Inactivation of apple pectin methylesterase induced by dense phase carbon dioxide

The inactivation of apple pectin methylesterase (PME) with dense phase carbon dioxide (DPCD) combined with temperatures (35-55 degrees C) is investigated. DPCD increases the susceptibility of apple PME to the temperatures and the pressures have a noticeable effect on apple PME activity. A labile and stable fraction of apple PME is present and the inactivation kinetics of apple PME by DPCD is adequately described by a two-fraction model. The kinetic rate constants k L and k S of labile and stable fractions are 0.890 and 0.039 min (-1), and the decimal reduction times D L and D S are 2.59 and 58.70 min at 30 MPa and 55 degrees C. Z T representing temperature increase needed for a 90% reduction of the D value and the activation energy E a of the labile fraction at 30 MPa is 22.32 degrees C and 86.88 kJ /mol, its Z P representing pressure increase needed for a 90% reduction of the D value and the activation volume V a at 55 degrees C is 21.75 MPa and -288.38 cm (3)/mol. The residual activity of apple PME after DPCD exhibits no reduction or reactivation for 4 weeks at 4 degrees C.     

Effect of mild-heat and high-pressure processing on banana pectin methylesterase: a kinetic study

Pectin methylesterase (PME) was extracted from bananas and purified by affinity chromatography. The thermal-high-pressure inactivation (at moderate temperature, 30-76 degrees C, in combination with high pressure, 0.1-900 MPa) of PME was investigated in a model system at pH 7.0. Under these conditions, the stable fraction was not inactivated and isobaric-isothermal inactivation followed a fractional-conversion model. At lower pressure (< or =300-400 MPa) and higher temperature (> or =64 degrees C), an antagonistic effect of pressure and heat was observed. Third-degree polynomial models (derived from the thermodynamic model) were successfully used to describe the heat-pressure dependence of the inactivation rate constants.     

Structural changes of microbial transglutaminase during thermal and high-pressure treatment

The activity of microbial transglutaminase (MTG) and the corresponding secondary structure, measured by circular dichroism (CD), was analyzed before and after treatment at different temperatures (40 and 80 degrees C) and pressures (0.1, 200, 400, 600 MPa). Irreversible enzyme inactivation was achieved after 2 min at 80 degrees C and 0.1 MPa. Enzyme inactivation at 0.1, 200, 400, and 600 MPa and 40 degrees C followed first-order kinetics. The enzyme showed residual activity of 50% after 12 min at 600 MPa and 40 degrees C. Mobility of aromatic side chains of the enzyme molecule was observed in all temperature- and/or pressure-treated samples; however, high-pressure treatment at 600 MPa induced a loss of tertiary structure and a significant decrease in the alpha-helix content. The relative content of beta-strand substructures was significantly increased after 30 min at 600 MPa and 40 degrees C or 2 min at 0.1 MPa and 80 degrees C. We conclude that the active center of MTG, which is located in an expanded beta-strand domain, is resistant to high hydrostatic pressure and pressure-induced inactivation is caused by destruction of alpha-helix elements with a corresponding influence on the enzyme stability in solution.     

Thermal inactivation of pectin methylesterase,polygalacturonase, and peroxidase in tomato juice

Thermal inactivation kinetics have been determined for pectin methylesterase (PME), polygalacturonase (PG), and peroxidase (POD) in tomato juice. Two parameters, the inactivation rate constant (k) at a reference temperature and the activation energy for inactivation (E(a)), were determined for each enzyme. For PME and PG, the k and E(a) values reported here do not agree with those in several previously published reports. These differences can be explained either by the differences in pH values used for inactivation determinations or by inadequacies in the heating methods used in some previous studies. POD showed simple first-order inactivation kinetics and was less thermally stable than either PME or PG. When different cultivars of tomatoes were evaluated, there was no difference in the thermal inactivation kinetics of these enzymes.     

Kinetics of the stability of broccoli (Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during pressure/temperature treatments

The Brassicaceae plant family contains high concentrations of glucosinolates, which can be hydrolyzed by myrosinase yielding products having an anticarcinogenic activity. The pressure and temperature stabilities of endogenous broccoli myrosinase, as well as of the synthetic isothiocyanates sulforaphane and phenylethyl isothiocyanate, were studied in broccoli juice on a kinetic basis. At atmospheric pressure, kinetics of thermal (45-60 degrees C) myrosinase inactivation could be described by a consecutive step model. In contrast, only one phase of myrosinase inactivation was observed at elevated pressure (100-600 MPa) combined with temperatures from 10 up to 60 degrees C, indicating inactivation according to first-order kinetics. An antagonistic effect of pressure (up to 200 MPa) on thermal inactivation (50 degrees C and above) of myrosinase was observed indicating that pressure retarded the thermal inactivation. The kinetic parameters of myrosinase inactivation were described as inactivation rate constants (k values), activation energy (Ea values), and activation volume (Va values). On the basis of the kinetic data, a mathematical model describing the pressure and temperature dependence of myrosinase inactivation rate constants was constructed. The stability of isothiocyanates was studied at atmospheric pressure in the temperature range from 60 to 90 degrees C and at elevated pressures in the combined pressure-temperature range from 600 to 800 MPa and from 30 to 60 degrees C. It was found that isothiocyanates were relatively thermolabile and pressure stable. The kinetics of HP/T isothiocyanate degradation could be adequately described by a first-order kinetic model. The obtained kinetic information can be used for process evaluation and optimization to increase the health effect of Brassicaceae.     

Purification and thermal and high-pressure inactivation of pectinmethylesterase isoenzymes from tomatoes (Lycopersicon esculentum): a novel pressure labile isoenzyme

Tomato pectinmethylesterase (PME) was successfully purified by a two-step method consisting of affinity chromatography followed by cation exchange chromatography. According to this procedure, four different isoenzymes were identified representing molar masses around 34.5-35.0 kDa. Thermal and high-pressure inactivation kinetics of the two major isoenzymes of tomato PME were studied. A striking difference between their process stability was found. The thermostable isoenzyme was completely inactivated after 5.0 min at 70 degrees C, whereas for the thermolabile isoenzyme, temperatures at around 60 degrees C were sufficient for complete inactivation. The thermostable isoenzyme was also found to be pressure stable since no inactivation was observed after 5.0 min of treatment at 800 MPa and 20 or 40 degrees C. The thermolabile isoenzyme appeared to be pressure labile since it could be completely inactivated after 5.0 min of treatment at 700 MPa and 20 degrees C or 650 MPa and 40 degrees C. Inactivation kinetics at pH 6.0 could be accurately described by a first-order model.     

Effect of temperature and/or pressure on tomato pectinesterase activity

The activity of tomato pectinesterase (PE) was studied as a function of pressure (0.1-900 MPa) and temperature (20-75 degrees C). Tomato PE was rather heat labile at atmospheric pressure (inactivation in the temperature domain 57-65 degrees C), but it was very pressure resistant. Even at 900 MPa and 60 degrees C the inactivation was slower as compared to the same treatment at atmospheric pressure. At atmospheric pressure, optimal catalytic activity of PE was found at neutral pH and a temperature of 55 degrees C. Increasing pressure up to 300 MPa increased the enzyme activity as compared to atmospheric pressure. A maximal enzyme activity was found at 100-200 MPa combined with a temperature of 60-65 degrees C. The presence of Ca(2+) ions (60 mM) decreased the enzyme activity at atmospheric pressure in the temperature range 45-60 degrees C but increased enzyme activity at elevated pressure (up to 300 MPa). Maximal enzyme activity in the presence of Ca(2+) ions was noted at 200-300 MPa in combination with a temperature of 65-70 degrees C.     

Kinetic study of the irreversible thermal and pressure inactivation of myrosinase from broccoli (Brassica oleracea L. Cv. italica).

Thermal and pressure inactivation of myrosinase from broccoli was kinetically investigated. Thermal inactivation proceeded in the temperature range 30-60 degrees C. These results indicate that myrosinase is rather thermolabile, as compared to other food quality related enzymes such as polyphenol oxidase, lipoxygenase, pectinmethylesterase, and peroxidase. In addition, a consecutive step model was shown to be efficient in modeling the inactivation curves. Two possible inactivation mechanisms corresponding to the consecutive step model were postulated. Pressure inactivation at 20 degrees C occurred at pressures between 200 and 450 MPa. In addition to its thermal sensitivity, the enzyme likewise is rather pressure sensitive as compared to the above-mentioned food quality related enzymes. By analogy with thermal inactivation, a consecutive step model could adequately describe pressure inactivation curves. At 35 degrees C, pressure inactivation was studied in the range between 0. 1 and 450 MPa. Application of low pressure (<350 MPa) resulted in retardation of thermal inactivation, indicating an antagonistic or protective effect of low pressure.     

Effect of high-pressure treatment on lipoxygenase activity

Solutions of commercial soybean lipoxygenase (100 microgram/ML in 0.2 M citrate-phosphate and 0.2 M Tris buffer were subjected to pressures of 0.1, 200, 400, and 600 MPa for 20 mm. The enzyme was stable at atmospheric pressure (0.1 MPa) over a wide pH range (5-9). In citrate phosphate buffer, the enzyme had maximum stability over the pH range 58 in untreated samples and after treatment at 200 MPa, but with increasing pressure, the pH stability range become narrower and centered around pH 78. The enzyme was more sensitive to acid than alkali, and at pH 9, it lost virtually all activity after pressurization at 600 MPa for 20 mm in both buffers. The activity of the crude enzyme extracted from tomatoes treated at 200 and 300 MPa for 10 mm was not significantly different from that of the untreated tomatoes, while a pressure of 400 MPa for 10 mm caused a significant decrease in activity and treatment at 600 MPa led to complete and irreversible activity loss. Compared to unpressurized tomatoes, treatment at 600 MPa gave significantly reduced levels of hexanal, cis-3-hexenal, and trans-2-hexenal, which are important contributors to "fresh" tomato flavor, and this was attributed to the inactivation of lipoxygenase.     

Inactivation of orange pectinesterase by combined high-pressure and -temperature treatments: a kinetic study

Pressure and/or temperature inactivation of orange pectinesterase (PE) was investigated. Thermal inactivation showed a biphasic behavior, indicating the presence of labile and stable fractions of the enzyme. In a first part, the inactivation of the labile fraction was studied in detail. The combined pressure-temperature inactivation of the labile fraction was studied in the pressure range 0.1-900 MPa combined with temperatures from 15 to 65 degrees C. Inactivation in the pressure-temperature domain specified could be accurately described by a first-order fractional conversion model, estimating the inactivation rate constant of the labile fraction and the remaining activity of the stable fraction. Pressure and temperature dependence of the inactivation rate constants of the labile fraction was quantified using the Eyring and Arrhenius relations, respectively. By replacing in the latter equation the pressure-dependent parameters (E(a), k(ref)(T)()) by mathematical expressions, a global model was formulated. This mathematical model could accurately predict the inactivation rate constant of the labile fraction of orange PE as a function of pressure and temperature. In a second part, the stable fraction was studied in more detail. The stable fraction inactivated at temperatures exceeding 75 degrees C. Acidification (pH 3.7) enhanced thermal inactivation of the stable fraction, whereas addition of Ca(2+) ions (1 M) suppressed inactivation. At elevated pressure (up to 900 MPa), an antagonistic effect of pressure and temperature on the inactivation of the stable fraction was observed. The antagonistic effect was more pronounced in the presence of a 1 M CaCl(2) solution as compared to the inactivation in water, whereas it was less pronounced for the inactivation in acid medium.     

Effect of intrinsic and extrinsic factors on the interaction of plant pectin methylesterase and its proteinaceous inhibitor from kiwi fruit

A proteinaceous pectin methylesterase inhibitor (PMEI) was isolated from kiwi fruit (Actinidia chinensiscv. Hayward) and purified by affinity chromatography on a cyanogen bromide (CNBr) Sepharose 4B-orange PME column. The optimal pH of banana PME activity was 7.0, whereas that for carrot and strawberry PME activity was 9.0. The optimal pH for the binding between kiwi fruit PMEI and these PMEs was 7.0. The kiwi fruit PMEI has a different affinity for PME depending on the plant source. The inhibition kinetics of kiwi fruit PMEI to banana and strawberry PME followed a noncompetitive type, whereas that to carrot PME followed a competitive type. The kiwi fruit PMEI was mixed with banana, carrot, and strawberry PME to obtain PMEI-PME complexes, which were then subjected to thermal (40-80 degrees C, atmospheric pressure) or high-pressure (10 degrees C, 100-600 MPa) treatment. Experimental data showed that the PMEI-PME complexes were easily dissociated by both thermal and high-pressure treatments.     

Inactivation kinetics of purified tomato polygalacturonase by thermal and high-pressure processing

Tomato polygalacturonase (PG) was extracted from ripe tomatoes and purified by cation exchange and gel filtration chromatography. Cation exchange chromatography yielded two peaks with PG activity: the first peak was identified as PG2 (the heat labile form) and the second one as PG1 (the heat stable form). Both PG2 and PG1 presented a molar mass of 42 kDa when analyzed by SDS-PAGE and an isoelectric point >9.3. Thermal inactivation of purified tomato PG2, at pH 4.4, in the temperature range from 53 to 63 degrees C, followed first-order kinetics. Combined pressure-temperature inactivation of tomato PG2 was studied at 5-55 degrees C/100-600MPa. Under all pressure-temperature conditions, PG2 inactivation followed first-order kinetics. Purified tomato PG1, although more thermostable than PG2, showed a pressure stability very similar to that of PG2. These results indicate that high-pressure processing is an efficient alternative to inactivate tomato PG without the need for applying high temperatures.     

Pressure-induced denaturation of β-lactoglobulin in skim milk: effect of milk concentration

The effect of milk concentration (10-40% TS) on the kinetics of the pressure-induced denaturation of β-lactoglobulin (β-LG) was studied. The denaturation was found to be a second-order process at all milk concentrations and pressures. There was a change in pressure dependence of the rate constants for denaturation at about 300 MPa, and this effect became more pronounced as the milk concentration increased. At pressures ≥300 MPa, a small effect of milk concentration was observed, with small decreases in the rate of denaturation as the milk concentration was increased above 20% TS. This was attributed to the lower pH as the milk concentration was increased. In contrast, at 200 MPa, β-LG denaturation was markedly retarded as the milk solids concentration was increased. This was attributed to the increased lactose concentration at higher milk concentrations. This would promote β-LG dimerization at this pressure and this would stabilize the β-LG to denaturation.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Soybean lipoxygenase inactivation by pressure at subzero and elevated temperatures

Soybean lipoxygenase (LOX) inactivation [0.4 mg/mL in Tris-HCl buffer (0.01 M, pH 9)] was studied quantitatively under constant pressure (up to 650 MPa) and temperature (-15 to 68 degrees C) conditions and kinetically characterized by rate constants, activation energies, and activation volumes. The irreversible LOX inactivation followed a first-order reaction at all pressure-temperature combinations tested. In the entire pressure-temperature area studied, LOX inactivation rate constants increased with increasing pressure at constant temperature. On the contrary, at constant pressure, the inactivation rate constants showed a minimum around 30 degrees C and could be increased by either a temperature increase or decrease. On the basis of the calculated rate constants at 102 pressure-temperature combinations, an iso-rate contour diagram was constructed as a function of pressure and temperature. The pressure-temperature dependence of the LOX inactivation rate constants was described successfully using a modified kinetic model of Hawley.     

Denaturation of beta-lactoglobulin in pressure-treated skim milk

The kinetics of beta-lactoglobulin (beta-LG) denaturation in pressure-treated reconstituted skim milk samples over a wide pressurization range (100-600 MPa) and at various temperatures (10-40 degrees C) was studied. Denaturation was extremely dependent on the pressure and duration of treatment. At 100 MPa, no denaturation was observed regardless of the temperature or the holding time. At higher pressures, the level of denaturation increased with an increasing holding time at a constant pressure or with increasing pressure at a constant holding time. At 200 MPa, there was only a small effect of changing the temperature at pressurization. However, at higher pressures, increasing the temperature from 10 to 40 degrees C markedly increased the rate of denaturation. The two major genetic variants of beta-LG (A and B) behaved similarly to pressure treatment, although the B variant appeared to denature slightly faster than the A variant at low pressures (< or =400 MPa). The denaturation could be described as a second-order process for both beta-LG variants. There was a marked change in pressure dependence at about 300 MPa, which resulted in markedly different activation volumes in the two pressure ranges. Evaluation of the kinetic and thermodynamic parameters suggested that there may have been a transition from an aggregation-limited reaction to an unfolding-limited reaction as the pressure was increased.     

Model studies on the stability of folic acid and 5-methyltetrahydrofolic acid degradation during thermal treatment in combination with high hydrostatic pressure

Stability of folic acid and 5-methyltetrahydrofolic acid in phosphate buffer (0.2 M; pH 7) toward thermal (above 65 degrees C) and combined high pressure (up to 800 MPa)/thermal (20 up to 65 degrees C) treatments was studied on a kinetic basis. Residual folate concentration after thermal and high pressure/thermal treatments was measured using reverse phase liquid chromatography. The degradation of both folates followed first-order reaction kinetics. At ambient pressure, the estimated Arrhenius activation energy (E(a)) values of folic acid and 5-methyltetrahydrofolic acid thermal degradation were 51.66 and 79.98 kJ mol(-1), respectively. It was noticed that the stability of folic acid toward thermal and combined high pressure thermal treatments was much higher than 5-methyltetrahydrofolic acid. High-pressure treatments at room temperature or higher (up to 60 degrees C) had no or little effect on folic acid. In the whole P/T area studied, the rate constant of 5-methyltetrahydrofolic acid degradation was enhanced by increasing pressure, and a remarkable synergistic effect of pressure and temperature on 5-methyltetrahydrofolic acid degradation occurred at temperatures above 40 degrees C. A model to describe the combined pressure and temperature effect on the 5-methyltetrahydrofolic acid degradation rate constant is presented.     

Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk

Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.     

Inactivation of soybean trypsin inhibitors and lipoxygenase by high-pressure processing

Trypsin inhibitors (TIA), one of the antinutritional factors of soy milk, are usually inactivated by heat treatment. In the current study, high-pressure processing (HPP) was evaluated as an alternative for the inactivation of TIA in soy milk. Moreover, the effect of HPP on lipoxygenase (LOX) in whole soybeans and soy milk was studied. For complete LOX inactivation either very high pressures (800 MPa) or a combined temperature/pressure treatment (60 degrees C/600 MPa) was needed. Pressure inactivation of TIA was possible only in combination with elevated temperatures. For TIA inactivation, three process parameters, temperature, time, and pressure, were optimized using experimental design and response surface methodology. A 90% TIA inactivation with treatment times of <2 min can be reached at temperatures between 77 and 90 degrees C and pressures between 750 and 525 MPa.     

Effect of combined heat and high-pressure treatments on the texture of chicken breast muscle (pectoralis fundus)

Commercially supplied chicken breast muscle was subjected to simultaneous heat and pressure treatments. Treatment conditions ranged from ambient temperature to 70 degrees C and from 0.1 to 800 MPa, respectively, in various combinations. Texture profile analysis (TPA) of the treated samples was performed to determine changes in muscle hardness. At treatment temperatures up to and including 50 degrees C, heat and pressure acted synergistically to increase muscle hardness. However, at 60 and 70 degrees C, hardness decreased following treatments in excess of 200 MPa. TPA was performed on extracted myofibrillar protein gels that after treatment under similar conditions revealed similar effects of heat and pressure. Differential scanning calorimetry analysis of whole muscle samples revealed that at ambient pressure the unfolding of myosin was completed at 60 degrees C, unlike actin, which completely denatured only above 70 degrees C. With simultaneous pressure treatment at >200 MPa, myosin and actin unfolded at 20 degrees C. Unfolding of myosin and actin could be induced in extracted myofibrillar protein with simultaneous treatment at 200 MPa and 40 degrees C. Electrophoretic analysis indicated high pressure/temperature regimens induced disulfide bonding between myosin chains.