CN107043705A - 用于建立临床营养菌种资源库的益生菌筛选方法 - Google Patents

用于建立临床营养菌种资源库的益生菌筛选方法 Download PDF

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CN107043705A
CN107043705A CN201611263098.5A CN201611263098A CN107043705A CN 107043705 A CN107043705 A CN 107043705A CN 201611263098 A CN201611263098 A CN 201611263098A CN 107043705 A CN107043705 A CN 107043705A
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文姝
唐立
潘浩宇
李培培
袁杰力
刘银辉
郭艳杰
李华军
李明
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Abstract

本发明公开一种用于建立临床营养菌种资源库的益生菌筛选方法，首先是建立基于功能的定向收集体系提高初筛效率；进而建立了适宜的粪便标本运送保存体系，延长了粪便标本的有效运送时间2~4倍，保证了分离存活的益生菌菌种数量；最后按照特定的优选试验方法分别筛选出具有促进抑菌及生物拮抗功能、促进代谢功能和促进免疫功能功能的优良菌种，操作简单、筛选效率高，可用于建立临床营养菌种资源库，以便针对个体化差异进行益生菌复配选用，提高治疗效果。

Description

用于建立临床营养菌种资源库的益生菌筛选方法
技术领域
本发明涉及一种益生菌筛选方法，尤其是一种操作简单、筛选效率高的用于建立临床营养菌种资源库的益生菌筛选方法。
背景技术
益生菌是定植于人体肠道、生殖***内，能产生确切健康功效从而改善宿主微生态平衡、发挥有益作用的活性有益微生物的总称。人体、动物体内有益的细菌或真菌主要有：酪酸梭菌、乳杆菌、双歧杆菌、放线菌、酵母菌、枯草芽孢杆菌、植物乳杆菌等等。益生菌可治疗的疾病较多，如参与维生素的合成，参与胆固醇代谢，调节胆固醇相关性疾病；防治过敏（食物过敏，哮喘，鼻炎），预防湿疹；治疗抗生素相关腹泻、缓解IBS、IBD、细菌性***炎，泌尿生殖道感染、坏死性小肠结肠炎；控制高血压，调节中枢神经***功能和障碍；抗幽门螺旋菌感染；提高免疫抵抗感染；也可调节乳糖不耐症等等。由于单个菌种功能具有局限性，因此已有文献报道将以上各类微生物复配而成的复合活性益生菌治疗疾病效果更好。但是，现有益生菌的筛选方法复杂、效率低，故目前能够真正用于临床和营养的菌株有限，难以建立临床营养菌种资源库，临床复配没有理论根据，不能针对个体化差异进行调整，导致治疗效果不够理想。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种操作简单、筛选效率高的用于建立临床营养菌种资源库的益生菌筛选方法。
本发明的技术解决方案是：一种用于建立临床营养菌种资源库的益生菌
筛选方法，其特征在于依次按照如下步骤进行：
a. 分别采集代谢功能好、免疫力强及抗感染能力强的人群新鲜粪便为标本；
b. 用选择性培养基筛选出各种益生菌菌株；
c. 通过试验进一步筛选出具有促进抑菌、生物拮抗功能的优良益生菌菌株、具有促进代谢功能的优良益生菌菌株及促进免疫功能的优良益生菌菌株：
c.1. 根据基本生物特性进行筛选
进行对酸性环境和胆汁的耐受能力、细菌的粘附能力试验，将耐受能力好、粘附能力强的菌株作为初步筛选菌株进行下一步筛选；
c.2. 定植抗力功能体外筛选
对初步筛选的菌株进行抑菌实验，筛选能有效抑制病菌的益生菌菌株，所述病菌为大肠埃希菌、金黄色葡萄球菌、绿脓杆菌和白色念珠菌；
c.3.促进代谢功能体外筛选
通过检测初步筛选的菌株的胰蛋白酶、胃蛋白酶、胞外蛋白酶、肽酶、乙醇脱氢酶、乙醛脱氢酶等活性，筛选具有不同代谢功能的益生菌菌株；
c.4 促进免疫功能体外筛选
以PBMC作为体外免疫筛选的实验细胞，检测初步筛选的菌株对PBMC细胞因子分泌的影响，筛选免疫刺激功能强的益生菌菌株。
所述a步骤采集标本后向样本中添加益生菌培养基及羊血，4℃保存最多4天。
本发明首先是建立基于功能的定向收集体系提高初筛效率；进而建立了适宜的粪便标本运送保存体系，延长了粪便标本的有效运送时间2~4倍，保证了分离存活的益生菌菌种数量；最后按照特定的优选试验方法分别筛选出具有促进抑菌及生物拮抗功能、促进代谢功能和促进免疫功能功能的优良菌种，操作简单、筛选效率高，可用于建立临床营养菌种资源库，以便针对个体化差异进行益生菌复配选用，提高治疗效果。
具体实施方式
一种用于建立临床营养菌种资源库的益生菌筛选方法，依次按照如下步骤进行：
a. 分别采集代谢功能好、免疫力强及抗感染能力强的人群新鲜粪便为标本；如采集的标本需要长时间运输，则向样本中添加益生菌培养基及羊血，4℃
保存最多4天；
b. 用选择性培养基筛选出各种益生菌菌株；
c. 通过试验进一步筛选出具有促进抑菌、生物拮抗功能的优良益生菌菌株、具有促进代谢功能的优良益生菌菌株及促进免疫功能的优良益生菌菌株：
c.1. 根据基本生物特性进行筛选
进行对酸性环境和胆汁的耐受能力、细菌的粘附能力试验，将耐受能力好、粘附能力强的菌株作为初步筛选菌株进行下一步筛选；
c.2. 定植抗力功能体外筛选
对初步筛选的菌株进行抑菌实验，筛选能有效抑制病菌的益生菌菌株，所述病菌为大肠埃希菌、金黄色葡萄球菌、绿脓杆菌和白色念珠菌；
c.3.促进代谢功能体外筛选体系
通过检测初步筛选的菌株的胰蛋白酶、胃蛋白酶、胞外蛋白酶、肽酶、乙醇脱氢酶、乙醛脱氢酶等活性，筛选具有不同代谢功能的益生菌菌株；
c.4 促进免疫功能体外筛选体系
以较易于分离培养且具有较好的免疫刺激反应的PBMC（外周血单个核细胞）作为体外免疫筛选的实验细胞，检测初步筛选的菌株对PBMC细胞因子分泌的影响，筛选免疫刺激功能强的益生菌菌株。
实验：
耐酸性能力低和耐酸性能力高的菌株分为两组，分别对大肠埃希菌和金黄色葡萄球菌进行抑菌实验，耐酸性能力低和耐酸性能力高的菌株抑菌能力有显著差别（P <0.05）。耐酸性能力与抑菌能力相关（大肠埃希菌R=0.465，p<0.05；金黄色葡萄球菌R=0.384，p<0.05）。因此，可以通过c.1初步筛选出具有一定抑菌能力的益生菌。

Claims (2)

1.一种用于建立临床营养菌种资源库的益生菌筛选方法，其特征在于依次
按照如下步骤进行：
a. 分别采集代谢功能好、免疫力强及抗感染能力强的人群新鲜粪便为标本；
b. 用选择性培养基筛选出各种益生菌菌株；
c. 通过试验进一步筛选出具有促进抑菌、生物拮抗功能的优良益生菌菌株、具有促进代谢功能的优良益生菌菌株及促进免疫功能的优良益生菌菌株：
c.1 根据基本生物特性进行筛选
进行对酸性环境和胆汁的耐受能力、细菌的粘附能力试验，将耐受能力好、粘附能力强的菌株作为初步筛选菌株进行下一步筛选；
c.2 定植抗力功能体外筛选
对初步筛选的菌株进行抑菌实验，筛选能有效抑制病菌的益生菌菌株，所述病菌为大肠埃希菌、金黄色葡萄球菌、绿脓杆菌和白色念珠菌；
c.3 促进代谢功能体外筛选
通过检测初步筛选的菌株的胰蛋白酶、胃蛋白酶、胞外蛋白酶、肽酶、乙醇脱氢酶、乙醛脱氢酶等活性，筛选具有不同代谢功能的益生菌菌株；
c.4 促进免疫功能体外筛选
以PBMC作为体外免疫筛选的实验细胞，检测初步筛选的菌株对PBMC细胞因子分泌的影响，筛选免疫刺激功能强的益生菌菌株。
2.根据权利要求1所述的用于建立临床营养菌种资源库的益生菌筛选方法，其特征在于：所述a步骤采集标本后向样本中添加益生菌培养基及羊血，4℃保存最多4天。
CN201611263098.5A 2016-12-30 2016-12-30 用于建立临床营养菌种资源库的益生菌筛选方法 Pending CN107043705A (zh)

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CN101649303A (zh) * 2009-05-08 2010-02-17 内蒙古蒙牛乳业（集团）股份有限公司 一种双歧杆菌及其应用和含有双歧杆菌的食品组合物
CN102373172A (zh) * 2011-11-03 2012-03-14 北京龙科方舟生物工程技术中心 一株屎肠球菌及其应用
CN102864091A (zh) * 2012-01-15 2013-01-09 河南科技大学 一种一菌多酶菌株及其筛选方法和应用
CN105112333A (zh) * 2015-08-31 2015-12-02 江南大学 一种具有良好肠道定殖能力的长双歧杆菌及筛选方法和应用

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Application publication date: 20170815