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.     

Optimizing conditions for thermal processes of soy milk

Mathematical and kinetic models were set up for heat-induced quality changes in soy milk, including inactivation of trypsin inhibitor activity (TIA) and degradation of thiamin, riboflavin, color, and flavor over a wide range of time-temperature combinations with particular interest in the ultrahigh-temperature (UHT) range. On the basis of these models, multiresponse optimization of the thermal processes for soy milk was carried out to obtain the following effects simultaneously: (1) maximum destruction of bacterial spores, (2) maximum inactivation of TIA, and (3) minimum degradation of sensory and nutritional qualities. By a suitable selection of high temperatures and extended heating times, for example, 143 degrees C/60 s, it is possible to use a single-step UHT process to produce a commercially sterile soy milk with satisfactory TIA inactivation, highly acceptable color and flavor, and thiamin retention between 90 and 93%.     

Elimination of trypsin inhibitor activity and beany flavor in soy milk by consecutive blanching and ultrahigh-temperature (UHT) processing

Soy foods contain significant health-promoting components but also may contain beany flavor and trypsin inhibitor activity (TIA), which can cause pancreatic disease if present at a high level. Thermal processing can inactivate TIA and lipoxygenase. Ultrahigh-temperature (UHT) processing is relatively new for manufacturing soy milk. Simultaneous elimination of TIA and soy odor by UHT processing for enhancing soy milk quality has not been reported. The objective was to determine TIA in soy milk processed by traditional, steam injection, blanching, and UHT methods and to compare the products with commercial soy milk products. Soybean was soaked and blanched at 70-85 degrees C for 30 s-7.5 min. The blanched beans were made into base soy milk. The hexanal content of the base soy milk was determined by gas chromatography to determine the best conditions for further thermal processing by indirect and direct UHT methods at 135-150 degrees C for 10-50 s using the Microthermics processor. Soy milk was also made from soaked soybeans by traditional batch cooking and steaming methods. Eighteen commercial products were selected from the supermarket. Residual TIA in soy milk processed by the traditional and steam injection to 100 degrees C for 20 min was approximately 13%. Blanching could inactivate 25-50% of TIAs of the raw soy milk. The blanch conditions of 80 degrees C and 2 min were selected for UHT processing because these conditions produced blanched soy milk without hexanal, indicating a complete heat inactivation of lipoxygenases. The TIA decreased with increased temperature and time of UHT heating. The maximal trypsin inhibitor inactivation was achieved by UHT direct and indirect methods with residual activities of approximately 10%. Some commercial soy milk products contained high TIAs. The results are important to the food industry and consumers. Kinetic analysis showed that heat inactivation (denaturation) of TIA, under the continuous processing conditions of the Microthermics processor, followed first-order reaction kinetics, and the activation energy of the inactivation was 34 kJ/mol.     

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.     

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.     

Homogenization conditions affect the oxidative stability of fish oil enriched milk emulsions: oxidation linked to changes in protein composition at the oil-water interface

Fish oil was incorporated into milk under different homogenization temperatures (50 and 72 degrees C) and pressures (5, 15, and 22.5 MPa). Subsequently, the oxidative stability of the milk and changes in the protein composition of the milk fat globule membrane (MFGM) were examined. Results showed that high pressure and high temperature (72 degrees C and 22.5 MPa) resulted in less lipid oxidation, whereas low pressure and low temperature (50 degrees C and 5 MPa) resulted in faster lipid oxidation. Analysis of protein oxidation indicated that especially casein was prone to oxidation. The level of free thiol groups was increased by high temperature (72 degrees C) and with increasing pressure. Furthermore, SDS-PAGE and confocal laser scanning microscopy (CLSM) indicated that high temperature resulted in an increase in beta-lactoglobulin adsorbed at the oil-water interface. This was even more pronounced with higher pressure. Less casein seemed to be present at the oil-water interface with increasing pressure. Overall, the results indicated that a combination of more beta-lactoglobulin and less casein at the oil-water interface gave the most stable emulsions with respect to lipid oxidation.     

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.     

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.     

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.     

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.     

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 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.     

Stability of isoflavones in soy milk stored at elevated and ambient temperatures

Soy isoflavones are widely recognized for their potential health benefits. The increased use of traditional and new food products calls for the assessment of their stability during processing and storage. The present study examines the stability of genistein and daidzein derivatives in soy milk. Soy milk was stored at ambient and elevated temperatures, and the change in isoflavone concentration was monitored with time. Genistin loss in time showed typical first-order kinetics, with rate constants ranging from 0.437-3.871 to 61-109 days(-1) in the temperature ranges of 15-37 and 70-90 degrees C, respectively. The temperature dependence of genistin loss followed the Arrhenius relation with activation energies of 7.2 kcal/mol at ambient temperatures and 17.6 kcal/mol at elevated temperatures. At early stages of soy milk storage at 80 and 90 degrees C, the 6' '-O-acetyldaidzin concentration increased, followed by a slow decrease. The results obtained in this study can serve as a basis for estimating the shelf life of soy milk as related to its genistin content.     

Bioaccessibility of tocopherols, carotenoids, and ascorbic acid from milk- and soy-based fruit beverages: influence of food matrix and processing

A study was made of the effect of high-pressure processing (HPP) and thermal treatment (TT) on plant bioactive compounds (tocopherols, carotenoids, and ascorbic acid) in 12 fruit juice-milk beverages and of how the food matrix [whole milk (JW), skimmed milk (JS), and soy milk (JSy)] modulates their bioaccessibility (%). HPP (400 MPa/40 °C/5 min) produced a significant decrease in carotenoid and ascorbic acid bioaccessibility in all three beverages and maintained the bioaccessibility of tocopherols in JW and JS while decreasing it in JSy. TT (90 °C/30 s) produced a significant decrease in tocopherol and carotenoid bioaccessibility in all three beverages and increased the bioaccessibility of ascorbic acid. With regard to the food matrix, α-tocopherol and ascorbic acid bioaccessibility was greatest in JW beverages and lowest in JSy beverages, whereas no significant differences were found among the three beverages in terms of carotenoid bioaccessibility. HPP-treated samples showed higher tocopherol and carotenoid bioaccessibility than TT-treated samples, thus indicating that HPP combined with a milk matrix positively modulates the bioaccessibility of certain types of bioactive components of food, mainly those of a lipophilic nature.     

Comparison of effects of high-pressure processing and heat treatment on immunoactivity of bovine milk immunoglobulin G in enriched soymilk under equivalent microbial inactivation levels

Immunoglobulin-rich foods may provide health benefits to consumers. To extend the refrigerated shelf life of functional foods enriched with bovine immunoglobulin G (IgG), nonthermal alternatives such as high-pressure processing (HPP) may offer advantages to thermal processing for microbial reduction. To evaluate the effects of HPP on the immunoactivity of bovine IgG, a soymilk product enriched with milk protein concentrates, derived from dairy cows that were hyperimmunized with 26 human pathogens, was subjected to HPP or heat treatment. To achieve a 5 log reduction in inoculated Escherichia coli 8739, the HPP or heat treatment requirements were 345 MPa for 4 min at 30 degrees C or for 20 s at 70 degrees C, respectively. To achieve a 5 log reduction in natural flora in the enriched soymilk, the HPP or heat treatments needed were 552 MPa for 4 min at 30 degrees C or for 120 s at 78.2 degrees C, respectively. At equivalent levels for a 5 log reduction in E. coli, HPP and heat treatment caused 25% and no detectable loss in bovine IgG activity, respectively. However, at equivalent levels for a 5 log reduction in natural flora, HPP and heat resulted in 65 and 85% loss of bovine IgG activity, respectively. Results of combined pressure-thermal kinetic studies of bovine milk IgG activity were provided to determine the optimal process conditions to preserve product function.     

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.     

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 high-pressure-moderate-temperature processing on the volatile profile of milk

The effects of high hydrostatic pressure on volatile generation in milk were investigated in this study. Raw milk samples were treated under different pressures (482, 586, and 620 MPa), temperatures (25 and 60 degrees C), and holding times (1, 3, and 5 min). Samples submitted to heat treatments alone (25, 60, and 80 degrees C for 1, 3, and 5 min) were used for comparison. Trace volatile sulfur compounds were analyzed using solid-phase microextraction (SPME) and gas chromatography (GC) with pulsed-flame photometric detection (PFPD), whereas the rest of the volatile compounds were analyzed using SPME-GC with flame ionization detection (FID). Multivariate analysis of variance (MANOVA) and principal component analysis (PCA) were used to study the effect of pressure, temperature, and time on volatile generation. Relative concentration increases of 27 selected volatile compounds were compared to an untreated sample. It was found that pressure, temperature, and time, as well as their interactions, all had significant effects (P < 0.001) on volatile generation in milk. Pressure and time effects were significant at 60 degrees C, whereas their effects were almost negligible at 25 degrees C. The PCA plot indicated that the volatile generation of pressure-heated samples at 60 degrees C was different from that of heated-alone samples. Heat treatment tended to promote the formation of methanethiol, hydrogen sulfide, methyl ketones, and aldehydes, whereas high-pressure treatment favored the formation of hydrogen sulfide and aldehydes.     

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.     

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.