Application of culture-independent methods for monitoring Listeria monocytogenes inactivation on food products

When new food processing technologies are investigated as alternative to traditional thermal pasteurization processes, conventional cultivation-based methods are usually applied to evaluate microbial concentration before and after the treatment to determine the process efficiency. However, these standard methods lead to a typical underestimation of the microbes present in the sample, which may represent an issue when pathogenic strains have to be detected. Here, the efficiency of SC-CO₂ pasteurization treatment in the inactivation of Listeria monocytogenes spiked on cured ham skin surface was evaluated using plate counts, flow cytometry (FCM) coupled with SYBR-Green I (SYBR-I) and propidium iodide (PI), and propidium monoazide quantitative PCR (PMA-qPCR), at different process conditions. SC-CO₂ best performed at 12 MPa, 45 and 50 °C, resulting in a 7.5 log reduction of cultivable cells quantified by plate counts after 15 min of treatment, while FCM and PMA-qPCR revealed a 4 log and 2 log reduction of intact cells, respectively. This striking difference between culture-based and culture-independent quantification methods was independent from treatment time and indicated that a large fraction of the cells lost cultivability after treatment but maintained an intact membrane, likely entering in a so-called Viable But Not Culturable (VBNC) state. Our study highlights the usefulness of FCM and PMA-qPCR to assess the viability status of microbial populations and support their application in microbiological quality control in the food industry, in particular when mild pasteurization technologies are used.