Inactivation of Bacillus cereus spores by isochoric freezing

Spores of psychrotrophic Bacillus cereus pose a challenge to chilled food preservation due to their resilience against chemical and physical treatments, both thermal and non-thermal. Isochoric freezing (IF) emerges as a promising alternative, preserving food at subzero temperatures without the detrimental effects of ice crystal formation while simultaneously inactivating microorganisms. However, its effectiveness against highly resistant bacterial spores, particularly those of B. cereus, remained unclear. This study compared the inactivation efficiency of IF to traditional isobaric freezing against both vegetative cells and spores of B. cereus under various conditions. Our results demonstrated complete inactivation (an impressive 8.6 log reduction) of B. cereus spores after just 4 days of cold storage at -15 degrees C and 150 MPa using IF treatment. Interestingly, the chosen encapsulation method significantly impacted the inactivation rate. Heat-sealed samples achieved a substantially higher log reduction compared to filter-sealed samples, as confirmed by statistically significant differences (p < 0.05). Scanning electron microscopy (SEM) revealed striking changes in the morphology of filter-sealed spores treated with IF, showing their exosporium adhering together with blurred edges. Transmission electron microscopy (TEM) provided even deeper insights, showcasing structurally incomplete spores with less dense and homogeneous inner cores compared to untreated samples. While the precise mechanism behind this inactivation remains under investigation, one possible hypothesis suggests that the inactivation might involve spore germination during storage. These findings underscore the potential of IF as a promising alternative for sterilizing spores in frozen foods, offering a gentler approach compared to conventional methods.