CN114786504A - 记忆/学习能力维持和/或改善用组合物以及含有所述组合物的食品、药物和饲料 - Google Patents
记忆/学习能力维持和/或改善用组合物以及含有所述组合物的食品、药物和饲料 Download PDFInfo
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- CN114786504A CN114786504A CN202080066097.XA CN202080066097A CN114786504A CN 114786504 A CN114786504 A CN 114786504A CN 202080066097 A CN202080066097 A CN 202080066097A CN 114786504 A CN114786504 A CN 114786504A
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Abstract
本发明的目的是提供对于随衰老发生的记忆/学习能力的下降的预防和/或改善有效的药物、饮食品和饲料。提供一种新的微生物,其具有预防和/或改善随衰老发生的记忆/学习能力的下降的效果。更具体地,所述微生物的特征在于属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属。仍更具体地,所述微生物的特征在于属于以下的任一种:嗜酸乳杆菌、加氏乳杆菌、母鸡乳杆菌、约氏乳杆菌、德氏乳杆菌德氏亚种、链状双歧杆菌、假长双歧杆菌、两歧双歧杆菌、短双歧杆菌、长双歧杆菌、嗜热链球菌、肠膜明串珠菌右旋葡聚糖亚种、乳明串珠菌、乳酸片球菌和戊糖片球菌。
Description
技术领域
本发明涉及随老化(衰老)发生的记忆和学习能力的下降的预防或改善剂和/或具有随老化(衰老)发生的记忆和学习能力的下降的预防或改善作用的饮食品。
背景技术
在日本,2010年,65岁以上的老年人口的百分比已超过总人口的21%,国家已进入超老龄化社会。预计老年人的百分比将持续增加,并且据说到2060年将达到40%。在这种情况下,解决例如医疗保健和福利的问题等增加的老年人口的问题是当务之急。
随着老年人口的增加,中老年龄层中例如记忆和学习能力的下降和痴呆等认知损害的增加已成为一项问题。一般认为脑中神经元的微观结构随着年龄而变化,并且通过异常蛋白质的沉积和细胞脱落使脑萎缩,这导致了认知功能的下降。除了年龄相关的变化之外,由于脑梗塞、脑出血、或异常蛋白质的过度累积等,认知功能下降至病理状态的状态是痴呆。
截至2012年,估计约有462万人,即65岁以上的老年人的15%患有痴呆,并且有约400万的称为可能痴呆的轻度认知损害患者。据说日本目前正在经历“800万痴呆患者的时代”。
虽然已经开发出用于痴呆的治疗剂,但目前的治疗剂用于延缓进展的目的,尚未实现根本治疗。对于健康老年人或定位为痴呆前的状态的具有轻度认知损害的老年人,提出可以通过例如改变饮食习惯或锻炼等适当的干预来预防或改善例如记忆和学习能力等认知功能的下降,并且甚至可以降低痴呆症的发病风险(Psychiatria et NeurologiaJaponica 111,26-30,(2009))。因此,可在日常生活中连续摄取并且高度安全和无副作用的饮食品备受关注。
例如乳酸菌等用于食品的微生物广泛用于例如奶酪、酸奶和腌渍物等发酵食品和乳酸菌饮料等,并且由于相对低的供应成本和高适口性,已在世界范围内长期大量生产。近来,微生物作为还具有有益于健康的健康作用的健康食品受到关注,例如包括预防便秘和改善腹泻等肠道调节作用、预防感染和免疫刺激作用。关于与衰老或认知功能的相关性,例如,已公开了加氏乳杆菌(Lactobacillus gasseri)和长双歧杆菌(Bifidobacteriumlongum)对于年龄相关的代谢异常是有效的(专利号5155961和专利号5155960),并且罗伊氏乳杆菌(Lactobacillus reuteri)DSM 17938株对于青少年哺乳动物的认知功能的发育是有效的(JP-T-2015-503913,如本文所用的术语“JP-T”是指已公布的PCT专利申请的日本语翻译)。
然而,尚未阐明微生物对随老化(衰老)发生的记忆和学习能力的下降的作用。
线虫秀丽隐杆线虫(Caenorhabditis elegans)(以下称为C.elegans)可使用由302个神经元组成的简单神经回路感知不同的外部刺激并进行不同的应答。由于秀丽隐杆线虫将与其过去的培养条件关联的环境温度记忆为舒适温度,它们在温度梯度上迁移至该温度并在该温度附近等温移动。例如,在将已在23℃下培养的秀丽隐杆线虫置于17℃至23℃的温度梯度上而无任何食物时,秀丽隐杆线虫迁移至大约23℃。此外,当将培养温度从23℃转换至17℃时,已经获得23℃的记忆的虫在约4小时内获得新温度17℃的记忆,并在温度梯度中迁移至约17℃(Proc.Natl.Acad.Sci.USA,72,4061-4065(1975);Genetics,169,1437-1450(2005);J.Neurosci.Methods,154,45-52(2006))。这种行为称为趋温性,并已知作为反映例如记忆和学习等神经功能的可塑性的优秀模型系统(Curr.Opin.Neurobiol.,17,712-719(2007))。
在发明人已进行的用激光杀死各神经元的实验中,已鉴定了对趋温性行为关键的神经元,并且已知趋温性行为是基于非常简单的神经回路(Nature,376,344-348(1995))。此外,发明人的研究组通过全基因组微阵列分析、分子遗传学方法和成像技术分析了热记忆的形成过程。结果发现,当秀丽隐杆线虫记忆培养温度时,秀丽隐杆线虫通过转录因子HSF-1由全身感知环境温度。此外,已显示温度感知导致基因表达的变化,因此,热信号通过雌性激素雌激素在作为神经回路中的热感觉神经元的AFD神经元中作为记忆累积(Nat.Neurosci.,14,984-92(2011))。
此外,作为寻找其中负责记忆的代表性转录因子的CREB的秀丽隐杆线虫直向同源物起作用的细胞的结果,发明人发现CREB直向同源物仅在AFD神经元中起作用(EMBO Rep.,12,855-862(2011))。这表明,在秀丽隐杆线虫的所有神经元中,AFD神经元是唯一通过CREB具有热记忆的神经元。这些研究阐明了形成热记忆的一部分分子机制。
尽管从进化的观点来看,线虫是在系统发育上与哺乳动物相距甚远的生物体,但细胞水平的分子机制有很多共同之处。已报道已知与哺乳动物中记忆相关的分子也涉及线虫中的趋温性。例如,有很多报道:CREB(线虫中的CRH-1),其是与哺乳动物中记忆相关的因子,涉及线虫中热记忆的获得(EMBO Rep.,12,855-862(2011));血清素,其是一种与哺乳动物中记忆相关的脑内递质(transmitter),调节线虫中的趋温性(PLoS One.,8,1-10(2013));钙调磷酸酶(线虫中的RCAN-1),其是与哺乳动物中记忆相关的因子,涉及线虫中的趋温性行为(J.Mol.Biol.,427,3457-3468(2015));等等。
此外,已报道案例,其中已观察到对线虫中神经系统有效的组分也表现对哺乳动物中的脑-神经系统的作用。即,例如,已报道,口服摄取α-硫辛酸改善线虫和哺乳动物中联想学习行为的年龄相关的下降(Neurobiol.Aging.,26,899-905(2005);J.Med.Food.,15,713-7(2012))以及口服姜黄素改善线虫和哺乳动物中年龄相关的认知损害(NeurobiolAging.,39,69-81(2016);Geroscience.,40,73-95(2018))。
因此,秀丽隐杆线虫的趋温性行为被认为是适用于分析温度感知和记忆的分子机制的模型系统。
现有技术文献
专利文献
专利文献1:特许5155961
专利文献2:特许5155960
专利文献3:特表2015-503913
非专利文献
非专利文献1:Katsuyoshi Mizukami,Psychiatria et Neurologia Japonica111,26-30,(2009)
非专利文献2:Hedgecock,E.M.&Russell,R.L.:Normal and mutant thermotaxisin the nematode Caenorhabditis elegans.Proc.Natl.Acad.Sci.USA,72,4061-4065(1975)
非专利文献3:Mohri,A.,Kodama,E.,Kimura,K.D.et al.:Genetic control oftemperature preference in the nematode Caenorhabditis elegans.Genetics,169,1437-1450(2005)
非专利文献4:Ito,H.,Inada,H.&Mori,I.:Quantitative analysis ofthermotaxis in the nematode Caenorhabditis elegans.J.Neurosci.Methods,154,45-52(2006)
非专利文献5:Mori,I.,Sasakura,H.&Kuhara,A.:Worm thermotaxis:a modelsystem for analyzing thermosensation and neuralplasticity.Curr.Opin.Neurobiol.,17,712-719(2007)
非专利文献6:Mori,I.&Ohshima,Y.:Neural regulation of thermotaxis inCaenorhabditis elegans.Nature,376,344-348(1995)
非专利文献7:Sugi,T.,Nishida,Y.,&Mori,I.:Regulation of behavioralplasticity by systemic temperature signaling in Caenorhabditiselegans.Nat.Neurosci.,14,984-92(2011)
非专利文献8:Nishida,Y.,Sugi,T.,Nonomura,M.et al.:Identification ofthe AFD neuron as the site of action of the CREB protein in Caenorhabditiselegans thermotaxis.EMBO Rep.,12,855-862(2011)
非专利文献9:Li,Y.,Zhao,Y.,Huang,X.,et al.:Serotonin control ofthermotaxis memory behavior in nematode Caenorhabditis elegans.PLoS One.,8,1-10(2013)
非专利文献10:Li,W.,Bell,H.W.,Ahnn,J.,Lee,S.K.:Regulator ofCalcineurin(RCAN-1)Regulates Thermotaxis Behavior in Caenorhabditiselegans.J.Mol.Biol.,427,3457-3468(2015)
非专利文献11:Murakami,S.,Murakami,H.:The effects of aging andoxidative stress on learning behavior in C.elegans.Neurobiol.Aging.,26,899-905(2005)
非专利文献12:Cui,Y.,Shu,Y.,Zhu,Y.,Shi,Y.,Le,G.:High-fat diets impairspatial learning of mice in the Y-maze paradigm:ameliorative potential ofα-lipoic acid.J.Med.Food.,15,713-7(2012)
非专利文献13:Miyasaka,T.,Xie,C.,Yoshimura,S.,et al.:Curcumin improvestau-induced neuronal dysfunction of nematodes.Neurobiol.Aging.,39,69-81(2016)
非专利文献14:Sarker,M.R.,Franks,S.F..:Efficacy of curcumin for age-associated cognitive decline:a narrative review of preclinical and clinicalstudies.Geroscience.,40,73-95(2018)
发明内容
发明要解决的问题
本发明涉及提供具有预防和/或改善随老化(衰老)发生的记忆和学习能力的下降的效果的新的微生物。
用于解决问题的方案
从使用可日常摄取的食品材料来预防或改善活体中的不同功能障碍的观点,本发明人已专注于用于食品的微生物及其中所含组分,并已进行了深入研究以解决该问题。结果是,本发明人已发现了通过多种不同属的微生物的细胞或培养物来预防和/或改善随老化(衰老)发生的记忆和学习能力的下降的作用,从而完成了本发明。即,本发明具有以下特征。
(1)一种记忆和学习能力维持和/或改善用组合物,其含有属于乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)、链球菌属(Streptococcus)、明串珠菌属(Leuconostoc)和片球菌属(Pediococcus)的任何的一种以上的微生物的细胞或培养物。
(2)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述乳杆菌属是选自嗜酸乳杆菌(Lactobacillus acidophilus)、加氏乳杆菌(Lactobacillusgasseri)、母鸡乳杆菌(Lactobacillus gallinarum)、约氏乳杆菌(Lactobacillusjohnsonii)和德氏乳杆菌德氏亚种(Lactobacillus delbrueckii subsp.Delbrueckii)的一种以上。
(3)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述乳杆菌属不是选自以下的一种以上:嗜酸乳杆菌(Lactobacillus acidophilus)、母鸡乳杆菌(Lactobacillus gallinarum)、短乳杆菌(Lactobacillus brevis)、干酪乳杆菌(Lactobacillus casei)、卷曲乳杆菌(Lactobacillus crispatus)、德氏乳杆菌(Lactobacillus delbrueckii)、德氏乳杆菌德氏亚种(Lactobacillus delbrueckiisubsp.delbrueckii)、发酵乳杆菌(Lactobacillus fermentum)、加氏乳杆菌(Lactobacillus gasseri)、瑞士乳杆菌(Lactobacillus helveticus)、约氏乳杆菌(Lactobacillus johnsonii)、乳酸乳杆菌(Lactobacillus lactis)、莱氏曼氏乳杆菌(Lactobacillus leichmannii)、副干酪乳杆菌(Lactobacillus paracasei)、戊糖乳杆菌(Lactobacillus pentosus)、植物乳杆菌(Lactobacillus plantarum)、罗伊氏乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、清酒乳杆菌(Lactobacillus sakei)和唾液乳杆菌(Lactobacillus salivarius)。
(4)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述双歧杆菌属是选自链状双歧杆菌(Bifidobacterium catenulatum)、假长双歧杆菌(Bifidobacterium pseudolongum)、两歧双歧杆菌(Bifidobacterium bifidum)、短双歧杆菌(Bifidobacterium breve)和长双歧杆菌(Bifidobacterium longum)的一种以上。
(5)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述双歧杆菌属不是选自以下的一种以上:青春双歧杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacterium animalis)、链状双歧杆菌(Bifidobacteriumcatenulatum)、假链状双歧杆菌(Bifidobacterium pseudocatenulatum)、两歧双歧杆菌(Bifidobacterium bifidum)、短双歧杆菌(Bifidobacterium breve)、假长双歧杆菌(Bifidobacterium pseudolongum)、长双歧杆菌(Bifidobacterium longum)和长双歧杆菌婴儿亚种(Bifidobacterium longum subspecies infantis)。
(6)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述链球菌属是嗜热链球菌(Streptococcus thermophilus)。
(7)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述链球菌属不是选自以下的一种以上:无乳链球菌(Streptococcus agalactiae)、咽峡炎链球菌(Streptococcus anginosus)、海豚链球菌(Streptococcus iniae)、中间链球菌(Streptococcus intermedius)、马链球菌(Streptococcus equi)、口腔链球菌(Streptococcus oralis)、解没食子酸链球菌(Streptococcus gallolyticus)、解没食子酸链球菌解没食子酸亚种(Streptococcus gallolyticus subspecies gallolyticus)、星座链球菌(Streptococcus constellatus)、戈登链球菌(Streptococcus gordonii)、唾液链球菌(Streptococcus salivarius)、血链球菌(Streptococcus sanguis)、嗜热链球菌(Streptococcus thermophilus)、猪链球菌(Streptococcus suis)、兽疫链球菌(Streptococcus zooepidemicus)、远缘链球菌(Streptococcus sobrinus)、肺炎链球菌(Streptococcus pneumoniae)、酿脓链球菌(Streptococcus pyogenes)、链球菌噬菌体(Streptococcus phage)、粪链球菌(Streptococcus faecalis)、屎链球菌(Streptococcusfaecium)、牛链球菌(Streptococcus bovis)、缓症链球菌(Streptococcus mitis)和变异链球菌(Streptococcus mutans)。
(8)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述明串珠菌属是选自肠膜明串珠菌右旋葡聚糖亚种(Leuconostoc mesenteroidessubsp.dextranicum)和乳明串珠菌(Leuconostoc lactis)的一种以上。
(9)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述明串珠菌属不是选自肠膜明串珠菌右旋葡聚糖亚种(Leuconostoc mesenteroidessubsp.dextranicum)、肠膜明串珠菌(Leuconostoc mesenteroides)和乳明串珠菌(Leuconostoc lactis)的一种以上。
(10)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述片球菌属是选自乳酸片球菌(Pediococcus acidilactici)和戊糖片球菌(Pediococcuspentosaceus)的一种以上。
(11)根据(1)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述片球菌属不是选自乳酸片球菌(Pediococcus acidilactici)和戊糖片球菌(Pediococcuspentosaceus)的一种以上。
(12)根据(2)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述乳杆菌属是选自嗜酸乳杆菌SBT1921株、加氏乳杆菌SBT2056株、母鸡乳杆菌SBT0316株、约氏乳杆菌SBT0307株和德氏乳杆菌德氏亚种SBT0413株的一种以上。
(13)根据(4)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述双歧杆菌属是选自链状双歧杆菌SBT11114株、假长双歧杆菌SBT2922株、两歧双歧杆菌SBT10550株、短双歧杆菌SBT10532株和长双歧杆菌SBT10527株的一种以上。
(14)根据(6)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述链球菌属是嗜热链球菌SBT0118株。
(15)根据(8)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述明串珠菌属是选自肠膜明串珠菌右旋葡聚糖亚种SBT0096株和乳明串珠菌SBT2561株的一种以上。
(16)根据(10)所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述片球菌属是选自乳酸片球菌SBT3331株和戊糖片球菌SBT2229株的一种以上。
(17)根据(1)至(16)中任一项所述的记忆和学习能力维持和/或改善用组合物,其中所述记忆和学习能力维持和/或改善用是预防和/或改善随老化(衰老)发生的记忆和学习能力的下降。
(18)根据(17)所述的记忆和学习能力维持和/或改善用组合物,其中所述随老化(衰老)发生的记忆和学习能力的下降不包括随脑疾病、记忆损害和精神疾病发生的记忆和学习能力的下降或记忆能力的下降。
(19)根据(1)至(18)中任一项所述的记忆和学习能力维持和/或改善用组合物,其含有微生物的细胞作为活性成分,而不是微生物的培养上清液中所含有的肽组分。
(20)一种记忆和学习能力维持和/或改善用食品、记忆和学习能力维持和/或改善用药物或记忆和学习能力维持和/或改善用饲料,其特征在于,其含有根据(1)至(19)中任一项所述的记忆和学习能力维持和/或改善用组合物。
(21)具有保藏编号NITE P-02980的嗜酸乳杆菌SBT1921株、具有保藏编号NITE P-02981的母鸡乳杆菌SBT0316株、具有保藏编号NITE P-02982的约氏乳杆菌SBT0307株、具有保藏编号NITE P-02983的链状双歧杆菌SBT11114株、具有保藏编号NITE P-02984的假长双歧杆菌SBT2922株、具有保藏编号NITE P-02985的两歧双歧杆菌SBT10550株、具有保藏编号NITE P-02986的短双歧杆菌SBT10532株、具有保藏编号NITE P-02987的长双歧杆菌SBT10527株、具有保藏编号NITE P-02988的嗜热链球菌SBT0118株、具有保藏编号NITE P-02989的肠膜明串珠菌右旋葡聚糖亚种SBT0096株、具有保藏编号NITE P-02990的乳明串珠菌SBT2561株、具有保藏编号NITE BP-02991的乳酸片球菌SBT3331株或具有保藏编号NITEBP-02992的戊糖片球菌SBT2229株。
发明的效果
根据本发明,可预防和/或改善随老化(衰老)发生的记忆和学习能力的下降。本发明对于改善担忧其记忆和学习能力的下降的老年人的日常生活中的生活质量是有效的。
附图说明
[图1]说明使用秀丽隐杆线虫的趋温性试验的概述的图。
[图2]显示秀丽隐杆线虫的随年龄的记忆指数的变化和已摄取乳酸菌的老年成虫的记忆指数的图。
具体实施方式
本说明书中的“记忆和学习能力的维持和/或改善”优选是指随老化(衰老)发生的记忆和学习能力的下降的预防和/或改善,并且更优选是指随老化(衰老)发生的记忆能力的下降的预防和/或改善。此外,本说明书中的“随老化(衰老)发生的记忆和学习能力的下降”或“随老化(衰老)发生的记忆能力的下降”优选不包括随以下发生的记忆和学习能力的下降或记忆能力的下降:痴呆如阿尔茨海默病和亨廷顿病、脑疾病包括基底神经节疾病如帕金森病、记忆损害如健忘症、记忆丧失和科萨科夫综合征以及精神疾病如重度抑郁症、焦虑症、双相情感障碍、精神分裂症、睡眠障碍和应激障碍。
本说明书中的“组合物”优选是指药物组合物或食品组合物。
本发明的记忆和学习能力维持和/或改善用组合物的特征在于,含有属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属和片球菌属的任何微生物作为活性成分。属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属和片球菌属的微生物优选为嗜酸乳杆菌种、加氏乳杆菌种、母鸡乳杆菌种、约氏乳杆菌种、德氏乳杆菌德氏亚种、链状双歧杆菌种、假长双歧杆菌种、两歧双歧杆菌种、短双歧杆菌种、长双歧杆菌种、嗜热链球菌种、肠膜明串珠菌右旋葡聚糖亚种、乳明串珠菌种、乳酸片球菌和戊糖片球菌种。进一步优选嗜酸乳杆菌SBT1921株、加氏乳杆菌SBT2056株、母鸡乳杆菌SBT0316株、约氏乳杆菌SBT0307株、德氏乳杆菌德氏亚种SBT0413株、链状双歧杆菌SBT11114株、假长双歧杆菌SBT2922株、两歧双歧杆菌SBT10550株、短双歧杆菌SBT10532株、长双歧杆菌SBT10527株、嗜热链球菌SBT0118株、肠膜明串珠菌右旋葡聚糖亚种SBT0096株、乳明串珠菌SBT2561株、乳酸片球菌SBT3331株和戊糖片球菌SBT2229株,但对微生物没有特别限制,只要微生物是具有本发明的记忆和学习能力维持和/或改善作用的、属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属的那些即可。
属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属的微生物可单独使用或者可以适当地与两种以上的乳酸菌组合使用。此外,属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属的微生物可根据用于培养乳酸菌的一般方法培养。对于培养基,可使用各种培养基,例如乳培养基、含有乳组分的培养基以及不含有乳组分的半合成培养基。这样的培养基的实例包括还原脱脂乳培养基(reduced skim milk medium)、MRS介质等。
通过例如离心等细胞收集手段从获得的培养物分离的细菌细胞可直接用作本发明的活性成分。也可使用浓缩、干燥或冷冻干燥等之后的细菌细胞,也可以使用通过加热干燥等获得的死细胞。作为细菌细胞,不仅可以使用单纯分离的细菌细胞,还可使用培养物、悬浮液、其他含有细菌细胞的产品以及通过用酶或物理手段处理细菌细胞而获得的细胞质或细胞壁部分(fraction)。培养物等的形式的实例不仅包括使用通常用于培养乳酸菌的培养基如作为合成培养基的MRS培养基(由DIFCO制造)和还原脱脂乳培养基的培养物,还包括乳制品如奶酪、发酵乳和乳制品乳酸菌饮料等,但对形式没有特别限制。
对于本发明的记忆和学习能力维持和/或改善剂的配制,可以在适当地混合制剂可接受的赋形剂、稳定剂、矫味剂等之后进行浓缩或冷冻干燥,或者可以通过加热干燥来获得死细胞。这些的干燥物、浓缩物和糊状物也包括在内。此外,在不损害属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属的微生物的细菌细胞或培养物的记忆和学习能力维持和/或改善作用的范围内,赋形剂、粘合剂、崩解剂、润滑剂、调味剂、悬浮剂、涂布剂或任何其他药物可以被混合用于配制。作为剂型,片剂、胶囊剂、颗粒剂、散剂、撒布剂(dusting powder)、糖浆剂等是可接受的,并且这样的剂型期望口服施用。
本发明的记忆和学习能力维持和/或改善用组合物可以混入任何饮食品中,并且可以在饮食品的生产过程中添加至原材料中。饮食品的实例包括乳制品如奶酪、发酵乳、乳制品乳酸菌饮料、乳酸菌饮料、黄油和人造黄油,饮料如乳饮料、果汁和软饮料,加工蛋制品如果冻、糖果、布丁和蛋黄酱,糕点-面包类如黄油蛋糕,不同种类的乳粉、婴儿食品、营养组合物等,但对饮食品没有特别限制。此外,本发明的记忆和学习能力维持和/或改善用组合物可混入饲料中。与饮食品类似,所述组合物可以混入任何饲料中,并且可以在饲料的生产过程中添加至原材料中。
当属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属的微生物的细胞或培养物通过混入记忆和学习能力维持和/或改善用组合物或记忆和学习能力维持和/或改善用饮食品、营养组合物或饲料等的材料或所述材料的加工产品中来使用时饮食品,对属于乳杆菌属、双歧杆菌属、链球菌属、明串珠菌属或片球菌属的微生物的细胞或培养物的比例没有特别限制,并且可以根据生产的容易度或优选的日剂量适当地进行调整。尽管比例是考虑施用的受试者的症状、年龄等来分别地决定的,但在一般成人的情况下,例如,可以以可摄取10至200g的加氏乳杆菌的培养物等或0.1至5,000mg的其细菌细胞自身的方式调整混入量等。通过以这种方式摄入,可以发挥期望的效果。
作为本发明中预防和/或改善随老化(衰老)发生的记忆和学习能力的下降的乳酸菌的筛选方法,进行了使用秀丽隐杆线虫的趋温性试验。通常,在检测食品组分和药物的生理功能时,使用高等动物如小鼠、大鼠、狗和猴。然而,所述使用需要大量的劳动力、时间和成本,并且应充分注意伦理方面。作为动物研究的替代方法,还进行了使用从动物组织等分离的培养细胞的试验。
秀丽隐杆线虫是一种生活在土壤中的非寄生性多细胞生物,鉴于作为实验动物容易处理,并且由于已全部阐明构成身体的约1000个体细胞的细胞谱系,以及还由于秀丽隐杆线虫具有神经系统,被广泛用作不同研究领域中的模式生物。特别地,秀丽隐杆线虫在细胞死亡、神经、发育、衰老等的不同研究领域中具有很大贡献。近年来,使用作为用于神经退行性疾病的模型的秀丽隐杆线虫,已寻找对于预防或改善各种神经退行性疾病如阿尔茨海默病、肌萎缩性脊髓侧索硬化症(amyotrophic lateral sclerosis,ALS)、帕金森病和亨廷顿病有效的药物和食品组分。
已知,秀丽隐杆线虫具有简单的神经回路,但将饮食的存在或不存在与秀丽隐杆线虫所处环境中的盐浓度和温度等信息相关联进行记忆-学习。因此,秀丽隐杆线虫可用作用于评价认知功能如联想学习和记忆的模型系统(Nature,376,344-348(1995))。
以下基于实施例更详细地说明本发明,但本发明不被解释为限于这些实施例。
[试验例1]预防和/或改善随老化(衰老)发生的认知功能的下降的乳酸菌的评价
作为本说明书中使用的秀丽隐杆线虫,使用作为野生型的N2株,并根据“线虫实验室手册”进行培养。
1.试验方法
(1)待测细菌
对表1中所示的细菌菌株进行筛选。
[表1]
待测细菌
(2)试验程序
(i)评价样本的制备
在表2中的条件下培养上述(1)中的待测细菌。将培养的细菌细胞离心(7,000×g,4℃,15分钟),丢弃上清液。采用生理盐水洗涤细菌细胞后,测定细菌细胞的湿重。将细菌细胞再次悬浮在NG缓冲液(0.3%NaCl、1mM CaCl2、1mM MgSO4、25mM磷酸盐缓冲液[pH6.0])中,至浓度为0.05g/ml,并且将悬浮液各自以200μl的体积涂布在秀丽隐杆线虫-培养板(NGM板)上以获得样本板。作为对照,使用通常用作秀丽隐杆线虫的食物的大肠杆菌(Escherichia coli)OP50株。
[表2]
用于培养待测细菌的培养条件
(ii)摄取乳酸菌的老年成虫的制备
将通过漂白(bleaching)处理收集的秀丽隐杆线虫的卵接种在已接种有OP50的板上,并在23℃下静置三天,从而获得年轻成虫。用NG缓冲液收集成虫,移至已接种有乳酸菌的板或已接种有OP50的板上,在23℃下培养四天,从而获得老年成虫。
(iii)通过趋温性试验筛选
根据Ito H.等人(J.Neurosci.Methods,154,45-52(2006))描述的方法如下进行趋温性试验。准备两个恒温槽。通过将U形铝架(21.5cm×60cm×1.5cm厚)的一个支柱浸入低温(5℃)的恒温槽中,并将另一个支柱浸入高温(35℃)的恒温槽中,在铝架上形成约5℃至35℃的线性温度梯度。以使20℃的点位于中间的方式将方形盘中的琼脂板置于铝架上。将年轻成虫或老年成虫在盘的中间释放,并评价一个小时后的温度梯度中的秀丽隐杆线虫的分布。关于记忆和学习能力,将板沿温度梯度方向等分为八段,并将培养温度(23℃)附近的两段中的秀丽隐杆线虫的百分比评价作为记忆指数(memory index)(图1)。此外,通过Dunnett多重检验,使用摄取OP50的老年成虫作为对照,评价存在或不存在显著性差异(当p<0.05时,确定存在显著性差异)。
2.试验结果
筛选的结果示于图2。与年轻成虫的记忆指数相比,老年成虫的记忆指数下降。
摄取以下的老年成虫的记忆指数与摄取OP50的老年成虫的记忆指数相比显著增加:嗜酸乳杆菌SBT1921株、加氏乳杆菌SBT2056株、母鸡乳杆菌SBT0316株、约氏乳杆菌SBT0307株、德氏乳杆菌德氏亚种SBT0413株、链状双歧杆菌SBT11114株、假长双歧杆菌SBT2922株、两歧双歧杆菌SBT10550株、短双歧杆菌SBT10532株、长双歧杆菌SBT10527株、嗜热链球菌SBT0118株、肠膜明串珠菌右旋葡聚糖亚种SBT0096株、乳明串珠菌SBT2561株、乳酸片球菌SBT3331株和戊糖片球菌SBT2229株。即,发现摄取所述细菌细胞可预防和/或改善随老化(衰老)发生的记忆和学习能力的下降。
(实施例1)记忆和学习能力维持和/或改善用剂(颗粒剂)的生产
将母鸡乳杆菌SBT0316株以5重量%接种至可食用合成培养基(含有0.5%酵母提取物和0.1%胰蛋白酶解酪蛋白胨(trypticase peptone))中,在38℃下培养15小时,然后通过离心来收集细菌细胞。将回收的细菌细胞冷冻干燥,从而获得细菌细胞的冷冻干燥粉末。将1克的冷冻干燥粉末与5g的乳糖混合,将混合物成形为颗粒,以获得本发明的记忆和学习能力维持和/或改善用剂。
(实施例2)记忆和学习能力维持和/或改善用剂(散剂)的生产
根据第13次修订的日本药典指南通用配方(Japanese Pharmacopoeia GuidelineGeneral Formulation)“散剂”的规定,将400g的乳糖(日本药典)和600g的马铃薯淀粉(日本药典)添加至10g的实施例1中获得的母鸡乳杆菌SBT0316株的冷冻干燥粉末并均匀混合,从而生产本发明的记忆和学习能力维持和/或改善用剂。
(实施例3)记忆和学习能力维持和/或改善用营养组合物的生产
将母鸡乳杆菌SBT0316株以5重量%接种至可食用合成培养基(含0.5%酵母提取物和0.1%胰蛋白酶解酪蛋白胨)中,在38℃下培养15小时。然后通过离心收集细菌细胞,并将收集的细菌细胞冷冻干燥。向40g的维生素C或40g的含等量维生素C和柠檬酸的混合物、100g的砂糖和60g的含等量玉米淀粉和乳糖的混合物,添加40g的该冷冻干燥粉末的冷冻干燥粉末并混合。将混合物包装在袋中,从而生产本发明的记忆和学习能力维持和/或改善用营养组合物。
(实施例4)记忆和学习能力维持和/或改善用饮料的生产
在将1g的在实施例3中获得的母鸡乳杆菌SBT0316株溶解于699g的去离子水后,将混合物加热至40℃,然后使用超分散机(ULTRA-TURRAX T-25;由IKA Japan KK制造)、以9,500rpm搅拌和混合20分钟。在添加100g的麦芽糖醇、2g的酸味剂、20g的还原淀粉糖浆、2g的香料和176g的去离子水后,用混合物填充100ml玻璃瓶,在95℃下灭菌15秒,然后紧密塞住。从而生产十瓶(100ml)本发明的记忆和学习能力维持和/或改善用饮料。
(实施例5)记忆和学习能力维持和/或改善用饲料的生产
将十二千克的大豆粉、14kg的脱脂乳粉、4kg的大豆油、2kg的玉米油、23.2kg的棕榈油、14kg的玉米淀粉、9kg的面粉、2kg的麦麸、5kg的维生素混合物、2.8kg的纤维素和2kg的矿物质混合物混合并在120℃下灭菌四分钟。然后,混入10kg的实施例3中获得的母鸡乳杆菌SBT0316株。从而生产本发明的记忆和学习能力维持和/或改善用饲料。
(实施例6)记忆和学习能力维持和/或改善用发酵乳的生产
将母鸡乳杆菌SBT0316株以5重量%接种至可食用合成培养基(添加0.5%酵母提取物、0.1%胰蛋白酶解酪蛋白胨)中,并在37℃下培养17小时,然后通过离心收集细菌细胞并冷冻干燥。将5克的冷冻干燥的细菌细胞、1700g的脱脂乳粉、300g的葡萄糖和7695g的去离子水混合,并通过将混合物保持在95℃2小时来进行加热灭菌。将所得物冷却至37℃,并接种300g的乳酸菌起种(starter)(Lb.casei)。搅拌和混合后,在保持在37℃的培养箱中发酵混合物,直至pH达到4.0。在pH达到4.0后,将所得物冷却至10℃以下,从而生产10kg的本发明的记忆和学习能力维持和/或改善用发酵乳。
保藏编号
(1)嗜酸乳杆菌SBT1921株(保藏编号:NITE P-02980,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(2)母鸡乳杆菌SBT0316株(保藏编号:NITE P-02981,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(3)约氏乳杆菌SBT0307株(保藏编号:NITE P-02982,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(4)链状双歧杆菌SBT11114株(保藏编号:NITE P-02983,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(5)假长双歧杆菌SBT2922株(保藏编号:NITE P-02984,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(6)两歧双歧杆菌SBT10550株(保藏编号:NITE P-02985,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(7)短双歧杆菌SBT10532株(保藏编号:NITE P-02986,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(8)长双歧杆菌SBT10527株(保藏编号:NITE P-02987,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(9)嗜热链球菌SBT0118株(保藏编号:NITE P-02988,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(10)肠膜明串珠菌右旋葡聚糖亚种SBT0096株(保藏编号:NITE P-02989,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(11)乳明串珠菌SBT2561株(保藏编号:NITE P-02990,保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(12)乳酸片球菌SBT3331株(保藏编号:NITE ABP-02991,接收日期:2020年7月13日,原保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
(13)戊糖片球菌SBT2229株(保藏编号:NITE ABP-02992,接收日期:2020年7月13日,原保藏日期:2019年6月26日),保藏单位:邮政编码:292-0818日本国千叶县木更津市上总镰足2-5-8,国家技术评估学会,专利微生物保藏中心(NPMD)。
PCT/RO/134表
Claims (13)
1.一种记忆和学习能力维持和/或改善用组合物,其含有属于乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)、链球菌属(Streptococcus)、明串珠菌属(Leuconostoc)和片球菌属(Pediococcus)的任何的一种以上的微生物的细胞或培养物。
2.根据权利要求1所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述乳杆菌属(Lactobacillus)是选自嗜酸乳杆菌(Lactobacillus acidophilus)、加氏乳杆菌(Lactobacillus gasseri)、母鸡乳杆菌(Lactobacillus gallinarum)、约氏乳杆菌(Lactobacillus johnsonii)和德氏乳杆菌德氏亚种(Lactobacillus delbrueckiisubsp.Delbrueckii)的一种以上。
3.根据权利要求1所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述双歧杆菌属(Bifidobacterium)是选自链状双歧杆菌(Bifidobacterium catenulatum)、假长双歧杆菌(Bifidobacterium pseudolongum)、两歧双歧杆菌(Bifidobacteriumbifidum)、短双歧杆菌(Bifidobacterium breve)和长双歧杆菌(Bifidobacteriumlongum)的一种以上。
4.根据权利要求1所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述链球菌属(Streptococcus)是嗜热链球菌(Streptococcus thermophilus)。
5.根据权利要求1所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述明串珠菌属(Leuconostoc)是选自肠膜明串珠菌右旋葡聚糖亚种(Leuconostocmesenteroides subsp.dextranicum)和乳明串珠菌(Leuconostoc lactis)的一种以上。
6.根据权利要求1所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述片球菌属(Pediococcus)是选自乳酸片球菌(Pediococcus acidilactici)和戊糖片球菌(Pediococcus pentosaceus)的一种以上。
7.根据权利要求2所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述乳杆菌属(Lactobacillus)是选自嗜酸乳杆菌(Lactobacillus acidophilus)SBT1921株、加氏乳杆菌(Lactobacillus gasseri)SBT2056株、母鸡乳杆菌(Lactobacillusgallinarum)SBT0316株、约氏乳杆菌(Lactobacillus johnsonii)SBT0307株和德氏乳杆菌德氏亚种(Lactobacillus delbrueckii subsp.Delbrueckii)SBT0413株的一种以上。
8.根据权利要求3所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述双歧杆菌属(Bifidobacterium)是选自链状双歧杆菌(Bifidobacterium catenulatum)SBT11114株、假长双歧杆菌(Bifidobacterium pseudolongum)SBT2922株、两歧双歧杆菌(Bifidobacterium bifidum)SBT10550株、短双歧杆菌(Bifidobacterium breve)SBT10532株和长双歧杆菌(Bifidobacterium longum)SBT10527株的一种以上。
9.根据权利要求4所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述链球菌属(Streptococcus)是嗜热链球菌(Streptococcus thermophilus)SBT0118株。
10.根据权利要求5所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述明串珠菌属(Leuconostoc)是选自肠膜明串珠菌右旋葡聚糖亚种(Leuconostocmesenteroides subsp.dextranicum)SBT0096株和乳明串珠菌(Leuconostoc lactis)SBT2561株的一种以上。
11.根据权利要求6所述的记忆和学习能力维持和/或改善用组合物,其特征在于,所述片球菌属(Pediococcus)是选自乳酸片球菌(Pediococcus acidilactici)SBT3331株和戊糖片球菌(Pediococcus pentosaceus)SBT2229株的一种以上。
12.一种记忆和学习能力维持和/或改善用食品、记忆和学习能力维持和/或改善用药物或记忆和学习能力维持和/或改善用饲料,其特征在于,其含有根据权利要求1至11中任一项所述的记忆和学习能力维持和/或改善用组合物。
13.具有保藏编号NITE BP-02991的乳酸片球菌(Pediococcus acidilactici)SBT3331株或具有保藏编号NITE BP-02992的戊糖片球菌(Pediococcus pentosaceus)SBT2229株。
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