CN116634889A - 通过益生菌菌株改善蛋白质消化和氨基酸生物利用度 - Google Patents
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Abstract
本发明涉及细菌菌株用于在受试者中改善蛋白质消化和/或提高氨基酸生物利用度的用途,其中所述细菌菌株是物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物。
Description
技术领域
本发明涉及物种动物双歧杆菌乳酸亚种(Bifidobacterium animalissubsp.lactis)、副干酪乳杆菌(Lacticaseibacillus paracasei)、乳酸乳球菌(Lactococcus lactis)、嗜酸乳杆菌(Lactobacillus acidophilus)、植物乳杆菌(Lactiplantibacillus plantarum)和/或鼠李糖乳杆菌(Lacticaseibacillusrhamnosus)的细菌菌株或其混合物用于在受试者中改善蛋白质消化和/或提高氨基酸生物利用度的用途。本发明进一步涉及以组合物(包括食物产品、食物成分、功能性食物、膳食补充剂以及药学上可接受的配制品)的形式施用的如本发明所述的细菌菌株的用途。
背景技术
蛋白质因其营养价值和在食物结构中的作用而在我们的日常饮食中必不可少。蛋白质是非常活跃的分子,在体内发挥着多种作用。一些蛋白质充当酶,意味着它们通过使身体发生化学反应起到生化催化剂的作用。一些蛋白质是激素,另一些是激素受体(使细胞识别激素),并且还有一些是转运蛋白(负责某些物质从细胞外到细胞内或反之的运输)。
氨基酸是蛋白质的主要成分,并且尽管一些氨基酸可以由人体产生,但是一些氨基酸无法由身体产生(因而被称为必需氨基酸),因此,确保我们的饮食提供我们所需要的所有氨基酸以便合成新陈代谢所需的蛋白质是必要的。
膳食蛋白质的营养质量取决于其氨基酸组成和氨基酸生物利用度。胃肠道中蛋白质的消化率和消化速率影响蛋白质生物利用度和蛋白质源的氨基酸吸收(van der Wielen等人2017)。已证明膳食氨基酸的可用性是餐后肌肉蛋白质代谢的重要调节剂(Koopman等人2009)。
可持续和植物性食品选择的市场不断扩大。然而,与动物蛋白和乳蛋白(即乳清蛋白)相比,植物蛋白的消化率通常较低(FAO 2012)。这是由于蛋白质溶解度较低以及植物基质中发现的抗营养因子(Gilani等人2012)。植物蛋白的较低消化率导致氨基酸的生物利用度较低,这可能对个体的营养状况、免疫功能、肌肉质量和肌肉力量产生影响(Wu 2016)。
儿童期、女性的妊娠和哺乳期以及老年人对蛋白质的需求增加(Wu 2016)。在儿童中,蛋白质质量在支持生长方面发挥作用(Ghosh 2016),而在老年人中,蛋白质质量是预防肌肉减少的重要因素(Baum等人2016)。一些研究表明,运动后摄入蛋白质剂量后,血液中氨基酸的快速增加(高氨基血)改善了对阻力训练的适应(Koopman等人2009;等人2017),并因此为运动员提供了益处。因此,改善植物蛋白的消化率和消化速率对个体的营养和健康状况具有重要益处。
发明目的
本发明的目的是改善蛋白质消化率和来自蛋白质(特别是来自植物蛋白,并且更特别地来自豆类蛋白质)的氨基酸的生物利用度。
特别地,本发明的目的是:(1)改善植物蛋白的营养价值,(2)提高植物蛋白的消化,(3)使用益生菌菌株改善植物蛋白的生物可及性,(4)改善来自植物蛋白的氨基酸和肽的可用性,和/或(5)改善人类和动物对植物蛋白的利用。
发明内容
本发明基于本文所述的研究,这些研究令人惊讶地证明物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的菌株或其混合物可以改善受试者的蛋白质消化并提高氨基酸生物利用度。
因此,在一个方面,本发明提供了细菌菌株用于改善受试者中的蛋白质消化和/或提高氨基酸生物利用度的用途,其中所述细菌菌株是物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物。
在根据本发明的另一方面,该蛋白质是植物蛋白。
在本发明的另一方面,该植物蛋白是豆类蛋白。
植物蛋白可以是大豆蛋白、豌豆蛋白、蚕豆蛋白、鹰嘴豆蛋白和/或扁豆蛋白。
在另外的方面,细菌菌株是菌株B420、Bl-04、Lpc-37、Ll-23、NCFM、Lp-115和/或HN001或其混合物。
在又另外的方面,根据本发明的细菌菌株以组合物(如食物产品、食物成分、功能性食物、膳食补充剂以及药学上可接受的配制品)的形式施用。
附图说明
图1.基线处(黑条)和模拟上GI道消化后(灰色条)大豆蛋白(A)和豌豆蛋白(B)中可溶性蛋白占总蛋白的百分比。x轴示出了对照和测试的益生菌菌株。在基线值处未检测到对照和益生菌处理之间的差异。这些值是三次重复的平均值。*P<0.05,**P<0.01,***P<0.001,****P<0.0001,消化后,益生菌处理与对照之间具有统计学显著差异。
图2.体外消化大豆蛋白(A-B)和豌豆蛋白(C-D)后的游离氨基氮(FAN)含量。图A、图C:基线处(SSF)和模拟上GI道消化的肠期后(SIF)收集的样品中FAN(mg)/克蛋白质的浓度。图B、图D:模拟消化后,与对照相比的相对FAN浓度。在基线值处未检测到对照和益生菌处理之间的差异。这些值是三次重复的平均值。*P<0.05,**P<0.01,***P<0.001,****P<0.0001,益生菌处理与对照之间具有统计学显著差异。图中呈现的P>0.05值表明有提高的趋势。
图3.大豆蛋白基线处(SSF)和体外消化后(SIF),总游离氨基酸(AA;A)、必需氨基酸(EAA;B)、支链氨基酸(BCAA、C)和游离亮氨酸(D)的浓度。在基线值处未检测到对照(无益生菌)和益生菌处理之间的差异。*P<0.05,**P<0.01,***P<0.001,益生菌处理与对照之间具有统计学显著差异。
图4.豌豆蛋白基线处(SSF)和体外消化后(SIF),总游离氨基酸(AA;A)、必需氨基酸(EAA;B)、支链氨基酸(BCAA、C)和游离亮氨酸(D)的浓度。在基线值处未检测到对照(无益生菌)和益生菌处理之间的差异。*P<0.05,**P<0.01,**P<0.001,益生菌处理与对照之间具有统计学显著差异。图中呈现的P>0.05值表明,与对照相比,浓度更高的趋势。
图5.基线处(SSF)和用冻干益生菌粉末的豌豆蛋白模拟消化的肠期后(SIF)收集的样品中的游离氨基氮(FAN,模拟单元中的mg/g蛋白质)。在基线值处未检测到豌豆蛋白对照(对照=无益生菌的消化)和益生菌处理之间的差异。*p<0.05,**p<0.01,***p<0.001,****p<0.0001,消化后,益生菌处理与对照之间具有统计学显著差异。
具体实施方式
如本发明所述的结果表明,细菌菌株改善了蛋白质消化率和来自蛋白质(即豆类源植物蛋白)的氨基酸的生物利用度。在模拟人上胃肠(GI)道中消化的条件下,体外研究了细菌菌株对植物蛋白消化的影响。
以下陈述了本发明的详细方面。在单独的部分中部分地讨论了一些详细方面。这是为了便于参考,并且绝对不是限制性的。下面描述的所有实施例同样适用于本发明的所有方面,除非上下文另有特别规定。
细菌
本发明各方面使用的细菌菌株是物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物。在一个特定方面,动物双歧杆菌乳酸亚种是菌株B420或Bl-04;在另一方面,副干酪乳杆菌是菌株Lpc-37;在另一方面,乳酸乳球菌是菌株L1-23;在另一方面,嗜酸乳杆菌是菌株NCFM;在另一方面,植物乳杆菌是菌株Lp-115;在另一方面,鼠李糖乳杆菌是菌株HN001。这些菌株可商购自杜邦营养生物科学公司(DuPont Nutrition Biosciences ApS)。
这些细菌菌株也由杜邦营养生物科学公司(丹麦哥本哈根K区Langebrogade街1号,邮编DK-1411)(Langebrogade 1,DK-1411Copenhagen K,Denmark)根据布达佩斯条约保藏在莱布尼茨学院德国微生物及细胞培养物保藏中心(Leibniz-Institut DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH(D SMZ))(德国布伦瑞克因霍芬街7B号,邮编38124(Inhoffenstrasse 7B,38124Braunschweig,Germany)),其中它们以以下登录号记录:
1.菌株Lp-115(DGCC4715);于2009年2月9日以登录号DSM22266保藏。
2.菌株Lpc-37(DGCC4981);于2017年10月5日以登录号DSM32661保藏。
3.菌株B420(DGCC420);于2015年6月30日以登录号DSM32073保藏。
4.菌株L1-23(DGCC8656);于2021年2月23日以登录号DSM33830保藏。
5.菌株Bl-04(DGCC2908);于2020年5月19日以登录号DSM33525保藏。
6.菌株NCFM(DGCC8698);于2021年3月15日以登录号DSM33840保藏。
7.菌株HN001(DGCCl460);于2021年5月7日以登录号DSM22876保藏。
优选地,在本发明中使用的细菌菌株是通常被认为是安全的(GRAS)并且优选地被GRAS批准的细菌菌株。GRAS是美国食品和药物管理局(FDA)的标志数据:专家认为添加到食物中的化学物质或物质是安全的,并且因此免除了通常的联邦食品,药品和化妆品法案(FFDCA)食品添加剂耐受性要求。
因此,在第一方面,本发明提供了物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物,其用于在受试者中改善蛋白质消化和/或提高氨基酸生物利用度中使用。
当与无益生菌的消化相比时,细菌菌株使大豆蛋白的消化速率改善了10%-38%,豌豆蛋白的消化速率改善了15%。通过增加蛋白质溶解度和/或增加更容易从GI道吸收的蛋白质消化终产物的浓度来改善消化率。
在另一方面,本发明提供了物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物,其用于在受试者中改善植物蛋白消化和/或提高氨基酸生物利用度。
在本发明的另一方面,该植物蛋白是豆类蛋白。
在另一方面,根据本发明的植物蛋白是大豆蛋白、豌豆蛋白、蚕豆蛋白、鹰嘴豆蛋白和/或扁豆蛋白。
在本发明的另一方面,蛋白质呈粉末形式。
当用于本发明各方面时,细菌菌株适用于人类和/或动物消费。技术人员将容易了解用于食品和/或农业工业并且通常被认为适用于人类和/或动物消费的特定菌株。
任选地,当用于本发明各方面时,细菌菌株是益生细菌。术语“益生细菌”被定义为包括任何非致病细菌,其当以活菌向宿主施用足够量时,细菌赋予宿主健康益处。若要分类为“益生菌”,细菌必须存活地通过宿主消化道的上部。它们是非致病性的、无毒的,并且一方面经由与消化道中的常居菌群的生态相互作用以及另一方面通过它们以积极的方式影响宿主生理和免疫系统的能力发挥其对健康的有益作用。当施用于宿主足够数量的益生细菌时,益生细菌具有前进通过肠道的能力,从而保持生存力并在宿主胃肠道的管腔和/或壁中发挥其主要作用。然后,它们短暂地形成常居菌群的一部分,并且这种定殖(或短暂定殖)允许益生细菌发挥有益的作用,如抑制存在于菌群中的潜在致病性微生物以及与包括免疫系统的肠内宿主相互作用。
因此,在本发明的特定方面,根据本发明使用的细菌菌株是益生菌菌株。
组合物
术语“组合物”在广义上用于意指某物的组成方式,即其一般组成。在本发明各方面,组合物可以基本上由选自物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和鼠李糖乳杆菌的单一菌株组成。
可替代地,组合物可以包含根据本发明的细菌菌株连同其它组分,如生物和化学组分、活性成分、代谢物、营养素、纤维、益生元等。
在一个方面,本发明提供了细菌菌株用于在受试者中改善蛋白质消化和/或提高氨基酸生物利用度的用途,其中所述细菌菌株是物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物,并且其中所述细菌菌株以组合物(如食物产品、食物成分、功能性食物、膳食补充剂以及药学上可接受的配制品)的形式施用。
在特定方面,根据本发明的组合物进一步包含益生元。
在本发明的又另外的方面,根据本发明的细菌菌株以106至1012,例如108至1012个菌落形成单位(CFU)/剂量,任选地1010 CFU/剂量的量存在于组合物中。
虽然不要求组合物包含任何支持物、稀释剂或赋形剂,但可以以本领域技术人员熟悉的方式添加和使用这样的支持物、稀释剂或赋形剂。合适的赋形剂的实例包括但不限于微晶纤维素、水稻麦芽糊精、二氧化硅和硬脂酸镁。本发明的组合物还可包含冷冻保护剂组分(例如,葡萄糖、蔗糖、乳糖、海藻糖、抗坏血酸钠和/或其他合适的冷冻保护剂)。
术语“组合物”和“配制品”可互换使用。
用于本发明各方面的组合物可以采取固体、液体、溶液或悬浮液制剂的形式。固体制剂的实例包括但不限于:可以是可湿性的、喷雾干燥的或冷冻干燥/冻干的片剂、丸剂、胶囊、颗粒剂和粉末。组合物可以含有调味剂或着色剂。组合物可以配制用于立即释放、延迟释放、修饰释放、持续释放、脉冲释放或控制释放应用。
举例来说,如果本发明的组合物以片剂形式使用,片剂也可含有以下中的一种或多种:赋形剂,如微晶纤维素、乳糖、柠檬酸钠、碳酸钙、磷酸氢钙和甘氨酸;崩解剂,如淀粉(优选玉米、土豆或木薯淀粉)、乙醇酸淀粉钠、交联羧甲基纤维素钠和某些复合硅酸盐;制粒粘合剂,如聚乙烯吡咯烷酮、羟丙基甲基纤维素(HPMC)、羟丙基纤维素(HPC)、蔗糖、明胶和阿拉伯树胶;润滑剂,如硬脂酸镁、硬脂酸、山嵛酸甘油酯和滑石。
用于制备组合物的其他可接受载剂的实例包括例如水、盐溶液、醇、硅酮、蜡类、凡士林、植物油、聚乙二醇、丙二醇、脂质体、糖类、明胶、乳糖、直链淀粉、硬脂酸镁、滑石、表面活性剂、硅酸、粘性石蜡、芳香油、脂肪酸甘油单酯和脂肪酸甘油二酯、羟甲基纤维素、聚乙烯吡咯烷酮等。
对于水性悬浮液和/或酏剂,可将本发明的组合物与各种甜味剂或调味剂、调色物质或染料进行组合,与乳剂和/或悬浮剂进行组合,以及与稀释剂(如水、丙二醇和甘油及其组合)进行组合。
为了说明的目的,下文列出了可用于本发明各方面的组合物的特定非限制性实例。这些包括但不限于食物产品、食物成分、功能性食物、膳食补充剂、药物组合物和药物。
食物产品
本发明的组合物可以采取食物产品的形式。此处,术语“食物”在广义上使用,并且包括人类的食物和饮物,也包括动物的食物和饮物(即,饲料)。优选地,食物产品适用于并且设计用于人类食用。
根据用途和/或应用方式和/或施用方式,食物可以呈液体、固体或悬浮液的形式。
当呈食物产品的形式时,组合物可以包含以下中的一种或多种或与以下中的一种或多种结合使用:营养上可接受的载剂、营养上可接受的稀释剂、营养上可接受的赋形剂、营养上可接受的辅剂、营养活性成分。
举例来说,本发明的组合物可以采取下列之一的形式:
果汁;含有乳清蛋白的饮料:保健茶或草药茶、可可饮料、乳饮料、乳酸菌饮料、酸奶和/或饮料酸奶、奶酪、冰淇淋、水冰、甜点、糖果、饼干、蛋糕、蛋糕混合物或蛋糕馅料、休闲食品、水果馅料、蛋糕或甜甜圈糖霜、即食烘焙馅料奶油、饼干馅料、即用烘焙馅料、卡路里降低的馅料、成人营养饮料、酸化大豆/果汁饮料、营养或保健棒、饮料粉末、钙强化豆奶或钙强化咖啡饮料。
任选地,在产品是食物产品的情况下,细菌副干酪乳杆菌应该通过正常的“最迟销售”日期或“截止”日期(在该日期期间食物产品由零售商出售)自始至终保持有效。优选地,有效时间应超过这样的日期,直到食物腐败变质变得明显时的正常新鲜期结束。期望的时间长度和正常保质期将随食料而变化,并且本领域普通技术人员将认识到,保质期时间取决于食料的种类、食料的大小、储存温度、加工条件、包装材料和包装设备而变化。
食物成分
本发明的组合物可采取食物成分和/或饲料成分的形式。
如本文所用,术语“食物成分”或“饲料成分”包括作为或可以添加到功能性食物或食料中作为用于人类和动物的营养和/或健康补充剂的组合物。
根据用途和/或应用方式和/或施用方式,食物成分可以呈液体、悬浮液或固体的形式。
功能性食物
本发明的组合物可以采取功能性食物的形式。
如本文所用,术语“功能性食物”意指不仅能够提供营养作用,还能够向消费者递送另外的有益作用的食物。
因此,功能性食物是具有掺入其中的组分或成分(如本文所述的那些)的普通食物,其赋予食物特定的功能-例如医学或生理学益处-而不是纯粹的营养作用。
虽然对于功能性食物没有法律定义,大多数关注此领域的各方都同意它们是作为具有超过基本营养作用的特定健康作用而面市的食物。
一些功能性食物是保健营养品。此处,术语“保健营养品”意指不仅能够提供营养作用和/或味道满足,还能够向消费者递送治疗(或其他有益)作用的食物。保健营养品跨越了在食物和药之间的传统分界线。
膳食补充剂
本发明的组合物可以采取膳食补充剂的形式,或者本身可以与膳食补充剂组合使用,这些膳食补充剂在本文中也称为食物补充剂。
如本文所用,术语“膳食补充剂”是指用于摄入的含有旨在增加膳食的营养价值或健康益处(以补充膳食)的“膳食成分”的产品。“膳食成分”可包括(但不限于)以下物质中的一种或任何组合:细菌、益生菌(例如,益生细菌)、维生素、矿物质、药草或其他植物制品、氨基酸、供人们用来通过增加总膳食摄取来补充饮食的膳食物质、浓缩物、代谢物、成分或提取物。
膳食补充剂可以许多形式存在,如片剂、胶囊、软胶囊(soft gels)、凝胶胶囊(gelcaps)、液体或粉末。一些膳食补充剂可以帮助确保从膳食中摄取足够的必需营养素;另一些可能有助于降低疾病风险。
药物组合物(配制品)
本发明的组合物可用作药品或用于制备药品。此处,术语“药品”以广义使用并且涵盖用于人类的药品以及用于动物的药品(即,兽医应用)。在优选的方面,药品是用于人类使用。
药品可以用于治疗目的-其本质上可以是治疗的、姑息的或预防的。
药品可以呈压缩片剂、片剂、胶囊、软膏、栓剂或可饮用溶液的形式。
当用作药品或用于制备药品时,本发明的组合物可与以下中的一种或多种结合使用:药学上可接受的载剂、药学上可接受的稀释剂、药学上可接受的赋形剂、药学上可接受的辅剂、药物活性成分。
根据用途和/或应用方式和/或施用方式,药品可以呈液体的形式或作为固体。
药物
本发明的组合物可以采取药物的形式。
如本文所用,术语“药物”涵盖在人类和兽医学中供人类和动物两者使用的药物。此外,如本文所用,术语“药物”意指提供治疗、预防和/或有益作用的任何物质。如本文所用,术语“药物”不一定限于需要上市许可证(marketing approval)的物质,而是可以包括可以用于化妆品、保健营养品、食物(包括例如饲料和饮料)、益生菌培养物和天然补救剂的物质。此外,如本文所用,术语“药物”涵盖设计用于掺入动物饲料(例如牲畜饲料和/或宠物食物)中的产品。
医疗食物
本发明的组合物可采取医疗食物的形式。
所谓“医疗食物”,意指如下食物,该食物被配制成在有或没有医生监督的情况下食用或施用并且旨在用于特定的膳食管理或状况,针对该膳食管理或状况,基于公认的科学原理,通过医学评估建立独特的营养需求。
剂量
本发明的组合物每剂量或每克组合物可包含106至1012个菌落形成单位(CFU)的一种或多种细菌菌株,并且更特别地,每剂量或每克组合物可包含108至1012CFU的一种或多种细菌菌株。任选地,组合物每剂量或每克组合物包含约1010CFU的一种或多种细菌菌株。
一种或多种细菌菌株可以以每剂量约106至约1012CFU的细菌菌株,优选地每剂量约108至约1012CFU的细菌菌株的剂量施用。术语“每剂量”意指每天或每次摄入(优选每天)向受试者提供这种数量的细菌。例如,如果将细菌施用在食物产品中,例如在酸奶中,则酸奶可含有约106至1012CFU的细菌菌株。然而,可替代地,这种数量的细菌可以分成多次施用,每次施用由较小量的微生物负荷组成-只要受试者在任何特定时间(例如每24h期间)接受的细菌菌株的总量是约106至约1012CFU的细菌,任选地108至约1012CFU的细菌。
根据本发明,至少一种细菌菌株的有效量可以是至少107CFU的细菌/剂量,任选地约108至约1012CFU的细菌/剂量,例如约1010CFU的细菌/剂量。
作用/受试者/医学适应症
在一个实施例中,如本文所用,术语“受试者”意指哺乳动物,包括例如牲畜(例如,牛、马、猪和绵羊)和人,以及鱼类,如鲑鱼。在一个实施例中,受试者是食物摄入量减少的人类个体、能量和/或蛋白质和/或氨基酸摄入量减少的个体、蛋白质和/或能量需求/需要高的个体、严格素食者、素食者、弹性素食者、肌肉损失的个体、肌肉减少的个体、肌肉萎缩的个体、运动员和身体活跃的个体(physically active individuals)。
在另一个实施例中,受试者是儿童、孕妇、哺乳期女性或老年人。
儿童应理解为出生至21岁之间,特别地出生至18岁之间,更特别地出生至14岁之间的人类。
对于老年人,可以理解是任何60岁或以上的人类。
在另外的实施例中,根据本发明的受试者是需要改善生长、增加胴体质量和/或增加肌肉质量的动物,如牲畜、宠物和伴侣动物、赛用动物(racing animal)(如,赛马和赛骆驼)。
益生元
在一个实施例中,本发明的细菌菌株和组合物可进一步组合或包含一种或多种纤维和/或益生元。
益生元被定义为被宿主微生物选择性利用从而赋予健康益处的底物。这些通常是通过选择性刺激一种或有限数量的细菌的生长和/或活性而有益地影响宿主的健康、从而改善宿主健康的成分。益生元可以应用于口服途径,但是它也可以应用于其他微生物定殖部位。典型地,益生元是碳水化合物(如寡糖),但是该定义不排除非碳水化合物(如多酚)或多不饱和脂肪酸或可以被有限数量的细菌选择性利用以赋予健康益处的其他成分。最普遍的益生元形式在营养上被归类为可溶性纤维。在某种程度上,许多形式的膳食纤维表现出一定水平的益生元作用。
合适的益生元的实例包括藻酸盐、黄原胶、果胶、槐豆胶(LBG)、菊粉、瓜尔胶,半乳寡糖(GOS)、果寡糖(FOS)、聚右旋糖10(即,)、乳糖醇、L-阿拉伯糖、D-木糖、L-鼠李糖、D-甘露糖、L-岩藻糖、肌醇、山梨醇、甘露醇、木糖醇、果糖、角叉菜胶、藻酸盐、微晶纤维素(MCC)、甜菜碱、低聚乳果糖、大豆寡糖、异麦芽酮糖(Palatinose TM)、异麦芽寡糖、葡萄糖寡糖、木寡糖、甘露寡糖、β-葡聚糖、纤维二糖、棉子糖、龙胆二糖、蜜二糖、木二糖、环糊精、异麦芽糖、海藻糖、水苏糖、潘糖、出芽短梗霉聚糖、毛蕊花糖、半乳甘露聚糖、(人类)乳寡糖和所有形式的抗性淀粉。
本发明的方法实施例
为免生疑问,如本发明所述的用于在受试者中改善蛋白质消化和/或提高氨基酸生物利用度的细菌菌株和组合物也可以用于方法中。
因此,在一个方面,本发明提供了在受试者中改善蛋白质消化和/或增加氨基酸生物利用度的方法,其中所述细菌菌株是物种动物双歧杆菌乳酸亚种、副干酪乳杆菌、乳酸乳球菌、嗜酸乳杆菌、植物乳杆菌和/或鼠李糖乳杆菌的细菌菌株或其混合物。
实例
提供以下实例以证明和进一步说明本发明的特定实施例和方面,而不应被解释为限制本发明的范围。
材料与方法
模拟上GI道模型中的蛋白质消化
在细菌特异性生长培养基中制备所选益生菌菌株的过夜培养物。使细菌生长至对数后期,并通过离心(10分钟,4000x g,4℃)收获。将含有细菌的沉淀洗涤三次并悬浮在0.9%NaCl中。测量光密度(OD600)并使用事先通过流式细胞术确定的每种菌株的OD600/浓度曲线来计数细菌数量。测试的菌株是以下商业益生菌:动物双歧杆菌乳酸亚种B420、动物双歧杆菌乳酸亚种Bl-04、嗜酸乳杆菌NCFM、鼠李糖乳杆菌HN001、副干酪乳杆菌Lpc-37、植物乳杆菌Lp-115和乳酸乳球菌L1-23。
模拟中使用的蛋白质粉末是:大豆蛋白分离物(XT 219D,杜邦公司(DuPont);86.7%蛋白质)和豌豆蛋白分离物(TRUPROTM 2000,杜邦公司;84.9%蛋白质)。进行12kGy的γ辐照以灭活蛋白质粉末中的固有细菌。
蛋白质的体外消化是根据模拟健康成人上胃肠道条件的静态标准化方法进行的(Minekus等人2014,Brodkorb等人2019)。简而言之,静态消化过程在以下三个阶段中进行模拟:口腔阶段,在此期间将用益生菌处理(2x108个细菌,20mL)或不含细菌的蛋白质粉末(2g,20mL)与模拟唾液(SSF)和淀粉酶(A3176,西格玛奥德里奇公司(Sigma-Aldrich),德国)混合。将pH调节至6.5,并将瓶子置于磁力搅拌器(200rpm)(在水浴中在37℃)上2分钟。然后将模拟胃液(SGF)与胃蛋白酶(P7012,西格玛奥德里奇公司,德国)添加至瓶子中,并将pH调节至2.8,并在37℃在水浴中在连续磁力搅拌下孵育2h。在胃部阶段之后,添加模拟肠液(SIF),然后添加胰酶(P3292,西格玛奥德里奇公司,德国)和胆汁溶液(B8631,西格玛奥德里奇公司,德国)。将pH调节至6.8,并且在水浴中在37℃伴随搅拌继续孵育2h。所有益生菌处理和对照均在三个生物学重复中进行。
此外,在如上所述的消化模型的类似条件下,以冻干益生菌培养物(1x109个细菌,20mL)的形式测试了益生菌对豌豆蛋白消化的功效。所选益生菌菌株是动物双歧杆菌乳酸亚种B420、动物双歧杆菌乳酸亚种Bl-04、嗜酸乳杆菌NCFM、副干酪乳杆菌Lpc-37(Lacticaseibacillus paracasei ssp.paracasei Lpc-37)和植物乳杆菌Lp-115(Lactiplantibacillus plantarum ssp.plantarum Lp-115)。
在基线时从模拟唾液(SSF样品)以及消化结束时从模拟小肠液(SIF)进行采样。将样品在4℃以10,000x g离心30分钟,并将上清液小心分离,等分并立即冷冻在-80℃。
微生物分析
平板计数法用于确定每个阶段后消化液中的细菌存活。在蛋白胨水中进行适当的10倍连续稀释后,将每个样品铺板在MRS板上,并在37℃孵育24h以进行菌落计数。将板在配有两个Anaerogen袋(Oxoid,赛默科技公司(Thermo Scientific),德国)的MitsubishiAnaeropack罐(赛默科技公司,美国)中在37℃无菌孵育。在随后三天每天对菌落进行计数,并通过将3天后获得的菌落总数除以基线时接种的细菌总数来计算存活百分比。
蛋白质溶解度测定
BCA(二辛可宁酸)测定(Thermo ScientificTM PierceTM BCA蛋白质测定,罗克福德,伊利诺伊州)用于测量可溶性蛋白质含量。将样品稀释100倍。在EnSight多模式读板器(珀金埃尔默公司(PerkinElmer))中在OD562 nm处测量吸光度。试剂盒随附的牛血清白蛋白用作蛋白质标准品。每个样品一式三份进行分析。
游离氨基氮测定
使用OPA(邻苯二甲醛)方法测量游离氨基氮(FAN)以评估样品中的蛋白质水解程度。将样品稀释10倍。根据制造商的标准方案,使用手动测定程序(K-PANOPA试剂盒,Megazyme公司,爱尔兰)定量FAN。在340nm波长处测量吸光度。每个样品一式两份进行分析。
游离氨基酸分析
如Greene等人(2009)所述,配合一些改进,使用自动化柱前衍生程序(用邻苯二甲醛(OPA))和反相高效液相色谱法(HPLC)来测定消化样品中的游离氨基酸。简言之,通过添加正缬氨酸作为内标来制备样品,然后提取氨基酸,并用0.1%三氟乙酸沉淀蛋白质,将样品在4℃孵育2h。离心后,将上清液的等分试样转移到Ultrafree-mc 10000NMWL微型离心机过滤装置(默克公司(Merck KGaA),达姆施塔特,德国)中,并以10000x G离心约1h。滤液用于分析。Agilent 1260Infinity II(安捷伦公司(Agilent),瓦尔德布龙,德国)色谱系统(由四元泵、柱温箱(column oven)、可编程注射器和二极管阵列检测器组成)用于氨基酸的衍生、分离和检测。在注射器针(injector needle)中用OPA和3-巯基丙酸试剂(各10mg/ml,安捷伦5061-3335)在pH 10.2的0.4M硼酸盐缓冲液(安捷伦5061-3339)中的混合物衍生样品。在40℃,在Agilent Zorbax Eclipse Plus C18柱(2.1x50mm,1.8μm,)上进行OPA氨基酸衍生物的分离。由10mM磷酸钠-10mM硼酸钠组成的缓冲溶液(pH 8.2)用作流动相A,并且乙腈、甲醇和水(45:45:10)的混合物用作流动相B。流速为0.42ml/min的以下A和B的梯度洗脱用于分离:0-0.2min,A=98%并且B=2%;0.2-7.7min,A线性降低至43%,并且B线性增加至57%;7.8-8.3min,A=0%并且B=100%;8.4-9min,A=98%并且B=2%。在338nm处检测OPA衍生物,并使用内标法进行定量。
结果
结果表明,细菌菌株改善了植物蛋白消化率和来自植物蛋白的氨基酸的利用度。细菌菌株使大豆蛋白的消化速率改善了10%-38%,豌豆蛋白的消化速率改善了15%,甚至高达18%(与无菌株的消化相比)。通过增加蛋白质溶解度和/或增加更容易从GI道吸收的蛋白质消化终产物的浓度来改善消化率。
1)益生菌菌株通过增加蛋白质在上GI道中的溶解度来改善植物蛋白的消化率
蛋白质的溶解度与蛋白质的消化率性质有关。可溶性蛋白质更容易通过胃肠道中的消化酶获得。在基线时和模拟消化后测量可溶性蛋白质含量。
与无益生菌的消化相比,菌株B420和L1-23改善了大豆蛋白的溶解度(图1A)。L1-23、Lp-115与L1-23的组合、B1-04和NCFM改善了豌豆蛋白的溶解度(图1B)。
总体而言,益生细菌显著改善了来自豆类源的蛋白质的溶解度(图1)。
所有益生细菌在消化条件下存活,并在消化后存活。
2)益生菌菌株改善上GI道中植物蛋白的消化
在基线时和模拟消化后测量样品中的游离氨基氮(FAN)浓度来评估蛋白质的消化速率。植物蛋白消化后,所有益生菌菌株处理和对照处理中的FAN浓度均增加,这证实在上GI道模拟消化后水解增加。
与对照相比,在益生菌菌株存在下,大豆蛋白的消化导致更高的FAN浓度。增幅最大的由菌株B1-04、HN001和NCFM产生,其次是Lp-115、Lpc-37和B420(图2A-图2B)。
与无益生菌的对照相比,用NCFM、B420和Lp-115与LL-23的组合的豌豆蛋白的消化导致更高的FAN(图2C-图2D)。此外,用更高剂量的冻干益生菌(用NCFM、B420、B1-04和Lpc-37)进行的豌豆蛋白的消化导致显著更高的FAN(图5)。
总体而言,益生细菌显著改善了来自豆类源的蛋白质的消化速率(图2)。益生菌在其作用方面显示出特异性,某些细菌对大豆蛋白更有效,而有些细菌对豌豆蛋白更有效。较高剂量的益生菌导致更高的蛋白质水解速率。
3)益生菌菌株增加植物蛋白消化后游离氨基酸的浓度
在基线时和模拟消化后测量样品中游离氨基酸的浓度。体外消化植物蛋白后,所有处理中总游离氨基酸(AA)、必需氨基酸(EAA)和支链氨基酸(BCAA)的浓度均增加。与对照相比,用NCFM、HN001、Bl-04、B420、Lpc-37和Ll-23处理导致大豆蛋白中游离AA、EAA、BCAA和亮氨酸的水平增加(图3)。与对照相比,用LL-23和Lpc-37处理导致豌豆蛋白中游离AA、EAA、BCAA和亮氨酸水平增加(图4)。与对照相比,用Bl-04处理导致豌豆蛋白中游离BCAA水平更高(图4C)。
在用益生菌进行蛋白质消化期间,游离氨基酸的释放示出于表1和表2中。
表1.比较益生菌大豆蛋白消化物与对照消化物从基线到消化后的可溶相中游离氨基酸含量的绝对变化。将比较报告为相对于对照处理的比率,其中比率为1.0表示没有差异,比率<1表示低于对照,比率>1表示高于对照。在括号中报告了估计的标准误差。
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aEAA,必需氨基酸;bBCAA,支链氨基酸。
*与无益生菌的对照消化相比,具有统计学显著差异,p<0.05
表2.比较益生菌豌豆蛋白消化物与对照消化物从基线到消化后的游离氨基酸含量的绝对变化。将比较报告为相对于对照处理的比率,其中比率为1.0表示没有差异,比率<1表示低于对照,比率>1表示高于对照。在括号中报告了估计的标准误差。
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aEAA,必需氨基酸;bBCAA,支链氨基酸。
*与无益生菌的对照消化相比,具有统计学显著差异,p<0.05
将上述说明书中提及的所有出版物通过引用并入本文。在不脱离本发明的范围和精神的情况下,本发明的所描述的方法和系统的各种修改和变化对于本领域的技术人员将是显而易见的。尽管已经结合特定优选实施例描述了本发明,但应当理解,如要求保护的本发明不应不适当地限制于这样的特定实施例。实际上,对于生物化学和生物技术或相关领域的技术人员显而易见的用于实施本发明的所描述的实施例的各种修改旨在落入以下权利要求的范围内。
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R,Kerksick CM,Campbell BI,Cribb PJ,Wells SD,Skwiat TM,PurpuraM,Ziegenfuss TN,Ferrando AA,Arent SM,Smith-Ryan AE.International society ofsports nutrition position stand:protein and exercise.Journal of theInternational Society of Sports Nutrition.2017;14(1):1-25.
Minekus M,Alminger M,Alvito P,Ballance S,Bohn TO,Bourlieu C,CarriereF,Boutrou R,Corredig M,Dupont D,Dufour C.A standardised static in vitrodigestion method suitable for food-an international consensus.Food&function.2014;5(6):1113-24.
Brodkorb A,Egger L,Alminger M,Alvito P,R,Ballance S,Bohn T,Bourlieu-Lacanal C,Boutrou R,Carrière F,Clemente A.INFOGEST static in vitrosimulation of gastrointestinal food digestion.Nature protocols.2019;14(4):991-1014.
Greene J,Henderson JW Jr.,Wikswo JP.(2009)Rapid and precisedetermination of cellular amino acid flux rates using HPLC with automatedderivatization with absorbance detection.Agilent technologies,applicationnote 5990-3283EN.
PCT/RO/134表
(原件为电子形式)
(此页不是国际申请的一部分,也不算作国际申请页)
Claims (15)
1.细菌菌株用于在受试者中改善蛋白质消化和/或提高氨基酸生物利用度的用途,其中所述细菌菌株是物种动物双歧杆菌乳酸亚种(Bifidobacterium animalissubsp.lactis)、副干酪乳杆菌(Lacticaseibacillus paracasei)、乳酸乳球菌(Lactococcus lactis)、嗜酸乳杆菌(Lactobacillus acidophilus)、植物乳杆菌(Lactiplantibacillus plantarum)和/或鼠李糖乳杆菌(Lacticaseibacillusrhamnosus)的细菌菌株或其混合物。
2.根据权利要求1所述的用途,其中所述蛋白质是植物蛋白。
3.根据权利要求2所述的用途,其中所述植物蛋白是豆类蛋白。
4.根据权利要求2所述的用途,其中所述植物蛋白是大豆蛋白、豌豆蛋白、蚕豆蛋白、鹰嘴豆蛋白和/或扁豆蛋白。
5.根据前述权利要求中任一项所述的用途,其中所述蛋白质呈粉末形式。
6.根据权利要求1所述的用途,其中所述细菌菌株是益生细菌菌株。
7.根据权利要求1所述的用途,其中所述动物双歧杆菌乳酸亚种物种的细菌菌株是菌株B420和/或菌株B1-04。
8.根据权利要求1所述的用途,其中所述副干酪乳杆菌物种的细菌菌株是菌株Lpc-37。
9.根据权利要求1所述的用途,其中所述乳酸乳球菌物种的细菌菌株是菌株L1-23。
10.根据权利要求1所述的用途,其中所述嗜酸乳杆菌物种的细菌菌株是菌株NCFM。
11.根据权利要求1所述的用途,其中所述植物乳杆菌物种的细菌菌株是菌株Lp-115。
12.根据权利要求1所述的用途,其中所述鼠李糖乳杆菌物种的细菌菌株是菌株HN001。
13.根据前述权利要求中任一项所述的用途,其中所述细菌菌株以组合物,如食物产品、食物成分、功能性食物、膳食补充剂以及药学上可接受的配制品,的形式施用。
14.根据权利要求12所述的用途,其中所述组合物进一步包含益生元,如纤维。
15.根据前述权利要求中任一项所述的用途,其中所述受试者是食物摄入量减少的人类个体、能量和/或蛋白质和/或氨基酸摄入量减少的个体、蛋白质和/或能量需求/需要高的个体、严格素食者、素食者、弹性素食者、肌肉损失的个体、肌肉减少的个体、肌肉萎缩的个体、运动员和身体活跃的个体。
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EP20214191.7A EP4014755A1 (en) | 2020-12-15 | 2020-12-15 | Improving protein digestion and amino acid bioavailability by probiotic strains |
EP20214191.7 | 2020-12-15 | ||
PCT/EP2021/085506 WO2022128927A1 (en) | 2020-12-15 | 2021-12-13 | Improving protein digestion and amino acid bioavailability by probiotic strains |
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CN116634889A true CN116634889A (zh) | 2023-08-22 |
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CN202180084640.3A Pending CN116634889A (zh) | 2020-12-15 | 2021-12-13 | 通过益生菌菌株改善蛋白质消化和氨基酸生物利用度 |
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US (1) | US20240108668A1 (zh) |
EP (2) | EP4014755A1 (zh) |
JP (1) | JP2023552860A (zh) |
KR (1) | KR20230118939A (zh) |
CN (1) | CN116634889A (zh) |
AU (1) | AU2021399012A1 (zh) |
CA (1) | CA3201727A1 (zh) |
WO (1) | WO2022128927A1 (zh) |
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Publication number | Priority date | Publication date | Assignee | Title |
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US20020090416A1 (en) * | 1999-03-09 | 2002-07-11 | Philip Connolly | Method of enhancing absorption and utilization of protein |
CN103188952B (zh) * | 2010-08-16 | 2016-01-13 | 杜邦营养生物科学有限公司 | 提高益生菌存活率的方法 |
CN104968780A (zh) * | 2012-09-20 | 2015-10-07 | 普洛特拉有限公司 | 用于治疗肥胖和肥胖相关病症的益生菌组合物和方法 |
MA45327A (fr) * | 2016-05-13 | 2019-03-20 | Sofar Spa | Utilisation de probiotiques pour améliorer l'absorption des protéines |
-
2020
- 2020-12-15 EP EP20214191.7A patent/EP4014755A1/en not_active Withdrawn
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2021
- 2021-12-13 CN CN202180084640.3A patent/CN116634889A/zh active Pending
- 2021-12-13 JP JP2023535816A patent/JP2023552860A/ja active Pending
- 2021-12-13 US US18/257,612 patent/US20240108668A1/en active Pending
- 2021-12-13 AU AU2021399012A patent/AU2021399012A1/en active Pending
- 2021-12-13 KR KR1020237023544A patent/KR20230118939A/ko unknown
- 2021-12-13 CA CA3201727A patent/CA3201727A1/en active Pending
- 2021-12-13 WO PCT/EP2021/085506 patent/WO2022128927A1/en active Application Filing
- 2021-12-13 EP EP21824406.9A patent/EP4262437A1/en active Pending
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US20240108668A1 (en) | 2024-04-04 |
EP4262437A1 (en) | 2023-10-25 |
EP4014755A1 (en) | 2022-06-22 |
KR20230118939A (ko) | 2023-08-14 |
WO2022128927A1 (en) | 2022-06-23 |
CA3201727A1 (en) | 2022-06-23 |
AU2021399012A1 (en) | 2023-06-22 |
JP2023552860A (ja) | 2023-12-19 |
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