CN113930380B - 一种促进栖粪杆菌增殖的方法 - Google Patents
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Abstract
本发明公开了一种促进栖粪杆菌增殖的方法，通过薄荷脑对肠道菌群进行干预实验，本发明首次验证了薄荷脑具有调节肠道微生物，提高栖粪杆菌Faecalibacterium丰度的作用，因此不仅可将薄荷脑作为促进栖粪杆菌Faecalibacterium增殖的促进剂，用于栖粪杆菌Faecalibacterium的体外大规模发酵生产，还可制成肠道菌群调节药物，用于改善肠道菌群紊乱，并辅助治疗与栖粪杆菌Faecalibacterium相关的如脂肪肝、痛风、肝硬化、克罗恩、便秘、抑郁症和2型糖尿病等疾病。
Description
技术领域
本发明属于肠道益生菌增殖促进剂技术领域，具体涉及一种促进栖粪杆菌增殖的方法。
背景技术
薄荷脑，也叫薄荷醇，是一种萜类有机化合物，化学式为C10H20O。薄荷脑系由薄荷的叶和茎中所提取，白色晶体，为薄荷和欧薄荷精油中的主要成分。薄荷醇一般有两种异构体（D型和L型），天然的薄荷醇主要为左旋异构体（L-薄荷醇），薄荷脑一般指消旋的薄荷醇（DL-薄荷醇）。薄荷脑可用作牙膏、香水、饮料和糖果等的赋香剂。在医药上用作刺激药，作用于皮肤或粘膜，有清凉止痒作用。其酯也可用于香料和药物。
人体肠道中的微生物群落与宿主之间的关系极为密切，很多科学家认为肠道菌群可以看作是人体的另一个器官，肠道菌群失调与多种临床疾病密切相关，可作为多种疾病的治疗靶点之一，有望通过调控肠道微生物的群落结构，用于辅助治疗某些相关疾病。
研究报道，肠道菌群中益生菌栖粪杆菌属Faecalibacterium与多种肠道疾病或代谢性疾病密切相关。如文献：任士萌，非酒精性脂肪性肝病患者肠道菌群改变，中公开了非酒精性脂肪性肝病（NAFLD）患者体内的肠道菌群与健康人群相比有丰度变化，NAFLD患者存在肠道菌群结构紊乱，即包括Faecalibacterium在内的部分肠道菌与NAFLD以及相关的炎症反应相关。文献Guo, Zhuang et al. Intestinal Microbiota Distinguish GoutPatients from Healthy Humans.Scientific Reports, 2016中报道了在痛风患者体内，其肠道微生物Faecalibacterium prausnitzii等被几乎耗尽，且肠道菌群变化特征与2型糖尿病患者和肝硬化类似，即Faecalibacterium菌属与痛风、肝硬化、糖尿病等疾病相关，并且也有相关研究报道Faecalibacterium菌属与抑郁程度呈负相关，与便秘具有一定关联。目前关于薄荷脑在调节肠道微生物，改善肠道环境，以肠道微生物作为靶点进行相关疾病的辅助治疗等均未见相关报道。
发明内容
本发明的目的在于提供了一种促进栖粪杆菌增殖的方法，通过薄荷脑对肠道菌群进行干预实验，本发明首次验证了薄荷脑具有调节肠道微生物，提高栖粪杆菌Faecalibacterium丰度的作用，因此不仅可制成促进栖粪杆菌Faecalibacterium增殖的促进剂，用于栖粪杆菌Faecalibacterium的体外大规模发酵生产，还可制成肠道菌群调节药物，用于改善肠道菌群紊乱，并辅助治疗与栖粪杆菌Faecalibacterium相关的如脂肪肝、痛风、肝硬化、克罗恩、便秘、抑郁症和2型糖尿病等疾病。
本发明的目的之一在于提供了薄荷脑在制备促进栖粪杆菌Faecalibacterium增殖的促进剂中的用途。
进一步地，所述促进剂中还包括GAM培养基。
本发明的目的之二在于提供了一种促进栖粪杆菌Faecalibacterium增殖的方法，所述方法包括：配制GAM培养基，并将薄荷脑用PBS缓冲液溶解得到薄荷脑溶液，将所述薄荷脑溶液加入GAM培养基中至薄荷脑终浓度为质量分数2%，得到薄荷脑-GAM培养基，采用所述薄荷脑-GAM培养基培养栖粪杆菌Faecalibacterium。
本发明的目的之三在于提供了薄荷脑在制备增加栖粪杆菌Faecalibacterium丰度的药物中的用途。
进一步地，所述薄荷脑通过增加栖粪杆菌Faecalibacterium的丰度，达到改善脂肪肝、痛风、肝硬化、克罗恩、便秘、抑郁症和/或2型糖尿病的作用。
本发明的目的之四在于提供了薄荷脑在制备辅助治疗脂肪肝的药物中的用途。
本发明的目的之五在于提供了薄荷脑在制备辅助治疗痛风的药物中的用途。
本发明的目的之六在于提供了薄荷脑在制备辅助治疗肝硬化的药物中的用途。
本发明的目的之七在于提供了薄荷脑在制备辅助治疗抑郁症的药物中的用途。
本发明的目的之八在于提供了薄荷脑在制备辅助治疗2型糖尿病的药物中的用途。
进一步地，所述药物中还包括辅料。
进一步地，所述药物的剂型为颗粒剂、片剂、胶囊剂、粉剂、丸剂或溶液剂。
与现有技术相比，本发明的有益效果是：本发明通过体外模拟肠道环境并以粪便中收集得到的肠道菌群作为样本，进行薄荷脑干预实验，观察薄荷脑对于肠道菌群的影响，首次验证了薄荷脑具有促进肠道益生菌栖粪杆菌Faecalibacterium增殖的作用，因此不仅可将薄荷脑作为栖粪杆菌的增殖促进剂，用于体外促进栖粪杆菌大规模增殖发酵生产，还可制成调节肠道菌群的药物，用于改善肠道菌群紊乱，并辅助治疗与栖粪杆菌Faecalibacterium相关的如脂肪肝、痛风、肝硬化、克罗恩、便秘、抑郁症和2型糖尿病等疾病。
附图说明
图1为本发明实施例1中试验组和对照组中栖粪杆菌Faecalibacterium的丰度测定和显著性分析结果；
图2为本发明实施例2中经薄荷脑干预前后栖粪杆菌Faecalibacterium的生长曲线图。
具体实施方式
下面将结合本发明中的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1
由于来源于人体的肠道菌群无法完全在小鼠体内定殖，因此直接取人体粪便经处理后作为肠道菌群样品，在体外模拟人体内肠道环境，观察经薄荷脑干预后的肠道菌群变化，具体操作过程如下：
1、粪便样品处理
（1）PBS缓冲液配制
以1L PBS缓冲液为例，用天平称取下列药品，倒入1000ml烧杯中，其中包括：KH2PO40.24 g，Na2HPO4·12H2O 2.90 g，NaCl 8.00 g，KCl 0.20 g。加入超纯水并利用磁力加热搅拌器使各物质完全溶解，然后用pH计和盐酸或氢氧化钠溶液调节缓冲液的pH值至7.4±0.05。
将上述溶液转移至容量瓶中，用超纯水定容至刻度线，塞上盖子后上下颠倒混匀。定容后的溶液倒入洁净的1L玻璃瓶中，放入高压灭菌锅，121℃灭菌15分钟。迅速拧紧盖子后取出。待降至室温后，将溶液存于4℃冰箱中。
（2）肠道菌群样品制备
以10g为例，用天平称量10 g粪便样品至离心管中，用移液枪取适量上述PBS缓冲液加至离心管中充分混匀。然后将完全混匀的样品平均分装至新50ml离心管中，每管再加适量PBS缓冲液。
混匀后在生物安全柜内进行过滤，将稀释后的样品依次通过20目、50目、100目、200目的滤网。收集滤液至离心管中，6000G，4℃离心15min，弃上清。称量沉淀后，用上述PBS缓冲液将沉淀溶解并定容至终浓度5%，得到肠道菌群样品。
2、薄荷脑干预实验
（1）基础培养基配制
配制用于培养肠道菌群的基础培养基-GAM培养基，以1000ml为例。称取60 g改良GAM肉汤药品，倒入1000ml烧杯中。量取800ml超纯水倒入烧杯中，并放到磁力加热搅拌器上搅拌至完全溶解。将上述完全溶解的溶液引流至1000ml容量瓶，并用少量超纯水冲洗烧杯后倒入容量瓶，重复3次。将容量瓶用超纯水定容至刻度线，塞上盖子后上下颠倒10次，确保溶液充分混匀。
将定容后的溶液倒入洁净玻璃瓶中，每1000ml的瓶子内放500ml培养基，防止高压灭菌时喷出。将装有培养基的玻璃瓶瓶盖拧松，放入脉冲高压灭菌锅，使用液体程序，121℃灭菌15分钟。灭菌完后，立即把瓶盖拧紧，冷却至室温。
（2）薄荷脑-GAM培养基配制
于无菌操作台上，用移液枪取上述GAM培养基分装至玻璃管中，每管2ml。接着用1ml移液器取薄荷脑溶液（薄荷脑购于盛恒生物科技有限公司，用PBS缓冲液溶解至质量百分浓度为：5%），加入上述装有GAM培养基的玻璃管中，每管2ml，作为试验组，使薄荷脑的终浓度为2%。同时还设置有空白对照组，每组3个重复，分组具体如下：
试验组：2ml GAM培养基+2ml 薄荷脑溶液+1mL PBS缓冲液；
对照组：2ml GAM培养基+3mL PBS缓冲液；
确认好每个玻璃管盖拧紧后，转移至厌氧箱传递箱中，放置之前需用消毒液消毒处理。放入厌氧箱后，拧松瓶盖，置换O212个小时。
将上述处理后得到的肠道菌群样品分别接种至试验组（薄荷脑-GAM培养基）以及空白对照组（GAM培养基-PBS缓冲液）中，每管1ml。于厌氧箱中培养72小时后，观察菌群的生长状况，拍照留存。
接着分别将试验组样品和对照组样品，10000 rpm，3 min离心，弃上清，将沉淀用液氮处理，并送至武汉艾康健生物科技有限公司，分别测定试验组和对照组中的肠道菌群如Faecalibacterium和Lachnospiracea的丰度，即不同菌群在检测到的所有菌种中所占的百分比，丰度测定结果如表1所示。
并对部分肠道菌群经薄荷脑干预前后的丰度变化进行显著性分析，分析结果如图1所示，其中**表示P＜0.01，即丰度变化有极显著差异；*表示P＜0.05，即丰度变化有显著差异。
表1 薄荷脑干预前后的菌群丰度变化
结果显示，经薄荷脑干预后，肠道菌群中栖粪杆菌Faecalibacterium的丰度相比于对照组有极显著增高。然而经薄荷脑干预的其他肠道微生物如Lachnospiracea的丰度则与对照组无明显差异。结果说明，薄荷脑具有促进栖粪杆菌Faecalibacterium增殖的作用，因此可作为体外生长发酵促进剂，促进栖粪杆菌Faecalibacterium增殖，进而大量生产发酵目的菌剂。同时薄荷脑通过在体内极显著上调栖粪杆菌Faecalibacterium的丰度，可改善肠道紊乱，并辅助治疗脂肪肝、痛风、肝硬化、克罗恩、便秘、抑郁症和/或2型糖尿病等相关疾病。
实施例2
为观察薄荷脑对于促进栖粪杆菌Faecalibacterium的动态变化，绘制相应的生长曲线，观察经薄荷脑干预前后栖粪杆菌Faecalibacterium的生长曲线变化，方法如下：
准备100μl无菌枪头，需在高压灭菌锅内进行115℃、20mins的灭菌。移液枪、无菌96孔板事先用酒精消毒后放入生物安全柜进行30分钟的紫外灭菌。再转移至厌氧箱内进行取样测定OD600值
培养液充分摇匀再进行取样，每个培养瓶取三次100μl样品的量，分别纵向添加，再横向稀释三至五组连续的梯度，每个培养瓶使用一根无菌枪头。以此类推，待取样完成后，对所有的培养液进行测定。
使用系统:Tecan i-control , 2.0.10.0，设定系统数值如下表：
根据系统导出不同时间段的OD600值，结果如表2所示。
表2 薄荷脑对栖粪杆菌Faecalibacterium的作用
根据上述数据，以培养时间为X轴，测定的OD600值的百分数为Y值，分别绘制试验组和对照组的生长曲线，结果如图2所示。结果显示，试验组和对照组内的Faecalibacterium在8-24h开始进入对数生长期，24-32h为稳定期，32h后进入衰亡期。对比试验组和对照组的生长曲线可以得出，在薄荷脑的培养下，Faecalibacterium生长得更加旺盛，说明，薄荷脑对Faecalibacterium的生长有促进作用。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。
Claims (1)
1.薄荷脑在制备促进栖粪杆菌Faecalibacterium增殖的促进剂中的用途，所述促进剂中还包括GAM培养基，促进栖粪杆菌Faecalibacterium增殖的方法包括：配制GAM培养基，并将薄荷脑用PBS缓冲液溶解得到薄荷脑溶液，将所述薄荷脑溶液加入GAM培养基中至薄荷脑终浓度为质量分数2%，得到薄荷脑-GAM培养基，采用所述薄荷脑-GAM培养基培养栖粪杆菌Faecalibacterium。
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