Polyphenol Oxidase (PPO) and Pectin Meth ylesterase (PME) inactivation by means of High Pressure Carbon Dioxide (HPCD)

Abstract

HPCD is a promising technology to inactivate the enzymes responsible for the juice spoilage, such as PPO and PME. In order to understand the mechanism that induces this inactivation, a study using two commercial enzymes (PPO from mushroom and PME from Aspergillus niger) was carried out. The effect of pressure, temperature, exposure time and ratio CO2/enzyme ratio loaded in the reactor were studied. The experimental results (residual activity) were fitted to a kinetic model that served to develop a complete kinetic study: the kinetic constants, activation volume and activation energy were calculated, as well as the pressure and temperature sensitivity parameters (ZP and ZT, respectively). The changes in the tertiary structure of the enzymes after different treatments were analyzed by fluorescence spectroscopy running different tests: intrinsic fluorescence measurement, KI quenching and ANS binding experiments. In the case of PPO, the experimental results revealed that this enzyme inactivation kinetics fitted the two fraction model, which indicates the presence of labile and stable isoenzymes. Exposure time (2 to 15 minutes), temperature (25 to 45°C) and pressure (50 to 200bar) were the studied experimental conditions that led to different physical states for the CO2 (gas, liquid and supercritical). Despite the different experimental combinations of pressure and temperature tried, a similar inactivation pattern was observed: a sudden decrease in activity (more than 75% of the total activity loss was observed within the first 2 minutes) was followed by a slowed decay. At constant temperature, higher inactivation rates were observed the higher the pressure, obtaining an almost complete inactivation at 200bar after 5 minutes regardless the temperature. When temperature was increased, much faster inactivation rates were observed. ZP and ZT were in the range 69-78bar and 27-40°C, respectively. In the case of the commercial PME, the use of supercritical CO2 (pressure 60-180bar, temperature 40-55°C and times up to 75 minutes) increased dramatically the PME inactivation rate, showing that pressure had a limited effect on PME inactivation but temperature had an important effect. The pressure and temperature sensitivity parameters (ZP and ZT) confirmed that trend, being in the range from 276 to 450bar and 8.7°C, respectively. The experimental data fitted the first order model and the inactivation kinetics study of PME was completed with the calculations of the activation energy and volume of activation. The ratio CO2/volume of enzyme (g/mL) loaded in the reactor was found to be critical for both enzymes. It was seen that ratios higher than 3 did not improve the inactivation kinetics, being a waste of CO2 from the economic point of view. Bellow that critical value, the inactivation of the enzyme strongly depended on pressure and temperature. In both cases the structure of the enzyme was dramatically affected after exposure to HPCD, as revealed by the fluorescence spectroscopy analysis that showed significant changes in the tertiary structure of the enzyme, which were compatible with the losses in activity observed.