Inactivation kinetics of polyphenol oxidase (PPO) by a two-stage method which was additionally pressurized at ambient temperature after carbon dioxide microbubbles (MB-CO2) was mixed with a solution at low temperature and pressure (two-stage MB-CO2) was analyzed. The PPO inactivation efficiency of the two-stage MB-CO2 treatment was higher than that of heat treatment. A decrease in the decimal reduction time (D value) and activation energy during PPO inactivation using the two-stage MB-CO2 treatment was observed as the temperature of the heating coil and the pressure in the mixing vessel increased. The temperature rise required for a 90% reduction in the D value was increased. Furthermore, the pressure rise required for a 90% reduction in the D value and activation volume increased with the temperature of the heating coil. These results showed that PPO inactivation using the two-stage MB-CO2 treatment could be achieved with less energy than heat treatment. (C) 2012 Elsevier Ltd. All rights reserved.