C. Weemaes

Effects of high pressure on enzymes related to food quality

An important issue in the application of high pressure technology in the food preservation/processing industry is regulatory approval, which focuses upon microbiological and toxicological safety of food products. High pressure preservation processes should reduce the microbial load to the same level achieved by traditional technologies, while delivering higher-quality products. Combination of high pressure with other treatments (e.g., mild temperature elevation, refrigerated storage, acidification) during processing and storage is a likely route the food industry will take, as the inactivation of bacterial spores and some pressure-resistant enzymes at room temperature cannot be achieved by pressure alone. Several authors suggest that the most safe and economically feasible use of high pressure is in combination with moderately elevated temperature (Gould & Sale, 1970; Knorr, 1993). From the point of view of those who define hazard analysis of critical control points (HACCP) guidelines (risk analysis method carried out to guarantee safety and quality of food products throughout the production chain) or “novel food” regulations (directives that have to be taken into account when introducing new products, new ingredients, or new processing techniques on the market), and also from an engineering point of view, methods to determine the impact of pressure processes on food safety and quality are indispensable. A variety of methods could be evaluated for this purpose: (1) the in situ method, in which the change of an intrinsic food component is measured before and after treatment; (2) the physical mathematical method, in which the actual temperature-pressure-time profile is combined with the kinetics of the attribute under consideration; or (3) the use of process history indicators, which are devices that mimic the change of the attribute under consideration when submitted to the same temperature-pressure-time profile. All these methods rely largely on kinetic models. In this context, quantitative kinetic studies on the combined effect of pressure and temperature on several aspects of food safety and quality (pathogenic/spoilage microorganisms, enzymes related to food quality, nutritional and sensorial quality characteristics) are required. From an engineering point of view, such kinetic data are very important for evaluation of process uniformity, for process validation, and for process optimization.

Kinetics of combined pressure-temperature inactivation of avocado polyphenoloxidase

Irreversible combined pressure-temperature inactivation of the food quality related enzyme polyphenoloxidase was investigated. Inactivation rate constants (k) were obtained for about one hundred combinations of constant pressure (0.1-900 MPa) and temperature (25-77.5 degrees C). According to the Eyring and Arrhenius equation, activation volumes and activation energies, respectively, representing pressure and temperature dependence of the inactivation rate constant, were calculated for all temperatures and pressures studied. In this way, temperature and pressure dependence of activation volume and activation energy, respectively, could be considered. Moreover, for the first time, a mathematical model describing the inactivation rate constant of a food quality-related enzyme as a function of pressure and temperature is formulated. Such pressure-temperature inactivation models for food quality-related aspects (e.g., the spoilage enzyme polyphenoloxidase) form the engineering basis for design, evaluation, and optimization of new preservation processes based on the combined effect of temperature and pressure. Furthermore, the generated methodology can be used to develop analogous kinetic models for microbiological aspects, which are needed from a safety and legislative point of view, and other quality aspects, e.g., nutritional factors, with a view of optimal quality and consumer acceptance.

Kinetic analysis and modelling of combined high-pressure-temperature inactivation of the yeast Zygosaccharomyces bailii.

Eight foodborne yeasts were screened for sensitivity to high-pressure (HP) inactivation under a limited number of pressure-temperature combinations. The most resistant strains were Zygoascus hellenicus and Zygosaccharomyces bailii. The latter was taken for a detailed study of inactivation kinetics over a wide range of pressures (120-320 MPa) and temperatures (-5 to 45 degrees C). Isobaric and isothermal inactivation experiments were conducted in Tris-HCl buffer pH 6.5 for 48 different combinations of pressure and temperature. Inactivation was biphasic, with a first phase encompassing four to six decades and being described by first-order kinetics, followed by a tailing phase. Decimal reduction times (D) were calculated for the first-order inactivation phase and their temperature and pressure dependence was described. At constant temperature, D decreased with increasing pressure as expected. At constant pressure, D showed a maximum at around 20 degrees C, and decreased both at lower and at higher temperatures. A mathematical expression was developed to describe accurately the inactivation of Z. bailii as a function of pressure and temperature under the experimental conditions employed. A limited number of experiments in buffer at low pH (3-6) suggest that the model is, in principle, applicable at low pH. In apple and orange juice however, higher inactivation than predicted by the model was achieved.

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 °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.

Kinetics for Isobaric−Isothermal Inactivation of Bacillus subtilis α-Amylase

Isobaric−isothermal inactivation of Bacillus subtilis α‐amylase (BSA, 15 mg/mL in Tris‐HCl at pH 8.6) in the pressure range 1–750 MPa and the temperature range 25–85 °C could be accurately described by a first‐order kinetic model. The kinetic parameters (k, Ea, and Va) were calculated at different pressure and temperature levels. At reference temperature (40 °C) and reference pressure (500 MPa), isobaric−isothermal inactivation was characterized by an Ea value of 74.8 kJ/mol, a Va value of −23.6 cm3/mol, and an inactivation rate constant of 0.0343 min−1. The influence of 15% glycerol on thermal and pressure−temperature stability of BSA was investigated. In both cases, a stabilizing effect of this additive was found, since the kref value was significantly reduced. Furthermore, a pressure−temperature kinetic diagram, indicating the possible synergistic and antagonistic effects of pressure and temperature on the inactivation of BSA, was constructed. Based on this diagram, a model describing the dependence of the inactivation rate constant on pressure and temperature, in the pressure range 250–750 MPa, was formulated.

Thermal and combined pressure--temperature inactivation of orange pectinesterase: influence of pH and additives.

Inactivation of commercially available orange pectinesterase (PE) was investigated under isothermal and isothermal-isobaric conditions. In both cases, inactivation data could be accurately described by a fractional conversion model. The influence of enzyme concentration, pH, Ca(2+) concentration, and sucrose on the inactivation kinetics was studied. Enzyme stability against heat and pressure increased by increasing enzyme concentration. An increased Ca(2+) concentration caused sensitization to temperature and increased the residual fraction active PE after thermal treatment. To the contrary, in the case of pressure treatment, decreasing Ca(2+) concentrations increased pressure inactivation. The remaining fraction active PE after pressure treatment was not influenced by the addition of Ca(2+) ions. Acidification accelerated thermal as well as pressure-temperature inactivation, whereas in the presence of sucrose an increased temperature and pressure stability of orange PE was observed. Sucrose had no influence on the remaining activity after thermal treatment, but it increased the residual fraction after pressure treatment. The remaining fraction was for all additives studied independent of the pressure and temperature level applied except for the inactivation in an acid medium, when a decrease of the residual fraction was observed with increasing temperature and pressure.

Kinetic Parameters for Pressure−Temperature Inactivation of Bacillus subtilis α‐Amylase under Dynamic Conditions

Pressure−temperature inactivation kinetics of two enzymatic model systems based on Bacillus subtilis α‐amylase (BSA), previously determined under isobaric−isothermal conditions, were studied under more realistic, i.e. dynamic, process conditions. The proposed kinetic model was found to accurately describe non‐isobaric/non‐isothermal inactivation of BSA in absence as well as in presence of 15% glycerol. Furthermore, the effect of pressure cycling on the inactivation of BSA (in absence of glycerol) was investigated. Multiple application of pressure seemed to exert a more pronounced inactivation effect as compared to a single‐cycle process of the same total time. This additional effect, however, could be completely attributed to the more extensive temperature variation occurring during pressure cycling.

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.

>Kinetics for heat and pressure‐temperature inactivation of bacillus subtilis α‐amylase

Abstract The kinetic parameters for thermal inactivation of Bacillus subtilis α‐amylase (15 mg/ml in Tris HCI buffer at pH 8.6) were determined from isothermal experiments using a two step linear regression method. At ambient pressure, the inactivation of Bacillus subtilis α‐amylase could be described by a first order kinetic model. The activation energy (Ea) was 266 kJ/mole and the rate constant (kref) at reference temperature (50°C) 6.3∗10−5 min−1. Subsequently the inactivation due to combined pressure and temperature was investigated in a pressure range of 0 to 550 MPa and a temperature range of 40 to 80°C. The kinetic parameters for pressure‐temperature inactivation of BSA were estimated applying a non‐linear regression method on a first order kinetic model. Ea‐values were found to decrease and kref‐values to increase with increasing pressure. Furthermore, the influence of glycerol on the thermal and pressure‐temperature stability of Bacillus subtilis α‐amylase was investigated. In both cases, glycero...

Thermal and pressure‐temperature denaturation kinetics of bacillus subtilis α‐amylase: A study based on gel electrophoresis

Abstract The commercially available enzyme Bacillus subtilis α‐amylase was characterized by a molecular weight of about 55 kDa and contained isozymes with pI values ranging in a narrow zone (4.6–5.3). Furthermore, the sample was confirmed to contain no disulfide bonds. Upon thermal denaturation in the presence of sodium dodecyl sulfate, a band at half molecular weight was noticed, indicating that the native enzyme might be a dimeric form. Thermal as well as pressure‐temperature denaturation kinetics were investigated using gel electrophoresis and both could accurately be described by a first order kinetic model. For thermal denaturation an activation energy of 283 kJ/mole was calculated. As far as pressure‐temperature denaturation is concerned, activation energy and activation volume at a constant pressure of 5500 bar and a constant temperature of 40°C were calculated respectively as 77.6 kJ/mole and ‐23.2 cm3/mole. These values were compared with those for thermal and pressure‐temperature inactivation of...