High thermal resistance and inactivation behaviour of Clostridioides difficile spores in milk

The presence of Clostridioides difficile spores in milk and dairy products may pose a potential food safety concern due to their high resistance to physical and chemical treatments. In this study, the effect of relatively mild heat treatments, including pasteurization-relevant conditions, on the inactivation of C. difficile spores in milk was evaluated. The thermal inactivation kinetics of C. difficile spores, including strains and milk-derived isolates, were investigated over the temperature range of 65-85 °C using log-linear, Weibull, and biphasic models. Spore inactivation increased with temperature, reaching ~ 7.0 log reduction at 85 °C but remaining below 2.2 log at ≤ 77.5 °C after 60 min. D-values decreased from 33.44 to 50.04 min at 75 °C to 9.55-10.85 min at 85 °C, with z-values ranging from 13.96 to 18.38 °C. Although temperature strongly affected inactivation rates, secondary modelling indicated that the observed non-linear behaviour could not be explained by temperature alone. At lower temperatures, all models provided similar fits, whereas at higher temperatures the Weibull and especially the biphasic model better described the non-linear survival patterns, indicating heterogeneity in heat resistance within the spore population. The results demonstrated that conventional pasteurization conditions were insufficient for complete elimination of C. difficile spores in milk. Overall, this study provides important data on the thermal resistance of C. difficile spores in a dairy matrix and may support the optimization of heat treatment strategies to improve microbiological safety in milk and dairy products.