Polyphenol oxidase (PPO) causes the browning reaction in fruits and vegetables and deteriorates the quality. Thermal treatment for enzyme inactivation may result in defects as opposed to high pressure CO₂ (HPCD) processing. In this study, the changes in activity, dissociation, aggregation and conformation of purified PPO from thermal and HPCD treated juice were investigated. HPCD exhibited inactivation of PPO at 55–65 °C whereas thermal processing alone at the same temperature resulted in PPO still showing activity. Under thermal treatment at 25 and 65 °C, the browning degree was higher (0.39 and 0.24) than for HPCD-treated juice (0.23 and 0.12). Fluorescence and circular dichroism spectral results indicated that HPCD induced large decreases in intensities, revealing a rearrangement of the secondary structure and destruction of the native configuration of the PPO molecule. The particle size distribution (PSD) pattern revealed structural modification leading to initial dissociation and subsequent aggregation of PPO after HPCD treatment. Polyacrylamide gel electrophoresis (PAGE) analysis exhibited that molecular size of protein was 40 kDa. In conclusion, the HPCD method was found to be more effective than thermal treatment to inactivate PPO. Structural modifications provided better insights into the phenomena of activation and inactivation of PPO.

In this study, the changes in enzyme activities, total polyphenols, phenolic profile, and physicochemical properties from thermally (25–75 °C) and high-pressure carbon dioxide (HP-CO₂) (25–65 °C/20 MPa)-treated apple juice were investigated. The HP-CO₂ exhibited complete inactivation of polyphenol oxidase (PPO) at 65 °C, whereas PPO was still active at 75 °C under thermal processing (TP). Similarly, the relative activity of peroxidase (POD) significantly decreased by 71% at 65 °C under HP-CO₂ processing, whereas TP was less effective. HP-CO₂ and TP treatments at 65 °C reduced the browning degree (BD) value to 0.47 and 0.89, respectively. Thus, HP-CO₂ inhibits the browning reactions caused by PPO and POD enzymes at each operating temperature. The concentration of epicatechin and catechin increased significantly with increasing temperature above 45 °C in TP-treated juices. HP-CO₂ treatment increased the same phenolic compounds at 35 °C and 9 MPa, whereas high-temperature and -pressure conditions caused insignificant changes in concentration of epicatechin and catechin. Changes in others phenolic compounds were insignificant under TP and HP-CO₂ treatment. Overall, HP-CO₂ is a promising technology to get high-quality juices with lower enzyme activity.