PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk

Zihang Shi; Xiefei Li; Xiankang Fan; Jue Xu; Qing Liu; Zhen Wu; Daodong Pan

doi:10.3389/fmicb.2022.984506

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk

Front Microbiol. 2022 Sep 7:13:984506. doi: 10.3389/fmicb.2022.984506. eCollection 2022.

Authors

Zihang Shi^{1

2}, Xiefei Li^{1

2}, Xiankang Fan^{1

2}, Jue Xu^{1

2}, Qing Liu³, Zhen Wu^{1

2}, Daodong Pan^{1

2}

Affiliations

¹ State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China.
² Key Laboratory of Animal Protein Food Processing Technology of Zhejiang Province, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, China.
³ Nanjing Dairy Group, Nanjing, China.

Abstract

The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 10¹¹ and 10⁶ CFU/mL, with R ² > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

Keywords: PMA-qPCR; fermented milk; lactic acid bacteria; pheS gene; viable bacteria numbers.