高通量测序技术对杀菌型益生菌含乳饮料菌群结构分析

Structural analysis of sterilization type probiotic milk containing beverage bacteria by high-throughput sequencing technology

目的分析杀菌型益生菌含乳饮料中菌群组成结构,并探究宣称添加益生菌的真实性和合规性。方法本研究以高通量测序技术为基础,从市售11批次杀菌型益生菌含乳饮料中全基因组提取,进行V3/V4变异区序列的聚合酶链式反应(polymerase chain reaction,PCR)扩增后,整理及统计样品测序序列数目,操作分类单元(operational taxonomic unit,OUT)归类,生成稀释曲线、Alpha多样性、聚类、丰度分布曲线与分析菌群结构和菌种标示合规性。结果共得到39231条优质序列,分布在210~518 bp范围内;共注解到16个门、37个纲、71个目、129个科、265个属;从门分类水平主要为厚壁菌门(Firmicutes 91.66%),从纲的分类水平主要为芽孢杆菌纲(Bacill 90.43%),从目的分类水平主要为乳杆菌目(Lactobacillales 82.74%);优势菌种为德氏乳杆菌保加利亚亚种、嗜热链球菌;同时产品有检出携带致病性非益生菌菌群。11种产品标签标示与实际检测到菌株完全一致的仅有1批次。结论本研究建立的高通量测序技术能准确、快速地探究杀菌型益生菌含乳饮料中微生物菌群多样性和潜在的致病性,市售产品益生菌种类丰富,但同质化程度较高,产品间菌群分布均匀度差异大。

Objective To analyze the composition and structure of microbial communities in sterilization type probiotic milk containing beverages,and explore the authenticity and compliance of the claimed addition of probiotics.Methods Based on high-throughput sequencing technology,this study extracted the whole genome from 11 batches of sterilization type probiotic milk containing beverages on the market.After the polymerase chain reaction(PCR)amplification of the V3/V4 variant region sequence,it sorted out and counted the number of sample sequencing sequences,operational taxonomic unit(OUT)was classified,generates dilution curves,Alpha diversity,clustering,abundance distribution curves was generated and flora structure was analyzed,and bacterial marking compliance was analyzed.Results A total of 39231 high-quality sequences were obtained,distributed in the range of 210‒518 bp;a total of 16 gates,37 classes,71 orders,129 families,and 265 genera were annotated;the classification level of Filmicutes(91.66%),the classification level of the class was mainly Bacillus(90.43%),and the classification level from the classification level was mainly Lactobacillales(82.74%);the dominant bacteria were the Bulgarian subspecies of Lactobacillus,Streptococcus thermophilus;at the same time,the product had been detected to carry pathogenic non-probiotic flora.There was only one batch of 11 kinds of product labels that were completely consistent with the actual detected strains.Conclusion The high-throughput sequencing technology established in this study can accurately and quickly explore the diversity and potential pathogenicity of microbial flora in sterilization type probiotic milk containing beverages.There are rich types of probiotics in commercially available products,but the degree of homogenization is high,and the distribution uniformity of flora between products is large.