塔什库尔干地区传统手工乳制品中乳酸菌多样性及其功能分析

Diversity and functions of lactic acid bacteria in traditional handmade dairy products in Tashkurgan region

摘要:

【背景】传统手工制作乳制品是塔吉克族农牧地区人们最喜爱的食品，蕴含着十分丰富的乳酸菌资源，亟待开发利用。【目的】通过解析塔吉克族农牧地区传统自制乳制品中乳酸菌的多样性和功能特性，为挖掘和保护其中的微生物资源提供保障，同时为评估传统奶酪的安全性提供理论基础。【方法】采用高通量测序技术对自制乳制品中细菌16S rRNA基因的V3−V4可变区进行测序分析，利用传统培养法从MRS、Lee氏和M17培养基中筛选出可培养疑似乳酸菌，经生理生化试验及16S rRNA基因序列同源性分析技术对其进行属种鉴定，解析塔县地区传统乳制品中微生物的种类及其基因功能信息。【结果】四份乳制品中共鉴定出37个细菌属65个细菌种，其中奶酪和酸奶中的优势细菌属为Lactobacillus和Streptococcus，优势乳酸菌种为瑞士乳杆菌，并且奶酪中含少许致病菌。在此基础上，利用传统培养法从MRS、M17和Lee氏培养基筛选出38株可培养疑似乳酸菌，经生理生化试验及16S rRNA基因序列同源性分析技术对其进行属种鉴定。38株疑似乳酸菌分别为3株枯草芽孢杆菌(Bacillus subtilis)、1株芽孢杆菌(Bacillus luti)、1株副炭疽杆菌(Bacillus paranthracis)、9株副干酪乳杆菌(Lactobacillus paracasei)、3株霍氏肠球菌(Enterococcus hormeachei)、1株溶血性葡萄球菌(Staphylococcus heamolyticus)、1株肺炎克雷伯菌(Klebsiella pneumoniae)、10株假肠膜明串珠菌(Leuconostoc pseudomesenteroides)、2株肠系膜明串珠菌(Leuconostoc mesenteroides)，4株植物乳杆菌(Lactobacillus plantarum)、1株塞内加尔醋杆菌(Acetobacter senegalensis)、1株乳肠球菌(Enterococcus durans)和1株粪肠杆菌(Enterobacter faecalis)。其中明串珠菌属(Leuconostoc)为优势菌属，其次为乳杆菌属(Lactobacillus)，归类为7个属13个种。在乳酸菌的属种鉴定上，传统培养法能够更准确地将乳酸菌鉴定到种水平。从38株乳酸菌中选取代表性的11株菌进行抑菌特性试验，11株菌对大肠杆菌(Escherichia coli)和枯草芽孢杆菌(Bacillus subtilis)均有抑制作用。奶酪微生物基因(clusters of orthologous groups of proteins COG)功能预测结果显示，奶酪中的微生物富含与氨基酸转运和代谢有关的基因。将各个样品的微生物群落组成与功能组成进行比较，发现各个样品的细菌群落组成虽存在明显差异，但其功能组成却具有高度相似性。【结论】以新疆塔县4个传统手工乳品为研究对象，采用现代分子生物学技术和常规培养技术对4种传统手工乳品进行菌种鉴定，发现其菌种组成、多样性均不高，并且有部分致病菌。这2种方法相结合能够更全面地了解奶酪中的微生物菌群结构，为实现乳品的工业生产奠定理论基础，并保护了塔吉克牧民地区微生物中丰富的资源。同时为评估乳酸菌安全性提供了理论基础。

Abstract:

[Background] Traditional handmade dairy products are the favorite food in Tajik agricultural and pastoral areas. They contain abundant lactic acid bacterial resources which need to be exploited and utilized. [Objective] By analyzing the diversity and functions of lactic acid bacteria in traditional homemade dairy products from Tajik agricultural and pastoral areas, we aim to aid in the exploration and protection of microbial resources and provide a theoretical basis for evaluating the safety of traditional dairy products. [Methods] The V3–V4 variable region of bacterial 16S rRNA gene was subjected to high-throughput sequencing. The culturable suspected lactic acid bacteria were screened by the conventional culture methods in the MRS, Lee’s, and M17 media and identified by physiological and biochemical tests and 16S rRNA gene sequence homology. Furthermore, the microbial species and gene function information in the dairy products in Tajik autonomous county of Taxkorgan were analyzed. [Results] There were 37 bacterial genera and 65 bacterial species in the 4 dairy samples, among which Lactobacillus and Streptococcus were the dominant bacterial genera in cheese and yogurt, and Lactobacillus helveticus was the dominant species. A small amount of pathogenic bacteria existed in the cheese samples. Moreover, 38 culturable suspected lactic acid bacterial strains were identified from MRS, M17, and Lee’s media, including 3 strains of Bacillus subtilis, 1 strain of B. luti, 1 strain of B. paranthracis, 9 strains of Lactobacillus paracasei, 3 strains of Enterococcus hormeachei, 1 strain of Staphylococcus heamolyticus, 1 strain of Klebsiella pneumoniae, 10 strains of Leuconostoc pseudomesenteroides, 2 strains of Leuconostoc mesenteroides, 4 strains of Lactobacillus plantarum, 1 strain of Acetobacter senegalensis, 1 strain of Enterococcus durans, and 1 strain of Enterobacter faecalis. Among them, the dominant genus was Leuconostoc, followed by Lactobacillus. The 38 strains were classified into 7 genera and 13 species. The results indicated that the conventional culture method can accurately identify lactic acid bacteria to the species level. Representative 11 strains of lactic acid bacteria were selected from 38 strains for antibacterial tests, and all 11 the strains had inhibitory effects on Escherichia coli and B. subtilis. The COG (clusters of orthologous groups of proteins) function prediction showed that the microorganisms in cheese carried rich genes involved in amino acid transport and metabolism. Although there were significant differences in the bacterial community composition between samples, their functions were highly similar. [Conclusion] The diversity of lactic acid bacteria in four traditional handmade dairy products in Tajik autonomous county of Taxkorgan, Xinjiang was studied by combining the molecular biological methods and the conventional culture method. The richness and diversity of lactic acid bacteria in the dairy products were low, and there were some pathogenic bacteria. The combination of the two methods in this study comprehensively revealed the microbial composition in the dairy products. The findings provided basic data for the industrial production and protection of the abundant microbial resources in this area and laid a foundation for evaluating the safety of lactic acid bacteria in traditional dairy products.