市售益生菌产品中15株乳杆菌的精准鉴定及毒力基因和耐药基因研究

Accurate Identification of 15 Strains of Lactobacillus in Commercially Available Probiotics and Study on Virulence Genes and Drug Resistance Genes

关键词:  益生菌产品  乳杆菌  精准鉴定  毒力基因  耐药基因

Key words:  probiotics  lactobacillus  accurate identification  virulence genes  drug resistance genes

摘要:

目的 随着微生物鉴定技术的发展及菌种分类命名规则改变,或由于传代次数增加、基因突变、质粒转移等因素引起基因组稳定性发生变化,导致市售益生菌产品实际菌名与产品标签明示不一致,造成菌名混乱的状况。本研究探讨采用多种手段对13批市售益生菌产品中15株乳杆菌进行鉴定研究,比较不同鉴定技术结果差异,旨在探索建立一套能够快速、精准鉴定乳杆菌的标准方法。同时,采用全基因组测序技术(WGS)和生物信息学方法对乳杆菌进一步开展安全性评价。方法 参照《GB 4789.35-2023 食品安全国家标准 食品微生物学检验 乳酸菌检验》方法对13批市售益生菌产品中15株乳杆菌进行分离纯化,分别采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)(简称MS)、16S rRNA基因测序技术(简称16S)和WGS 3种技术对15株乳杆菌进行鉴定,同时,利用WGS和生物信息学方法,对其进行序列组装、基因预测和功能注释,深入挖掘乳杆菌菌株中毒力基因和耐药元件等信息。结果 采用MALDI-TOF MS、16S rRNA基因测序技术和WGS技术,15株乳杆菌中分别有4株、7株、5株菌鉴定结果与标签明示菌名不一致。A04标签菌名为嗜酸乳杆菌,3种测序技术鉴定结果均为副干酪乳杆菌,提示该批产品存在菌名混乱的情况。Venn图结果分析MS与16S技术有5株菌鉴定结果不一致；16S技术与WGS技术有6株菌鉴定结果不一致；MS与WGS技术有3株菌鉴定结果不一致。采用WGS和生物信息学方法研究发现,15株乳杆菌中均未检测到耐药元件和毒力元件。结论 本研究从市售13批益生菌产品中分离到15株乳杆菌,采用MALDI-TOF MS、16S rRNA基因测序技术和WGS测序进行鉴定分析,证实了5批次市售益生菌产品存在实际菌名与产品标签明示不一致的现象。3种鉴定技术鉴定结果存在一定差异,WGS通过对益生菌整个基因组序列测序,可以获得完整的基因组信息,同时对乳杆菌的毒力基因和耐药元件进行精准解析,因此,可以作为乳杆菌鉴定及基因组安全性分析的标准方法,实现对市售益生菌产品的跟踪评价和监管。

Abstract:

OBJECTIVE With the development of microbial identification technology and the change of species classification and nomenclature rules, or because of the increase in the number of passages, Gene Mutation, plasmid transfer and other factors caused changes in genome stability, the actual bacterial names of probiotic products on the market are not consistent with the product labels, resulting in confusion of bacterial names. In this study, 15 strains of Lactobacillus from 13 batches of probiotics were identified by various methods, and the results of different identification methods were compared, the aim of this study was to establish a rapid and accurate standard method for the identification of Lactobacillus. Meanwhile, Whole Genome Sequencing (WGS) was used to further evaluate the safety of Lactobacillus Whole Genome Sequencing. METHODS According to the method of " GB 4789.35-2023 National food safety standard-Food microbiological examination-Lactic acid bacteria test, " 15 strains of Lactobacillus were isolated and purified from 13 batches of commercial probiotic products. The 15 strains of Lactobacillus were identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), 16 S rRNA gene sequencing (16 S ) and WGS, respectively. At the same time, WGS and bioinformatics methods were used to sequence assembly, gene prediction and functional annotation. In-depth excavation of information such as virulence genes and drug resistance elements of Lactobacillus strains. RESULTS MALDI-TOF MS, 16S rRNA gene sequencing and WGS technology were used to identify 4,7 and 5 strains of Lactobacillus in 15 strains, respectively. The identification results were inconsistent with the label name. A04 was identified as Lactobacillus acidophilus, and the identification results of three sequencing techniques were all Lactobacillus paracasei, suggesting that there was confusion in the names of the products. Venn diagram analysis showed that the identification results of 5 strains were inconsistent between MS and 16 S technology. The identification results of 6 strains of bacteria by 16 S technology and WGS technology were inconsistent. The identification results of 3 strains of bacteria by MS and WGS were inconsistent. Using WGS and bioinformatics methods, it was found that no drug resistance elements and virulence elements were detected in 15 strains of Lactobacillus. CONCLUSION In this study, 15 strains of Lactobacillus were isolated from 13 batches of commercially available probiotic products. MALDI-TOF MS, 16 S rRNA gene sequencing and WGS sequencing were used for identification and analysis. It was confirmed that the actual bacterial name of 5 batches of commercially available probiotic products was inconsistent with the product label. There are some differences in the identification results of the three identification techniques. WGS can obtain complete genomic information by sequencing the whole genome sequence of probiotics, and accurately analyze the virulence genes and drug resistance elements of Lactobacillus. Therefore, WGS can be used as a standard method for identification and genomic safety analysis of Lactobacillus, and realize the tracking evaluation and supervision of commercially available probiotic products.