CN115444867A - 短双歧杆菌207-1的应用 - Google Patents
短双歧杆菌207-1的应用 Download PDFInfo
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- CN115444867A CN115444867A CN202211273110.6A CN202211273110A CN115444867A CN 115444867 A CN115444867 A CN 115444867A CN 202211273110 A CN202211273110 A CN 202211273110A CN 115444867 A CN115444867 A CN 115444867A
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- lactobacillus
- bifidobacterium breve
- bifidobacterium
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Abstract
本申请涉及短双歧杆菌或其后代,或包含所述短双歧杆菌或其后代的组合物在制备药物或食品中的用途。本申请还涉及一种提高受试者胃肠道中短双歧杆菌的浓度的方法。所述短双歧杆菌在抗氧化方面具有较高的潜能。
Description
技术领域
本申请涉及短双歧杆菌或其后代,或包含所述短双歧杆菌或其后代的组合物在制备药物或食品中的用途。本申请还涉及一种制备抗氧化剂的方法,以及一种预防和/或抑制氧化应激的方法。
背景技术
人体氧化作用对于许多生命体来说是必须的,是维持正常的生命活动的必须能量来源,然而伴随着氧化作用的进行也会产生一些对生物分子有破坏作用的活性氧,这些活性氧会产生大量以氧为中心的自由基,这些活性氧与生物体的衰老及疾病的产生有着密切的关联。
抗氧化活性物质通过抑制自由基的产生,清除、熄灭自由基来抑制自由基参与的过氧化反应即为抗氧化作用(antioxidation)。抗氧化物质主要抑制ROS的产生,抑制过氧化氢的生成,减少DNA的氧化损伤, 抑制脂质过氧化。生物体内天然存在着抗氧化的机制,主要包括两大类, 一是抗氧化酶类,主要包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POX)及谷胱甘肽过氧化物酶(GSH-PX)等;另一类则是非酶类抗氧化剂,主要是一些天然小分子有机物,包括维生E、维生C、谷胱甘肽(GSH)、一氧化氮(NO)、β-胡萝卜素等。近年来,合成抗氧化剂对健康的安全性及其长效性愈发受到质疑,而天然抗氧化剂由于具有天然、高效、低毒的特点而逐渐为人们所关注和利用。
随着人们对益生菌功效认识的深入,越来越多的实验显示了益生菌在宿主抗氧化方面的益生作用。研究显示,部分益生菌可通过产生超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)来清除羟自由基和过氧化氢,从而实现抗氧化的功能。因此,利用益生菌的抗氧化活性来达到清除体内自由基和过氧化物是一种疾病预防的简便而有效的方法。
此外,益生菌能否对机体发挥以上益生作用,还取决于菌株能否耐受机体胃肠道的防御机制,如胃液中的低pH值环境和小肠中胆汁酸等,而以活菌的状态顺利到达肠道,粘附于肠上皮细胞,并在肠道内定植,产生有益代谢产物并与宿主发生相互作用。因此,筛选能够进入肠道、具有高生存性的菌株,也是益生菌开发的首要考虑的问题。
根据益生菌的科学共识:目前研究表明,不同种的益生菌基因组差别较大,即便是同种益生菌的不同菌株之间也存在差异性。国际乳品联合会(International DairyFederation,IDF)发布的《IDF公报 No.513/2021益生菌菌株水平鉴定指导文件》文件也指出,益生菌的功能性和安全性具有菌株水平特异性。由于同种益生菌的不同菌株含有或表达不同的功能基因,可发挥不同的益生功效,因此益生菌需要在微生物菌株水平上进行表征和描述。这种菌株特异性的差异也在本发明的相关实验研究结果中有所体现:即便是同一来源、同一种属的不同菌株,其生理特性和益生功效也存在显著差异。
因此,一种具有抗氧化功效的菌株将在益生菌生产和应用中具有广泛的应用潜能。
发明内容
本专利申请人在前期筛选到了一株益生菌—短双歧杆菌207-1,其微生物保藏编号为:GDMCC No.60962。在后续的研究过程中,意外的发现了该菌株在抗氧化方面具有突出的功效。因此,本申请的菌株和包含其的组合物在制备预防和/或抑制氧化应激的药物或食品中具有巨大潜能。
因此,在第一方面,本申请提供了短双歧杆菌(Bifidobacterium breve)或其后代,或包含所述短双歧杆菌或其后代的组合物在制备药物或食品中的用途,所述药物或食品用于在受试者中预防和/或抑制氧化应激,或者预防和/或治疗由氧化应激所引起的疾病或症状;
其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.60962。
在某些实施方案中,所述预防和/或抑制氧化应激,或者预防和/或治疗由氧化应激所引起的疾病或症状是通过下述的任意一种或多种方式:降低受试者体内丙二醛含量,降低受试者体内8-表氢氧-异前列腺素含量,降低受试者体内肝组织蛋白质羰基含量,提高超氧化物歧化酶活性。
在某些实施方案中,所述疾病是亨廷顿舞蹈症。在某些实施方案中,所述症状是由氧化应激所引起的衰老。
在本文中,术语“药物”涵盖用于人类的药物以及用于动物的药物 (即兽医应用)。在某些实施方案中,所述药物用于人。
在某些实施方案中,所述药物组合物包含短双歧杆菌的制剂。
在某些实施方案中,所述药物组合物包含药学上可接受的载体。
在某些实施方案中,所述药物组合物被配制用于口服施用。
在某些实施方案中,所述药物或食品是靶向胃肠释放的药物,或者是在胃肠中受控释放的药物。
在某些实施方案中,所述药物是丸剂、粉剂、胶囊剂、片剂(例如,泡腾片剂)、盖膜剂、口溶性颗粒剂、液体剂、栓剂或灌肠剂的形式。
在某些实施方案中,所述药物或食品单独使用,或与其他抗真菌剂、止痛药、抗炎药、促愈合剂、或保湿剂联合使用。
在文本中,“食品”一词是广义的,包括人类的食物和饮物,也涵盖动物的食物和饮物(即饲料)。在某些实施方案中,所述食品适合于以及设计用于人类进食。
可以理解的是,根据用途、应用方式或施用方式的不同,本申请的食品可以是液体、固体、悬浮液或粉末的形式。
在某些实施方案中,所述食品选自固体饮料,糖果或果汁,或者,所述食品为乳制品(例如,酸奶,风味发酵乳,乳酸菌饮料,奶酪)。
在某些实施方案中,所述食品是膳食补充剂。
如本文中所使用的,术语“膳食补充剂”是指能够向消费者提供有益效果(例如,营养效果、预防效果、治疗效果或其他有益效果)的可食用的产品。在本文中,膳食补充剂涵盖保健食品、特医食品、营养品、补剂等产品。
在某些实施方案中,所述膳食补充剂被配制用于口服施用。
在某些实施方案中,食品还可包括(但不限于)下列物质中的一种或任何组合:益生菌(例如,益生细菌),碳水化合物(例如,膳食纤维),蛋白质(例如,酶),脂类物质(例如,脂肪),维生素,矿物质。
在某些实施方案中,食品还可包括免疫调节剂。
在某些实施方案中,食品还可包括植物成分(例如黄酮类、多酚类植物提取物等),乳汁替代物,或者短双歧杆菌或其后代的代谢物或提取物。
在某些实施方案中,还可将本发明的菌株与不同的甜味剂或调味剂、调色物质、稳定剂、助流剂、填充剂等食品中可接受的辅料进行组合。
在某些实施方案中,所述食品还包含益生元。
在某些实施方案中,所述益生元选自低聚果糖、低聚半乳糖、低聚木糖、低聚异麦芽糖、大豆低聚糖、菊粉、螺旋藻、节旋藻、云芝多糖, 胡萝含氮多糖、酪蛋白水解物、α-乳清蛋白、乳铁蛋白,或其任何组合。
在某些实施方案中,所述食品是丸剂、粉剂、胶囊剂、片剂(例如,泡腾片剂)、盖膜剂、口溶性颗粒剂、液体剂的形式。
在某些实施方案中,所述受试者为哺乳动物。在某些实施方案中,所述哺乳动物选自鼠,猪,兔,猴,羊,人。
在某些实施方案中,所述药物或食品中短双歧杆菌以106至1012CFU/剂量的量存在。
在某些实施方案中,所述药物或食品中短双歧杆菌以108至 1012CFU/剂量的量存在。
可以理解的是,本领域技术人员有能力根据受试者的具体情况而施用对受试者有效量的副干酪乳杆菌。
在某些实施方案中,所述组合物包含所述短双歧杆菌或其后代,以及选自以下的微生物:细菌,真菌,或其任何组合。
在某些实施方案中,所述微生物是益生菌。
在某些实施方案中,所述微生物是酵母。
在某些实施方案中,所述酵母选自酿酒酵母(Saccharomyces cerevisiae),布拉氏酵母(Saccharomyces boulardii),马克斯克鲁维酵母 (Kluyveromyces marxianus),或其任何组合。
在某些实施方案中,所述细菌选自乳酸杆菌属(Lactobacillus spp.),双歧杆菌属(Bifidobacterium spp.),芽孢杆菌属(Bacillus spp.),丙酸杆菌属(Propionibacterium spp.),链球菌属 (Streptococcus spp.),乳球菌属(Lactococcusspp.),片球菌属 (Pediococcus spp.),肠球菌属(Enterococcus spp.),葡萄球菌属(Staphylococcus spp.),或其任何组合。
在某些实施方案中,所述乳酸杆菌属的细菌选自:副干酪乳杆菌,嗜酸乳杆菌(Lactobacillus acidophilus),短乳杆菌(Lactobacillus brevis),詹氏乳杆菌(Lactobacillus jensenii),惰性乳杆菌(Lactobacillus iners),干酪乳杆菌(Lactobacillus casei),卷曲乳杆菌(Lactobacillus crispatus),弯曲乳杆菌(Lactobacillus curvatus),德氏乳杆菌 (Lactobacillus 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),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),唾液乳杆菌(Lactobacillus salivarius),或其任何组合。
在某些实施方案中,所述双歧杆菌属的细菌选自:动物双歧杆菌(Bifidobacterium animalis),两歧双歧杆菌(Bifidobacterium bifidum),短双歧杆菌(Bifidobacterium breve),婴儿双歧杆菌(Bifidobacterium infantis),长双歧杆菌(Bifidobacterium longum),青春双歧杆菌 (Bifidobacterium adolescentis),或其任何组合。
在某些实施方案中,所述芽孢杆菌属的细菌选自:枯草芽孢杆菌 (Bacillussubtilis),凝结芽孢杆菌(Bacillus coagulans),或其任何组合。
在某些实施方案中,所述丙酸杆菌属的细菌选自:谢氏丙酸杆菌(Propionibacterium shermanii),费氏丙酸杆菌(Propionibacterium freudenreichii),产丙酸丙酸杆菌(Propionibacterium acidipropionici),或其任何组合。
在某些实施方案中,所述链球菌属的细菌选自:嗜热链球菌 (Streptococcusthermophilus),唾液链球菌(Streptococcus salivarius),或其任何组合。
在某些实施方案中,所述乳球菌属的细菌为乳酸乳球菌 (Lactococcus lactis)。
在某些实施方案中,所述肠球菌属的细菌选自:粪肠球菌 (Enterococcusfaecalis),屎肠球菌(Enterococcus faecium),蒙氏肠球菌 (Enterococcus mundtii),或其任何组合。
在第二方面,本申请提供了一种制备抗氧化剂的方法,所述方法包括培养短双歧杆菌(Bifidobacterium breve)或其后代,其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.60962。
在某些实施方案中,所述制备抗氧化剂的方法包括:破碎所述细菌的细胞,收取破碎后的成分。
在某些实施方案中,所述制备抗氧化剂的方法包括:培养所述短双歧杆菌,破碎所述细菌的细胞,离心并收取破碎后的上清液。
在本文中,“抗氧化剂(Antioxidants)”是指能够抑制氧化或过氧化作用的物质。在某些实施方案中,抗氧化剂能够抑制自由基的产生、清除存在的自由基或者阻断自由基参与氧化反应,来抑制过氧化反应。
在某些实施方案中,抗氧化剂用于制备食品。在此类实施方案中,抗氧化剂用于防止或延缓食品氧化,或者用于提高食品的稳定性,或者用于延长食品的贮存期。
在某些实施方案中,抗氧化剂用于制备药物。在此类实施方案中,抗氧化剂用于预防和/或抑制氧化应激,或者预防和/或治疗由氧化应激所引起的疾病或症状。
在第三方面,本申请提供了一种提高受试者胃肠道中短双歧杆菌的浓度的方法,所述方法包括向受试者给予治疗有效量的短双歧杆菌或其后代,或包含所述短双歧杆菌或其后代的组合物;
其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.60962;
在某些实施方案中,所述组合物如第一方面所定义。
在某些实施方案中,所述受试者如第一方面所定义。
在第四方面,本申请提供了一种预防和/或抑制氧化应激的方法,所述方法包括施用有效量的短双歧杆菌(Bifidobacterium breve)或其后代,或包含所述短双歧杆菌或其后代的组合物;
其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.60962。
在某些实施方案中,所述组合物如第一方面所定义。
在某些实施方案中,所述方法包括在体外对宿主细胞施用有效量的短双歧杆菌(Bifidobacterium breve)或其后代,或包含所述短双歧杆菌或其后代的组合物。
在某些实施方案中,所述方法包括在受试者体内施用有效量的短双歧杆菌(Bifidobacterium breve)或其后代,或包含所述短双歧杆菌或其后代的组合物。
在某些实施方案中,所述受试者为哺乳动物。在某些实施方案中,所述哺乳动物选自鼠,猪,兔,猴,羊,人。
术语定义
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
如本文中所使用的,术语“氧化应激(Oxidative Stress,OS)”是由体内氧化与抗氧化作用失衡所导致的。通常,这种失衡是由过氧化物或自由基在体内产生或积累造成的,氧化应激被认为是导致衰老和疾病的一个重要因素,与很多疾病相关(例如,亨廷顿舞蹈症)。
如本文中所使用的,术语“益生元”是指能够促进至少一种益生菌菌株生长或扩增的物质。在某些实施方案中,益生元能够促进至少一种益生菌菌株在受试者体内(例如,在人体胃肠道中)生长或扩增。在某些优选的实施方案中,所述益生元能够促进短双歧杆菌在人体胃肠道中生长或扩增。
如本文中所使用的,术语“后代”是指微生物通过生长(例如培养基中培养生长)而产生的子代细胞。可以理解的是,在微生物的培养过程中,遗传物质可能会发生变化(例如,1个或几个碱基的突变,置换或缺失),而不同菌株在同一功能的有无会有显著的区分(参见,例如,IPAinternational probiotics association)。因此,在某些实施方案中,所述后代与其所衍生自的菌株相比,具有相同的功能(例如,能够预防和/或抑制氧化应激,或者预防和/或治疗由氧化应激所引起的疾病或症状)。在某些实施方案中,所述后代与其所衍生自的菌株相比,具有相同的生理生化特性(例如,菌落形态相同,具有相同的16s DNA)。在某些实施方案中,所述短双歧杆菌的后代为革兰阳性菌,一端或两端膨大或分叉,无芽孢。在某些实施方案中,所述短双歧杆菌的后代具有显著地降低小鼠血清中的丙二醛含量,降低血清中8-表氢氧- 异前列腺素含量,提高血清中SOD活力,降低肝组织中的蛋白质羰基含量的功能。
如本文中所使用的,术语“药学上可接受的载体”是指在药理学和/或生理学上与受试者和活性成分相容的载体,其是本领域公知的 (参见例如Remington'sPharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:MackPublishing Company,1995),并且包括但不限于:pH调节剂,表面活性剂,佐剂,离子强度增强剂。例如,pH调节剂包括但不限于磷酸盐缓冲液;表面活性剂包括但不限于阳离子,阴离子或者非离子型表面活性剂,例如Tween-80;离子强度增强剂包括但不限于氯化钠。
如本文中所使用的,术语“膳食补充剂”是指能够向消费者提供有益效果(例如,营养效果、预防效果、治疗效果或其他有益效果)的可食用的产品。在本文中,膳食补充剂涵盖保健食品、特医食品、营养品、补剂等产品。
如本文中所使用的,术语“食品”包括人类的食物和饮物,也涵盖动物的食物和饮物(即饲料)。在某些实施方案中,所述食品适合于以及设计用于人类进食。
如本文中所使用的,术语“药物”涵盖了人类医学和兽医学中供人类和动物两者使用的药物,同时涵盖了用于掺入动物饲料(例如牲畜饲料和/或宠物食物)中的药物。此外,本文中所使用的术语“药物”意指提供治疗、预防和/或有益效果的任何物质。
如本文中所使用的,术语“CFU(Colony-Forming Units)”是指产品中细菌、真菌、酵母等微生物的群落总数,通常用作活菌数计算。
如本文中所使用的,术语“CFU/剂量”意指每天或每次向受试者提供的组合物/食品/药物组合物中细菌存在的量。例如,在某些实施方案中,所述食品中短双歧杆菌以106至1012CFU/剂量的量存在(例如108至1012CFU/剂量)。在此种实施方案中,如果将短双歧杆菌施用在食品中(例如,在固体饮料、酸奶中),则每天或每次向受试者提供的食品(例如固体饮料、酸奶)可含有约106至1012CFU的短双歧杆菌。当然,可替代地,这种细菌的量的可以分成多次施用,只要受试者在任何特定时间内(例如每24小时期间)接受的短双歧杆菌的总量是从约106至约1012CFU的细菌,即满足上述的食品中短双歧杆菌以106至 1012CFU/剂量的量存在(例如108至1012CFU/剂量)。
如本文中所使用的,术语“病原菌”是指能入侵宿主引起感染的微生物,包括病毒,细菌,真菌。通常,它们能产生致病物质,造成宿主感染。在某些实施方案中,所述病原菌是能够引起哺乳动物(例如,小鼠,人)感染的微生物。在某些实施方案中,所述病原菌是能够引起哺乳动物(例如,小鼠,人)胃肠道感染的细菌。
如本文中所使用的,术语“自由基”也称为“游离基”,是指化合物的分子在光热等外界条件下,共价键发生均裂而形成的具有不成对电子的原子或基团。如本文中所使用的,术语“自由基的氧化反应”是指自由基发生的氧化反应。为了维持生命,人体无时无刻不在发生氧化反应。然而,某些氧化反应对人体是有害的,例如细胞膜中的磷脂被氧化,会使细胞损伤,可能导致细胞功能失常甚至死亡。
发明的有益效果
本专利申请人在前期筛选到了一株益生菌—短双歧杆菌207-1,其微生物保藏编号为:GDMCC No.60962。在后续的研究过程中,意外的发现了该菌株在抗氧化方面具有突出的功效。同时,在前期研究中已发现该菌株对胃酸和胆盐有良好的耐受性,并在肠道中有良好的定植能力。因此,本申请的菌株和包含其的组合物在制备治疗氧化应激的药物或食品中具有巨大潜能,例如,有潜力应用于亨廷顿舞蹈症。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将理解,下列实施例仅用于说明本发明,而不是对本发明的范围的限定。根据优选实施方案的下列详细描述,本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
关于生物材料保藏的说明
短双歧杆菌207-1(Bifidobacterium breve 207-1)已在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心 (GDMCC,Guangdong Microbial CultureCollection Center)进行保藏,其具有保藏号GDMCC No.60962,且保藏时间为2020年1月15日。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明,否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如,本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术,可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis),《分子克隆:实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL),第2次编辑(1989);《当代分子生物学实验手册》 (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑,(1987));《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司):《PCR 2:实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、 B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995)),以及《动物细胞培养》(ANIMAL CELLCULTURE)(R.I.弗雷谢尼 (R.I.Freshney)编辑(1987))。
另外,实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。本领域技术人员知晓,实施例以举例方式描述本发明,且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.短双歧杆菌207-1的抗氧化特性(降低脂质过氧化)的动物实验
本专利申请人在前期筛选到了一株益生菌—短双歧杆菌207-1,并申请了专利。在后续的研究过程中,意外的发现了短双歧杆菌207-1在抗氧化方面具有突出的功效。因此,本实施例选取了同一菌株库中的另外6株菌(均为短双歧杆菌)作为对比,与本申请的短双歧杆菌207-1 共同进行实验(这些菌株均在前期的益生菌特性研究中表现出来较为良好的耐酸耐胆盐特性及较好的细胞粘附性)。
本申请所涉及到所有短双歧杆菌均源自于汤臣倍健自有菌株库,该菌株库所有菌株均分离于四川大学华西妇产儿童医院出生的正常足月新生儿粪便样本中。其中,新生儿的纳入标准为:居住于成都市五城区;胎龄37-42周,出生体重在2500-4000g之间,婴儿无先天性异常或出生缺陷。排除标准为:母亲在产前一个月内使用过抗生素;父母有艾滋病、结核病、乙肝等传染性疾病;新生儿因新生儿肺炎等严重疾病无法收集粪便。
收集婴儿出生后1-4月内的新鲜粪便于无菌采便管。采样后立即暂存于4℃,由采样人员低温送至实验室随即进行粪便样品的稀释培养,如不能立即操作,则厌氧4℃保存,当日进行培养,随后通过平板法分离纯化,得到单菌株,并采用梅里埃的API 50CH和16SrDNA测序鉴定分离菌株的具体种属,分别编号(205-1、205-4、206-5、206-6、207- 1、207-39、207-42)并保藏于汤臣倍健自有菌株库中。
1.1实验1:血清中丙二醛(MDA)的含量测定
1.1.1实验方法:选用老龄SPF级雌性小鼠80只,共分为8组,每组10只,包括空白对照组10只,益生菌干预组7组,每组10只。空白对照组每天灌胃无菌水1.0ml,益生菌干预组每天灌胃菌液1.0ml (0.2g菌粉用无菌水定容至1.0mL,活菌浓度为109CFU/mL),分组喂养41天后测各项指标.各组均给予维持饲料。其中,上述菌粉的制作方法为:菌株经MRS培养基复苏后,用液体MRS培养至对数期末尾,离心收集菌体,真空冻干并加入麦芽糊精等辅料调整活菌浓度后,即得菌粉。
样品采集:由老龄小鼠眼内眺静脉丛取血0.5mL,3000r/min离心 10min。使用MDA试剂盒(南京建成生物工程研究所)批号: 20211009按下表操作:
表1.各组试剂加入情况
试剂 | 标准管 | 标准空白管 | 测定管 | 测定空白管 |
10nmol/mL标准品(mL) | 0.100 | —— | —— | —— |
无水乙醇(mL) | —— | 0.100 | —— | —— |
血清(mL) | —— | —— | 0.100 | —— |
混合试剂(mL) | 4.00 | 4.00 | 4.00 | 4.00 |
混匀后,95℃水浴(开盖煮沸)50min,取出后3500r/min离心10 min,蒸馏水调,532nm处比色。
MDA含量(nmol I mL)=(测定管吸光强度-测定空白管吸光度)/(标准管吸光强度-标准空白管吸光度)×10nmol/mL×样品测试前稀释倍数
所得数据为计量资料,若干预组MDA含量明显低于空白对照组,且差异有显著性(P<0.05),可判定该干预组有降低脂质过氧化的作用。
1.1.2实验结果
干预前后各组体重无差异,说明短双歧杆菌207-1对小鼠正常发育无不良影响。实验前老龄小鼠血清丙二醛含量在各干预组与空白对照组间比较,异均无显著性(P>0.05),说明给予样品前老龄小鼠血清丙二醛含量在各组较为均衡。动物实验证明短双歧杆菌207-1可显著降低老龄小鼠血清中的丙二醛含量(表2),从而可判定短双歧杆菌207-1有降低脂质过氧化的作用,即短双歧杆菌207-1有抗氧化的作用。同一菌株库内其他短双歧杆菌干预后与空白对照组相比血清丙二醛含量无统计学差异,说明短双歧杆菌207-1抗氧化的能力存在菌株特异性。
表2.各干预组对老龄小鼠血清丙二醛的影响
1.2实验2:血清中8-表氢氧-异前列腺素的含量测定
1.2.1实验方法:选用老龄SPF级雌性小鼠80只,共分为8组,每组10只,包括空白对照组10只,益生菌干预组7组,每组10只。空白对照组每天灌胃无菌水1.0ml,益生菌干预组每天灌胃菌液1.0ml (0.2g菌粉用无菌水定容至1.0mL),分组喂养41天后测各项指标。各组均给予维持饲料。
样品采集:老龄小鼠眼内眺静脉丛取血,3000r/min离10min。使用8-IsoprostaneKIA Kit(酶联免疫试剂盒)(Cayman Chemical Company)批号:0627993,按如下操作:取上清液0.01mL,用EIA缓冲液稀释15倍备用。
绘制8-表氢氧-异前列腺素标准曲线,测定并计算血清中8-表氢氧- 异前列腺素浓度。所得数据为计量资料,若干预组8-表氢氧-异前列腺素含量明显低于空白对照组,且差异有显著性(P<0.05),可判定该干预组有降低脂质过氧化的作用。
1.2.2实验结果
干预前后各组体重无差异,说明短双歧杆菌207-1对小鼠正常发育无不良影响。动物实验证明短双歧杆菌207-1可显著降低老龄小鼠血清中8-表氢氧-异前列腺素浓度(表3),从而可判定短双歧杆菌207-1有降低脂质过氧化的作用,即双歧杆菌207-1有抗氧化的作用。同一菌株库内其他短双歧杆菌干预后与空白对照组相比血清8-表氢氧-异前列腺素浓度无统计学差异,说明短双歧杆菌207-1抗氧化的能力存在菌株特异性。
表3.各干预组对老龄小鼠血清8-表氢氧-异前列腺素的影响
实施例2.短双歧杆菌207-1的抗氧化特性(降低肝组织蛋白质羰基含量)的动物实
验
2.1实验方法:选用老龄SPF级雌性小鼠80只,共分为8组,每组10只,包括空白对照组10只,益生菌干预组7组,每组10只。空白对照组每天灌胃无菌水1.0ml,益生菌干预组每天灌胃菌液1.0ml (0.2g菌粉用无菌水定容至1.0mL),分组喂养41天后处死并检测各项指标。各组均给予维持饲料。
取一定量肝组织,在冰冷的生理盐水中漂洗,以去掉表面的血迹,滤纸拭干,使用来自南京建成生物工程研究所的蛋白质羰基试剂盒(批号:20211009)按照重量体积比1:9的比例加入试剂一,在冰浴条件下机械匀浆,将匀浆液用离心机3000r/min离心10min,取上清即10%肝组织匀浆进行测定。取10%肝组织匀浆450μL,加入50μL试剂二,室温放置10min后,以11000r/min的转速,离心10min,取上清液待测。同时取部分上清用考马斯亮蓝试剂盒测定蛋白含量。若干预组蛋白质羰基明显低于空白对照组,且差异有显著性(P<0.05),可判定该干预组有降低蛋白质过氧化的作用。
2.2实验结果
干预前后各组体重无差异,说明短双歧杆菌207-1对小鼠正常发育无不良影响。动物实验证明短双歧杆菌207-1可显著降低老龄小鼠肝组织蛋白质羰基含量(表4),从而可判定双歧杆菌207-1有抗氧化的作用。同时,同一菌株库内其他短双歧杆菌干预后与空白对照组相比肝组织蛋白质羰基含量无统计学差异,说明短双歧杆菌207-1抗氧化的能力存在菌株特异性。
表4.各干预组对老龄小鼠肝组织蛋白质羰基含量的影响
实施例3.短双歧杆菌207-1的抗氧化特性(抗氧化酶活力测定)的动物实验
实验1:血清超氧化物歧化酶(SOD)活力测定
实验方法:选用老龄SPF级雌性小鼠80只,共分为8组,每组10 只,包括空白对照组10只,益生菌干预组7组,每组10只。空白对照组每天灌胃无菌水1.0ml,益生菌干预组每天灌胃菌液1.0ml(0.2g菌粉用无菌水定容至1.0mL),分组喂养41天后测各项指标。各组均给予维持饲料。
由老龄小鼠眼内毗静脉丛取血0.5mL,3000r/min离心10min,之后使用SOD试剂盒(南京建成生物工程研究所)批号:20210929测定 SOD活力。若干预组SOD活力明显高千空白对照组,且差异有显著性 (P<0.05),可判定该干预组有提高抗氧化物酶的作用。
实验结果:干预前后各组体重无差异,说明短双歧杆菌207-1对小鼠正常发育无不良影响。如下表(表5)所示,经动物实验证实,经口给予老龄小鼠样品41d后,与空白对照组比较短双歧杆菌207-1干预组血清SOD活力升高,有显著差异(P<0.05)。即短双歧杆菌207-1能提高老龄小鼠的血清SOD活力。同时,同一菌株库内其他短双歧杆菌干预后与空白对照组相比血清SOD活力无统计学差异,说明短双歧杆菌207-1抗氧化的能力存在菌株特异性。
表5.短双歧杆菌207-1对老龄小鼠血清SOD活力的影响
3.结论:
短双歧杆菌207-1活菌制剂灌服实验大鼠后,能够显著地能降低小鼠血清中的丙二醛含量,能降低血清中8-表氢氧-异前列腺素含量,能提高血清中SOD活力,能降低肝组织中的蛋白质羰基含量,从而能有效地起到抗氧化作用。
尽管本发明的具体实施方式已经得到详细的描述,但本领域技术人员将理解:根据已经公布的所有教导,可以对细节进行各种修改和变动,并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
Claims (10)
1.短双歧杆菌(Bifidobacterium breve)或其后代,或包含所述短双歧杆菌或其后代的组合物在制备药物或食品中的用途,所述药物或食品用于在受试者中预防和/或抑制氧化应激,或者预防和/或治疗由氧化应激所引起的疾病或症状;
其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.60962。
2.权利要求1所述的用途,其中,所述药物具有选自下列的一项或多项特征:
(1)所述药物是靶向胃肠释放的药物,或者是在胃肠中受控释放的药物;
(2)所述药物还包含药学上可接受的载体;
(3)所述药物是丸剂、粉剂、胶囊剂、片剂(例如,泡腾片剂)、盖膜剂、口溶性颗粒剂、液体剂、栓剂或灌肠剂的形式。
3.权利要求1或2所述的用途,其中,所述食品具有选自下列的一项或多项特征:
(1)所述食品是膳食补充剂;
(2)所述食品还包含益生元;
优选地,所述益生元选自低聚果糖、低聚半乳糖、低聚木糖、低聚异麦芽糖、大豆低聚糖、菊粉、螺旋藻、节旋藻、云芝多糖,胡萝含氮多糖、酪蛋白水解物、α-乳清蛋白、乳铁蛋白,或其任何组合;
(3)所述食品选自固体饮料,糖果或果汁,或者,所述食品为乳制品(例如,酸奶,风味发酵乳,乳酸菌饮料,奶酪);
(4)所述食品是丸剂、粉剂、胶囊剂、片剂(例如,泡腾片剂)、盖膜剂、口溶性颗粒剂、液体剂的形式。
4.权利要求1-3任一项所述的用途,具有选自下列的一项或多项特征:
(1)所述受试者为哺乳动物;优选地,所述哺乳动物选自鼠,猪,兔,猴,羊,人;
(2)所述药物或食品中短双歧杆菌以106至1012CFU/剂量的量存在。
(3)所述药物或食品中短双歧杆菌以108至1012CFU/剂量的量存在。
5.权利要求1-4任一项所述的用途,其中,所述组合物包含所述短双歧杆菌或其后代,以及选自以下的微生物:细菌,真菌,或其任何组合;
优选地,所述微生物是益生菌;
优选地,所述微生物是酵母;
优选地,所述酵母选自酿酒酵母(Saccharomyces cerevisiae),布拉氏酵母(Saccharomyces boulardii),马克斯克鲁维酵母(Kluyveromyces marxianus),或其任何组合。
6.权利要求5所述的用途,其中,所述细菌选自乳酸杆菌属(Lactobacillus spp.),双歧杆菌属(Bifidobacterium spp.),芽孢杆菌属(Bacillus spp.),丙酸杆菌属(Propionibacterium spp.),链球菌属(Streptococcus spp.),乳球菌属(Lactococcusspp.),片球菌属(Pediococcus spp.),肠球菌属(Enterococcus spp.),葡萄球菌属(Staphylococcus spp.),或其任何组合。
7.权利要求6所述的用途,其中,所述用途具有选自下列的一项或多项特征:
(1)所述乳酸杆菌属的细菌选自:副干酪乳杆菌,嗜酸乳杆菌(Lactobacillusacidophilus),短乳杆菌(Lactobacillus brevis),詹氏乳杆菌(Lactobacillusjensenii),惰性乳杆菌(Lactobacillus iners),干酪乳杆菌(Lactobacillus casei),卷曲乳杆菌(Lactobacillus crispatus),弯曲乳杆菌(Lactobacillus curvatus),德氏乳杆菌(Lactobacillus 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),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),唾液乳杆菌(Lactobacillus salivarius),或其任何组合;
(2)所述双歧杆菌属的细菌选自:动物双歧杆菌(Bifidobacterium animalis),两歧双歧杆菌(Bifidobacterium bifidum),短双歧杆菌(Bifidobacterium breve),婴儿双歧杆菌(Bifidobacteriuminfantis),长双歧杆菌(Bifidobacterium longum),青春双歧杆菌(Bifidobacterium adolescentis),或其任何组合;
(3)所述芽孢杆菌属的细菌选自:枯草芽孢杆菌(Bacillus subtilis),凝结芽孢杆菌(Bacillus coagulans),或其任何组合;
(4)所述丙酸杆菌属的细菌选自:谢氏丙酸杆菌(Propionibacterium shermanii),费氏丙酸杆菌(Propionibacterium freudenreichii),产丙酸丙酸杆菌(Propionibacteriumacidipropionici),或其任何组合;
(5)所述链球菌属的细菌选自:嗜热链球菌(Streptococcus thermophilus),唾液链球菌(Streptococcus salivarius),或其任何组合;
(6)所述乳球菌属的细菌为乳酸乳球菌(Lactococcus lactis);
(7)所述肠球菌属的细菌选自:粪肠球菌(Enterococcus faecalis),屎肠球菌(Enterococcus faecium),蒙氏肠球菌(Enterococcus mundtii),或其任何组合。
8.一种制备抗氧化剂的方法,所述方法包括培养短双歧杆菌(Bifidobacteriumbreve)或其后代,其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.60962;
优选地,所述方法包括:培养所述短双歧杆菌,破碎所述短双歧杆菌的细胞,离心并收取破碎后的上清液。
9.一种提高受试者胃肠道中短双歧杆菌的浓度的方法,所述方法包括向受试者给予治疗有效量的短双歧杆菌或其后代,或包含所述短双歧杆菌或其后代的组合物;
其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.60962;
优选地,所述组合物如权利要求5-7任一项所定义;
优选地,所述受试者为哺乳动物;优选地,所述哺乳动物选自鼠,猪,兔,猴,羊,人。
10.一种预防和/或抑制氧化应激的方法,所述方法包括施用有效量的短双歧杆菌(Bifidobacterium breve)或其后代,或包含所述短双歧杆菌或其后代的组合物;
其中,所述短双歧杆菌保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNo.60962;
优选地,所述组合物如权利要求5-7任一项所定义。
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