CN1244684C - 能够预防病原细菌和轮状病毒引起的腹泻的乳杆菌菌株 - Google Patents
能够预防病原细菌和轮状病毒引起的腹泻的乳杆菌菌株 Download PDFInfo
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Abstract
本发明涉及乳杆菌科的新微生物,特别是乳杆菌属的微生物,它们可用于预防或治疗由轮状病毒和病原细菌引起的腹泻。具体而言,本发明涉及使用该微生物制备可摄食的支持物和含有该支持物的组合物。
Description
本发明涉及乳杆菌属的新微生物,它们可用于预防或治疗分别由病原细菌和轮状病毒引起的腹泻。具体而言,本发明涉及使用该微生物制备可摄食的支持物和含有该支持物的组合物。
产生乳酸作为主要代谢成分的生物早已为人所知。这些细菌分别可见于牛奶和牛奶加工厂、活的或腐烂的植物,也存在于人和动物的肠道中。这些微生物统称为“乳酸细菌”,它们代表了非同质性的组群,包括例如乳球菌属、乳杆菌属、链球菌属、双歧杆菌属,片球菌属等。
乳酸细菌已被用作保存食物的发酵剂,这利用了其低pH和在发酵活动中产生的发酵产物的作用来抑制腐败细菌的生长。为此,乳酸细菌已被用来制备多种不同的食品,如乳酪、酸奶和其他牛奶发酵制品。
最近,乳酸细菌引起广泛关注是因为发现某些菌株在摄入后对人和动物具有有利作用。具体而言,已经发现乳杆菌属或双歧杆菌属的特定菌株能够在肠粘膜上定居,帮助维持人和动物的健康。
在这方面,EP 0 768 375公开了双歧杆菌属的特定菌株,它们可以植入肠道菌群,也可以附着肠细胞。据报道,这些双歧杆菌帮助免疫调节,能够竞争性排斥病原菌对肠道细胞的附着,从而有助于维持个体健康。
最近几年,研究集中在乳酸细菌作为益生剂(probiotic agent)的潜在用途。益生菌是活的微生物制品,它通过保存肠道内的天然菌群促进个体健康。只要微生物制品的有效微生物和其作用方式是已知的,该微生物制品通常被认为是益生菌。人们认为,益生菌附着在肠道粘膜上,定居在肠道内,同样防止有害微生物附着于肠粘膜上。其作用的一个重要前提是它们必须以合适和活的形式到达肠道粘膜,而不会在胃肠道前部被破坏,特别是在胃中低pH的影响下。
在这方面,WO 97/00078公开了一种称为乳杆菌GG(ATCC 53103)的特异菌株,它是益生菌。该微生物特别是用在防止或治疗食物引起的过敏反应的方法中,它与已经用胃蛋白酶和/或胰蛋白酶水解处理过的食物一起给予服用者。选用的乳杆菌菌株据称具有附着和定居特性,具有蛋白酶***,因此,食物中含有的蛋白质被该特异性乳杆菌菌株分泌的蛋白酶进一步降解。该文献中讨论的方法最终导致蛋白质原料被肠道吸收,不再有明显的过敏物质。
并且,EP 0 577 903提及使用具有取代幽门螺杆菌能力的乳酸菌制备用于治疗或预防由幽门螺杆菌引起的溃疡的支持物,幽门螺杆菌被认为是导致溃疡的病因。
在WO 99/29833中报导,一种菌株LMG P 17806携带具有限定大小的3个质粒。据报导该菌株可以防止肠道细胞被鼠伤寒沙门氏菌侵袭。
了解到乳酸菌的某些菌株可能提供的有利特性,本领域希望获得其他对人和/或动物的健康有益的乳酸菌。
因此,本发明意欲提供对人和/或动物具有新的有益特性的细菌菌株。
通过提供新的微生物,即属于乳杆菌属、具有能够防止引起腹泻的病原细菌对肠道的定居并防止轮状病毒对肠道上皮细胞的感染的性状的乳酸菌,实现了本发明的目的。根据优选实施方案,乳杆菌菌株可以附着于宿主生物的肠道粘膜并能够基本在其上定居。
根据另一个优选实施方案,乳杆菌菌株可以在0.4%的胆汁盐存在时生长,因此很容易通过胃肠道并基本保持存活。
根据另一个优选实施方案,乳酸菌选自鼠李糖乳杆菌或类干酪乳杆菌,优选类干酪乳杆菌,更优选类干酪乳杆菌CNCMI-2116。
已经证实,本发明的微生物具有下列特性:它们是革兰氏阳性,触酶阴性,NH3形成精氨酸阴性,二氧化碳产生阴性,它们产生L(+)乳酸,能够在胆汁盐浓度达约0.4%时生长,可以基本上防止轮状病毒对上皮细胞的感染。
新微生物可以用来制备多种可摄食的支持物材料,如牛奶、酸奶、凝乳、发酵牛奶、牛奶基发酵产品、发酵谷物基产品、牛奶基粉末、婴儿配方食品,在支持物中的含量可以为约105cfu/g到约1011cfu/g。在本发明中,cfu代表菌落形成单位,其定义是通过在琼脂平板上的微生物计数获得的细菌细胞数量。
本发明也提供含有至少一种具有上述特性的乳杆菌菌株的食品或药物组合物。
为了制备本发明的食品组合物,至少一种本发明的乳杆菌菌株被包含在合适的支持物中,含量为约105cfu/g到约1011cfu/g,优选约106cfu/g到约1010cfu/g,更优选约107cfu/g到约109cfu/g。
在制备药物时,产品可以制备成丸剂、液体细菌悬液、干的口服补剂,湿的口服补剂、干的管饲产品或湿的管饲产品,加入的乳杆菌菌株的量为约1012cfu/g,优选约107cfu/g到约1011cfu/g,更优选约107cfu/g到约1010cfu/g。
新微生物在个体肠道中的活性通常是剂量依赖的。即,通过摄入上述食品原料或药物组合物导入的新微生物越多,微生物的保护和/或治疗活性越高。因为新微生物对人和动物没有害处,已从婴儿的粪便中分离出了该微生物,可以大量导入该微生物,使得个体肠道的大部分都被该新微生物定居。
附图说明:
图1显示评价细菌菌株的轮状病毒保护特性的细胞培养物筛选。
图2显示乳酪乳杆菌菌株CNCMI-2116(ST11)在不同生长培养基中的酸化。
图3显示乳酪乳杆菌菌株ST11在10°测定的30天存活率。
图4显示源自骨髓的小鼠附着细胞与系列稀释的ST11温育后,其中的IL-12和IL-10mRNA图谱。
图5显示由于IL-4的产生减少导致的Th2分化的结果。
图6显示细胞培养试验的结果,其中培养的ST11细胞用于抑制病原性大肠杆菌对上皮细胞的附着的试验中。
图7显示细胞培养试验的结果,其中ST11培养物上清用于抑制病原性大肠杆菌对上皮细胞的附着的试验中。
图8显示细胞培养试验的结果,其中培养的ST11细胞用于抑制鼠伤寒沙门氏菌对上皮细胞的侵袭的试验中。
图9显示细胞培养试验的结果,其中ST11培养物上清用于抑制鼠伤寒沙门氏菌对上皮细胞的侵袭的试验中。
在实现本发明的广泛研究过程中,发明人研究了婴儿粪便,从其中分离了多种不同细菌菌株。随后考察了这些菌株防止已知会引起腹泻的细菌对上皮细胞的定居的能力。
筛选了包括乳杆菌属、乳球菌属和链球菌属的几个细菌属的腹泻抑制特性。该试验基本上以代表人类病毒性腹泻的主要病原因子的三种轮状病毒血清型(血清型G1、G3和G4)和代表引起腹泻的病原微生物的病原性大肠杆菌和鼠伤寒沙门氏菌在感染个体中进行。
多种乳酸菌在合适的培养基如MRS、Hugo-Jago或M17培养基中在约30到40℃的温度(相应于其最佳生长温度)下生长。达到静止生长后,离心收集细菌,重悬于生理盐水溶液。不同试验之间,细菌细胞冰冻保存(-20℃)。
轮状病毒实验:
通过汇合细胞单层感染制备各种轮状病毒原种。轮状病毒在感染前温育。细胞以20组织培养物感染剂量感染。为了评价抗轮状病毒特性,使用了两种不同的方法。第一种方法中,检查各种细菌菌株与轮状病毒的直接相互作用,在第二种方法中,在细菌中筛选与细胞轮状病毒受体相互作用的菌株。第一种方法包括用不同的轮状病毒株接触各种细菌悬液,在合适的培养基中温育。然后,将病毒-细菌培养物施加到人类未分化结肠癌细胞HT-29的细胞单层上,继续温育。然后分析病毒复制。第二种方法包括首先将各种细菌悬液与人类未分化结肠癌细胞HT-29一起温育,然后加入病毒。继续温育后,分析病毒复制。轮状病毒复制可以通过感染细胞中轮状病毒代表的组织-免疫染色方便地分析。若以轮状病毒加指定细菌接种的细胞培养物中感染细胞数较仅接种轮状病毒的细胞中减少了90%,轮状病毒抑制效果可归因于给定细菌。
在最初分离的总共260株不同的细菌菌株中,只有9株可以基本上抑制轮状病毒复制。这些不同的细菌被确定属于乳杆菌属的鼠李糖乳杆菌或类干酪乳杆菌。一种称为干酪乳杆菌ST11的菌株已根据布达佩斯条约保藏,保藏号为NCC 2461(I-2116),这种菌株在预防轮状病毒对人类细胞的感染方面特别有效。而且,该菌株显示良好的生长特性,这可由不同培养基的酸化证实。该菌株在约10℃的低温保存期间的存活率也表现良好,这使得它非常适合包括在冰箱保存的食品或药物组合物中。
抗病原细菌实验:
为了评价抗细菌特性,选择了下列方法。一种方法是,分别考察本发明培养的乳杆菌属菌株防止引起腹泻的病原菌附着于肠道细胞或侵入肠道细胞。为此,肠道细胞与病原菌和本发明培养的乳杆菌属菌株接触,分别评价附着率或侵入率。第二种方法是,本发明的乳杆菌属菌株的细胞培养物上清与病原微生物一起加入肠道细胞,分别评价附着率或侵入率。实验过程显示培养的乳杆菌和上清对防止对肠道细胞的附着和侵入都极其有效,这表明新微生物分泌的代谢化合物可能与抗病原细菌引起的腹泻活性有关。
除了上述发现,还发现该菌株出人意料地也表现出抗过敏特性,所述菌株对不同免疫介体的合成有影响。
通常认为,体液免疫应答和过敏反应是由具有2型表型(Th2)的CD4+T细胞介导的。Th2细胞的特征在于产生高水平白介素4(IL-4),这种细胞因子对IgE的分泌是必需的,而IgE是参与过敏反应的主要抗体类别。
Th2细胞的分化为IFN-γ抑制,后者是由互斥的Th1亚类CD4+T细胞产生的。所述Th1细胞又受白介素12(IL-12)的强烈诱导。与此相对,另一种细胞因子IL-10已被证实对Th1细胞的增殖具有强烈的抑制作用,因此被认为在免疫抑制机制中发挥作用。
总之,IL-12和IL-10通过影响Th1亚类的发育都对CD4+T细胞的发育具有强烈的调节作用。IL-12是诱导Th1分化的关键调节细胞因子,从而抑制Th2应答的产生。因此,抑制Th2细胞的主要途径见于辅助细胞对IL-12合成的刺激。
众所周知,革兰氏阴性细菌的某些成分如LPS在附着细胞如巨噬细胞和树突细胞中诱导高水平IL-12。因此,现已发现革兰氏阴性细菌可以强烈促进CD4+T细胞向Th1表型的分化。
微生物ST11作为本发明乳杆菌属菌株的实例,已经测试了其在诱导参与调控CD4+T细胞分化的细胞因子中的可能作用。具体而言,已经研究了ST11对经历Th2分化的CD4+T细胞表型的作用。
为此,ST11诱导源自骨髓的小鼠附着细胞中编码这两种调节细胞因子的mRNA合成的能力与其他四株乳杆菌菌株和革兰氏阴性细菌对照(大肠杆菌K12)进行了比较。细胞与109到107cfu/ml系列稀释的细菌温育6小时后,通过半定量RT-PCR测定mRNA。
尽管所有乳杆菌菌株都可以诱导IL-12mRNA一定程度的转录,但ST11是最强的诱导者,因为,即使在最低细菌剂量下,也可检测到强PCR信号。实际上,ST11诱导IL-12mRNA转录的能力同大肠杆菌一样强。IL-10mRNA的诱导一般弱于IL-12mRNA,只有在较高的细菌剂量下才可检测到信号。但是,与其他乳杆菌菌株和大肠杆菌对照相比,ST11仍是IL-10的最强诱导者。
因此,ST11被认为可以有效地诱导参与CD4+T细胞分化的免疫调节细胞因子。其诱导IL-12的能力强使其成为抑制Th2应答的候选者,其明显的IL-10诱导能力防止了炎症应答。
除了上述发现之外,也确定了ST11是否对经历Th2分化的CD4+T细胞具有抑制作用,对Th1功能具有正效应。使用了成熟的细胞分化培养***,其中前体CD4+T细胞经多克隆活化和调节经历Th1或Th2分化,具体取决于在培养基中提供的共刺激因素的类型。Th1/Th2分化在7天的原代培养期间诱导,然后细胞在仅含培养基的继代培养中再刺激2天,通过测定上清中产生的细胞因子类型(IFN-γvs.IL-4)评价特异表型(Th1或Th2)。
已知源自BALB/c背景的小鼠的前体CD4+T细胞在中性条件(第一代培养物中只有培养基)下激活后优先向优势Th2表型分化(在第二代培养上清中IL-4高IFN-γ低)。这种表型在第一代培养物中加入针对IL-4的封闭单克隆抗体后可以完全逆转到Th1表型(高IFN-γ低IL-4)。
为了研究ST11对Th2抑制的可能作用,来自BALB/c小鼠的纯化前体CD4+T细胞在第一代培养时在存在骨髓附着细胞作为辅助细胞的情况下激活。这些细胞在仅有培养基时或在存在1mg/ml LPS或108cfu/ml ST11或108cfu/ml其他乳杆菌时共培养。此时,洗涤细胞,再次纯化CD4+T,在仅有培养基的第二代培养中再刺激。分化CD4+T细胞产生的细胞因子在2天后测定。不出所料,在仅有培养基存在时分化的细胞显示主要是Th2表型。加入ST11至第一代培养中强烈地调节Th2分化的结果,因为它导致IL-4的产生减少了8倍。抑制的幅度类似于在存在LPS时分化的细胞培养物中观察到的结果。与此相对,其他乳杆菌菌株对IL-4水平没有明显可见的作用。有趣的是,IFN-γ水平在第一代培养中加入ST11时没有增加。
总之,ST11特异性阻止经历Th2分化的CD4+T细胞产生IL-4,但并不显著增加IFN-γ的分泌。ST11不增加IFN-γ产生可能是由于其诱导IL-10的能力,因此尽管其具有抗Th2活性,但它保持了低炎症效应。
因此,结果显示,ST11是一种具有良好抗Th2谱的乳杆菌菌株,这使其成为具有抗过敏的益生活性的细菌的优秀候选者。
以下通过实施例介绍本发明。
培养基和溶液:
MRS(Difco)
Hugo-Jago(胰胨30g/l(Difco),酵母提取物10g/l(Difco),乳糖5g/l(Difco),磷酸二氢钾6g/l,牛肉提取物2g/l(Difco),琼脂2g/l(Difco))
M17(Difco)
M199(Seromed)
Ringer溶液(Oxoid)
PBS(氯化钠8g/l,氯化钾0.2g/l,磷酸氢二钠1.15g/l)磷酸二氢钾0.2g/l)
胰蛋白酶-EDTA溶液(Seromed)
人轮状病毒Wa(G1血清型)和猿猴轮状病毒SA-11(G3血清型)获自美国费城儿童医院P.A.Offit。DS-1×RRV重排病毒获自美国NIHBethesda,A.Kapikian。血清型4人轮状病毒Hochi获自德国慕尼黑大学P.Bachmann。大肠杆菌DAEC C 1845获自西雅图的华盛顿大学,大肠杆菌JPN15获自美国马里兰大学的疫苗开发中心。鼠伤寒沙门氏菌SL1344获自美国加州斯坦福大学微生物学系。
实施例1
由婴儿粪便中分离乳酸菌
由16名15到27日龄健康婴儿的尿布收集新鲜粪便。将1g新鲜粪便至于厌氧条件下转运至实验室,取样后2小时进行微生物分析,在Ringer溶液中系列稀释,在选择培养基上铺平板。MRS琼脂培养基加抗生素(phosphomycine 80μg/ml,磺胺甲恶唑93μg/ml,甲氧苄啶5μg/ml)37℃温育48小时以分离乳酸菌。随机挑取菌落并纯化。对分离物进行生理和遗传鉴定。
实施例2
在细胞培养物中测定抗轮状病毒活性
选择包括乳杆菌属、乳球菌属、链球菌属的几个细菌属,测定细胞培养物抑制实验中具有抗轮状病毒活性的成员。乳球菌属由包括两个亚种(乳酸乳球菌乳亚种和乳酸乳球菌乳脂亚种)的单一种(乳酸乳球菌)代表。总共测定了30个菌株。链球菌属由具有45个菌株的单一种(嗜热链球菌)代表。明串珠菌属和丙酸杆菌属仅由单一种(6个菌株)代表,肠球菌和链球菌属各由两个种代表,总共有17个菌株。
总共测定了260个细菌菌株的轮状病毒抑制活性。
第一种方法:
30微升细菌悬液(平均含有3×106细菌)与70微升M199培养基混合,培养基中补加有10%胰蛋白
磷酸培养液(Flow)和5%胰蛋白酶-EDTA溶液(Seromed)(HT-29细胞时1∶4稀释),在补充M199培养基中另外还含有100微升病毒。病毒细菌混合物物在4℃温育1小时,37℃1小时。在96孔微量板上长成单层的人未分化结肠癌细胞HT-29用磷酸缓冲盐水(PBS;pH7.2)洗涤3次。将病毒细菌混合物施加到细胞上,微量板在二氧化碳培养箱(Heraeus)中温育18小时。如下所述分析病毒复制。
第二种方法:
30微升细菌悬液(平均含有3×106细菌)与70微升M199培养基混合,培养基中补加有10%胰蛋白
磷酸培养液(Flow)和5%胰蛋白酶-EDTA溶液(Seromed)(HT-29细胞时1∶4稀释),直接施加到微量板上的细胞中。37℃温育1小时后,补充M199培养基中的病毒100微升加入到微量板中的细胞。在二氧化碳培养箱(Heraeus)中继续温育18小时。如下所述分析病毒复制。
轮状病毒复制通过如下所述的感染细胞中轮状病毒蛋白的组织-免疫染色分析。
感染后1天,弃去微量板中的细胞培养基,细胞用无水乙醇固定10分钟。弃去乙醇,培养板在PBS缓冲液中洗涤3次。兔(获自Lausanne大学ISREC)中产生并在PBS中1∶2000稀释的50微升抗轮状病毒血清(主要针对VP6蛋白)加入到各孔中,37℃温育1小时,加盖防止孔干燥。然后弃去抗血清,培养板用PBS洗涤3次。PBS中1∶500稀释的山羊中产生并与过氧化物酶偶联的50微升抗兔免疫球蛋白G(IgG)(GAR-IgG-PO;Nordic)加入到各孔中,培养板在37℃温育1小时。弃去血清,培养板再用PBS洗涤3次。然后将100微升下列底物混合物加入到各孔中:10毫升0.05M Tris-盐酸(pH7.8),1毫升过氧化氢(30%suprapur在水中1∶600稀释;Merck)和200微升3-氨基-9-乙基咔唑(0.1g/10ml乙醇以200微升等份保存在-80℃;A-5754;Sigma)。培养板室温温育至少30分钟。弃去底物,向各孔中加入200微升水终止反应。感染细胞病灶以倒置显微镜(Diavert;Leitz)计数。
只有极少细菌菌株与轮状病毒相互作用。最初选定的260株细菌细胞中只有9种在至少一种方法中抑制轮状病毒复制。类干酪乳杆菌NCC2461(ST11)具有极高的抗血清型1轮状病毒、血清型3轮状病毒SA11和血清型4轮状病毒Hochi活性。
实施例3
培养Caco-2细胞
在病原菌抑制实验中,细胞系Caco-2用作肠道模型。该细胞系具有肠道细胞的典型特征,如极化、肠道酶的表达,产生特定结构多肽等。
细胞生长在三种不同的支持物上,即在塑料平皿(25cm3,Corning)上生长和增殖,在去脂和无菌的6孔玻璃平板(22×22mm,Corning)上进行附着实验,在24孔玻璃平板(Corning)上进行抑制实验。
培养2天后,每日更换培养基(DMEM)。在使用前,培养基中加入100U/ml青霉素/链霉素,1μg/ml amphoterine和20%56℃灭活30分钟的FCS。在含有90%空气和10%二氧化碳的气氛中37℃培养。细胞每6天***一次。通过用PBS中的0.25%胰蛋白酶和3mM EDTA,pH7.2处理使细胞同孔壁脱离。为了中和胰蛋白酶的作用,向得到的细胞悬液中加入等体积FCS,混合物离心(1000rpm 10分钟),将细胞沉淀再次置于培养物中。大约3.5×105细胞被转移到新的培养瓶中,培养到细胞单层汇合。
实施例4
培养细菌
ST11:
细菌菌株在含有15%甘油的MRS培养基中-20℃保存。菌株在厌氧条件下在MRS培养基中生长,每隔24小时转移到新培养基中,抑制实验之前共转移两次。实验中使用2×109cfu/ml的浓度。
20000rpm离心1小时收集上清,然后检查所得上清中是否存在细菌。
大肠杆菌:
使用两种大肠杆菌菌株大肠杆菌DAECC1845(扩散附着大肠杆菌)和大肠杆菌JPN15(EPEC:致肠病大肠杆菌)。
解冻后第一次传代在CFA-Muller Hinton琼脂上进行,该培养基适于细菌表达附着因子。
每次实验之前,细菌细胞在37℃温育,每隔24小时转移到新鲜培养基中,共转移两次。由于JPN15含有氨苄青霉素抗性基因,生长过程中使用该抗生素选择。
沙门氏菌:
鼠伤寒沙门氏菌SL1344用于该实验,使用前将其生长于LB培养基中。
实施例5
大肠杆菌抑制实验
两次传代到新鲜培养基中后,病原菌株用10μCi/ml C14-乙酸盐在LB培养基中标记。在该培养基中将菌株37℃温育18小时。
然后将细菌悬液离心(1041g,15min),以除去上清中剩余的C14-乙酸盐。细胞沉淀悬浮于PBS中洗涤,细胞悬液的浓度为约108细胞/ml,其中含有1%无菌甘露糖。抑制甘露糖抑制非特异性附着。
不同病原菌株(大肠杆菌)与Caco-2细胞单层接触(37℃,10%二氧化碳,90%空气)3小时。使用上清(20000rpm离心40分钟得到)进行同样的实验。
作为对照,病原菌与Caco-2细胞单层接触,但分别不同时加入ST11或培养物上清。
温育3小时后,更换培养基,将单层用PBS洗涤3次。每一洗涤步骤包括20×PBS溶液搅动,以便基本上除去所有非特异性附着。然后加入1毫升碳酸钠裂解细胞,37℃温育40分钟。匀浆后,取一试样(250微升)在5毫升闪烁液(Hionicfluor Packcard)中稀释并计数(Packard 2000)。病原细胞对Caco-2细胞的附着百分比根据对照计算,对照设定为100%(附着,或侵入见实施例6)。
实施例6
沙门氏菌抑制实验
沙门氏菌是侵入上皮细胞并在其中增殖的细菌。为了确定ST11的抑制活性,鼠伤寒沙门氏菌SL1344如上所述在含有C14乙酸盐的培养基中温育,如实施例5所述进行实验。
温育后,Caco-2细胞用PBS洗涤,以便除去所有非附着细胞。然后将培养基加入到含有庆大霉素(20μg/ml),37℃继续温育1小时。庆大霉素不会进入肠道细胞,因此,杀死所有细胞外微生物,而已经侵入肠道细胞内的沙门氏菌则存活下来。用PBS洗涤细胞两次后,细胞通过加入无菌蒸馏水裂解,如实施例4所述测定发射活性。
实验5和6的结果见图6到9。由结果可见,培养的ST11细胞和培养物上清在防止引起腹泻的微生物对肠道细胞的附着和侵袭方面非常有效。
实施例7
ST11的特性
ST11已经经受了模拟胃液的温育。模拟胃液通过将胃蛋白酶(3g/l)悬浮于无菌盐水(0.5%w/v)中并用浓盐酸调节pH为2.0到3.0来制备。ST11在上述培养基中以不同量生长,微生物的抗性也被确定。
结果总结在下表1中。
表1
pH | Cfu/ml,T0 | Cfu/ml,T1 | Cfu/ml,T15 | Cfu/ml,T30 | Cfu/ml,T60 |
2.0 | 2.0×109 | 1.8×109 | 1.2×109 | 3.7×108 | 7.0×103 |
3.0 | 2.0×109 | 1.9×109 | 1.7×109 | 1.7×109 | 8.4×108 |
ST11具有根据Ed.B.J.B.Wood和W.H.Holzapfel,Blackie A & P在乳酸细菌属公开的方法限定的下列特性。
-革兰氏阳性
-触酶阴性
-氨形成精氨酸阴性
-二氧化碳产生阴性
-L(+)乳酸产生
-在浓度高达约0.4%的胆汁盐存在下生长
实施例8
在不同条件下ST11的生长
ST11在基于番茄的培养基(4%番茄粉在蒸馏水中重配)中37℃培养不同时间,培养基中补加有蔗糖(0,0.5,1或2%)或大豆蛋白胨(0.5%)或葡萄糖(0.5%)。结果见图2。
再将2.5%的ST11加入由稻米淀粉(3%)、小麦淀粉(2%)或蔗糖(3%)组成的培养基中,37℃温育直至pH达到4.4。冷却后,加入或不加入维生素C将产物包装,并储存在10℃。
实施例9
ST11诱导小鼠附着细胞中IL-12和IL-10mRNA合成
由8周龄无病原菌C57BL/6小鼠股骨和胫骨分离骨髓细胞,并在RPMI培养基(Gibco)中以2×106细胞/毫升的浓度在5%二氧化碳气氛下37℃培养12小时,培养基中含有10%胎牛血清,1mM L-谷氨酰胺,12mM Hepes,0.05mM 2-巯基乙醇,100U/ml青霉素和100μg/ml链霉素(所有试剂来自Gibco)。非附着细胞通过用温热培养基3次连续洗涤除去,收获留下的附着细胞,以106细胞/ml的浓度在细菌存在或不存在时温育6小时。以前已经确定,6小时是小鼠附着细胞响应于LPS合成细胞因子mRNA的最佳时间点。细菌以109到107cfu/ml的不同浓度加入。细菌如前所述生长和保存。
6小时培养期末,离心分离细胞,使用TRIzol试剂盒(GibcoBRL,目录号15596-018)按照厂商说明裂解细胞。通过异丙醇沉淀分离总RNA,使用200单位逆转录酶(Superscript II,BRL)在40微升反应体积中42℃90分钟将RNA逆转录成cDNA,反应溶液中含有200mM TrispH8.3,25mM KCl,1μg/ml寡聚d(T)15(Boehringer Mannheim)和40U/ml Rnasin(Promega)。PCR引物和条件如Kopf等所述(实验医学杂志,1996Sep 1;184(3):1127-36)。样品中cDNA的量使用管家基因(β-2-微球蛋白)特异性引物校准。PCR产物在2%琼脂糖凝胶上分离,在紫外光下分析条带。
如图4所示,ST11对IL-12和IL-10mRNA显示最强烈的诱导作用,与阳性对照(大肠杆菌)中观察到的水平相当。在最低细菌浓度下可以最清楚地看到差异。
实施例10
通过ST11抑制IL-4合成
CD4+T细胞使用Miltenyi Biotec(目录号492-01)的MiniMACS试剂盒从物特异病原菌的BALB/c小鼠脾脏纯化。CD4+T细胞以2×105细胞/ml在RPMI培养基中培养,培养基中含有10%胎牛血清,1mM L-谷氨酰胺,12mM Hepes,0.05mM 2-巯基乙醇,100U/ml青霉素和100μg/ml链霉素,在一周期间通过与培养板结合的抗CD3(克隆2C11)和CD28(克隆37.51)单克隆抗体(两种抗体均来自Pharmingen)交叉结合而被激活。在第一代培养中,CD4+T细胞与作为辅助细胞的骨髓附着细胞(如上分离)和108cfu/ml ST11或cfu/ml La1或1mg/mlLPS或仅培养基共同培养。此后,洗涤细胞,使用MiniMACS试剂盒再次纯化CD4+T细胞,在仅含培养基的第二代培养中再次刺激。分化CD4+T细胞产生的细胞因子在2天后使用夹心ELISA在上清中测定(试剂盒来自Endogen和Pharmingen)。
结果示于图5。在仅含培养基时分化的细胞显示以高水平IL-4为特征的优势Th2表型。加入ST11至第一代培养物中强烈地调节Th2分化的结果,因为它导致IL-4的产生降低8倍。这种抑制的幅度类似于来自在LPS存在时分化的细胞的培养物中观察到的结果。与此相对,其他乳杆菌菌株对IL-4水平没有可检测的结果。有趣的是,IFN-γ水平在第一代培养中加入ST11时没有增加。
由以上可见,利用微生物的有利特性,本发明的菌株可以制备成食品和/或药物载体。
实施例11
在Guatemala市郊的一个社区进行的临床试验中测试了ST11菌株影响该地区大部分儿童患过的急性雨季腹泻病的传递和发病的能力。本研究招募了总共203名35到70个月龄的儿童,他们在29天的进食期间接受了1010活微生物(ST11)或对照(安慰剂)。选择样品和安慰剂的儿童由于营养不良在重量-年龄和高度-年龄方面具有典型缺陷。
在学龄前儿童进食试验开始之前,进行了体外和体内安全性评价研究。体外研究显示了类似于食品中应用的其他乳杆菌的抗生素抗性特征,没有由于降解粘蛋白和解聚胆汁盐形成生物源氨的可能。在包括42个成年志愿者的安慰剂对照临床研究中,在监测的可能表型如肠胃气胀、每日排便次数和粪便稠度中,ST11耐受良好,没有诱发任何副作用;血清中急性期蛋白水平相对于可能的炎症反应没有出现任何值得关注之处。
样品和安慰剂在瑞士Konolfingen的Nestle Product TechnologyCentre制造厂包装成酏剂(sachet),低温海运到Guatemala。每10克酏剂由巧克力味赋形剂和0.2克ST11(1010cfu)组成,安慰剂则是0.2克奶粉。巧克力味赋形剂由可可粉、糖、大豆卵磷脂、香草和肉桂组成。酏剂保存在4℃到6℃,使用前2小时取出。使用前,酏剂必需溶解在100毫升Nestle提供的水中,这种水中不含任何细菌污染。
根据应用的方法,腹泻定义为24小时期间排出3次或更多次液体或不定形粪便。腹泻事件定义为表现出腹泻特征(24小时腹泻***3次)的事件。其持续时间自3次指标粪便首次的时刻到首次出现成形粪便或24小时未***的时刻。对于儿童,前一事件终止后至少48小时才计算为新的事件。如果不是这样,则计算为同一事件的持续,使用总的持续事件来评价。一个病例是在29天的观察期内,经历了一次或多次有记录的事件的儿童。腹泻事件的强度根据产生的松散粪便总数评价。事件严重性的因素包括粪便中带血、粘液或脓,以及有发烧和呕吐症状。每24小时排便7次的强度或需要在诊所、健康中心或医院中由专业人员干预也被归入严重事件。
当预警***监视腹泻事件时,取腹泻粪便的样品进行显微镜检查,并培养以鉴定该事件可能的病原体。检查样品中的轮状病毒抗原、贾第鞭毛虫和E.Histolytica,样品是痢疾,检查细菌病原体包括志贺氏菌、沙门氏菌、气单胞菌、类志贺邻单胞菌、大肠杆菌和可能的霍乱弧菌。
在研究期间,收集产物样品以检查施用期间包括的微生物的活力。可以证实,在整个研究期间,酏剂内微生物都是活的,所以研究结束时,酏剂用水重配时仍能提供1010活微生物。
该研究揭示,与对照组(安慰剂)相比,含有益生微生物的样品可以降低腹泻的出现约30%。然而,相对于没有接受样品或安慰剂的正常人群,对照组中腹泻的发生也降低了。后一发现可能部分归因于接受了额外的有价值营养和无污染的水的儿童。但是,由于该研究是在自然环境中进行的,很显然ST11可以减少体内腹泻的发生。
申请人或代理人档案号 | 国际申请号 |
关于微生物保藏的说明
(细则13之二)
A.对说明书第4页.第23-25行所述的微生物的说明, |
B.保藏事项 其他保藏在补充页中二 |
保藏单位名称道 国立微生物保藏中心 |
保藏单位地址(包括邮政编码和国名) 28,Rue du Cocteur Roux F-75724 Paris Cedex 15法国 |
保藏日期 1999年1月12日 保藏编号NCC 2461(I-2116) |
C.补充说明(必要时) 本栏内容有补充页二 |
D.本说明是为下列指定国作的(如果说明下是为所有指定国而作的 |
E.补充说明(必要时) |
下列说明将随后向国际局提供(写出说明的类别,例如:“保藏的编号”) |
PCT·R( )/134 長(1992年7月)
Claims (4)
1.类干酪乳杆菌CNCMI-2116的培养物的上清液用于制备可摄入的支持物的用途。
2.权利要求1的用途,其中支持物材料是选自牛奶、酸奶、凝乳、发酵牛奶、牛奶基发酵产品、冰激凌、发酵谷物基产品、牛奶基粉末、婴儿配方食品的食品组合物。
3.含有类干酪乳杆菌CNCMI-2116的培养物上清液的食品或药物组合物。
4.权利要求3的组合物,它选自牛奶、酸奶、凝乳、乳酪、发酵牛奶、牛奶基发酵产品、冰激凌、发酵谷物基产品、牛奶基粉末、婴儿配方食品、片剂、液体细菌悬液、干的口服补剂、湿的口服补剂、干的管饲产品或湿的管饲产品。
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EP1034788A1 (en) * | 1999-03-11 | 2000-09-13 | Société des Produits Nestlé S.A. | Lactic acid bacteria strains capable of preventing diarrhea |
EP1034787A1 (en) * | 1999-03-11 | 2000-09-13 | Société des Produits Nestlé S.A. | Lactobacillus strains preventing diarrhea caused by pathogenic bacteria |
PL207930B1 (pl) * | 2000-05-25 | 2011-02-28 | Nestle Sa | Zastosowanie wyizolowanego probiotycznego szczepu bakterii kwasu mlekowego o aktywności probiotycznej, kompozycja pokarmowa i izolowany szczep bakterii kwasu mlekowego |
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ES2460891T3 (es) * | 2005-10-06 | 2014-05-14 | Probi Ab | Uso de lactobacilus para el tratamiento de enfermedades autoinmunes |
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- 2000-03-02 JP JP2000603691A patent/JP3765983B2/ja not_active Expired - Fee Related
- 2000-03-02 US US09/936,542 patent/US6887465B1/en not_active Expired - Lifetime
- 2000-03-02 EP EP00909292A patent/EP1165105A1/en not_active Withdrawn
- 2000-03-02 NZ NZ513804A patent/NZ513804A/en unknown
- 2000-03-02 BR BR0008920-6A patent/BR0008920A/pt not_active Application Discontinuation
- 2000-03-02 AU AU31629/00A patent/AU779789B2/en not_active Ceased
- 2000-03-02 IL IL14508000A patent/IL145080A0/xx active IP Right Grant
- 2000-03-02 CN CNB00807402XA patent/CN1244684C/zh not_active Expired - Fee Related
- 2000-03-02 WO PCT/EP2000/001798 patent/WO2000053202A1/en active IP Right Grant
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2001
- 2001-08-23 IL IL145080A patent/IL145080A/en not_active IP Right Cessation
- 2001-09-04 NO NO20014298A patent/NO20014298L/no not_active Application Discontinuation
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2002
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Also Published As
Publication number | Publication date |
---|---|
PL203281B1 (pl) | 2009-09-30 |
IL145080A0 (en) | 2002-06-30 |
CZ20013269A3 (cs) | 2002-04-17 |
RO121700B1 (ro) | 2008-02-28 |
US6887465B1 (en) | 2005-05-03 |
JP3765983B2 (ja) | 2006-04-12 |
CN1350462A (zh) | 2002-05-22 |
AU779789B2 (en) | 2005-02-10 |
NZ513804A (en) | 2001-09-28 |
HK1046864B (zh) | 2006-10-27 |
JP2002537867A (ja) | 2002-11-12 |
CA2364440A1 (en) | 2000-09-14 |
EP1165105A1 (en) | 2002-01-02 |
NO20014298D0 (no) | 2001-09-04 |
BR0008920A (pt) | 2001-12-18 |
MXPA01009017A (es) | 2002-04-24 |
IL145080A (en) | 2006-12-10 |
WO2000053202A1 (en) | 2000-09-14 |
AU3162900A (en) | 2000-09-28 |
NO20014298L (no) | 2001-11-05 |
HK1046864A1 (en) | 2003-01-30 |
HUP0200374A2 (en) | 2002-06-29 |
PL350776A1 (en) | 2003-02-10 |
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