Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR

Abstract

This study describes a procedure that allows specific detection and enumeration of viable bacteria in four species of lactic acid bacteria ( Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei subsp. casei and Lactobacillus acidophilus) and of Bifidobacterium lactis, mixed in fermented milk products. The procedure is based on the combined use of propidium monoazide (PMA), able to distinguish between viable and irreversibly damaged cells, with species-specific quantitative real-time PCR (RTi-PCR). Loss of viability of the species in a fermented milk through storage at 4 °C was similarly ( P < 0.05) detected by PMA–RTi-PCR and selective plate counts. Furthermore, comparison of results obtained by both methods showed a Pearson linear correlation of 0.995. The enumeration of viable bacteria by PMA–RTi-PCR could be performed in 3 h, whereas enumeration by selective plate counts required three days. The procedure developed is a fast method for the identification, enumeration and discrimination of viability of lactic acid bacteria and bifidobacteria mixed in fermented milk products.