食品与发酵工业 >

2025 , Vol. 51 >Issue 22: 360 - 367

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.042014

分析与检测

复合益生菌产品中副干酪乳酪杆菌活菌定量的PMA-qPCR方法研究

  • 焦颖欣 ,
  • 郭立峥 ,
  • 宋程羽 ,
  • 宋智泉 ,
  • 冯会粉 ,
  • 迮晓雷 ,
  • 葛媛媛 ,
  • 姚粟
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  • 1(中国食品发酵工业研究院有限公司,中国工业微生物菌种保藏管理中心,北京,100015)
    2(汤臣倍健股份有限公司,广东 广州,510663)
第一作者:硕士研究生(姚粟教授级高级工程师为通信作者,E-mail:milly@china-cicc.org)

收稿日期: 2024-12-31

  修回日期: 2025-04-07

  网络出版日期: 2025-12-15

基金资助

中国轻工集团有限公司科技创新基金项目(ZQ2023JC-QN04);中国食品发酵工业研究院有限公司强院工程项目

收起

Development of a PMA-qPCR method for quantification of viable Lacticaseibacillus paracasei in composite probiotic products

  • JIAO Yingxin ,
  • GUO Lizheng ,
  • SONG Chengyu ,
  • SONG Zhiquan ,
  • FENG Huifen ,
  • ZE Xiaolei ,
  • GE Yuanyuan ,
  • YAO Su
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  • 1(China National Research Institute of Food and Fermentation Industries Co.Ltd., China Center of Industrial Culture Collection, Beijing 100015, China)
    2(BYHEALTH Institute of Nutrition & Health, Guangzhou 510663, China)

Received date: 2024-12-31

  Revised date: 2025-04-07

  Online published: 2025-12-15

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摘要

复合益生菌产品的活菌数是评估其质量和功效的关键参数。然而,如何在复合益生菌产品中实现特定菌种活菌数的精准检测是目前行业内面临的技术难题。叠氮溴化丙锭(propidium monoazide,PMA)结合荧光定量PCR(quantitative PCR,qPCR)技术,可有效区分死活菌,精确检测多菌种体系中目标菌种的活菌数。该研究以副干酪乳酪杆菌(Lacticaseibacillus paracasei)为研究对象,通过比较7种常用DNA提取方法的效率并优化PMA前处理条件,建立了基于PMA-qPCR的活菌定量检测方法,并对其特异性和适用性进行了验证。结果表明,采用珠式研磨仪破碎法一步式提取DNA时,其提取效率优于其他方法。当PMA浓度为50 μmol/L,暗孵育5 min并曝光15 min时,此条件能够有效区分死活菌。该方法的定量限为8.07×103 CFU/mL。特异性和适用性验证结果表明,所建立的PMA-qPCR方法特异性较好,能够在复合益生菌产品中精确测定L. paracasei的活菌数,并能有效识别未标示添加量的样品中是否存在活性L. paracasei。该方法为复合益生菌产品的质量控制和行业标准化提供了可靠的技术支持,为益生菌产品的质量监管提供了新的技术选择。

关键词： 副干酪乳酪杆菌; 活菌定量; PMA-qPCR技术; 复合益生菌产品; 质量控制

本文引用格式

焦颖欣 , 郭立峥 , 宋程羽 , 宋智泉 , 冯会粉 , 迮晓雷 , 葛媛媛 , 姚粟 . 复合益生菌产品中副干酪乳酪杆菌活菌定量的PMA-qPCR方法研究[J]. 食品与发酵工业, 2025 , 51(22) : 360 -367 . DOI: 10.13995/j.cnki.11-1802/ts.042014

Abstract

The viable cell count of probiotics in a composite probiotic product is a key parameter for evaluating its quality and efficacy.However, achieving accurate detection of the viable cell count of specific strains within composite probiotic products remains a technical challenge in the industry.Propidium monoazide (PMA) combined with quantitative PCR (qPCR) technology can effectively distinguish between live and dead bacteria and accurately quantify viable bacteria for targeted strains in a multi-strain system.In this study, Lacticaseibacillus paracasei was selected as the research object.By comparing the efficiency of seven commonly used DNA extraction methods and optimizing the PMA pre-treatment conditions, a PMA-qPCR-based viable cell quantification method was established, and its specificity and applicability were verified.Results showed that the DNA extraction efficiency was superior to other methods when using a bead mill homogenizer for one-step DNA extraction.When the PMA concentration was 50 μmol/L, with 5 min dark incubation and 15 min of exposure to light, this condition effectively distinguished between live and dead bacteria.The limit of quantitation of the method was 8.07×103 CFU/mL.The specificity and applicability verification results indicated that the established PMA-qPCR method had good specificity, capable of accurately determining the viable cell count of L.paracasei in composite probiotic products and effectively identifying the presence of viable L.paracasei in samples without labelled addition.This method provides reliable technical support for the quality control and industry standardization of composite probiotic products and offers a new technical option for the quality supervision of probiotic products.

Key words： Lacticaseibacillus paracasei; viable bacterial quantification; PMA-qPCR technology; composite probiotic products; quality control

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