Influence of pH, benzoic acid, EDTA, and glutathione on the pressure and/or temperature inactivation kinetics of mushroom polyphenoloxidase.

The pressure and/or temperature inactivation of mushroom polyphenoloxidase (PPO) in the absence and presence of EDTA, benzoic acid, and glutathione was studied on a kinetic basis. In addition, the effect of pH was evaluated. The temperature stability of PPO at atmospheric pressure increased with increasing pH up to pH 6.5. The pressure stability of PPO at room temperature (25 degrees C) also increased with increasing pH. EDTA slightly increased the thermal stability of the enzyme but did not alter the pressure stability of PPO. Benzoic acid protected the enzyme toward temperature but caused sensitization toward pressure when used at a concentration of 50 mM. Glutathione produced sensitization to both temperature and pressure. It is suggested that the sensitizing effect of glutathione is due to an interaction with a disulfide bond of the enzyme.

High pressure, thermal, and combined pressure-temperature stabilities of alpha-amylases from Bacillus species.

Three different alpha-amylases from Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis, were mutually compared with respect to thermal stability, pressure stability, and combined pressure-temperature stability. Measurements of residual enzyme activity and residual denaturation enthalpy showed that the alpha-amylase from B. licheniformis has by far the highest thermostability and that the two other alpha-amylases have thermostabilities of the same order of magnitude. FTIR spectroscopy showed that changes in the conformation of the alpha-amylases from B. amyloliquefaciens, B. subtilis, and B. licheniformis due to pressure occurred at about 6.5, 7.5, and 11 kbar, respectively. It seemed that, for the enzymes studied, thermal stability was correlated with pressure stability. As to the resistance under combined heat and high pressure, the alpha-amylase from B. licheniformis was much more stable than the alpha-amylases from B. amyloliquefaciens and B. subtilis, the latter two being about equally stable. It appears that under high pressure and/or temperature, B. licheniformis alpha-amylase is the most resistant among the three enzymes studied.

Potential Bacillus subtilis alpha-amylase-based time-temperature integrators to evaluate pasteurization processes.

Thermal inactivation kinetics of Bacillus subtilis alpha-amylase (BSA) in different environmental conditions was studied by performing isothermal experiments. As a response property, residual enzymic activity and residual heat of enzyme deterioration were chosen. A comparison of processing values determined from the read-out of a system with actual integrated processing values revealed the potentials of these systems as time-temperature integrators to be used in the pasteurization domain (temperatures of 70 to 100 degrees C) for target attributes with z-values ranging from 6 to 12 degrees C.

Evaluation of the integrated time-temperature effect in thermal processing of foods.

In this review, current methods used to evaluate the integrated impact of time and temperature upon preserving a food product by a heat treatment are considered. After identifying the basic premise any preservation scheme shall meet, the central role of a feasible description for the heat activation kinetics of microorganisms, their spores, and other quality attributes are stressed. Common concepts to quantify a thermal process are presented. Shortcomings of the prevalent evaluation methods are highlighted and attention is given to the development, restrictions, and possibilities of time-temperature-integrators as "new" evaluation tools to measure the impact of a "classical" in-pack heat treatment and more modern heating techniques such as continuous processing of solid/liquid mixtures on foods.

Theoretical Consideration on the Influence of the z-Value of a Single Component Time/Temperature Integrator on Thermal Process Impact Evaluation.

The allowed difference in z-value between a single component time/temperature integrator (SCTTI) and target attribute to measure the impact of a thermal process with a given accuracy was examined theoretically. For isothermal heating profiles, the issue and the degree of over- or underestimation of the actual process-value can be predicted as a function of z-value difference and reference temperature. The closer the processing temperature approaches reference temperature, the larger the allowed difference in z-value. As target attributes are characterized by a higher z-value, the allowed z-value difference between SCTTI and target attribute increases. For non-isothermal heating profiles, over- or underestimation depends in addition on the temperature history of the product. In order to obtain a safe estimate of the impact value, whatever the shape of the time/temperature profile, a new approach is suggested based on a SCTTI with a z-value below the target z-value in combination with the use of a reference temperature above or equal to the maximum processing temperature.

DSC and protein-based time-temperature integrators: Case study of alpha-amylase stabilized by polyols and/or sugar.

Differential scanning calorimetry (DSC) was used as a tool for rapid assay of the thermostability of two Bacillus sp. alpha-amylases and horseradish peroxidase as a function of the concentration of glycerol, sorbitol, and sucrose. In this screening study, the DSC peak temperature proved to be a good measure of protein thermostability. By means of isothermal heating experiments, the kinetics of heat decay of B. amyloliquefaciens alpha-amylase were studied by following the course of the DSC peak area (heat exchange (DeltaH/wt)) as a function of time. The high stability of this enzyme in the presence of polyolic alcohols or carbohydrates allowed working at temperatures as high as 127 degrees C. The results of this study can have particular relevance with regard to research on and development of protein-based time-temperature integrators (TTls) for evaluating heat pasteurization or sterilization treatments of foods or pharmaceutical products. The use of the DSC peak area (DeltaH.wt) as TTI-response was validated in experiments with a time-variable temperature profile. Finally, it was shown how the results of such non-isothermal experiments can even be used for (re-) estimation of the protein decay kinetic parameters (k, E(A)). (c) 1994 John Wiley & Sons, Inc.

Convenience of immobilized Bacillus licheniformis alpha-amylase as time-temperature-integrator (TTI).

For the immobilization of Bacillus licheniformis alpha-amylase to porous glass beads, the performances of three possible linking agents, glutaric dialdehyde, benzoquinone and s-trichlorotriazine were assessed in respect of the protein yield, the enzymic activity and the thermostability of the immobilized enzyme. These three properties are to be evaluated in view of the possible use of the enzyme preparations as time-temperature-integrators (TTIs) for assessing the severity of heat pasteurization or sterilization processes of food or pharmaceuticals. All three linkers improved the enzyme's resistance to irreversible heat inactivation to a similar extent and in each case biphasic inactivation kinetics were observed, whereas the dissolved B. licheniformis alpha-amylase showed a simple first order decay. The immobilization yield, measured as protein per carrier weight, did not differ markedly for the three linkers, although the enzymic activity of the glutaric dialdehyde-linked enzyme was lower than that of the benzoquinone- and s-trichlorotriazine-linked preparations.

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase.

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis.

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction.