Rapid quantification of viable probiotic Lactobacillus species in Indian fermented dairy products using propidium monoazide-real time PCR assay.

Abstract

Accurate identification and quantification of probiotic species are essential for ensuring regulatory compliance, verifying product authenticity, and assessing efficacy. Therefore, this study aimed to develop a rapid assay for quantifying viable Lactobacillus spp. in fermented probiotic milk products using propidium monoazide (PMA) combined with quantitative PCR (qPCR). The PMA-qPCR assay was initially optimized in buffer system using Response Surface Methodology (RSM), with standard curves generated from recombinant plasmid DNA (2-10 log copies), showing high linearity (R2 > 0.99). The optimized PMA-qPCR method was found reliable (R2 values of >0.99) for quantification and effectively quantified viable Lactobacillus spp. in buffer system, exhibiting a strong positive correlation (r = 0.999) between the mean copy number of probiotic bacterial DNA and mean viable probiotic count. Subsequently, the assay was further validated in dairy matrices, such as milk, fermented milk, and Dahi, demonstrating good correlation with plate counts (r = 0.876-0.998). The method achieved over 86% accuracy and 2-3% precision in detecting two Lactobacillus strains. These findings indicate that developed PMA-qPCR assay offers high precision, specificity, and significantly faster than traditional culture-based methods, offering dairy stakeholders with an efficient alternative for quantifying viable probiotic Lactobacillus in milk matrices.