Use of qPCR for the analysis of population heterogeneity and dynamics during Lactobacillus delbrueckii spp. bulgaricus batch culture

Abstract

Direct molecular methods such as real-time polymerase chain reaction (qPCR) and propidium monoazide (PMA)-qPCR have been successfully used for quantifying viable microorganisms in the food industry. This study attempted to use qPCR and PMA-qPCR for quantifying Lactobacillus delbrueckii spp. bulgaricus sp1.1 physiological states. The qPCR standards of the 16S rRNA gene were employed to calibrate the qPCR assay, which contributed to an amplification efficiency of 98.42%. The number of copies of the 16S rRNA gene was linearly related to cell density, and this linear relationship was used to construct a quantitative curve (R2=0.9981) with a detection limit of 15.1 colony-forming units mL−1·reaction−1. qPCR in combination with an optimal PMA concentration (60 μM) helped in discriminating and quantifying the viable cells, without any interference by heat-killed cells. Compared with the conventional methods, the population heterogeneity of viable, culturable, dormant-like and membrane-permeabilized cells were well identified and quantified using qPCR during L. delbrueckii spp. bulgaricus sp1.1 batch culture. Despite the restriction in the enumeration of lysed cells, qPCR-based methods facilitated reliable identification and quantification of bacterial physiological states and provided additional knowledge on the dynamics of L. delbrueckii spp. bulgaricus sp1.1 physiological states.

Keywords

• Lactobacillus delbrueckii spp. bulgaricus
• batch culture
• PMA-qPCR
• cell viability
• physiological heterogeneity