Important factors that influence the survival of Listeria monocytogenes during the minimal thermal processing of freshly shredded cabbage.

摘要

Listeria monocytogenes is widespread in nature and is often isolated from the soil or decaying plant debris. Therefore, this pathogen is likely to be found on raw produce with relatively high frequency. Several key factors (attachment, growth and thermal resistance) important for the survival of Listeria spp. on minimally processed cabbage have been examined. Attachment of cells to cabbage tissues was rapid ( 5 min) for all 24 tested strains with some strains showing greater proficiencies to attach to both cut and intact cabbage surfaces. In all cases, cut tissues were preferred by all strains with an increase of 1.0 to 1.2 log in colony forming units (CFU) per cm2 over intact tissues. Scanning electron microscopy analysis confirmed the presence of these cells along cut edges and within crevices. Increasing the contact time resulted in the formation of cell clusters and microcolonies. Attached cells for 24 different Listeria strains were shown to survive at relatively high levels for up to 28 days on intact cabbage, albeit with a general decreasing trend in CFUs over time. In contrast, most strains showed moderate growth (1.0 to 1.5 log CFU/cm2 increase) on cut cabbage tissues stored at 5°C over the same time period. The application of a mild thermal treatment (50°C for 3 min) on shredded cabbage resulted in a significant increase (P 0.05) in CFUs for all tested strains after refrigerated storage at 5°C for 21 days. This increase exceeded 3.0 log CFU/g for two L. monocytogenes strains. Cellular changes resulting in heat resistant phenotypes were examined using a transposon library constructed in Lm568 (serotype 1/2a). Sixteen thermotolerant mutants were isolated with Tn917 insertions in a diverse array of genes. The seven mutants displaying the highest level of thermal resistance at 52°C were also exposed to higher temperatures (56°C and 60°C). Five of these mutants continued to be significantly more (P 0.05) thermal resistant than the wild type. The interrupted genes for the 16 mutants were determined to be involved in; transport, metabolism, replication and repair, general stress, and structural properties such as cell wall and membrane composition, and surface structures. An isogenic mutant with a 462 bp deletion in a putative treA gene was constructed and shown to be devoid of phosphotrehalase activity. The mutant showed subsequent intracellular accumulation of high levels (22.8 mug/mg cell protein) of trehalose-6-phophate (T6P) when grown in the presence of trehalose. A concomitant build up of trehalose (11.6 mug/mg protein) was also detected, presumably through the dephosphorylation of T6P. This mutant was also shown to be significantly (P 0.05) more resistant to osmotic stress and desiccation in comparison to Lm568. The work presented in this thesis will help to advance our understanding of how Listeria contaminate, survive and proliferate in a vegetable processing system implementing a mild thermal treatment. Moreover, these data may help elucidate the mechanisms involved in enhanced thermal resistance in L. monocytogenes. As such, this information may lead to new strategies for the implementation of remedial measures to reduce the risk of foodborne illness pertaining to fresh cut, minimally processed fruits and vegetables.