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

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 <i>pheS</i> gene and 16S rRNA gene sequence similarities in five species of LAB, namely <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i>, <i>Lactiplantibacillus plantarum</i>, <i>Streptococcus thermophilus</i>, <i>Lactobacillus helveticus</i>, and <i>Lactococcus lactis</i> subsp. <i>lactis</i>, the <i>pheS</i> gene resolved species identities better and was thus selected to design specific primers and probes. The <i>pheS</i> 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 <i>R</i> ² > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (<i>P</i> < 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.