Polyphenoloxidase deactivation kinetics during ohmic heating of grape juice

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Journal of Food Engineering 85 (2008) 410–417 www.elsevier.com/locate/jfoodeng

Polyphenoloxidase deactivation kinetics during ohmic heating of grape juice _ ß_ier a,*, Hasan Yildiz b, Taner Baysal a Filiz Ic b

a Ege University, Faculty of Engineering, Food Engineering Department, Bornova, 35100 Izmir, Turkey Celal Bayar University, Faculty of Engineering, Food Engineering Department, Muradiye, Manisa, Turkey

Received 6 April 2006; received in revised form 1 August 2007; accepted 2 August 2007 Available online 8 August 2007

Abstract The heating method affects the temperature distribution inside a food, and directly modifies the time–temperature relationship for enzyme deactivation. Fresh grape juice was ohmically heated at different voltage gradients (20, 30, and 40 V/cm) from 20 °C to temperatures of 60, 70, 80 or 90 °C and the change in the activity of polyphenoloxidase enzyme (PPO) was measured. The critical deactivation temperatures were found to be 60 °C or lower for 40 V/cm, and 70 °C for 20 and 30 V/cm. Various kinetic models for the deactivation of PPO by ohmic heating at 30 V/cm were fitted to the experimental data. The simplest kinetic model involving one step first-order deactivation was better than more complex models. The activation energy of the PPO deactivation for the temperature range of 70–90 °C was found to be 83.5 kJ/mol. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Ohmic heating; Enzyme; Grape juice; Polyphenoloxidase; Kinetic

1. Introduction Mechanical damage during harvesting and transportation, and some processes including slicing, dicing, pulping, etc. lead to enzymatic browning and colour degradation in many fruits and vegetables. Heating treatment is often used to deactivate the appropriate enzymes (Yemenicioglu & Cemeroglu, 1998). Indicator enzymes can be used to determine the blanching efficiency of fruit and vegetables. One of these indicator enzymes is polyphenoloxidase (PPO) (Yemenicioglu & Cemeroglu, 1998). PPO (E.C.1.14.18.1) is an oxidoreductase enzyme and contains copper. It is also known as catecholoxidase, cathecholase, diphenyloxidase, o-diphenolase, phenolase. PPO causes browning by catalysing the oxidation of monophenolic compounds to o-diphenols and o-dihydroxy compounds to o-kinons (Van Loey, *

Corresponding author. Tel.: +90 232 3880110x3021; fax: +90 232 3427592. _ ß_ier). E-mail address: fi[email protected] (F. Ic 0260-8774/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2007.08.002

Verachtert, & Hendrickx, 2002). PPO catalyses the oxidation of o-phenolic substrates to o-quinones, which are subsequently polymerized to dark-coloured pigments. This metallo-enzyme which is widely distributed in plants, is considered to be the main contributor to browning, discoloration and darkening in fruits and vegetables (Billaud, Brun-Merimee, Louarme, & Nicolas, 2004). Enzymatic browning can be inhibited by using chemicals such as ascorbic acid, sulphites, sodium diethyl dithiocarbamate, etc., or heat treatment (Ilhami, Kufrevioglu, & Munir, 2005; Kim, Kim, & Park, 2005). The deactivation of PPO by thermal treatment has been reported as the most effective method to control enzymatic browning Weemaes, Ludikhuyze, Van den Broeck, Hendrickx, and Tobback (1998). Tate, Luh, and York (1964) have suggested that heat deactivation treatments should be rapid since slow blanching processes might result in activation of the PPO in the plant tissue rather than deactivation. The effect of heat treatment on enzyme activity has been described by several mathematical models. The deactivation of enzymes was evaluated by kinetic models by fitting

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Nomenclature area of cross section of the electrodes (m2) relative residual activity (dimensionless) enzyme activity (u) completely deactivated form of enzyme native form of enzyme partially active enzyme species activation energy current (A) irreversibly deactivated isozyme forms kinetic constants (1/min) pre-exponential term of the Arrhenius equation and kinetic constant at reference temperature (1/min) L distance between the electrodes (m) M.E.E. mean estimated error N1,N2 native isozyme forms R ideal gas constant (8314.34 m3 Pa/kg mol K)

A a C D E E* Ea I I1,I2 k1,k2 kref

either a single deactivation curve at one temperature (Bryjak, Ciesielski, & Zbicinski, 2004; Leksawasdi, Breuer, Hauer, Rosche, & Rogers, 2003) or the simultaneous evaluation of deactivation curves obtained at different temperatures (Cruz, Vieira, & Silva, 2006; Illeova, Polakovic, Stefuca, Acai, & Juma, 2003; Ladero, Ferrero, Vian, Santos, & Garcia-Ochoa, 2005). Some researchers also used multi-temperature evaluation for the kinetic studies of more complex enzyme systems (Polakovic & Bryjak, 2002). The simplest model applied in kinetic studies of isothermal enzyme deactivation has been the first-order kinetics. For the enzyme structures having isozymes, biexponential models leading to completely deactivated isozyme forms were also used (Cruz et al., 2006; Polakovic & Bryjak, 2002). Ladero et al. (2005) compared the models to describe the thermal deactivation of complex enzyme structures. They applied statistical criteria including correlation coefficient, residual sum of squares, F value, standard error of estimation and physical criteria to check the suitability of the model. In recent years there has been an increase in studies on deactivation of enzymes by novel techniques such as high hydrostatic pressure, manothermosonication, electrical treatments, etc. (Castro, Macedo, Teixeira, & Vicente, 2004; Castro, Teixeira, Salengke, Sastry, & Vicente, 2004; Icier, Yildiz, & Baysal, 2005; Van Loey et al., 2002; Vercet, Sanchez, Burgos, Montanes, & Lopez Buesa, 2002). Ohmic heating is based on the passage of electrical current through a food product that has electrical resistance. The electrical energy is converted to heat. Instant heating occurs depending on the current passing through the food material. The uniform heat generation that results gives uniform temperature distribution, especially for liquid foods. Ohmic heating is used in a wide range of applications such as preheating, blanching, pasteurization, sterilization,

R.M.S. residual mean of squares R.S.S. residual sum of squares S.E.E. standard error of estimation Std. error standard error for parameters T temperature (°C), in Eq. (6) (K) t time (min) V voltage applied (V) a ratio between the initial activity of isozyme form N1 to the total initial activity (dimensionless) b activity ratio between enzyme species (dimensionless) r electrical conductivity (S/m) Subscripts 0 initial conditions (at zero holding time) i enzymatic i species

extraction of food products (Leizerson & Shimoni, 2005; Lima & Sastry, 1999; Mizrahi, 1996). Its advantages compared to conventional heating include maintaining the colour and nutritional value of food, short processing time and higher yield (Castro, Teixeira, et al., 2004; Icier, 2005; Icier et al., 2005; Leizerson & Shimoni, 2005; Vikram, Ramesh, & Prapulla, 2005; Wang & Sastry, 2002; Yildiz, 2004). There have been limited researches on the effect of ohmic heating on enzymes. Castro, Macedo, et al. (2004) compared the deactivation of different enzymes samples heated with ohmic or conventional heating. They showed that the electrical field applied during ohmic heating caused the faster deactivation than the conventional heating. Leizerson and Shimoni (2005) found that ohmic heating reduced pectinmethylesterase activity by 98%. Icier, Yildiz, and Baysal (2006) reported that peroxidase in pea puree was deactivated in a shorter time by ohmic heating as compared to conventional heating. They determined the enzyme activity qualitatively and suggested that ohmic heating caused less browning than conventional heating. In this study, the effects of ohmic heating at pasteurization temperatures range on PPO activity in fresh squeezed grape juice were investigated. The objectives were to measure the effect of voltage gradient, temperature and holding time on the PPO activity in grape juice ohmically heated to 70–90 °C and to fit models to the deactivation kinetics. 2. Material and methods 2.1. Material Organic seedless Sultana grapes were used. They were obtained from a single vineyard located in Manisa, Turkey. The samples were immediately transferred to laboratory and stored at 4 °C.

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Approximately 150 g samples from the grape bulk were taken. Each sample was washed prior to the process. The grapes were removed from bunches and then the excess water was removed. The grape juice was produced by using a juice extractor (Kamet 04369.01, Germany). A 19 ml sample of fresh grape juice was immediately transferred to the cell of ohmic heater, and electrical heating was carried out. 2.2. Electrical treatment A static ohmic heating system including an isolated power supply, microprocessor board and a Pyrex test cell was used. Teflon coated electronic temperature sensors (Omega Eng. Inc., Stanford, CT) with a compression fitting were used to measure the temperature at the different sections of the sample in the test cell. Temperature uniformity was checked during previous heating experiments by measuring the temperatures at seven different locations in the test cell. Since the temperature variation at different points inside the test cell was ±1 °C during heating, the ohmic heating process was assumed as uniform. Therefore, only the temperature in the center of the test cell was measured. The microprocessor board monitored the current and voltage applied at constant time intervals of 1 s. Details of ohmic heating system used are given by Icier (2003). In the first part of the study, the grape juices were heated from 20 °C to 60, 70 and 80 °C at 20 and 40 V/cm, and from 20 °C to 60, 70, 80, and 90 °C at 30 V/cm. The changes in PPO activity with temperature were measured. The heating rate was expressed as temperature increase per unit of time (°C/s). Electrical conductivity (S/m) was calculated from voltage and current data using the following equation (Icier & Ilicali, 2005); LI r¼ AV

ð1Þ

In the second part of the study, the grape juices were heated to 60, 70, 80 or 90 °C at 30 V/cm, and were held at these temperatures for 5, 10, 15, 20 and 25 min. During the holding period, a constant voltage gradient was applied and temperature was kept constant within ±0.5 °C by manual on–off control of the power supply. The heating experiments were replicated three times. The residual PPO activities were determined immediately. Previous research have shown an effect of the electric field on enzyme deactivation over and above any thermal deactivation so control experiments with conventional heating to the same temperatures and with the same holding times were not performed (Baysal, Icier, & Yildiz, 2006; Espachs-Barroso, Van Loey, Hendrickx, & Martı’n-Belloso, 2006; Icier et al., 2006; Mizrahi, Kopelman, & Perlman, 1975; Van Loey et al., 2002; Yeom, Streaker, Zhang, & Min, 2000.). 2.3. Determination of PPO activity The PPO activity was determined by the method given in Shin, Froderman, and Flurkey (1997) with some modifi-

cations. Just after the ohmic heating, 6 ml of heated grape juice was added to 9 ml of buffer solution having a pH of 7.0 at 4 °C, and was shaken. The solution was centrifuged at 4000 rpm for 30 min at 4 °C by using a refrigerated benchtop centrifuge (Hettich Zentrifugen Universal, Tuttlingen, Germany). The upper phase was filtered through glass wool, and stored at 4 °C for 30 min until the spectrophotometric analyses. Reactivation was not detected during this storage period. Spectrophotometric analyses were carried out at 4 °C by using Varian Cary 50 Scan (Australia) model spectrophotometer, at 435 nm for 10 min in 0.1 s interval. The absorbance of 2 ml of buffer and 1 ml of catechin (5 mM) solution was used as the blank. The absorbance of the solutions containing 1 ml of buffer, 1 ml of catechin and 1 ml of filtered sample was determined. The PPO activity was expressed as units (1 unit = change in absorbance per minute/10). Enzyme activity analyses were replicated three times for each heating run. 2.4. Kinetic model Four different kinetic models were fitted to the isothermal deactivation of PPO during ohmic heating. Models 1 and 2 involved completely deactivation of enzyme whereas models 3 and 4 included degradation to a denatured form with some residual activity. Model 1: The enzyme deactivation is assumed to be a one step irreversible first-order reaction. k1

E!D The kinetic equation is a¼

C ¼ ek1 t C0

ð2Þ

where a is the relative enzyme activity, which was the ratio of the current (C) and initial (C0; at zero holding time) values of enzyme activity. The coefficient k1 represented the first-order rate constant for a one step irreversible transition of the native enzyme into an inactive form. Model 2: The second mechanism is parallel one step irreversible reactions of two native isozyme forms N1 (heat labile) and N2 (heat resistant) to irreversibly deactivated enzyme forms I1 and I2 (Polakovic & Bryjak, 2002); k1

N 1 ! I 1;

k2

N2 ! I2

The kinetics are given by C ¼ C 01 ek1 t þ C 02 ek2 t

ð3Þ

C01 and C02 are the initial enzyme activities of heat labile and resistant isozyme forms, respectively (Cruz et al., 2006). The a value, which is the initial ratio of the isozyme form N1 to the total enzyme activity (N1/(N1 + N2)), was used to

_ßier _ et al. / Journal of Food Engineering 85 (2008) 410–417 F. Ic

describe the initial distribution of the isozyme forms. It should be independent of the temperature (Cruz et al., 2006). Model 3: The third mechanism is a one step deactivation to a partially active enzyme species (E*) (Ladero et al., 2005);

2.5. Statistical analysis Statistical evaluation and non-linear regression analyses were performed using SPSS Ver.11.0.1 statistical package (SPSS, 2001). The statistical criteria applied to discriminate among the kinetic models were R2 (correlation coefficient), adjusted R2, residual sum of squares (R.S.S., the lower the better), mean estimated error (MEE), the F value, and standard errors (std. error) for each coefficient. The confidence level used to determine statistical significance was 95%. The physical criteria used to compare models were that parameters at a given temperature must not be negative and that the activation energy of kinetic constants must be positive. Kinetic modeling was performed on three holding temperatures (70, 80 and 90 °C) at 30 V/cm; three replicated enzyme activity analyses for each of three replicated heating runs were performed giving nine enzyme activity values for each holding time. Thus a total of 54 (9  6) data points were used for determination of kinetic constants at each temperature.

k1

E ! E : The kinetic equation of model 3 is a¼

C ¼ ð1  bÞek1 t þ b C0

ð4Þ

b is the ratio of the activity of the natural enzyme to the partly inactive form. Model 4: The last kinetic model involves two first-order reactions in series with residual enzyme activity of the intermediate and final enzyme forms (Ladero et al., 2005); k1

k2

E ! E1 ! E2 The kinetic equation of model 4 is   C k1 k2 ¼ 1 þ b1  b2 a¼ ek1 t C0 k2  k1 k2  k1 k1  ðb  b2 Þek2 t þ b2 k2  k1 1

3. Results and discussion The ohmic heating rate of grape juice is given in Fig. 1. The time required to heat the grape juice from 30 to 80 °C at 20 V/cm was 2.2 and 3.7 times longer than at 30 and 40 V/cm, respectively. The changes in electrical conductivity of grape juice with temperature during ohmic heating at three different voltage gradients are given in Fig. 2. The electrical conductivity increased as the temperature increased. Between 55 and 75 °C, the electrical conductivity at 40 V/cm was slightly higher than that at 20 or 30 V/cm. Similar effects of voltage gradient on electrical conductivity of fruit juice and purees has been previously reported (Icier & Ilicali, 2004, 2005). In addition, Castro, Teixeira, et al. (2004) also reported an increase of electrical conductivity with field strength for strawberry pulps and strawberry filling during ohmic heating. The changes in PPO activity at different voltage gradients and temperatures are given in Fig. 3. As the temperature

ð5Þ

where b1 and b2 are activity ratios between enzyme species for the first and second step of the deactivation, respectively. 2.4.1. Temperature dependency The temperature dependency of the rate constant k1 was fitted to the Arrhenius equation (Eq. (6)) for all kinetic models:    k 1 ¼ k 1;ref e

Ea R

1 1 T T ref

413

ð6Þ

Tref (reference temperature) was taken to be the average temperature of the ohmic heating experiments (Tref = 80 °C).

Temperature (ºC)

85 75 65 55 45 35 25 0

20

40

60

80

100

120

Time (s) Fig. 1. Ohmic heating curves of grape juice at different voltage gradients. (s) 20 V/cm; (–) 30 V/cm and () 40 V/cm.

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Electrical conductivity (S/m)

414

0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 30

40

50

60

70

80

Temperature (ºC) Fig. 2. The changes in electrical conductivity of grape juice with temperature during ohmic heating at different voltage gradients. (s) 20 V/cm; (–) 30 V/cm and () 40 V/cm.

3.0

Enzyme activity (Unit)

2.5 2.0 1.5 1.0 0.5 0.0

0

20

40

60

80

100

Temperature (ºC) Fig. 3. The changes in PPO activity of grape juices heated to different temperatures at different voltage gradients. (–N–) 20 V/cm; (–j–) 30 V/cm and (––) 40 V/cm.

increased from 20 to 60 °C, enzyme activity increased at all voltage gradients. The critical temperature at which the deactivation starts was lower at 40 V/cm than that at 20 or 30 V/cm. The enzyme activity was also found to be significantly lower at 40 V/cm than at 20 or 30 V/cm at 70 and 80 °C. Yang, Li, and Zhang (2004) reported similar deactivation of pepsin activity as a function of applied electric field strength, electrical conductivity, and pH. They also reported that higher electrical conductivity increased the deactivation of pepsin by pulsed electric field at the same temperature. As shown in Fig. 3, the maximum PPO activity was detected at 70 °C for 20 and 30 V/cm. At 40 V/cm the maximum activity was at 60 °C but a lack of data at lower temperatures meant that the critical temperature may be lower than 60 °C. Similar results were reported in a previous study where the maximum PPO activity was found at 70 °C for 35 V/cm (Icier et al., 2005). The critical deactivation temperature of PPO was reported as 68 °C in apple (Yemenicioglu, Ozkan, & Cemeroglu, 1997) and 45 and 60 °C in cocoa (Lee, Lee, & Karim, 1991).

Although the greatest deactivation was obtained with a voltage gradient of 40 V/cm, grape juice could not be heated to 90 °C without bubbling at this high voltage gradient. Based on these results, a voltage gradient of 30 V/cm was chosen for the isothermal experiments because it appeared to give practicable deactivation in the temperature range of interest (70–90 °C). In this temperature range, which corresponds to low temperature pasteurization of fruit juice, slight activity remained at very long deactivation times (Figs. 3 and 4). At 60 °C where the highest enzyme activity observed (Fig. 3), an small increase in the activity with holding time was also observed until deactivation started after 15 min (Fig. 4). (Margot, Flaschel, & Renken, 1997) reported similar results for the deactivation of trypsin at temperatures ranging from 55 to 70 °C. Cruz et al. (2006) found that the application of thermosonication gave an increase in the peroxidase enzyme activity in watercress in the temperature range of 40–80 °C. The increase in the enzyme activity at constant temperature can be explained by the change of conformation of the enzyme to give higher enzyme–substrate interaction and conse-

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415

3.5

Enzyme activity (unit)

3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

5

10

15 Time (min)

20

25

30

Fig. 4. The inactivation curves of PPO at different ohmic holding temperatures at 30 V/cm. (––) 60 °C; (–j–) 70 °C; (–N–) 80 °C and (––) 90°C. ( model 1, ( ) model 2, ( ) model 3 and ( ) model 4.

quently to an optimal consumption of the substrate (Cruz et al., 2006). Also the presence of an electric field can influence biochemical reactions by changing molecular spacing and increasing interchain reactions (Castro, Macedo, et al., 2004). As the ohmic holding temperature increased, the deactivation of the PPO enzyme increased for the same holding time (Fig. 4). The effect of temperature, holding time and their interaction on the activity of PPO was significant (p < 0.05). For the temperature range of 70–90 °C, similar deactivation curves were obtained in recent studies; for glucoamylase in Polakovic and Bryjak (2002), for urease in Illeova et al. (2003), for b-galactosidase in Ladero et al. (2005). Castro, Macedo, et al. (2004) suggested that the presence of the electric field might remove the metallic prosthetic groups present in the PPO enzyme, thus causing the enhancement of enzyme activity loss. Similarly Giner, Bailo, Gimeno, and Martin-Belloso (2005) suggested that any increase of electric field would yield considerable improvement in the effectiveness of pulsed electric field treatments in reducing pectinesterase activity. Moreover, in a previous study (Icier et al., 2005), the residual PPO activity in grape juice was 35% after 14 min at 70 °C with ohmic heating by applying 35 V/cm. In the present study at 70 °C and 30 V/cm, the value was approximately 50% which is very similar. Overall there was little deactivation at 60 °C so this data was eliminated from the kinetic modeling. For the temperature range of 70–90 °C, Table 1 summarizes the fitting of the four different kinetic models for the deactivation of PPO enzyme. A usual first step in analyzing the kinetics of deactivation of any enzyme is to check the suitability of first-order kinetics. Residual sum of squares for the first model decreased from 0.063 at 70 °C to 1.14  103 at 90 °C. The corresponding mean errors of estimate varied between 0.017 and 0.110 and the R2 values were high which confirmed the suitability of first-order kinetics. It is well known

)

that first-order kinetics corresponding to one step irreversible transition of native form is suitable for small monomeric enzymes, whereas the existence of intermediate forms and deviation from first-order kinetics can be expected for the enzymes having a more complex structure and high molecular weight (Illeova et al., 2003). For the temperature range studied, the using of a one step first-order model to a totally deactivated product proved adequate. The use of more complex models did not improve the fits, which was reflected in similar R.S.S. values for the kinetic models with a higher number of fitting parameters (Table 1). Standard errors for estimated coefficients confirmed the dominant effect of rate constant k1 and Co in the models (Tables 1 and 2). For model 2 and 4, the first step rate constants increased with temperature (Table 1). This indicated the enhanced activity of intermediate species with increasing temperature. However, large uncertainty and inconsistent trends in the second step rate constant, k2, with temperature in model 2 meant that the validity of the model was questionable. The consistency of model 2 was checked against deactivation data at different temperatures. Table 1 showed that the parameter a changed with temperature. However, this is inconsistent with the isozyme mechanism since the initial distribution of isozyme forms must be constant (Polakovic & Bryjak, 2002). Thus, the deactivation of PPO in this study could not be explained by different deactivation behaviours of two isozymes. The rate constant k1 explained most of the effect of temperature on the loss of activity in temperature range used. The temperature dependency of the rate constant k1 was fitted by the exponential Arrhenius function for all models (Table 2). For models 2 and 4, the temperature dependency of the other rate constant k2 was not estimated because of its inconsistent behaviour with temperatures. Although F values and adjusted R2 of the more complex models were higher than model 1, the improvement was marginal (Table 2). The comparative plot of the four models against the

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416

Table 1 Estimated kinetic parameters for the deactivation of PPO during ohmic heating with a voltage gradient of 30 V/cm

Table 2 The temperature dependency and the activation energies of rate constant k1 obtained by different kinetic models

Parameter

Model Ea, kJ/mol kref R2 (std. error) (std. error)

Adj. R2

S.E.E. F

1

0.802

0.604

0.567

4.047 0.321

0.860

0.721

0.502

6.157 0.252

0.866

0.732

0.435

6.470 0.189

0.996

0.992

0.038

260.546 0.001

Model 1 C0 (std. error) k1 (std. error) R2 R.S.S. M.E.E.

Temperature (°C) 70

80

90

2.534 (0.110) 0.059 (0.005) 0.978 0.063 0.110

1.622 (0.019) 0.268 (0.008) 0.999 1.43  103 0.019

0.509 (0.017) 0.291 (0.026) 0.994 1.14  103 0.017

2 3 4

83.45 (41.48) 91.10 (36.71) 80.98 (31.84) 44.83 (2.78)

0.169 (0.089) 0.178 (0.076) 0.190 (0.080) 0.332 (0.022)

R.M.S.

Confidence level for all parameters: 95%.

Model 2 C01 (std. error) C02 (std. error) a (std. error) k1 (std. error) k2 (std. error) R2 R.S.S. M.E.E.

1.267 (0.295) 1.267 (0.294) 0.500 (0.001) 0.059 (0.014) 0.059 (0.014) 0.978 0.063 0.102

0.804 (0.187) 0.818 (0.190) 0.497 (0.005) 0.268 (0.062) 0.268 (0.062) 0.999 1.43  103 0.018

0.488 (0.009) 0.022 (0.009) 0.957 (0.002) 0.339 (0.015) 0.005 (0.020) 0.999 1.96  105 0.001

Model 3 C0 (std. error) B (std. error) k1 (std. error) R2 R.S.S. M.E.E.

2.545 (0.160) 0.100 (0.001) 0.071 (0.010) 0.968 0.093 0.160

1.622 (0.022) 0.001 (0.008) 0.269 (0.012) 0.999 1.42  103 0.022

0.510 (0.003) 0.038 (0.003) 0.336 (0.007) 0.999 2.01  105 0.002

Model 4 C0 (std. error) b1 (std. error) b2 (std. error) k1 (std. error) k2 (std. error) R2 R.S.S. M.E.E.

2.520 (0.122) 0.800 (0.020) 0.010 (0.003) 0.216 (0.081) 0.069 (0.016) 0.980 0.058 0.015

1.620 (0.021) 0.270 (0.011) 0.006 (0.002) 0.322 (0.391) 0.510 (0.308) 0.999 9.14  104 0.003

0.510 (0.008) 0.216 (0.004) 0.019 (0.001) 0.514 (0.053) 0.115 (0.017) 0.999 2.68  104 0.016

experiment data does not show any obvious improvement (Fig. 4). For k1 in model 1, estimated rate constant at a reference temperature of 80 °C (kref) and the activation energy were 0.17 min1 and 83.5 kJ/mol, respectively. Weemaes et al. (1998) found that the activation energies of the rate constant for the heat deactivation of grape PPO as 166 ± 27 kJ/mol for the temperature range of 65–75 °C during heating in a water bath. Overall, for the temperature range used, it was concluded that the simplest model 1 was adequate to describe the PPO deactivation in grape juice by ohmic heating.

4. Conclusion The ohmic heating rate increases as the voltage gradient increases. The critical deactivation temperature at 40 V/cm was lower than that of at 20 and 30 V/cm probably because of the faster increase in electrical conductivity at higher voltage gradients causing higher deactivation in PPO. At constant voltage gradient a small increase in the activity with holding time was observed at 60 °C until the deactivation started after 15 min. The one step firstorder kinetic model was found to adequately describe the deactivation kinetics of PPO, for the temperature range of 70–90 °C. Acknowledgement This study was a part of the project financially supported by Ege University Science Foundation EBIL_ TEM-03/BIL/024, Turkey. References Baysal, T., Icier, F., & Yildiz, H. (2006). Experimental Investigation and Modeling of the effects of Ohmic Heating on the Quality of Some Fruit and Vegetable Products. Ege University Scientific Research Projects, _ Project No: 03/BIL/024, Izmir, Turkey, unpublished report. Billaud, C., Brun-Merimee, S., Louarme, L., & Nicolas, J. (2004). Effect of glutathione and Maillard reaction products prepared from glucose or fructose with glutathione on polyphenoloxidase from apple-I: Enzymatic browning and enzyme activity inhibition. Food Chemistry, 84, 223–233. Bryjak, J., Ciesielski, K., & Zbicinski, I. (2004). Modelling of glucoamylase thermal inactivation in the presence of starch by artificial neural network. Journal of Biotechnology, 114, 177–185. Castro, I., Macedo, B., Teixeira, J. A., & Vicente, A. A. (2004). The effect of electric field on important food-processing enzymes: Comparison of inactivation kinetics under conventional and ohmic heating. Journal of Food Science, 69(9), 696–701. Castro, I., Teixeira, J. A., Salengke, S., Sastry, S. K., & Vicente, A. A. (2004). Ohmic heating of strawberry products: Electrical conductivity measurement and ascorbic acid degradation kinetics. Innovative Food Science and Emerging Technologies, 5, 27–36. Cruz, R. M. S., Vieira, M. C., & Silva, C. L. M. (2006). Effect of heat and thermosonication treatments on peroxidase inactivation kinetics in watercress (Nasturtium officinale). Journal of Food Engineering, 72, 8–15.

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