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.     

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.     

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 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.     

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.     

Characterization of the temperature activation of pectin methylesterase in green beans and tomatoes

Low-temperature blanching of vegetables activates the enzyme pectin methylesterase (PME), which demethylates cell wall pectins and improves tissue firmness. This temperature activation of PME has been investigated by measuring the formation of methanol in intact tissue of green beans and tomatoes. Rates of methanol formation at temperatures of 35-65 degrees C were obtained by measuring the release of methanol from thin slices of tomato pericarp or green bean pod material. Activation energies of 112 and 97 kJ mol(-1) were calculated for PME activity in green beans and tomatoes, respectively. These activation energies indicate that the rate of pectin demethylation at 65 degrees C will be nearly 100 times that at 25 degrees C. PME activity was also determined titrimetrically using a solubilized form of the enzyme and purified pectin at temperatures from 30 to 60 degrees C. Under these conditions, much lower activation energies of 37 and 35 kJ mol(-1) were obtained for green beans and tomatoes, respectively. Methanol accumulation during heating of whole intact green beans was also determined and yielded an activation energy similar to that obtained with sliced beans. Whole green beans held at room temperature did not accumulate any methanol, but sliced or homogenized beans did. If whole beans were first heated to 45 degrees C and then cooled, methanol accumulation was observed at room temperature. These results indicate that two factors contribute to the observed high rate of pectin de-esterification during low-temperature blanching: (1) An irreversible change, causing PME to become active, occurs by heating to > or = 45 degrees C. (2) The high activation energy for pectin de-esterification means that the rate of de-esterification increases substantially with increasing temperature.     

Influence of critical storage temperatures on degradative pathways of pigments in green beans (Phaseolus vulgaris cvs. Perona and boby)

In this work a study of critical storage temperatures on pigment degradation of green beans (Phaseolus vulgaris, cvs. Perona and Boby) was conducted. In this way, green beans kept better quality at 4 degrees C than either 8 or 12 degrees C, maintaining a bright green color and good texture. Nevertheless, temperatures of 4 degrees C induced chilling injury (CI) after eight days of storage, which became evident when the pods were transferred to 20 degrees C. Cold storage temperatures, 12, 8, and 4 degrees C, produced different changes on the green beans chlorophyll profile. Green beans of both cultivars, Perona and Boby, stored at 4 and 12 degrees C showed a continuous degradation of chlorophyll pigments during storage, while samples stored at 8 degrees C showed an increase of chlorophyll content at the first 15 days. Carotenoid pigments also suffered different changes during cold storage. Perona was the green beans cultivar which maintained the higher level of lutein, mainly when samples were stored at the most suitable temperature (8 degrees C).     

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.     

Effect of ultrahigh-temperature continuous ohmic heating treatment on fresh orange juice

The scope of this study is the effect of ohmic heating thermal treatment on liquid fruit juice made of oranges. Effects of ohmic heating on the quality of orange juice were examined and compared to those of heat pasteurization at 90 degrees C for 50 s. Orange juice was treated at temperatures of 90, 120, and 150 degrees C for 1.13, 0.85, and 0.68 s in an ohmic heating system. Microbial counts showed complete inactivation of bacteria, yeast, and mold during ohmic and conventional treatments. The ohmic heating treatment reduced pectin esterase activity by 98%. The reduction in vitamin C was 15%. Ohmic-heated orange juice maintained higher amounts of the five representative flavor compounds than did heat-pasteurized juice. Sensory evaluation tests showed no difference between fresh and ohmic-heated orange juice. Thus, high-temperature ohmic-heating treatment can be effectively used to pasteurize fresh orange juice with minimal sensory deterioration.     

Comparative study on pressure and temperature stability of 5-methyltetrahydrofolic acid in model systems and in food products

A comparative study on the pressure and temperature stability of 5-methyltetrahydrofolic acid (5-CH(3)-H(4)folate) was performed in model/buffer systems and food products (i.e., orange juice, kiwi puree, carrot juice, and asparagus). Effects of pH and ascorbic acid (0.5 mg/g) on 5-CH(3)-H(4)folate stability in buffer systems were studied on a kinetic basis at different temperatures (from 65 to 160 degrees C) and different pressure/temperature combinations (from 100 to 700 MPa/from 20 to 65 degrees C). These studies showed that (i) the degradation of 5-CH(3)-H(4)folate in all model systems could be described by first-order reaction kinetics, (ii) the thermostability of 5-CH(3)-H(4)folate was enhanced by increasing pH up to 7, (iii) 5-CH(3)-H(4)folate was relatively pressure stable at temperatures lower than 40 degrees C, and (iv) ascorbic acid enhanced both the thermo- and barostabilities of 5-CH(3)-H(4)folate. In food products, temperature and pressure stabilities of 5-CH(3)-H(4)folate were studied at different temperatures (70-120 degrees C) and different pressure/temperature combinations (from 50 to 200 MPa/25 degrees C and 500 MPa/60 degrees C). 5-CH(3)-H(4)folate in orange juice and kiwi puree was relatively temperature (up to 120 degrees C) and pressure (up to 500 MPa/60 degrees C) stable in contrast to carrot juice and asparagus. Addition of ascorbic acid (0.5 mg/g) in carrot juice resulted in a remarkable protective effect on pressure (500 MPa/60 degrees C/40 min) and temperature degradation (120 degrees C/40 min) of 5-CH(3)-H(4)folate.     

Influence of thermal treatment on black currant (Ribes nigrum L.) juice aroma

The influence of thermal treatment on black currant juice aroma was investigated in temperature and time ranges relevant for black currant juice concentration processes, namely, 45, 60, 75, and 90 degrees C. Forty-nine aroma compounds were quantified, and the thermal treatment resulted in concentration increases of most terpenes, aldehydes, furans, and phenols, whereas the concentration of esters slightly decreased. Higher temperatures and longer exposure times had larger effects on the aroma compounds. Odor triangle tests showed no sensory difference between pasteurized juice and juice heated at 60 degrees C, whereas juice heated at 90 degrees C differed significantly from pasteurized juice. It is concluded that a 90 degrees C thermal treatment of black currant juice, which is in the temperature range used for conventional evaporation of black currant juice, has an effect on the aroma and sensory properties.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Air-liquid partition coefficients of aroma volatiles in frozen sugar solutions

Static headspace gas chromatography was used to measure air-solution partition coefficients of homologous series of methyl ketones and ethyl esters in aqueous sugar solutions. The measurements were performed in a temperature range from 25 to -25 degrees C. A rather unexpected temperature dependence of the partition coefficients was observed at subzero temperatures; that is, partition coefficients were found to increase in the temperature interval from 0 to -10 degrees C. A simple model, based on the freeze concentration effect, is proposed to explain the observed temperature dependence.     

Effect of storage temperature on the degradation of dimethoate in fortified orange and peach juices

The effect of storage temperature on dimethoate degradation in fortified orange and peach juices was studied. The insecticide was aseptically injected into packed orange and peach juices and stored at 40, 15, and 0 degrees C. Samples were taken at regular time intervals and were examined for dimethoate residues. The residues were determined with a simple gas chromatographic method; the recoveries of dimethoate from orange and peach juices were found to be from 88 to 114% for both products. The limits of determination were 0.004 and 0.003 mg/kg, respectively. From the experimental data, rate constants, half-lives, and activation energies for the decomposition of dimethoate in orange and peach juices were evaluated. During the storage of fruit juices in refrigerated rooms (0 degrees C) half-lives of dimethoate were found to be largely extended, being 1733 days for orange juice and 2310 days for peach juice. Corresponding times for storage at 15 degrees C were 533 days for both juices and for storage at 40 degrees C 24 days for orange juice and 24.6 days for peach juice. The activation energy for dimethoate in orange juice was 22.3 kcal/mol and for peach juice, 21. 2 kcal/mol.