Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

The significance of viable but non-culturable (VBNC) cells in the food industry is not well known, mainly because of the lack of suitable detection methodologies to distinguish them from dead cells. The study aimed at the selection of the method to differentiate dead and VBNC cells of Listeria monocytogenes in process wash water (PWW) from the fruit and vegetable industry. Different methodologies were examined including (i) flow cytometry, (ii) viability quantitative polymerase chain reaction (v-qPCR) using an improved version of the propidium monoazide (PMAxx) dye as DNA amplificatory inhibitor, and (iii) v-qPCR combining ethidium monoazide (EMA) and PMAxx. The results showed that the flow cytometry, although previously recommended, was not a suitable methodology to differentiate between dead and VBNC cells in PWW, probably because of the complex composition of the water, causing interferences and leading to an overestimation of the dead cells. Based on results obtained, the v-qPCR combined with EMA and PMAxx was the most suitable technique for the detection and quantification of VBNC cells in PWW. Concentrations of 10 μM EMA and 75 μM PMAxx incubated at 40°C for 40 min followed by a 15-min light exposure inhibited most of the qPCR amplification from dead cells. For the first time, this methodology was validated in an industrial processing line for shredded lettuce washed with chlorine (10 mg/L). The analysis of PWW samples allowed the differentiation of dead and VBNC cells. Therefore, this method can be considered as a rapid and reliable one recommended for the detection of VBNC cells in complex water matrixes such as those of the food industry. However, the complete discrimination of dead and VBNC cells was not achieved, which led to a slight overestimation of the percentage of VBNC cells in PWW, mostly, due to the complex composition of this type of water. More studies are needed to determine the significance of VBNC cells in case of potential cross-contamination of fresh produce during washing.