珠江口水产源乳酸菌资源库的构建及优良乳酸菌的高通量筛选  

Construction of a strain bank of lactic acid bacteria separated from aquatic livestock in the Pearl river estuary and the high-throughput exploration of excellent strains

作　　者：李嘉盛 刘复荣 刘明珠 张宏刚 崔金明 蒙海林 LI Jia-Sheng;LIU Fu-Rong;LIU Ming-Zhu;ZHANG Hong-Gang;CUI Jin-Ming;MENG Hai-Lin(Bioengineering Research Center,Guangzhou Institute of Advanced Technology,Chinese Academy of Sciences,Guangzhou 511458,China;Guangzhou GLAM Biotechnology Co.,Ltd,Guangzhou 510535,China)

机构地区：[1]广州中国科学院先进技术研究所,生物工程研究中心,广州511458 [2]广州格拉姆生物科技有限公司,广州510535

出　　处：《食品安全质量检测学报》2020年第16期5627-5634,共8页Journal of Food Safety and Quality

基　　金：国家自然科学基金项目(31670056,31870776,31971338);广东省科技计划项目(2019B030316016);广东省自然科学基金项目(2017A030313149);广州市科技计划项目(201803020035,201904010337);广州市珠江科技新星(201806010053)。

摘　　要：目的构建珠江口水产养殖动物源的乳酸菌资源库,开发一套较为完善的高通量筛选优良乳酸菌的方法。方法采集珠江口水域多种水产动物的肠道,研磨,稀释涂布于含溴甲酚紫及碳酸钙的MRS平板上,挑取有溶钙圈的单菌落,进行过氧化氢酶实验以排除产气菌。提取细菌的基因组作模板, PCR扩增16S rDNA基因,根据测序结果进行同源性比对分析确定乳酸菌的种属,建立乳酸菌库。进一步借助96孔板培养技术,将乳酸菌与水产致病菌(指示菌)混合培养,根据对指示菌的抑菌效果,确定具有优良抑菌活性的乳酸菌。结果从水产动物肠道中筛选出乳酸菌14属71种1638株,获得了15株对常见水产养殖致病菌(嗜水气单胞菌、创伤弧菌、副溶血弧菌等)具有高效广谱抑制效果的菌株。结论本研究建立的乳酸菌菌种资源库,弥补了国内目前水产养殖来源乳酸菌菌株储备缺乏的现状,有望提供各类性能优良菌株用于渔业、畜牧业和食品生产。Objective To construct a strain bank of lactic acid bacteria(LAB) separated from aquatic livestock in the Pearl river estuary, and develop a high-throughput screening technology for exploration of excellent strains from the bank. Methods Various kinds of aquatic livestock in the Pearl river estuary were sampled and the intestinal contents were collected and grinded. Samples were diluted and incubated on MRS plates containing bromocresol purple and calcium carbonate. Colonies with dissolved calcium were picked out and aerogenic bacteria were excluded by catalase test. With genomic DNA as template, 16 S rDNA of each strain was amplified by PCR and then sequenced. A bank of LAB strain was then constructed according to species identification via sequence homology analysis. Finally, LAB strains with high antibacterial activity were identified by co-culturing them with aquatic pathogenic bacteria, i.e. indicator bacteria, on 96-well plates. Results A total of 1638 LAB strains belonging to 14 genera and 71 species were collected from aquatic livestock, of which 15 strains showed broad-spectrum antibacterial activity against popular pathogenic bacteria of aquaculture, such as Aeromonas hydrophila, Vibrio vulnificus, and Vibrio parahaemolyticus. Conclusion The LAB strain bank constructed in this work not only alleviates the deficiency of repositories for aquaculture source LAB strain in China, but also provides potential excellent strains for various applications in fishing industry, stock farming, and food production.

关 键 词：乳酸菌 菌种库 水产养殖 高通量筛选 抑菌活性 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程]