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.     

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.     

Cross-linking of hen egg white lysozyme by microbial transglutaminase under high hydrostatic pressure: localization of reactive amino acid side chains

After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.     

Model studies on the influence of high hydrostatic pressure on the formation of glycated arginine modifications at elevated temperatures

Aqueous solutions of Nalpha-acetylarginine and glucose were reacted for 2 h with pressure application from 0 to 600 MPa and varying temperatures between 90 and 120 degrees C. After enzymatic deacetylation of the reaction products, the glycated amino acids were separated by means of a self-assembled preparative ion exchange chromatography system using ninhydrin detection. On the basis of the use of eight synthesized reference compounds known in the literature as posttranslational arginine modifications, first, the presence of several glycated amino acids could be excluded. On the other hand, N5-[[(1-carboxyethyl)amino]iminomethyl]ornithine [N7-(1-carboxyethyl)arginine; N7-CEA; 12] was identified as a previously unknown arginine modification based on LC-MS, NMR measurements, and synthesis. In addition, N5-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine (1) was identified as a further major reaction product. In further experiments, the formation of 1 and 12 was quantitatively followed at different pressures and/or temperatures. The results indicated that high hydrostatic pressure at elevated temperatures significantly increased the amounts of both arginine modifications. 2-Oxopropanal, known to form 1 in a reaction with arginine, was also quantified to explain the different yields observed after pressure application. A new formation mechanism leading to 12 by a reaction of the guanidine group or arginine with 2-oxopropanal is discussed.     

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.     

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.     

Conjugation of bovine serum albumin and glucose under combined high pressure and heat

The effect of combined heat and pressure on the Maillard reaction between bovine serum albumin (BSA) and glucose was investigated. The effects in the range of 60-132 °C and at 0.1-600 MPa on the lysine availability of BSA were investigated at isothermal/isobaric conditions. The kinetic results showed that the protein-sugar conjugation rate increased with increasing temperature, whereas it decreased with increasing pressure. The reaction followed 1.4th order kinetics at most conditions investigated. A mathematical model describing BSA-glucose conjugation kinetics as a function of pressure and temperature is proposed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to verify BSA-glucose conjugation and to identify the glucosylated sites. These indicated that the application of combined high pressure and high temperature resulted in significant differences in the progression of the Maillard reaction as compared to heat treatments at atmospheric pressure.     

Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment

The sulfhydryl (SH) content of egg white proteins (10% v/v or 9.64 mg of protein/mL) after heat (50-85 degrees C) and combined heat- and high-pressure treatments (100-700 MPa, 10-60 degrees C) was determined using 5',5-dithiobis (2-nitrobenzoic acid) (DTNB), both for the soluble fraction and the total protein fraction. Only irreversible changes were taken into account. Both physical treatments were performed at two pH levels: pH 7.6, corresponding to the pH of fresh egg white, and pH 8.8, corresponding to that of aged egg white. Both heat and combined heat- and high-pressure treatment resulted in an exposure of buried SH groups. These exposed SH groups were involved in the formation of disulfide bond stabilized protein aggregates, as shown by gel electrophoresis. Under severe processing conditions (above 70 degrees C at atmospheric pressure or above 500-600 MPa, depending on the temperature applied), a decrease in total SH content could be observed, probably due to the formation of disulfide bonds by oxidation, especially at alkaline pH when the thiolate anion was more reactive. The high degree of exposure of sulfhydryl groups, and subsequent oxidation and sulfhydryl-disulfide bond exchange reactions resulting in soluble aggregates, can explain why pressure-induced egg white gels are softer and more elastic than heat-induced ones. When pressure treatment was performed at low temperatures (e.g., 10 degrees C), a lower pressure was required to induce similar changes in the sulfhydryl content, as compared to higher temperatures (e.g., 25 degrees C), indicating an antagonistic effect between pressure and temperature in the domain studied (10-60 degrees C, 100-700 MPa). Treatment conditions resulting in extensive protein insolubilization were accompanied by a transfer of free sulfhydryl groups from the soluble to the insoluble protein fraction. These SH groups were mainly accessible to DTNB.     

Effect of high pressure on isomerization and degradation of lactose in alkaline media

The combined effects of temperature (60 degrees C) and high-pressure treatments (400 MPa for 3 h) on the isomerization-degradation of lactose (10%) in basic media were studied. The formation of isomeric disaccharides (lactulose and epilactose) and galactose decreased by the application of high pressure in aqueous sodium hydroxide 4 mM (pH 10.2) and 8 mM (pH 10.6), and sodium carbonate-bicarbonate buffer (pH 10.0). In addition, no substantial color development was observed in the sodium hydroxide systems for high and atmospheric pressure, whereas the application of high pressure led to a noticeable decrease of color development in the carbonate-bicarbonate system.     

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.     

Effect of high-pressure processing at elevated temperatures on thiamin and riboflavin in pork and model systems

High-pressure/high-temperature properties of vitamins in food are important with respect to the new pressure-assisted thermal sterilization method utilizing pressure-induced adiabatic temperature changes. Riboflavin, thiamin, and thiamin monophosphate (TMP) stabilities were assayed in the temperature range from 25 to 100 degrees C under normal pressure (0.1 MPa) and high pressure (600 MPa) in acetate-buffered (pH 5.5) model solutions, some with added fructose, hemoglobin, or ascorbic acid. Thiamin and riboflavin stabilities were also assayed in minced fresh pork fillet and in rehydrated pork reference material with and without pressure treatment at 600 MPa in the temperature range from 20 to 100 degrees C. In pork, the vitamins proved to be sufficiently stabile for high-pressure/high-temperature processing. Under similar conditions, vitamin decay in model solutions was up to 30 times faster, especially that of TMP. Thus, it appears that it may not be possible to draw conclusions for the pressure behavior of real food matrices from the results of investigations in food models. A further consequence is that caution is necessary when supplementing foods with synthetic B vitamins preceding high-pressure/high-temperature processing.     

Antioxidant activity of extracts of black sesame seed (Sesamum indicum L.) by supercritical carbon dioxide extraction

Antioxidant activities of extracts derived from sesame seed by supercritical carbon dioxide (SC-CO(2)) extraction and by n-hexane were determined using alpha,alpha-diphenyl-beta-picylhydrazyl (DPPH) radical scavenging and linoleic acid system methods. The highest extracted yield was given at 35 degrees C, 40 MPa, and a CO(2) flow rate of 2.5 mL min(-1) by an orthogonal experiment. The yields of extracts increased with increasing pressure, and yields at 40 and 30 MPa were higher than that by solvent extraction at 46.50%. Results from the linoleic acid system showed that the antioxidant activity follows the order: extract at 35 degrees C, 20 MPa > BHT > extract at 55 degrees C, 40 MPa > extract at 55 degrees C, 30 MPa > Trolox > solvent extraction > alpha-tocopherol. The SC-CO(2) extracts exhibited significantly higher antioxidant activities comparable to that by n-hexane extraction. The extracts at 30 MPa presented the highest antioxidant activities assessed in the DPPH method. At 20 MPa, the EC(50) increased with temperature, which indicated that the antioxidant activity was decreased in a temperature-dependent manner. The significant differences of antioxidant activities were found between the extracts by SC-CO(2) extraction and n-hexane. However, no significant differences were exhibited among the extracts by SC-CO(2) extraction. The vitamin E concentrations were also significantly higher in SC-CO(2) extracts than in n-hexane extracts, and its concentrations in extracts corresponded with the antioxidant activity of extracts.     

High-pressure effects on Maillard reaction between glucose and lysine

Glucose-lysine model systems prepared over a range of pH values (5-10) in unbuffered and buffered media were incubated at 60 degrees C either under atmospheric pressure or at 400 MPa. The results obtained showed that high pressure affected in different ways the different stages of the Maillard reaction and that such effects were strongly influenced by pressure-induced changes in the pH of the systems. In unbuffered media, at an initial pH < or =8.0, the formation of Amadori rearrangement products (ARP) was not considerably affected by pressure, whereas the intermediate and advanced stages of the Maillard reaction were suppressed, suggesting a retardation of the degradation of the ARP. In buffered media, at pH values < or =8.0, pressure slowed the Maillard reaction from the initial stages. These effects are attributed to the pH drop caused by the pressure-induced dissociation of the acid groups. In unbuffered and buffered media at initial pH = 10.2, high pressure accelerated the formation and subsequent degradation of ARP, leading to increased levels of intermediate and advanced reaction products.     

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.     

The effect of high hydrostatic pressure on the strawberry anthocyanins

Color stability of fruit juice made from strawberries (Fragaria x ananassa, cv. Elsanta) that were subjected to high hydrostatic pressure was studied by measuring the anthocyanin content. High hysrostatic pressure is a method of preservation of food alternative to heat treatment. It is therefore essential to assess the impact of high pressure on color molecules. Samples were pressurized under 200, 400, 600, and 800 MPa for 15 min at a temperature controlled between 18 and 22 degrees C. After application of pressure, the anthocyanin content of the strawberry juice was analyzed by HPLC-UV using a novel isocratic elution system. The high-pressure treated samples were kept at refrigerator temperature (4 degrees C), room temperature (20 degrees C), and 30 degrees C. Two pigments were identified and quantified: pelargonidin 3-glucoside and pelargonidin 3-rutinoside. The highest stability of the anthocyanins was found when strawberries were stored at a temperature of 4 degrees C. High-pressure treatment at 800 MPa led to the lowest losses, at 4 degrees C.     

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.     

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.     

Pressure-temperature phase transition diagram for wheat starch

Wheat starch suspensions in water (5% dry matter) were subjected to various pressures (0.1-600 MPa) and temperatures (-20 to 96 degrees C) for 15 min. The gelatinization rate was measured after treatment by using microscopic measurements of the loss of birefringence of the granules. This method was previously calibrated by differential scanning calorimetry. Curves of isogelatinization were found to be quite similar to a pressure-temperature (P-T) diagram of unfolding proteins. Results were first analyzed by considering the thermodynamic aspects related to the dT/dP curve shifts. On the basis of equations already shown for proteins, the P-T gelatinization diagram of wheat starch would show different kinds of thermal contributions, suggesting endothermic, athermic, or exothermic melting reactions. Second, as a practical consequence, these previous P-T areas corresponded to specific gelatinization conditions as confirmed by hydration evaluation measured by starch swelling index. Depending on the pressure-temperature conditions, gelatinization would involve hydration. Lowering the pressure and temperature resulted in a complete gelatinization with less hydration in comparison with a thermal treatment at atmospheric pressure. A hydration model based on an energetic approach was proposed.     

Changes in volatile flavor components of guava juice with high-pressure treatment and heat processing and during storage.

The changes in volatile flavor components of guava juice during pressure processing (25 degrees C, 600 MPa, 15 min), heat processing (95 degrees C, 5 min), and storage at 4 and 25 degrees C were evaluated by purge and trap/gas chromatography/mass spectrometry. Esters were the major volatile fraction in guava juice, and alcohols were the second. Pressure processing could maintain the original flavor distribution of the juice. Heat processing (95 degrees C, 5 min) caused decreases in the majority of flavor components in the juice when compared with freshly extracted juice. High-pressure treatment at 600 MPa for 15 min can effectively sterilize microbes but partially inactivate enzymes of guava juice; therefore, volatile components in pressure-treated juice gradually changed during storage periods. Pressure-treated guava juice showed increases in methanol, ethanol, and 2-ethylfuran with decreases in the other components during storage period. Nevertheless, the volatile distribution of 600 MPa treated guava juice was similar to that of freshly extracted juice when stored at 4 degrees C for 30 days.