Effects of Combined High Hydrostatic Pressure and Dense Phase Carbon Dioxide on the Activity,Structure and Size of Polyphenoloxidase

High hydrostatic pressure (HHP) may activate undesirable enzymes such as polyphenoloxidase (PPO). Carbon dioxide (CO₂) addition to HHP could increase enzyme inactivation. We investigated the inactivation of combined HHP and dense phase carbon dioxide process on activity, secondary conformation and size of pure PPO from mushroom. Solutions (2.35μM, in phosphate buffer pH 6.8) were treated with HHP alone (HHP), or 3.6% w/w of CO₂ was injected into the package (HHP+CO₂). Treatment conditions were 600 MPa, 20 °C, for 1, 3, 5, 7, and 9 min. HHP+CO₂ treatment significantly decreased residual enzyme activity (REA) to 30% to 12% after 1 to 9 min, respectively, whereas only HHP had no significant effect. Both HHP and HHP+CO₂ treatments caused changes in secondary conformations, however HHP+CO₂ changes were more extensive. Alpha‐helix fractions were reduced by 32% and 41%, while β sheet, turn and unordered increased by 63% and 213%, 100% and 71%, and 118% and 82% for HHP and HHP+CO₂, respectively after 9 min. The protein size in HHP+CO₂ samples was 5‐ to 6‐fold larger than that of Control and HHP treatment, and this increase was inversely correlated with REA. The best inactivation kinetics of HHP+CO₂ model was the 2‐fractional model with 2 simultaneous 1st‐order steps, contributing 70% and 30% to original enzyme activity, with k_labile = 12.15 min^?1 and k_stable = 0.07 min^?1, respectively. No recovery in activity, secondary conformation and size in all samples were observed after 1‐mo storage. Addition of CO₂ in HHP treatment can improve enzyme inactivation, and therefore product shelf‐life and quality.