Detection of Glycolytically Active Lacticaseibacillus paracasei Strain Shirota by Flow Cytometry Targeting the Efflux Activity of Fluorescent Dye: a Potential Tool for Quality Assessment of Probiotic Cells in Milk Products

To our knowledge, this is the first report to demonstrate that CF efflux requires uncompromised glycolytic activity in certain lactic acid bacteria. Compared with the cell properties currently widely used for cell viability assessment, such as intracellular esterase activity and membrane integrity, CF-efflux activity enables the accurate detection of culturable cells, especially in products stored for long periods at cold temperatures. The rapid and accurate detection of viable probiotic cells in dairy products is important for assessing product quality in manufacturing. Flow cytometry is widely used for the rapid analysis of bacterial cells. However, further investigation is needed into the optimum property to use it for assessing cell viability. Here, we proposed using the efflux activity of a fluorescent dye, carboxyfluorescein (CF), as an indicator of cell viability. CF is generated from 5(6)-carboxyfluorescein diacetate as a result of cleavage by intracellular esterase. It generally accumulates in the cell, but certain bacterial species are known to extrude it. We found here that the probiotic strain Lacticaseibacillus paracasei strain Shirota (LcS) also extruded CF in the presence of energy sources, such as glucose. To investigate the mechanism of its CF-efflux activity, we screened CF-efflux-negative mutants from a random mutagenesis LcS library and examined the whole genome for genes responsible for CF efflux. We identified a base substitution in the pfkA gene in the glycolytic pathway, and we demonstrated that intact pfkA was essential for CF efflux, indicating that CF-efflux-positive cells must have uncompromised glycolytic activity. We also confirmed that there was a good correlation between the rate of CF-efflux-positive cells and that of colony-forming cells of LcS in a fermented milk product, whereas other properties, such as esterase activity and cell membrane integrity, lost their correlation with the colony-forming activity after long storage. We propose that CF-efflux activity could be an appropriate indicator of cell viability in certain probiotic strains.IMPORTANCE To our knowledge, this is the first report to demonstrate that CF efflux requires uncompromised glycolytic activity in certain lactic acid bacteria. Compared with the cell properties currently widely used for cell viability assessment, such as intracellular esterase activity and membrane integrity, CF-efflux activity enables the accurate detection of culturable cells, especially in products stored for long periods at cold temperatures. These results indicate strongly that CF-efflux activity can be an adequate cell-viability indicator and that flow cytometric quantification could be an alternative to conventional CFU counting. Our findings should be especially informative for dairy/probiotic product manufacturing.