冷冻保护剂对粪悬液中活细菌的保护效果  

Protective Effect of Cryoprotectant on Viable Bacteria in Fecal Suspension

作　　者：付鑫超 陈傲蕾 张雅洁 庞小燕[1] Fu Xinchao;Chen Aolei;Zhang Yajie;Pang Xiaoyan(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai,200240)

机构地区：[1]上海交通大学生命科学技术学院,微生物代谢国家重点实验室,上海200240

出　　处：《基因组学与应用生物学》2020年第12期5586-5592,共7页Genomics and Applied Biology

基　　金：国家自然科学基金(81670603);上海交通大学医工交叉项目(YG2014ZD10)共同资助。

摘　　要：粪菌移植中常将制备好的供体粪悬液加入冷冻保护剂后-80℃冻存待用。但是,作为在粪悬液中主要发挥临床效果的肠道菌群,其活性在冻存前后的变化却很少被检测。本研究采集健康志愿者粪便样品,制作粪悬液后,将其分为3组:冻存前、冻存/无保护剂、冻存/甘油(10%)。采用细菌培养与菌群16S rRNA基因测序相结合的方法,比较新鲜粪悬液与-80℃冻存1个月的粪悬液中活细菌的数量和组成。结果表明,不加保护剂直接冻存会显著降低粪悬液中活细菌的数量,新鲜粪悬液中仅7.1%的细菌能保持活性,其菌群多样性也显著下降;而10%的甘油具有保护肠道菌群活性的作用,新鲜粪悬液中41.5%的细菌在冻存后仍有活性,其菌群的组成结构也与新鲜粪悬液相似,最优势的属为Escherichia/Shigella、Bifidobacterium、Megamonas、Klebsiella和Prevotella。本研究用10%甘油做保护剂冻存粪悬液是一种可行的保存粪悬液的方法,但也提示了发展更有效的保存方法的必要性。In fecal microbiota transplantation(FMT),the fecal suspension of donor is usually prepared and added with cryoprotectant,then frozen at -80℃ to be used.However,as the key functional material of FMT,the living bacterial community in fecal suspension is rarely detected before and after freezing.In this study,fecal samples were collected from healthy donors and suspensions were prepared and divided into three groups:fresh fecal suspension,frozen without cryoprotectant and frozen with 10% glycerol.Using traditional culture methods combined with 16S rRNA gene sequencing,the number and composition of viable bacteria in the three groups were compared.The results indicated that freezing without cryoprotectant significantly reduced the number and diversity of viable bacteria in fecal suspension.Only 7.1% bacteria remained alive in fecal suspension after direct frozen.10% glycerol showed protective effect of fecal microbiota.41.5% living bacteria in fresh fecal suspension remained alive after frozen with glyceral,meanwhile,the microbiota composition was also similar to that of fresh fecal suspension.The dominant genera of this two groups were both Escherichia/Shigella,Bifidobacterium,Megamonas,Klebsiella and Prevotella.This study indicated that using 10% glycerol as cryoprotectant was a feasible method to freeze fecal suspension,while,it also inferred the necessity of the development of more effective cryopreservation methods.

关 键 词：粪菌移植 粪悬液 活细菌 冻存保护剂 

分 类 号：R37[医药卫生—病原生物学]