Extended shelf-life (ESL) refrigerated ready-to-eat foods are thermally pasteurized to ensure food safety and stability. However, surviving psychrotrophic spores can still pose a challenge. Studies predicting their behavior often overlook sporulation conditions. This study investigated the effect of sporulation temperature on germination of three strains in specific nutrients (inosine and/or amino acids) with or without prior heat activation (80 °C, 10 min). Sporulation temperature variably affected germination, with stronger effects in moderately responsive strains and nutrients. Heat activation strongly stimulated germination, particularly in nutrients with poorer responses, mitigating differences induced by sporulation temperature. The influence of sporulation temperature on germination and growth in an ESL matrix at refrigeration temperatures (4 °C or 8 °C) in vacuum packaging after heat activation or commercial pasteurization (90 °C, 10 min) was also studied. The latter treatment increased germination rates of surviving spores; however, some strains suffered damage and lost viability upon germination at 4 °C but recovered and grew at 8 °C. These findings highlight the need to account for variability in spore recovery and outgrowth during quantitative risk assessments for psychrotrophic in ESL foods.
