Washing Protocol and Challenge Test for Listeria monocytogenes in Ready-to-Eat Salads.

Listeria monocytogenes is a food-borne pathogen able to cause severe diseases with high mortality. L. monocytegenes is commonly associated with ready-to-eat food products. Ready-to-eat salads are consumed worldwide, and their consumption continues to increase. Because they are minimally processed, there is a high risk of microbial contamination during post-harvest handling, storage and transportation. There is a limited data available on whether industrial washing or sanitation procedures are sufficient to ensure long-term control or reduction of L. monocytogenes in RTE salads during refrigerated storage. The aim of this study was to evaluate the effectiveness of a new washing protocol for RTE salads in the reduction of L. monocytogenes. To achieve this result a challenge test was carried out on three batches for each product type (“Iceberg lettuce” and “baby leaf”), with 21 units in each batch (100 g each), using L. monocytogenes as the target microorganism. Experimental contamination of L. monocytegenes was performed using two Laboratory collection strains and one field strain isolated from raw salad. The inoculum was prepared according to the EURL 2021 guidelines, with an initial bacterial load of 3 log₁₀ CFU/g for the first two batches and 1.5 log₁₀ CFU/g for the third batch. The combined washing treatment was performed using water for 2 minutes, followed by peracetic acid and lactic acid, each applied separately for 4 minutes. For Iceberg lettuce, the combined washing process proved effective, achieving a reduction of approximately 2-2.5 log₁₀ CFU/g of L. monocytogenes, with residual levels below the limit of detection (10 CFU/g) in batches with low inoculum levels. For baby-leaf lettuce, the combined washing process was also effective, resulting in a reduction of approximately 1.9-2.5 log₁₀ CFU/g, with non-detectable levels in batches with low contamination. Samples were packaged according to company procedures (ATM for Iceberg lettuce, Air for baby-leaf), stored at 7°C, and analyzed at 9 and 12 days to monitor post-wash L. monocytogenes behavior. Although washing substantially reduced L. monocytogenes to low or undetectable levels, the pathogen was not completely inactivated, and regrowth was observed during storage at 7°C in both Iceberg and baby-leaf lettuce. These findings demonstrated that washing procedure alone is not sufficient for RTE salads, as surviving L. monocytogenes may regrow during refrigerated storage at 7°C, more effective intervention strategies should be implemented to control its growth in RTE salads.