Inactivation and mechanism of Alicyclobacillus acidoterrestris by different modes of high hydrostatic pressure, including synergistic effects with essential oils

High hydrostatic pressure (HHP) is widely used to inactivate microorganisms in food, but achieving effective spore inactivation by conventional HHP remains challenging due to their resistance. The present study compared the effectiveness of continuous, intermittent and induced inactivation HHP techniques for inactivation of Alicyclobacillus acidoterrestris spores. Under optimal induced inactivation conditions (200 MPa/4 cycles, 600 MPa/2 cycles), reductions of 0.79 log CFU/mL for the highly resistant DSM3922 spores and 1.94 log CFU/mL for the less resistant DSM3924 spores were achieved. Mechanistic investigations revealed that: (Ⅰ) structural damage, including breakage, wrinkling, and concavity, was induced in spores post-HHP treatment; (Ⅱ) pulsation forces generated by repeated pressurization and depressurization during induced inactivation HHP reduced the D90 values of the spore aggregates to 99.37 μm for DSM3922 and 76.49 μm for DSM3924, enhancing the inactivation efficacy; (Ⅲ) the inner membrane (IM) and cortex were severely impaired, while nucleic acid and protein leakage from spores significantly increased after HHPs. To further enhance spore inactivation, Tea Tree, Nutmeg, Eucalyptus, and Litsea cubeba essential oils (EOs) were selected from twelve EOs as safe and effective antimicrobial substances that synergize with HHP. Combining 5% Litsea cubeba EO with HHP (200 MPa/4 cycles, 600 MPa/2 cycles) resulted in reductions of 2.57 log CFU/mL for DSM3922 and 5.08 log CFU/mL for DSM3924. These findings suggest that the combination of HHP and EOs could be a promising approach for inactivating A. acidoterrestris spores.