超高温(UHT)处理来处理的。
UHT处理是超高温处理或超热处理(均缩写为UHT),涉及在超过135℃(275℉)的温度下加热短时(约1-10秒)至少部分灭菌组合物,所述的温度是杀死乳中细菌芽孢所需的温度。例如,以这种方式使用超过135℃的温度处理的乳允许在必需的保持时间(2-5秒)内减少细菌载量,从而能够连续流操作。
存在两种主要类型的UHT系统:直接和间接系统。在直接系统中,产品是通过注入蒸汽或输入蒸汽处理的,而在间接系统中,产品是用板式换热器、管式换热器或刮面式换热器处理的。可以在处理产品制品中的任何步骤或多个步骤中使用UHT系统的组合。
HTST处理定义如下(高温/短时):设计巴氏消毒法以获得5个对数降低,杀死99.9999%数量的乳中的存活微生物。这被认为足以破坏几乎所有的酵母、霉菌和常见酸败菌,而且确保足以破坏常见的致病耐热生物。在HTST过程中,将乳加热至71.7℃(161℉)达15-20秒。
快速巴氏消毒法是易腐坏的饮料(如水果和蔬菜汁)、啤酒和乳制品的 热巴氏消毒法的一种方法。其是在装入容器之前进行的,以杀死酸败微生物,从而使得产品更安全并且延长它们的贮存期限。液体以可控的连续流流动,同时在71.5℃(160℉)至74℃(165℉)的温度下进行约15-30秒。
对于本发明的目的,术语“短时高温处理”应当例如包括高温短时(HTST)处理、UHT处理和快速巴氏消毒法。
由于这种热处理提供了具有改善的抗炎特性的非复制益生菌,本发明的喂养配方可以用于预防或治疗炎性障碍。
本发明的喂养配方可以治疗或预防的炎性障碍没有特别的限制。例如,它们可以选自急性炎症例如脓毒症;烧伤;和慢性炎症,例如炎性肠病,例如克隆病、溃疡性结肠炎、隐窝炎;坏死性小肠结肠炎;皮肤炎症,例如UV或化学诱导的皮肤炎症、湿疹、反应性皮肤;肠易激惹综合征;眼睛炎症;变态反应、哮喘;及其组合。
如果长期热处理用于使得益生微生物非复制,这种热处理可以在温度范围为约70-150℃下进行约3分钟-2小时、优选在温度范围为80-140℃下进行5分钟-40分钟。
然而现有技术通常教导通过长期热处理使得细菌非复制在执行它们的益生菌性质方面常常不如活细胞有效,本发明人能够证实热处理的益生菌在刺激免疫系统中优于它们的活对应物。
本发明还涉及包含益生微生物的喂养配方,所述的益生微生物是通过在至少约70℃下热处理至少约3分钟使得其非复制的。
通过体外免疫分析证实非复制益生菌的免疫增强作用。所用的体外模型使用来自人外周血单核细胞(PBMCs)的细胞因子分析,并且该模型是本领域广泛公认的测试免疫调节化合物的标准模型(Schultz等人,2003,Journal of Dairy Research 70,165-173;Taylor等人,2006,Clinical and Experimental Allergy,36,1227-1235;Kekkonen等人,2008,World Journal of Gastroenterology,14,1192-1203)。
数个作者/研究组已经使用体外PBMC分析例如根据它们的免疫特性(即它们的抗-或促-炎特性)来分类益生菌(Kekkonen等人,2008,World Journal of Gastroenterology,14,1192-1203)。例如,该分析已经显示了提 供预测益生候选物在结肠炎的小鼠模型中的抗炎作用(Foligne,B.等人,2007,World J.Gastroenterol.13:236-243)。而且,该分析常常用于临床试验的读出,并且显示出引起与临床结果相关的结果(Schultz等人,2003,Journal of Dairy Research 70,165-173;Taylor等人,2006,Clinical and Experimental Allergy,36,1227-1235)。
在过去的数十年内变态反应疾病稳定增加,并且目前它们被WHO认为是流行性的。以通常的方式,变态反应被认为源于免疫系统Th1和Th2响应之间的不平衡,从而导致强烈倾向于Th2介质的产生。因此,可以通过恢复免疫系统Th1和Th2臂之间的适当平衡来缓解、下调或预防变态反应。这意味着需要减少Th2响应或增强(至少短暂)Th1响应。后者是免疫增强响应的特征,通常伴随例如更高水平的IFNγ、TNF-α和IL-12(Kekkonen等人,2008,World Journal of Gastroenterology,14,1192-1203;Viljanen M.等人,2005,Allergy,60,494-500)。
因此,本发明的喂养配方使其用于治疗或预防与免疫防御缺乏相关的障碍。
因此,没有特别限制可以用本发明的喂养配方治疗或预防的与免疫防御缺乏相关的障碍。
例如,它们可以选自感染,特别是细菌、病毒、真菌和/或寄生物感染;吞噬细胞缺陷;低的严重免疫抑制水平,例如应激或免疫抑制药物、化学治疗或放射治疗诱导的那些;更低免疫活性的免疫系统的自然状态,例如新生儿那些;变态反应;及其组合。
本发明描述的喂养配方还使其增强婴儿对疫苗(特别是口服疫苗)的响应。
任何量的非复制微生物是有效的。然而,通常优选如果至少90%、优选至少95%、更优选至少98%、最优选至少99%、理想地至少99.9%、最理想所有的益生菌是非复制的。
在本发明的一个实施方案中,所有的微生物是非复制的。
因此,在本发明的喂养配方中,至少90%、优选至少95%、更优选至少98%、最优选至少99%、理想地至少99.9%、最理想所有的益生菌可以是非复制的。
所有益生微生物可以用于本发明的目的。
例如,益生微生物可以选自双歧杆菌属(bifidobacteria)、乳杆菌属(lactobacilli)、丙酸杆菌属(propionibacteria)或其组合,例如长双歧杆菌(Bifidobacterium longum)、乳双歧杆菌(Bifidobacterium lactis)、动物双歧杆菌(Bifidobacterium animalis)、短双歧杆菌(Bifidobacterium breve)、婴儿双歧杆菌(Bifidobacterium infantis)、青春双歧杆菌(Bifidobacterium adolescentis)、嗜酸乳杆菌(Lactobacillus acidophilus)、干酪乳杆菌(Lactobacillus casei)、类干酪乳杆菌(Lactobacillus paracasei)、唾液乳杆菌(Lactobacillus salivarius)、路氏乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、约氏乳杆菌(Lactobacillus johnsonii)、植物乳杆菌(Lactobacillus plantarum)、发酵乳杆菌(Lactobacillus fermentum)、乳乳球菌(Lactococcus lactis)、嗜热链球菌(Streptococcus thermophilus)、乳乳球菌(Lactococcus lactis)、双乙酰乳酸乳球菌(Lactococcus diacetylactis)、乳脂乳球菌(Lactococcus cremoris)、保加利亚乳杆菌(Lactobacillus bulgaricus)、瑞士乳杆菌(Lactobacillus helveticus)、德氏乳杆菌(Lactobacillus delbrueckii)、大肠杆菌(Escherichia coli)和/或其混合物。
本发明的喂养配方可以例如包含益生微生物,所述的益生微生物选自长双歧杆菌NCC 3001、长双歧杆菌NCC 2705、短双歧杆菌NCC 2950、乳双歧杆菌NCC 2818、约氏乳杆菌La1、类干酪乳杆菌NCC 2461、鼠李糖乳杆菌NCC 4007、路氏乳杆菌DSM17938、路氏乳杆菌ATCC55730、嗜热链球菌NCC 2019、嗜热链球菌NCC 2059、干酪乳杆菌NCC 4006、嗜酸乳杆菌NCC 3009、干酪乳杆菌ACA-DC 6002(NCC 1825)、大肠杆菌Nissle、保加利亚乳杆菌NCC 15、乳乳球菌NCC 2287或其组合。
所有这些菌株或是在布达佩斯条约(Budapest treaty)下保藏和/或是可商购获得的。
在布达佩斯条约下保藏的菌株如下:
长双歧杆菌NCC 3001: ATCC BAA-999
长双歧杆菌NCC 2705: CNCM I-2618
短双歧杆菌NCC 2950 CNCM I-3865
乳双歧杆菌NCC 2818: CNCM I-3446
类干酪乳杆菌NCC 2461: CNCM I-2116
鼠李糖乳杆菌NCC 4007: CGMCC 1.3724
嗜热链球菌NCC 2019: CNCM I-1422
嗜热链球菌NCC 2059: CNCM I-4153
乳乳球菌NCC 2287: CNCM I-4154
干酪乳杆菌NCC 4006: CNCM I-1518
干酪乳杆菌NCC 1825: ACA-DC 6002
嗜酸乳杆菌NCC 3009: ATCC 700396
保加利亚乳杆菌NCC 15: CNCM I-1198
约氏乳杆菌La1 CNCM I-1225
路氏乳杆菌DSM17938 DSM17938
路氏乳杆菌ATCC55730 ATCC55730
大肠杆菌Nissle 1917: DSM 6601
本领域那些技术人员将理解它们可以自由地组合本文描述的所有本发明特征,而不脱离所公开的本发明的范围。
根据下列实施例和附图,本发明的进一步的优点和特征是显而易见的。
图1A和B表示用“短时高温”处理的益生菌的抗炎免疫特性增强。
图2表示无抗炎性的益生菌株在用“短时高温”处理后变成抗炎性,即表现出显著的体外抗炎免疫特性。
图3A和B表示商购可获得的产品中使用的益生菌株在用“短时高温”处理后表现出增强的或新的体外抗炎免疫特性。
图4A和B表示乳品起始菌株(即Lc1起始菌株)在高温热处理后表现出增强的或新的体外抗炎免疫特性。
图5表示无抗炎性的益生菌株在用HTST处理处理后表现出体外抗炎免疫特性。
图6:活的和热处理(140℃,15秒)形式的益生菌株和乳品起始菌株产生的PBMC数据(IL-12p40、IFN-γ、TNF-α、IL-10)的主成分分析。每个
图7表示活的和热处理(85℃,20分钟)的菌株的IL-12p40/IL-10比例。相比本发明的“短时高温”处理,85℃热处理20分钟的所有菌株引起IL-12p40/IL-10比例的增加(图1、2、3、4和5)。
图8表示用热处理细菌刺激的人PBMCs中体外细胞因子分泌的增加。
图9表示在用盐水刺激(challenged)的OVA-致敏的小鼠(阴性对照)、用OVA刺激的OVA-致敏的小鼠(阳性对照)和用OVA刺激且用热处理或活的短双歧杆菌NCC2950处理的OVA-致敏的小鼠中观察到的腹泻强度的百分数。结果以腹泻强度百分数显示(由4组独立试验计算出的平均值±SEM),100%的腹泻强度对应于阳性对照组(变应原致敏和刺激)发生的症状。
实施例1:
方法学
细菌制剂:
活的益生菌对宿主免疫系统的健康益处通常被认为是菌株特异性的。体外诱导高水平IL-10和/或诱导低水平促炎细胞因子的益生菌(PBMC分析)已经显示是有效的体内抗炎菌株(Foligné,B.等人,2007,World J.Gastroenterol.13:236-243)。
使用数种益生菌株来研究热处理的益生菌的抗炎特性。这些菌株是长双歧杆菌NCC 3001、长双歧杆菌NCC 2705、短双歧杆菌NCC 2950、乳双歧杆菌NCC 2818、类干酪乳杆菌NCC 2461、鼠李糖乳杆菌NCC 4007、干酪乳杆菌NCC 4006、嗜酸乳杆菌NCC 3009、干酪乳杆菌ACA-DC 6002(NCC 1825)和大肠杆菌Nissle。还测试了数种起始培养菌株,包括某些商购菌株,用于产生NestléLc1发酵产物:嗜热链球菌NCC 2019、嗜热链球菌NCC 2059、保加利亚乳杆菌NCC 15和乳乳球菌NCC 2287。
细菌细胞是在5-15L生物反应器中的每种菌株的最佳条件下培养的。所有典型的细菌生长培养基是可用的。这类培养基是本领域技术人员已知的。当pH调至5.5时,连续加入30%碱溶液(NaOH或Ca(OH)2)。当足够时,通过用CO2气体处理顶部空间来维持缺氧条件。大肠杆菌是在标准的 需氧条件下培养的。
通过离心(5,000×g,4℃)收集细菌细胞,并且重悬于足够体积的磷酸盐缓冲液(PBS)中,以便达到终浓度约109-1010cfu/mL。部分制剂用15%甘油冻存于-80℃。另一部分细胞通过以下进行热处理:
-超高温:140℃,15秒;通过间接注入蒸汽。
-高温短时(HTST):74℃、90℃和120℃,15秒,通过间接注入蒸汽。
-水浴中的长时低温(85℃,20分钟)。
热处理后,将样品冻存于-80℃,直至使用。
细菌制剂的体外免疫分析:
评估活的和热处理的细菌制剂的免疫特性(即体外诱导人血细胞分泌特定细胞因子的能力)。从血液过滤器分离人外周血单核细胞(PBMCs)。通过细胞密度梯度分离后,收集单核细胞并且用Hank平衡盐溶液洗涤两次。然后将细胞重悬于补充有10%胎牛血清(Bioconcept,巴黎,法国)、1%L-谷氨酰胺(Sigma)、1%青霉素/链霉素(Sigma)和0.1%庆大霉素(Sigma)的Iscove改良的Dulbecco培养基(IMDM,Sigma)中。然后将PBMCs(7×105个细胞/孔)与活的和热处理的细菌(相当于7×106cfu/孔)在48孔板中培养36小时。活的和热处理的细菌的作用是在来自8位单独供体的PBMCs上测试的,分为两个独立试验。培养36小时后,将培养板冷冻并且保持在-20℃,直至细胞因子测量。细胞因子分析是在活的细菌和它们热处理的对应物中平行进行的(即在相同试验中在相同批次PBMCs上测试)。
按照供应商的说明,通过ELISA(R&D DuoSet Human IL-10,BD OptEIA Human IL12p40,BD OptEIA Human TNFα,BD OptEIA Human IFN-γ)测定培养36小时后细胞培养基的上清液中细胞因子(IFN-γ、IL-12p40、TNF-α和IL-10)的水平。IFN-γ、IL-12p40和TNF-α是促炎细胞因子,而IL-10是有效的抗炎介质。结果是以4位单独供体的平均值(pg/mL)+/-SEM表示的,并且是各用4位供体进行的两个单独试验的代表。计算每个菌株的IL-12p40/IL-10的比值,作为体内抗炎作用的预测值(Foligné,B.等人,2007,World J.Gastroenterol.13:236-243)。
将通过ELISA(参加上面)测定的每个菌株的细胞因子数值(pg/mL)转 移至BioNumerics v5.10软件(Applied Maths,Sint-Martens-Latem,Belgium)中。对每套数据进行主成分分析(PCA,尺寸度量技术)。该分析包括特征值减去平均值和除以特征值的方差(Subtraction of the averages over the characters and division by the variances over the characters were included in this analysis)。
结果
超高温(UHT)/高温短时(HTST)-样处理产生的抗炎特性
将在研的益生菌株进行一系列的热处理(超高温(UHT)、高温短时(HTST)和85℃,20分钟),并且将它们的免疫特性与活细胞的免疫特性进行体外比较。当与人PBMC温育时,活的微生物(益生菌和/或乳品起始培养物)诱导不同水平的细胞因子产生(图1、2、3、4和5)。这些微生物的热处理以温度依赖方式修饰了PBMC产生的细胞因子水平。“短时高温”处理(120℃或140℃,15秒)产生具有抗炎免疫特性的非复制细菌(图1、2、3和4)。事实上,UHT-样处理的菌株(140℃,15秒)诱导更少的促炎细胞因子(TNF-α、IFN-γ、IL-12p40),而维持或诱导额外的IL-10产生(相比活的对应物)。相比活细胞,任何UHT-样处理的菌株产生的IL-12p40/IL-10比例更低(图1、2、3和4)。HTST-样处理处理的细菌也证实了该观察,所述的HTST-样处理即120℃进行15秒(图1、2、3和4),或74℃和90℃进行15秒(图5)。热处理(UHT-样或HTST-样处理)对益生菌株(图1、2、3和4)和乳品起始培养物(图4)的体外免疫特性具有相似的作用。对活的和热处理(140℃,15秒)的益生菌和乳品起始菌株产生的PBMC数据的主成分分析表明,活菌株沿着x轴从低(左侧)到高(右侧)诱导物的促炎细胞因子全部延伸,这说明菌株表现出非常不同的体外免疫特性。热处理的菌株聚集在图的左侧,这表明热处理的菌株诱导的促炎细胞因子更少(图6)。通过比较,85℃热处理20分钟的细菌比活细胞诱导更多的促炎细胞因子和更少的IL-10,从而导致更高的IL-12p40/IL-10比例(图7)。
UHT-样和HTST-样处理增强或产生抗炎特性。
UHT和HTST处理的菌株表现出抗炎特性,不管它们各自最初的免疫特性(活细胞)。已知具有体内抗炎并且体外表现出抗炎特性的益生菌株(长 双歧杆菌NCC 3001、长双歧杆菌NCC 2705、短双歧杆菌NCC 2950、乳双歧杆菌NCC 2818)在“短时高温”处理后表现出增强的体外抗炎特性。如图1所示,UHT-样处理的双歧杆菌属菌株的IL-12p40/IL-10比例低于活的对应物的比例,从而表明UHT-样处理的样品的改善的抗炎特性。更引人注目地,无抗炎性的活菌株也证实了通过UHT-样和HTST-样处理产生的抗炎特性。活的鼠李糖乳杆菌NCC 4007和类干酪乳杆菌NCC 2461体外表现出高的IL-12p40/IL-10比例(图2和5)。两种活菌株显示出不能预防小鼠中TNBS-诱导的结肠炎。在“短时高温”处理(UHT或HTST)后鼠李糖乳杆菌NCC 4007和类干酪乳杆菌NCC 2461诱导的IL-12p40/IL-10比例急剧下降,达到与双歧杆菌属菌株获得的同样低的水平。这些低的IL-12p40/IL-10比例是由于低水平的IL-12p40产生并且组合无变化(鼠李糖乳杆菌NCC 4007)或急剧诱导IL-10分泌(类干酪乳杆菌NCC 2461)(图2)。
结果:
-UHT-样和HTST-样热处理可以增强活微生物的抗炎特性(例如长双歧杆菌NCC 2705、长双歧杆菌NCC 3001、短双歧杆菌NCC 2950、乳双歧杆菌NCC 2818)
-UHT-样和HTST-样热处理可以使非抗炎性活微生物产生抗炎特性(例如鼠李糖乳杆菌NCC 4007、类干酪乳杆菌NCC 2461、乳品起始物嗜热链球菌NCC 2019)
-从可商购获得的产品中分离的菌株(包括益生的大肠杆菌菌株)也证实了抗炎特性(图3A&B)。
UHT/HTST-样处理的影响类似于所有测试的益生菌和乳品起始物,例如乳酸杆菌属、双歧杆菌属和链球菌属。
UHT/HTST-样处理应用于数种表现出不同体外免疫特性的乳酸杆菌属、双歧杆菌属和链球菌属。UHT/HTST-样处理后的所有菌株比它们的活对应物诱导更少的促炎细胞因子(图1、2、3、4、5和6),这证实了UHT/HTST-样处理对产生的非复制细菌的免疫特性的作用可以概括为所有益生菌,特别是乳酸杆菌属和双歧杆菌属以及特定的大肠杆菌菌株和所 有乳品起始培养物,特别是链球菌属、乳球菌属和乳酸杆菌属。
实施例2:
方法学
细菌制剂:
五种益生菌株用于研究非复制益生菌的免疫增强特性:双歧杆菌属的3种(长双歧杆菌NCC3001、乳双歧杆菌NCC2818、短双歧杆菌NCC2950)和乳酸杆菌属的2种(类干酪乳杆菌NCC2461、鼠李糖乳杆菌NCC4007)。
细菌细胞生长在MRS上,于37℃分批发酵16-18小时,无pH控制。将细菌细胞旋转沉降(5,000×g,4℃),并且重悬于磷酸盐缓冲液中,之后在盐水中稀释以达到终浓度约10E10cfu/mL。将长双歧杆菌NCC3001、乳双歧杆菌NCC2818、类干酪乳杆菌NCC2461、鼠李糖乳杆菌NCC4007在85℃水浴中热处理20分钟。将短双歧杆菌NCC2950在90℃水浴中热处理30分钟。将热处理的细菌悬浮液等分并且冻存于-80℃直至使用。活细菌在PBS-甘油15%中存于-80℃直至使用。
细菌制剂的体外免疫分析
评估活的和热处理的细菌制剂的免疫特性(即体外诱导人血细胞分泌特定细胞因子的能力)。从血液过滤器分离人外周血单核细胞(PBMCs)。通过细胞密度梯度分离后,收集单核细胞并且用Hank平衡盐溶液洗涤两次。然后将细胞重悬于补充有10%胎牛血清(Bioconcept,巴黎,法国)、1%L-谷氨酰胺(Sigma)、1%青霉素/链霉素(Sigma)和0.1%庆大霉素(Sigma)的Iscove改良的Dulbecco培养基(IMDM,Sigma)中。然后将PBMCs(7×105个细胞/孔)与活的和热处理的细菌(相当于7×106cfu/孔)在48孔板中培养36小时。活的和热处理的细菌的作用是在来自8位单独供体的PBMCs上测试的,分为两个独立试验。培养36小时后,将培养板冷冻并且保持在-20℃,直至细胞因子测量。细胞因子分析是在活的细菌和它们的热处理对应物中平行进行的(即在相同试验中在相同批次PBMCs上测试)。
按照供应商的说明,通过ELISA(R&D DuoSet Human IL-10,BD OptEIA Human IL12p40,BD OptEIA Human TNF,BD OptEIA Human IFN-γ)测定培养36小时后细胞培养基上清液中细胞因子(IFN-γ、IL-12p40、 TNF-α和IL-10)的水平。IFN-γ、IL-12p40和TNF-α是促炎细胞因子,而IL-10是有效的抗炎介质。结果是以4位单独供体的平均值(pg/mL)+/-SEM表示的,并且是各用4位供体进行的两个单独试验的代表。
活的和热处理的短双歧杆菌NCC2950在预防过敏性腹泻中的体内作用
过敏性腹泻的小鼠模型用于测试短双歧杆菌NCC2950的促进Th1作用(Brandt E.B等人.JCI 2003;112(11):1666-1667)。致敏(间隔14天,于第0和14天分2次腹膜内注射卵白蛋白(OVA)和硫酸铝钾)后,将雄性Balb/c小鼠用OVA口服刺激6次(第27、29、32、34、36、39天),导致短暂的临床症状(腹泻)和免疫参数变化(总IgE、OVA特异性IgE、小鼠肥大细胞蛋白酶1即MMCP-1的血浆浓度)。在OVA致敏前(第-3、-2、-1、0天和第11、12、13和14天)和刺激期内(第23至39天)将活的或90℃热处理30分钟的短双歧杆菌NCC2950通过管饲法施用4天。使用每日细菌剂量约109集落形成单位(cfu)或等价的cfu/小鼠。
结果
热处理后诱导分泌“促炎”细胞因子
体外评估热处理的细菌菌株刺激人外周血单核细胞(PBMCs)分泌细胞因子的能力。在相同的体外分析中,将热处理的细菌刺激PBMCs后四种细胞因子的免疫特性和活细菌细胞诱导的四种细胞因子的免疫特性进行比较。
将热处理的制剂铺板并且评估不存在任何存活的计数。热处理的细菌制剂铺板后不产生集落。
当与人PBMCs温育时,活的益生菌诱导不同且菌株依赖水平的细胞因子产生(图8)。热处理的益生菌与它们的活对应物比较修饰了PBMCs产生的细胞因子水平。热处理的细菌比它们的活的对应物诱导更多的促炎细胞因子(TNF-α、IFN-γ、IL-12p40)。通过比较,热处理的细菌比活细胞诱导相似或更低量的IL-10。这些数据表明热处理的细菌比它们的活对应物更能刺激免疫系统,并且因此更能增强衰弱的免疫防御。换言之,体外数据说明了热处理后细菌菌株的增强的免疫增强作用。
为了说明热处理的短双歧杆菌NCC2950对免疫系统的增强作用(相比活细胞),在过敏性腹泻的动物模型中测试活的和热处理的短双歧杆菌NCC2950(菌株A)。
相比阳性对照组,用热处理的短双歧杆菌NCC2950处理后腹泻强度显著且持续下降(41.1%±4.8),而用活的短双歧杆菌NCC2950处理后腹泻强度仅降低20±28.3%。这些结果证实,热处理的短双歧杆菌NCC2950比其活的对应物表现出对过敏性腹泻的增强的保护作用。
结果显示,热处理后改善了益生菌增强免疫防御的能力。
实施例3-7:
可以制备下列婴儿或幼儿喂养配方:
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(该页不作为国际申请的一部分并且不认为是国际申请页)
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0-5-1 | 授权官员 |
Claims (19)
1.施用于从6个月开始的婴儿或幼儿的婴儿或幼儿喂养配方组合物,所述的配方组合物向婴儿或幼儿提供完整的营养并且包含益生微生物,其中益生微生物包括非复制的益生微生物,并且其中非复制的益生微生物是通过在71.5-150℃下高温处理1-120秒使其非复制的,其中益生微生物选自双歧杆菌属、乳杆菌属、丙酸杆菌属或其组合。
2.权利要求1的喂养配方组合物,其具有热量密度范围为62-68kcal/100mL,并且包含蛋白质源的量为1.9-3.5g/100kcal,碳水化合物源的量为12-13g/100kcal,以及脂质源的量为4-5g/100kcal。
3.权利要求1的喂养配方组合物,其包含0.15-0.25g LC-PUFA/100g脂肪酸,其中LC-PUFA可以选自ARA、DHA或其组合。
4.权利要求1的喂养配方组合物,其每100mL配方组合物包含1.5-2.5mg核苷酸。
5.权利要求1的喂养配方组合物,其包含益生微生物的量相应于106至1012cfu。
6.权利要求1-5中任意一项的喂养配方组合物,其中高温处理是在90-140℃进行1-120秒。
7.权利要求1-5中任意一项的喂养配方组合物,其中高温处理是在120-140℃进行1-120秒。
8.权利要求1-5中任意一项的喂养配方组合物,其中高温处理是在71.5-74℃进行15-30秒。
9.权利要求1-5中任意一项的喂养配方组合物,其中至少90%的益生菌是非复制的。
10.权利要求1-5中任意一项的喂养配方组合物,其中至少95%的益生菌是非复制的。
11.权利要求1-5中任意一项的喂养配方组合物,其中至少98%的益生菌是非复制的。
12.权利要求1-5中任意一项的喂养配方组合物,其中至少99%的益生菌是非复制的。
13.权利要求1-5中任意一项的喂养配方组合物,其中至少99.9%的益生菌是非复制的。
14.权利要求1-5中任意一项的喂养配方组合物,其中所有的益生菌是非复制的。
15.权利要求1-5中任意一项的喂养配方组合物,其中益生微生物选自长双歧杆菌、乳双歧杆菌、动物双歧杆菌、短双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、嗜酸乳杆菌、干酪乳杆菌、类干酪乳杆菌、唾液乳杆菌、路氏乳杆菌、鼠李糖乳杆菌、约氏乳杆菌、植物乳杆菌、发酵乳杆菌、乳乳球菌、嗜热链球菌、乳乳球菌、双乙酰乳酸乳球菌、乳脂乳球菌、保加利亚乳杆菌、瑞士乳杆菌、德氏乳杆菌、大肠杆菌和/或其混合物。
16.权利要求15的喂养配方组合物,其中益生微生物选自长双歧杆菌NCC 3001、长双歧杆菌NCC 2705、短双歧杆菌NCC 2950、乳双歧杆菌NCC 2818、约氏乳杆菌La1、类干酪乳杆菌NCC 2461、鼠李糖乳杆菌NCC4007、路氏乳杆菌DSM17938、路氏乳杆菌ATCC55730、嗜热链球菌NCC2019、嗜热链球菌NCC 2059、干酪乳杆菌NCC 4006、嗜酸乳杆菌NCC3009、干酪乳杆菌ACA-DC 6002(NCC 1825)、大肠杆菌Nissle、保加利亚乳杆菌NCC 15、乳乳球菌NCC 2287或其组合。
17.权利要求1-5中任意一项的喂养配方组合物,其每日剂量包含0.005mg-1000mg的非复制的微生物。
18.权利要求1-17中任意一项的喂养配方组合物在制备用于预防或治疗炎性障碍的产品中的用途。
19.权利要求1-17中任意一项的喂养配方组合物在制备用于预防或治疗与免疫防御缺乏相关的障碍的产品中的用途。
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PCT/EP2010/056397 WO2010130702A1 (en) | 2009-05-11 | 2010-05-11 | Infant and young children feeding formula comprising probiotics for infants and young children |
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CN2010800310277A Pending CN102762216A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的用于1-10岁儿童的全营养液 |
CN2010800309890A Pending CN102917714A (zh) | 2009-05-11 | 2010-05-11 | 包含非复制性益生菌微生物的婴儿米粉 |
CN2010800311547A Pending CN102939093A (zh) | 2009-05-11 | 2010-05-11 | 用于待施用于吞咽困难患者的热或冷食物和饮料的包含益生菌的即用型增稠剂 |
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CN201080031149.6A Pending CN103648510A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的口服营养补充物 |
CN201080031172.5A Pending CN103596578A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的营养平衡的标准管饲制剂 |
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CN2010800311636A Pending CN102883731A (zh) | 2009-05-11 | 2010-05-11 | 包含益生微生物的婴儿喂养配方 |
CN201080031174.4A Pending CN104220082A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的成年胃肠不耐患者专用营养制剂 |
CN2010800208141A Pending CN102595916A (zh) | 2009-05-11 | 2010-05-11 | 含有益生微生物的成长乳 |
CN2010800311674A Pending CN103052395A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的儿童口服营养增补剂 |
CN201080031173XA Pending CN102695519A (zh) | 2009-05-11 | 2010-05-11 | 含有益生微生物的早餐谷物 |
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CN2010800311759A Pending CN102869365A (zh) | 2009-05-11 | 2010-05-07 | 长双歧杆菌ncc2705(cncm i-2618)和免疫疾病 |
CN2010800311886A Pending CN102844037A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的管饲模块 |
CN2010800310277A Pending CN102762216A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的用于1-10岁儿童的全营养液 |
CN2010800309890A Pending CN102917714A (zh) | 2009-05-11 | 2010-05-11 | 包含非复制性益生菌微生物的婴儿米粉 |
CN2010800311547A Pending CN102939093A (zh) | 2009-05-11 | 2010-05-11 | 用于待施用于吞咽困难患者的热或冷食物和饮料的包含益生菌的即用型增稠剂 |
CN2010800311570A Pending CN102770145A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的手术和创伤患者专用医学营养 |
CN201080031149.6A Pending CN103648510A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的口服营养补充物 |
CN201080031172.5A Pending CN103596578A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的营养平衡的标准管饲制剂 |
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CN201080031174.4A Pending CN104220082A (zh) | 2009-05-11 | 2010-05-11 | 包含益生菌的成年胃肠不耐患者专用营养制剂 |
CN2010800208141A Pending CN102595916A (zh) | 2009-05-11 | 2010-05-11 | 含有益生微生物的成长乳 |
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CN201080031173XA Pending CN102695519A (zh) | 2009-05-11 | 2010-05-11 | 含有益生微生物的早餐谷物 |
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