Lipoxygenase inactivation in green beans (Phaseolus vulgaris L.) due to high pressure treatment at subzero and elevated temperatures

The kinetics of lipoxygenase (LOX) inactivation in green beans due to high-pressure treatment were studied in the pressure-temperature area of 0.1 up to 650 MPa and -10 up to 70 degrees C for systems with different levels of food complexity, i.e., in green bean juice and intact green beans (in situ study). For both systems, LOX was irreversibly inactivated by high-pressure treatment combined with subzero and elevated temperatures and the inactivation could be described as a first-order reaction. At ambient pressure, in situ LOX was less thermostable than in the juice at temperatures below 68 degrees C whereas the stability ranking was reverse at temperatures above 68 degrees C. At temperatures below 63 degrees C, sensitivity of the inactivation rate constants to temperature changes was on the same order of magnitude in the juice and in situ, while at higher temperature it was lower in situ. The pressure needed to obtain the same rate of LOX inactivation at a given temperature was lower in situ than in the juice. Application of high-pressure treatment at low/subzero temperature resulted in an antagonistic effect on LOX inactivation for both systems, whereas no such effect was found above room temperature. The pressure-temperature dependence of the LOX inactivation rate constants in green beans was successfully modeled.     

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.     

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.     

Thermal and high-pressure inactivation kinetics of polyphenol oxidase in Victoria grape must

The inactivation kinetics of polyphenol oxidase (PPO) in freshly prepared grape must under high hydrostatic pressure (100-800 MPa) combined with moderate temperature (20-70 degrees C) was investigated. Atmospheric pressure conditions in a temperature range of 55-70 degrees C were also tested. Isothermal inactivation of PPO in grape must could be described by a biphasic model. The values of activation energy and activation volume of stable fraction were estimated as 53.34 kJ mol(-1) and -18.15 cm3 mol(-1) at a reference pressure of 600 MPa and reference temperature of 50 degrees C, respectively. Pressure and temperature were found to act synergistically, except in the high-temperature-low-pressure region where an antagonistic effect was found. A third-degree polynomial model was successfully applied to describe the temperature/pressure dependence of the inactivation rate constants of the stable PPO fraction in grape must.     

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.     

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.     

Biogenic amine formation and nitrite reactions in meat batter as affected by high-pressure processing and chilled storage

Changes in biogenic amine formation and nitrite depletion in meat batters as affected by pressure-temperature combinations (300 MPa/30 min/7, 20, and 40 degrees C), cooking process (70 degrees C/30 min), and storage (54 days/2 degrees C) were studied. Changes in residual nitrite concentration in raw meat batters were conditioned by the temperature and not by the pressure applied. Cooking process decreased (P < 0.05) the residual nitrite concentration in all samples. High-pressure processing and cooking treatment increased (P < 0.05) the nitrate content. Whereas protein-bound nitrite concentration decreased with pressure processing, no effect was observed with the heating process of meat batters. High-pressure processing conditions had no effect on the rate of residual nitrite loss throughout the storage. The application of high pressure decreased (P < 0.05) the concentration of some biogenic amines (tyramine, agmatine, and spermine). Irrespective of the high processing conditions, generally, throughout storage biogenic amine levels did not change or increased, although quantitatively this effect was not very important.     

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.     

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.     

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.     

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.     

Kinetic study on the changes in the susceptibility of egg white proteins to enzymatic hydrolysis induced by heat and high hydrostatic pressure pretreatment

A kinetic study was conducted on the effect of heat pretreatment in the temperature range of 50-85 degrees C at atmospheric pressure and of high hydrostatic pressure pretreatment (100-700 MPa) at four temperatures (10, 25, 40, and 60 degrees C) on the susceptibility of egg white solutions (10% v/v, pH 7.6) to subsequent enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Both heat pretreatment at atmospheric pressure and high-pressure pretreatment resulted in an increase in degree of hydrolysis (DH) after 10 min of enzymatic reaction (DH10) of egg white solutions, as measured using the pH-stat method, which could be described by a fractional conversion model (based on an apparent first-order reaction kinetic model). The temperature dependence of the corresponding rate constants could be described by the Arrhenius equation. At elevated pressure, a negative apparent activation energy was obtained, implying an antagonistic effect of pressure and temperature. The pressure dependence of the rate constants could be described by the Eyring equation, and negative activation volumes were observed, which demonstrates the positive effect of pressure on the susceptibility of egg white solutions to subsequent enzymatic hydrolysis.     

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.     

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.     

Impact of pH on the kinetics of acrylamide formation/elimination reactions in model systems

The effect of pH on acrylamide formation and elimination kinetics was studied in an equimolar (0.1 M) asparagine-glucose model system in phosphate or citrate buffer, heated at temperatures between 120 and 200 degrees C. To describe the experimental data, a simplified kinetic model was proposed and kinetic parameters were estimated by combined nonlinear regression and numerical integration on the data obtained under nonisothermal conditions. The model was subsequently validated in a more realistic potato-based matrix with varying pH. By increasing acidity, the reaction rate constants at T(ref) (160 degrees C) for both acrylamide formation and elimination can significantly be reduced, whereas the temperature dependence of both reaction rate constants increases. The introduction of a lyophilized potato matrix (20%) did not affect the acrylamide formation reaction rate constant at reference temperature (160 degrees C) as compared to the asparagine-glucose model system; the elimination rate constant at T(ref), on the contrary, was almost doubled.     

Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position

The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.     

Tomato (Lycopersicon esculentum) pectin methylesterase and polygalacturonase behaviors regarding heat- and pressure-induced inactivation.

The combined high pressure/thermal (HP/T) inactivation of tomato pectin methyl esterase (PME) and polygalacturonase (PG) was investigated as a possible alternative to thermal processing classically used for enzyme inactivation. The temperature and pressure ranges tested were from 60 degrees C to 105 degrees C, and from 0.1 to 800 MPa, respectively. PME, a heat-labile enzyme at ambient pressure, is dramatically stabilized against thermal denaturation at pressures above atmospheric and up to 500-600 MPa. PG, however, is very resistant to thermal denaturation at 0.1 MPa, but quickly and easily inactivated by combinations of moderate temperatures and pressures. Selective inactivation of either PME or PG was achieved by choosing proper combinations of P and T. The inactivation kinetics of these enzymes was measured and described mathematically over the investigated portion of the P/T plane. Whereas medium composition and salinity had little influence on the inactivation rates, PME was found less sensitive to both heat and pressure when pH was raised above its physiological value. PG, on the other hand, became more labile at higher pH values. The results are discussed in terms of isoenzymes and other physicochemical features of PME and PG.     

Pressure and temperature stability of 5-methyltetrahydrofolic acid: a kinetic study

Detailed kinetic studies of [6S] and [6RS] 5-methyltetrahydrofolic acid (5-CH3-H4folate) degradation during thermal (from 60 to 90 degrees C) and high pressure/thermal (from 30 to 45 degrees C; from 200 to 700 MPa) treatments were carried out. The results confirmed that the temperature and pressure induced degradation kinetics of [6S] 5-CH3-H4folate were identical (within 95% confidence interval) with those of [6RS] 5-CH3-H4folate. Under equal processing conditions, the estimated degradation rate constants (k), activation energy (E(a)), and activation volume (V(a)) values of [6S] and [6RS] 5-CH3-H4folate were the same (95% confidence interval). The modified thermodynamic model proposed by Nguyen and co-workers (J. Agric. Food Chem. 2003, 51, 3352-3357) to describe the pressure and temperature dependence of the rate constant for folate degradation was reevaluated.     

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.     

Kinetics and mechanism of lactosylation of alpha-lactalbumin

The lactosylation of alpha-lactalbumin in aqueous solution was followed at pH(c) = 6.0, 6.3, 7.0, 7.3, and 7.9 and constant ionic strength (I = 0.080) at 50-60 degrees C by reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry (MS). The rate of the lactosylation reaction increased with increasing pH and with temperature most significantly at lower pH. The rate of lactosylation could be described by an acid dissociation curve corresponding to pK(a) of the epsilon-amino group of lysine in alpha-lactalbumin. From initial rates for conditions of excess of lactose, pseudo-first-order rate constants were calculated and further transferred into second-order rate constants by dividing with the lactose concentration. Second-order rate constants for protonated and unprotonated lysine in alpha-lactalbumin both showed Arrhenius behavior, and using transition-state theory, DeltaH# = 31 +/- 2 kJ/mol and DeltaS# = -266 +/- 48 J/(mol . K) were determined for the unprotonated form and DeltaH# = 158 +/- 49 kJ/mol and DeltaS# = 80 +/- 150 J/(mol . K) for the protonated form, respectively. On the basis of the marked differences in activation parameters, initial formation of a lactosylamine is suggested as rate-determining for reaction between lactose and a protonated lysine in alpha-lactalbumin, while subsequent water elimination to form a Schiff base becomes rate-determining for the unprotonated form.