基于纯培养方法和PacBio三代测序技术研究蒙古国传统酸马奶中乳酸菌多样性  

Lactic Acid Bacteria Diversity in Mongolia Traditional Koumiss based on Culture-dependent and PacBio Sequencing Technology

作　　者：刘文俊[1] 多拉娜 刘亚华 任冬艳 张和平[1] 孟和毕力格[1] Liu Wenjun;Duolana;Liu Yahua;Ren dongyan;Zhang Heping;Menghebilige(Inner Mongolia Agricultural University,Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Key Laboratory of Dairy Products Processing,Ministry of Agriculture and Rural Affairs,Inner Mongolia Key Laboratory,of Dairy Biotechnology and Engineering,Hohhot 010018)

机构地区：[1]内蒙古农业大学乳品生物技术与工程教育部重点实验室农业农村部奶制品加工重点实验室内蒙古自治区乳品生物技术与工程重点实验室,呼和浩特010018

出　　处：《中国食品学报》2019年第4期27-37,共11页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家现代农业产业技术体系建设项目(CARS-36);内蒙古自治区科技计划项目(2018);国家自然科学基金面上项目(31671871);内蒙古自治区自然科学基金面上项目(2016MS0316)

摘　　要：通过纯培养方法结合PacBio三代测序技术对采自蒙古国的12份传统酸马奶中的乳酸菌多样性进行研究。应用传统的纯培养技术从12份传统酸马奶样品中分离出62株乳酸菌,通过16S rRNA基因序列分析和系统进化关系研究,62株乳酸菌被鉴定为5个属,6个种,分别是:25株瑞士乳杆菌、20株植物乳杆菌、7株产马乳酒乳杆菌、5株屎肠球菌、3株嗜热链球菌和2株粪肠球菌。采用PacBio SMRT测序技术研究其中8份样品中乳酸菌的多样性,宏基因组测序得到的优势菌为乳酸菌,同时也检测到一些痕量的其它种属细菌。共检测到5个乳酸菌的属,分别是:乳杆菌属、乳球菌属、链球菌属、明串珠菌属和肠球菌属,20个乳酸菌的种,即:瑞士乳杆菌、乳酸乳球菌、副乳链球菌、棉子糖乳球菌、肠膜阴串珠球菌和产马乳酒乳杆菌。宏基因组测序相对含量表明:蒙古国酸马奶样品中乳酸菌优势属为乳杆菌属(95.56%),优势种为瑞士乳杆菌(94.64%),纯培养方法和宏基因组三代测序方法都表明瑞士乳杆菌为蒙古国传统酸马奶中的优势菌种。宏基因组测序技术除检测到纯培养获得的菌株外,还获得14个低丰度乳酸菌,如棉籽糖乳球菌、肠膜明串珠球菌和开菲尔乳杆菌等,这些菌在酸马奶中含量较少。通过PacBio SMRT测序技术可以全面了解酸马奶中乳酸菌的多样性,为通过纯培养方法获得低丰度乳酸菌提供指导。Biodiversity of lactic acid bacteriain 12 traditional koumiss samples collected from Mongolia were studied by using culture-dependent method and Pacific Biosciences(PacBio) single-molecule, real-time sequencing technology(SMRT) analysis in the present study. 62 strains of lactic acid bacteria were isolated from 12 traditional koumiss samples by using pure culture method. Based on the 16 S rRNA gene sequence and phylogenetic relationship analysis, these strains were identified as Lactobacillus helveticus(25 strains), Lactobacillus plantarum(20 strains), Streptococcus thermophilus(3 strains) and Enterococcus faecium(5 strains), Lactobacillus kefiranofaciens(7 strains) and Enterococcus faecalis(2 strains). In addition, we used PacBio SMRT sequencing technology found 5 genera of lactic acid bacteria, including Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Enterococcus, and 20 species of lactic acid bacteria,such as Lactobacillus helveticus, Lactococcus lactis, Streptococcus parauberis, Leuconostoc mesenteroides and Lactobacillus kefiranofaciens and so on. The dominant genus and speciesrespectively were Lactobacillus(94.64%) and Lactobacillus helveticus(94.64%) in these traditional koumiss samples. The culture-dependent method and PacBio SMRT sequencing technology both verified Lactobacillus helveticus was the dominant species in 8 traditional koumiss samples in the results of the study. In addition, PacBio SMRT sequencing technology also obtained traces of 14 low-abundance lactic acid bacteria such as Lactococcus raffinolactis, Leuconostoc mesenteroides, and Lactobacillus kefiri and so on. The diversity of lactic acid bacteria in koumiss can be comprehensively understood by PacBio SMRT sequencing technology, which could provide guidance for obtaining low abundance lactic acid bacteria through pure culture method.

关 键 词：酸马奶 乳酸菌 PacBio SMRT测序技术 生物多样性 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]