Effect of pressure, temperature, time and storage on peroxidase and polyphenol oxidase from pineapple

Abstract

The individual and combined effect of pressure, temperature, time and post-pressurisation storage on pineapple polyphenol oxidase and peroxidase activity was evaluated in the study. The experiments were carried out according to a 27-basic treatments design using the following levels of the independent variables: temperature: 12, 40 and 60°C; pressure: 0, 100, 300, 500 and 600 MPa; time: 15, 30 and 45 min.: storage: 24 h storage at 5°C or absence of storage after pressure treatment previously to the activity determination. Enzymatic extract including both peroxidase and polyphenol oxidase was obtained by blending internal part of the fruit with tris buffer (pH=6.5). After being filtrated the extract to be pressurised was divided into small portions in polietilene bags, which were then termosealed. The samples were frozen and kept in a freezer at-180°C and defrosted just before the pressurisation. Polyphenol oxidase activity was determined through an espectofometric method by measuring the absorbance resulted from the residual enzyme activity on catechol, used as substrate. Peroxidase was similarly determined but using in that case hydrogen peroxide as substrate and p-phenylenediamine as acceptor. A second-degree polynomial model was adjusted for each dependent variable polyphenol oxidase activity and peroxidase activity. Due to the restrictions in the aleatorisation procedure, a mixed model was adjusted, i.e., aleatory effects were used for the factor “day in which the experiment was carried out” and fixed effects for the polynomial coefficients. The aleatory effect for the day of treatment relates to the fact that the level of temperature was adjusted and maintained all day long for a set of three treatments carried out in each day of experiments. The statistical analysis of polyphenol oxidase has shown that there was a negative significant effect of storage on enzyme activity, i.e., the activity was lower for stored samples. No interaction among the independent variables was detected. Operational time presented a slight negative linear effect i.e., activity dropped linearly with time. Temperature and time did not result in linear and quadratic significant effects on polyphenol oxidase activity. The maximum polyphenol oxidase activity was verified for a temperature of 30.8°C and pressure of 274 MPa. Change in temperature had a higher effect on polyphenol oxidase activity than change in pressure. In the case of peroxidase, the analysis revealed that both time and storage of enzymatic extract after pressurisation did not affect significantly the enzymatic activity. However, the effect of interaction between temperature and pressure on peroxidase activity was significant. In those terms, for low temperatures the enzyme activity was almost constant with pressure increasing, while for high temperatures the peroxidase activity dropped abruptly with the increase of pressure. Curvature resulting from the quadratic effect was only observed for temperature in the case of peroxidase. The maximum peroxidase activity was verified at the temperature of 36.8°C and absence of operational pressure (0 MPa) inside the pressure vessel. The lowest level of enzymatic activity was achieved at the highest level of quantitative variables for both enzymes. Maximum reduction of enzymatic activities obtained were 60.08% for peroxidase, and 33.17% for polyphenol oxidase, in comparison to the original levels obtained for the enzymatic extract without treatment.