High-pressure inactivation of strawberry beverage-derived Bacillus spores in acidic environment: Impact of antimicrobial agents, temperature, pressure level and internal structure

This study investigated the efficacy of high hydrostatic pressure (HHP) for inactivating highly pressure-resistant Bacillus spores ( B. licheniformis , B. subtilis , and B. cereus ) isolated from an industrial strawberry beverage (pH 3.43 ± 0.01, 22.15 ± 0.20 °Bx). While HHP (500 MPa) combined with antimicrobials selectively enhanced B. subtilis spore inactivation, it was ineffective against B. licheniformis and B. cereus . Comparatively, HHP combined with moderate temperatures (25–50 °C) significantly inactivated spores of all three species. Mechanistic studies on B. subtilis revealed that elevated HHP temperatures caused progressive damage to the spore inner membrane (IM) and disruption of the multilayered coat structure, which led to spore inactivation. However, both transmission electron microscopy (TEM) and SYTO 16 staining demonstrated the limited cortex and/or SASPs degradation, indicating restricted HHP-induced germination under acidity. Moreover, over 400 MPa were crucial for IM damage and inactivation at 25 °C. In addition, spore coat defects and lower sporulation temperatures significantly increased HHP sensitivity. Our results demonstrated that HHP combined with moderate temperature effectively inactivated diverse Bacillus spores under acidity by primarily damaging the IM and coat structure, independent of significant germination. This study offered a promising non-thermal strategy for enhancing microbial safety without compromising quality. • Spores of Bacillus licheniformis , B. subtilis , and B. cereus were identified as pressure-resistant targets in an industrial strawberry beverage. • 500 MPa + nisin/DMDC effectively inactivated B. subtilis spores, but was ineffective against spores of B. licheniformis and B. cereus under pH 3.43. • HHP combined with 50°C effectively inactivated pressure-resistant Bacillus spores under acidity likely by directly damaging spore IM. • Spore coat defects and lower sporulation temperatures significantly increased HHP sensitivity of Bacillus spores under pH 3.43.