Enhancing Spore Inactivation: Low-Intensity Pulsed Electric Field Combined with Ohmic Heating and Germinant Pretreatment

Bacterial spores, as one of the most resistant microbial forms, are difficult to completely eliminate through conventional heat treatments such as pasteurization, allowing them to persist in food and pose a significant threat to microbial safety. This study employed a "germination-inactivation" strategy to inactivate <i>Alicyclobacillus acidoterrestris</i> (AAT) spores using a germinant under low-intensity pulsed electric fields (PEFs). Analysis of germination curves identified 40 mM L-valine as the most effective germinant. Results showed that after 4-h incubation with 40 mM L-valine followed by 210 s of 0.18 kV/cm PEF treatment, the synergistic effect of electric field and ohmic heating (OH) reduced AAT spore counts by 1.73 log units. In contrast, the control group treated with the same PEF parameters without a germinant showed only a 0.54 log unit reduction. These findings indicate that germination agents significantly reduce spore resistance. Subsequent experiments confirmed that L-valine-treated AAT spores underwent pronounced structural disruption under the combined effects of the electric field and OH, leading to leakage of intracellular components such as nucleic acids and proteins. This phenomenon was verified via scanning electron microscopy (SEM) and laser confocal microscopy. Additionally, both ROS levels and ATPase activity in spores were substantially reduced, further indicating that the combined electric field and OH synergistically disrupted the spore's external structure and internal macromolecules, leading to spore death. Thus, low-intensity PEF assisted by spore germination agents offers an energy-efficient and effective inactivation method, opening new avenues for spore inactivation research.