Mechanism of sublethal injury induced by refrigerated thawing on Escherichia coli O157:H7 in suspension and carrot.

Escherichia coli O157:H7 can survive freezing in a sublethally injured state, and its survival behavior may vary depending on thawing conditions; therefore, it is important to use appropriate thawing methods to ensure food safety. Refrigerated thawing, although recommended as safe, may pose microbiological risks in terms of formation and recovery of sublethally injured cells. To evaluate the risks, E. coli O157:H7 was frozen at -20°C for 24 h and subsequently thawed under different conditions (bacterial suspension: 4°C for 180 min, 25°C for 60 min, and 37°C for 30 min; carrots: 4°C for 16 h, 25°C for 2 h, and 37°C for 30 min). Sublethal injury and recovery ratios, cell membrane integrity, cell membrane potential, leakage of intracellular materials, and reactive oxygen species levels were ascertained, and confocal laser scanning microscopy analysis (CLSM) was performed. The ratio of sublethal injury was high when thawed at 4°C, reaching 99.44 ± 0.05% and 86.24 ± 6.37% with the bacterial suspension and carrot, respectively. Such injury was associated with increased membrane integrity, membrane depolarization, leakage of intracellular materials, and structural damage as confirmed by CLSM. Furthermore, sublethally injured cells recovered within 3 h; cell viability in carrot was confirmed immediately after freezing-thawing (FT) and even after microwave heating, suggesting that the risks associated with sublethally injured cells after FT may persist during subsequent handling. These results indicate that refrigerated thawing does not ensure microbiological safety and highlight the need to reassess thawing practices, taking into account sublethal injury in frozen foods.