Abstract: Bacterial spores are formed by cells of Clostridium, Bacillus and related genera as a response to a lack of one or more nutrients. These spores are dormant and resistant to inactivation treatments, including heat, radiation, preservatives, and high-pressure. Particularly, spores can germinate and develop in food products, resulting in the food spoilage or instances of foodborne illness. Bacillus subtilis spores are inactivated in food at high temperatures exceeding 100 ℃ during the traditional food processing. Nevertheless, high temperatures can pose a significant impact on food quality. This study aims to mitigate this temperature effect for the lower sterilization temperature. Potassium sorbate was integrated with PATS (pressure assisted thermal sterilization) into inactivate Bacillus subtilis spores. The release of important intraspore substances was established by spectrophotometry testing the OD₆₀₀ (optical density at 600 nm) value, OD₂₆₀ (optical density at 260 nm) value, OD₂₈₀ (optical density at 280 nm) value, and DPA (2,6-pyridine dicarboxylic acid) levels. And the spore structure was examined via SEM (scanning electron microscopy), flow cytometry, FTIR (Fourier transform infrared spectroscopy), and PSD (particle size distribution analysis). The addition of potassium sorbate was improved the sporicidal effect of PATS. The surviving spores plate counting demonstrated that the initial viable spore concentration was approximately 1.5×10⁸ CFU/mL. Treatment with 200 MPa-75 ℃ inactivated 2.63 lg CFU/mLspores, and the combined treatment of 200 MPa-75 ℃ and 2 g/L potassium sorbate inactivated 3.24 lg CFU/mLspores. The untreated spore suspensions displayed OD₆₀₀, OD₂₆₀, and OD₂₈₀ values of 1, 0.053, and 0.046, respectively. Following a spore suspension treatment of 200 MPa-75 ℃-2 g/L potassium sorbate, the OD₆₀₀ value declined by 0.413, while the OD₂₆₀ and OD₂₈₀ values increased to 0.401 and 0.290, respectively. It was indicated that the addition of potassium sorbate was enhanced the liberation of protein and nucleic acid in spores, leading to the reduced turbidity of spore suspensions. After treatment with 200 MPa-75 ℃, the DPA release increased by 51.31%, compared with the untreated spore suspensions. Additionally, the DPA release rate increased to 75.28% after treatment with 200 MPa-75 ℃-2 g/L potassium sorbate. This result further confirmed that the combination of PATS with potassium sorbate treatment was enhanced the permeability of the spore inner membrane, which was positively correlated with the addition of potassium sorbate and temperature. SEM images demonstrated that the addition of potassium sorbate to PATS caused significant spore damage and destruction of the cell wall, cortex and inner membrane, compared with the treatment with PATS alone. Further analysis through flow cytometry and PSD revealed that the damage on the inner membrane was worsened, while particle diameter significantly decreased, and specific surface area increased considerably after combined treatment. The FTIR disclosed the peak shift and change in intensity. The spore inner membrane phospholipids were transformed from a gel state to a liquid crystal state after PATS and potassium sorbate treatment, indicating the reduced the stability, compared with the untreated spores and PATS treatment alone. Simultaneously, the Na⁺ /K⁺-ATPase activity registered a significant decrease, when PATS was used in the combination with potassium sorbate. This activity was used to disturb the balance of the inner membrane potential, as well as the fundamental physiological and metabolic functions. The findings reveal that there was the synergistic bactericidal impact on Bacillus subtilis spores at lower temperatures, compared with the conventional inactivated spores, when PATS was combined with potassium sorbate. The synergistic bactericidal effect was also augmented with the increase of temperature and potassium sorbate concentration. The primary mechanism for the inactivation of Bacillus subtilis spores was attributed to the damage of the permeability and structural integrity of inner membrane. These findings have practical applications to promote the food sterilization, in order to reduce the adverse impact of high temperatures on food quality.