CN113355252A - 一种副干酪乳杆菌及其应用 - Google Patents
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Abstract
本发明属于生物保健领域,具体涉及一种副干酪乳杆菌及其在调节肠道菌群中的应用。所述副干酪乳杆菌为副干酪乳杆菌(Lactobacillus paracasei)WG‑7,其保藏编号为CGMCC No.15961。本发明的另一个目的在于提供所述副干酪乳杆菌WG‑7在调节肠道菌群中的应用,具体涉及在制备调节肠道菌群的药物和/或食品和/或保健品中的应用。本发明的副干酪乳杆菌WG‑7可有效地调节肠道菌群,促进机体微生态正向平衡,在基本不改变肠道菌群总数量的情况下,可有效提高肠道中双歧杆菌属、乳杆菌属中的一种或多种的数量;降低肠道中大肠杆菌、肠球菌中的一种或多种的数量。
Description
技术领域
本发明属于生物保健领域,具体涉及一种副干酪乳杆菌及其在调节肠道菌群中的应用,特别涉及在制备调节肠道菌群的药物和/或食品和/或保健品中的应用。
背景技术
早在2000年世界卫生组织提出“万病之源始于肠道”。肠道是人体内最大的微生态系统,居于其中的细菌多达100万亿个,对机体的健康意义重大,被称为人体的第二基因组。随着生活水平的提高、对品质生活的重视,人们对食品安全问题、饲用抗生素的残留及耐药性菌株问题日益关注,寻找安全的抗生素替代品提上日程。另外,因为生活节奏加快、工作压力增大、饮食不规律、睡眠不足,导致便秘、口臭、情绪焦虑、烦躁失眠等症状,这很大程度是因肠道菌群失衡使得其功能紊乱引起的。对于老年人,更需要关注肠道微生态平衡,因为随年龄增加,肠道逐渐老化,益生菌数量减少,有害菌增多,更易出现肠道相关疾病和代谢功能障碍系列问题。研究表明,人体内肠道菌群失衡使人体处于亚健康状态,甚至引发糖尿病和高血压等慢性疾病。因此,调节肠道菌群正向平衡越来越成为热点。
益生菌及其次级代谢产物均可通过改善宿主微生态平衡而发挥益生功效。益生菌制剂通过增殖机体有益菌,可与有害菌形成竞争优势而有益健康;也可分泌酶、营养物质及抑制有害菌的代谢产物,调整肠道内源微生物系统和预防系统,从而改善肠道生理机能。然而,现有益生菌调节肠道菌群的效果均不佳。
发明内容
为了解决现有技术中存在的肠道菌群失衡引起的功能紊乱问题,本发明在于提供一种副干酪乳杆菌WG-7及其在调节肠道菌群中的应用,特别涉及在制备调节肠道菌群的药物和/或食品和/或保健品中的应用。
本发明的技术方案如下:
一种副干酪乳杆菌,其名称为副干酪乳杆菌(Lactobacillus paracasei)WG-7,保藏编号为CGMCC No.15961,保藏日期:2018年6月19日,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:中国,北京,中国科学院微生物研究所。
本发明还提供一种所述的副干酪乳杆菌在调节肠道菌群中的应用。
进一步地,所述的副干酪乳杆菌在制备调节肠道菌群的药物和/或食品和/或保健品中的应用。
优选地,所述药物还包括药学上可接受的佐剂,所述药物和其药学上可接受的佐剂根据给药途径不同制备成散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂或混悬剂。
优选地,所述食品的类型为固体饮料、豆制品、果汁、乳制品、冰淇淋、糖果或饼干,所述食品还含有常规添加剂和/或营养强化剂,所述常规添加剂可以是香精香料、稳定剂、增稠剂和防腐剂中的一种或多种;所述营养强化剂可以是矿物质和/或维生素。
优选地,在所述药物、食品和保健品中含有所述副干酪乳杆菌的活菌体和/或死菌体作为活性成分。
更优选地,在所述药物、食品和保健品中含有所述副干酪乳杆菌的活菌体或者活菌体和死菌体的混合菌体作为活性成分。
优选地,所述混合菌体中活菌体的数量高于死菌体的数量。
优选地,以每克或每毫升所述药物和/或保健食品和/或食品为基准,所述副干酪乳杆菌添加量为106-1011CFU/g或CFU/mL,更优选为108-1010CFU/g或CFU/mL。
需要说明的是,尽管本发明将副干酪乳杆菌WG-7添加到所述药物和/或保健食品和/或食品中,并对个体进行食用,即可实现本发明目的,起到调节肠道菌群的作用。
本发明的有益效果:
本发明的副干酪乳杆菌WG-7(保藏编号为CGMCC No.15961)可有效地调节肠道菌群,促进机体微生态正向平衡。在基本不改变肠道菌群总数量的情况下,可有效提高肠道中双歧杆菌属、乳杆菌属中的一种或多种的数量;降低肠道中大肠杆菌、肠球菌中的一种或多种的数量;而对于其它菌群,例如,梭菌的数量无显著影响。
具体实施方式
下面将结合具体实施例对本发明做进一步详细的说明,但不限于此。
实施例1
菌株培养:副干酪乳杆菌WG-7,用MRS液体培养基37℃厌氧培养12h,4500r/min离心15min,菌体沉淀重悬于10%的脱脂乳中,配制不同浓度(分别为1×106CFU/mL、1×107CFU/mL、1×108CFU/mL)菌悬液。副干酪乳杆菌CG-C1(保藏编号:CGMCC No.12941,购于中国普通微生物菌种保藏管理中心)的以上述相同方法培养,得到浓度为1×108CFU/mL的副干酪乳杆菌CG-C1的菌悬液。
实验动物分组及处理:
实验动物分为5组,每组10只,副干酪乳杆菌WG-7设三个剂量组(分别为WG-7低剂量组、WG-7中剂量组和WG-7高剂量组)、阳性对照组(副干酪乳杆菌CG-C1)和空白组。WG-7低剂量、WG-7中剂量、WG-7高剂量组小鼠分别灌胃1×106CFU/mL、1×107CFU/mL、1×108CFU/mL的副干酪乳杆菌WG-7的菌悬液0.1mL/(kg·d)(以体质量计),阳性对照组小鼠灌胃1×108CFU/mL的副干酪乳杆菌CG-C1的菌悬液0.1mL/(kg·d)(以体质量计),空白组小鼠灌胃质量分数10%的脱脂乳0.1mL/(kg·d)(以体质量计)。每天一次,试验期间自由进食,记录各组小鼠体质量变化。
肠道中细菌基因组DNA样品收集:
在上述灌胃菌种前和灌胃菌种14天后,用装有1mL厌氧稀释液和无菌玻璃珠的离心管收集小鼠新鲜粪便0.1g,用漩涡振荡器均质完全后提取粪便中的细菌基因组DNA(酚仿法)。用1%琼脂糖进行DNA琼脂糖凝胶电泳检测。检测结果表现良好,所有粪便样品中细菌组DNA均成功提出,并且无明显降解现象。
粪便中肠道菌群含量的测定:
使用下表1中的特异性引物分别对步骤(1)中获得的粪便样品细菌组DNA进行实时定量PCR实验。实时定量PCR的反应体系为1μL的粪便基因组DNA,0.4μL的10μM上下游引物,10μL的SYBR Premix Ex Taq,0.4μL的ROX,7.8μL的无菌水;反应程序如下:预变性(95℃反应30s),变性(95℃反应5s),退火(按表1中的退火温度反应30s),延伸(72℃反应30s),PCR反应45个循环。将PCR扩增产物进行浓度为2%的琼脂糖凝胶电泳,用无菌手术刀将单一的目标条带切下,于干净的5mL离心管中,使用Zemo胶回收试剂盒并按说明书进行DNA回收。切胶回收的标准DNA模板利用QubitAssays测定各自的DNA浓度后,10倍稀释8个梯度作为定量PCR的标准样品。
按上述的定量PCR反应体系和条件,分别以各自的下表1中的特异性引物进行定量PCR反应,以各梯度稀释液中DNA的拷贝数浓度的对数为横坐标,以Ct值为纵坐标,制作下表1中不同目标细菌组的标准曲线。计算引物的扩增效率,公式为:扩增效率=10-(1/斜率)-1。以同样的PCR体系和条件对WG-7低剂量组、WG-7中剂量组、WG-7高剂量组、阳性对照组和空白组小鼠粪便样品基因组DNA进行PCR反应,得到Ct值。通过标准曲线,获得样品中该菌群的拷贝数浓度,根据取样时粪便质量,换算成每克粪便样品中含有该菌群的拷贝数对数(lgDNA拷贝数/克粪便),结果如表2所示。
表1为各PCR组所用引物序列、产物大小和退火温度:
表1
表2为各实验组中的小鼠粪便中各菌群的数量变化情况:
表2
注:表中数值表示方式为均值±标准差,单位为(lgDNA拷贝数)/(克粪便)。
如表2结果所示,灌胃本发明的副干酪乳杆菌WG-7共14天后,所有组别的小鼠粪便中总菌数量没有明显变化,无显著差异。与空白组和阳性对照组(副干酪乳杆菌CG-C1)相比,WG-7低剂量组、WG-7中剂量组、WG-7高剂量组灌胃14天后,小鼠粪便中的双歧杆菌数量和乳杆菌数量均明显上升;并且具有剂量效应。肠道中肠球菌和大肠杆菌数量过多容易引发腹泻、脱水等一系列肠道相关症状,中剂量组、高剂量组均可显著降低小鼠粪便中肠球菌和大肠杆菌数量,但中剂量组和高剂量组之间无明显差异。实验中,小鼠粪便中的梭菌属菌群无明显变化。由此可以说明,本发明的副干酪乳杆菌WG-7在不改变肠道总菌数量的同时,可增殖肠道中常见益生菌双歧杆菌和乳杆菌数量,减少潜在致病菌肠球菌和大肠杆菌数量,对梭菌属影响不明显,证明本发明的副干酪乳杆菌WG-7能够有效地调节肠道菌群,维持肠道微生态的正向平衡。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
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Claims (10)
1.一种副干酪乳杆菌,其特征在于,所述副干酪乳杆菌为副干酪乳杆菌(Lactobacillus paracasei)WG-7,其保藏编号为CGMCC No.15961。
2.一种权利要求1所述的副干酪乳杆菌在调节肠道菌群中的应用。
3.根据权利要求2所述的应用,其特征在于,在制备调节肠道菌群的药物和/或食品和/或保健品中的应用。
4.根据权利要求3所述的应用,其特征在于,所述药物还包括药学上可接受的佐剂,所述药物和其药学上可接受的佐剂根据给药途径不同制备成散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂或混悬剂。
5.根据权利要求3所述的应用,其特征在于,所述食品的类型为固体饮料、豆制品、果汁、乳制品、冰淇淋、糖果或饼干,所述食品还含有常规添加剂和/或营养强化剂。
6.根据权利要求3所述的应用,其特征在于,在所述药物、食品和保健品中含有所述副干酪乳杆菌的活菌体和/或死菌体作为活性成分。
7.根据权利要求6所述的应用,其特征在于,在所述药物、食品和保健品中含有所述副干酪乳杆菌的活菌体或者活菌体和死菌体的混合菌体作为活性成分。
8.根据权利要求7所述的应用,其特征在于,所述混合菌体中活菌体的数量高于死菌体的数量。
9.根据权利要求2-8任一项所述的应用,其特征在于,以每克或每毫升所述药物和/或保健食品和/或食品为基准,所述副干酪乳杆菌添加量为106-1011CFU/g或CFU/mL。
10.根据权利要求9所述的应用,其特征在于,以每克或每毫升所述药物和/或保健食品和/或食品为基准,所述副干酪乳杆菌添加量为108-1010CFU/g或CFU/mL。
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CN116121120A (zh) * | 2022-11-30 | 2023-05-16 | 天津小薇生物科技有限公司 | 具有抑菌作用的副干酪乳杆菌gf009、其后生元制备方法及其应用 |
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