Assessment of spoilage microbial communities in modified atmosphere-packed ready-to-eat salad during cold storage: A comparative study using MALDI-TOF MS identification and PacBio full-length 16S rRNA and ITS sequencing

Abstract

Effect of storage temperatures and modified atmosphere packaging (MAP) on the shelf-life and spoilage microbial dynamics of industrially produced mayonnaise-based potato salad was studied over two years (Spring 2021, Autumn 2021, Spring 2022). Three salad batches, stored at chilling and abuse temperatures ranging from 0 °C to 12 °C, were analyzed. Microbial communities were evaluated using ISO-standard microbiological methods, MALDI-TOF MS identification, and PacBio full-length 16S rRNA gene and fungal ITS amplicon sequencing. The results showed that sensory and microbiological quality strongly depended on storage temperature, MAP packaging, and salad batch. The recommended upper temperature limit of 6 °C did not preserve salad quality at the end of its shelf-life, while storage below 4 °C extended the seven-day shelf-life to 10 days. Distinct spoilage communities were identified across batches, dominated by Dellaglioa algida, Leuconostoc carnosum, and Latilactobacillus sakei (Spring 2021); Leuconostoc inhae and Leuconostoc gelidum (Autumn 2021); and Lactococcus carnosus (Spring 2022). The microbial communities of ingredients and processing facilities influenced salad microbiota. Comparative analysis showed that full-length 16S rRNA sequencing provided higher resolution, detecting species missed by culturing methods, including psychrotrophic and fastidious lactic acid bacteria. However, fungal community analysis, using full-length ITS amplicons, requires the selection of specific fungal primers. This study provides a comprehensive evaluation of microbial profiles in RTE salads under different storage conditions, highlighting the critical role of temperature and microbial management in preserving MAP salad safety and quality during extended shelf-life.