复合益生菌产品菌种鉴定及活菌定量检测方法

Study on methods of species identification and quantitative detection of viable cells in composite probiotics products

复合益生菌产品的菌种组成与活菌数是产品质量的关键,该研究以2种市售复合益生菌固体饮料及其添加的5种(6株)益生菌为研究对象,采用宏基因组测序分析方法及平均核苷酸一致性(average nucleotide identity,ANI)在种水平上精确鉴定产品的菌种组成;通过筛选菌种特异性引物,优化叠氮溴化丙锭-荧光定量PCR(propidium monoazide-quantitative PCR,PMA-qPCR)方法参数,快速定量产品中每种益生菌的活菌数。结果表明,产品1宏基因组数据分箱获得的4个bins分别被鉴定为动物双歧杆菌乳亚种、鼠李糖乳杆菌、发酵乳杆菌和短双歧杆菌,PMA-qPCR定量检测其活菌数分别为4.86×10^(9)、4.33×10^(9)、3.95×10^(8)和6.38×10^(6)CFU/g;产品2宏基因组数据分箱获得的4个bins分别被鉴定为鼠李糖乳杆菌、植物乳杆菌、发酵乳杆菌、动物双歧杆菌乳亚种,PMA-qPCR定量检测其活菌数分别为3.12×10^(9)、1.53×10^(9)、9.36×10^(8)和4.16×10^(8)CFU/g。PMA-qPCR方法定量2种产品乳酸菌的活菌总数与平板菌落计数结果相比,差异不显著(P>0.05);该研究在菌种水平上精确鉴定的产品微生物组成与产品声称一致。除短双歧杆菌外,其他5种菌的活菌数检测结果与实际添加量基本吻合。该研究在实现精确鉴定产品物种组成的同时,快速、特异、准确的定量了产品中每种乳酸菌的活菌数,为复合益生菌产品的质量控制提供了技术支持。

The composition of probiotic species and the number of viable cells are essential to the quality of composite probiotics products.Two commercial composite probiotic solid beverages and five probiotic species(six strains) contained in the products were studied in this research.The metagenomic sequencing and the average nucleotide identity(ANI) analysis were conducted to accurately identify probiotic composition at the species level.Through selection of species-specific PCR primers and optimization of key parameters of propidium monoazide-quantitative PCR(PMA-qPCR) method,the number of viable probiotic cells of each species were quantitatively detected.The results showed that four bins obtained from the metagenomic data of product 1 were identified as Bifidobacterium animalis subsp.lactis,Lactobacillus rhamnosus,Lactobacillus fermentum,and Bifidobacterium breve,and the number of viable cells detected by PMA-qPCR were 4.86×10^(9),4.33×10^(9),3.95×10^(8) and 6.38×10^(6) CFU/g respectively.Four bins obtained from metagenomic data of product 2 were identified as L.rhamnosus,L.plantarum,L.fermentum,and B.animalis subsp.lactis,and the number of viable cells were 3.12×10^(9),1.53×10^(9),9.36×10^(8) and 4.16×10^(8) CFU/g respectively.There were no significant differences(P>0.05) between the results of total viable counts in two products obtained by PMA-qPCR method and plate counting method.The probiotics identification results at the species level in this study are consistent with the products’ claims.Except for Bifidobacterium breve,the detection results of the number of viable cells for other five species were basically consistent with the products addition.The methods applied in this study can accurately identify the species composition,and can quickly,specifically and accurately quantify the number of viable cells of each probiotic species in the products,which could provide technical support for the quality control of composite probiotic products.