Effects of PEF and heat pasteurization on PME activity in orange juice with regard to a new inactivation kinetic model

Food Chemistry 165 (2014) 70–76

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Effects of PEF and heat pasteurization on PME activity in orange juice with regard to a new inactivation kinetic model E. Agcam a,⇑, A. Akyıldız a, G. Akdemir Evrendilek b a b

Department of Food Engineering, Faculty of Agriculture, Cukurova University, Balcali, Adana, Turkey Department of Food Engineering, Faculty of Engineering and Architecture, Abant Izzet Baysal University, Golkoy Campus, Bolu, Turkey

a r t i c l e

i n f o

Article history: Received 5 February 2014 Received in revised form 5 May 2014 Accepted 16 May 2014 Available online 27 May 2014 Keywords: Pulsed electric fields (PEF) Pectin methyl esterase (PME) Orange juice Inactivation kinetics Shelf-life

a b s t r a c t The inactivation kinetics of pectin methyl esterase (PME) during the shelf life (4 °C-180 days) of freshly squeezed orange juice samples processed by both pulsed electric fields (PEF) and heat pasteurization (HP) was evaluated in the study. The PME inactivation level after the PEF (25.26 kV/cm-1206.2 ls) and HP (90 °C-20 s) treatments were 93.8% and 95.2%, respectively. The PME activity of PEF-processed samples decreased or did not change, while that of HP samples increased during storage (p < 0.01). A kinetic model was developed expressing PME inactivation as a function of the PEF treatment conditions, and this enabled the estimation of the reaction rate constant (587.8–2375.4 s1), and the time required for a 90% reduction (De, 3917.7–969.5 s). Quantification of the increase in PEF energy to ensure a ten-fold reduction in De (ze, 63.7 J), activation electric fields (921.2 kV cm1mol1), and electrical activation energy (12.9 kJ mol1) was also carried out. Consequently, PEF processing was very effective for the inactivation of PME and for providing stability of orange juice during storage. Ó 2014 Published by Elsevier Ltd.

1. Introduction Fresh orange juice is one of the mostly consumed juices worldwide owing to its rich vitamin C content and fresh taste. Pectin methyl esterase (PME) is an enzyme with a major impact on orange juice that leads to quality losses due to its adverse effect on clarification of orange juice, or gelation of concentrated orange juice, if not inactive (Basak & Ramaswamy, 2001). The pectin content of the juice, which would lead to pectate (LMP) formation, is deesterified by PME enzyme. Pectate’s reaction with calcium ion causes gelation and cloud stability loss phenomenon (Kimball, 1991; Zhang et al., 2011). Cloud stability plays an important role in turbidity, flavor, aroma and characteristic colour of citrus juices (Baker & Cameron, 1999; Van Den Broeck, Ludikhuyze, Van Loey, & Hendrickx, 2000). Therefore, in order to control PME-induced quality losses, orange juice must be inactivated at high temperatures (Cameron, Niedz, & Grohmann, 1994). As PME is more thermally resistant than most vegetative spoilage microorganisms, the main aim of orange juice pasteurization is the inactivation of PME (Chen & Wu, 1998). Previous studies reported that 22%, 48%, 87%, 94%, and 95% inactivations of PME were obtained after thermal pasteurization at 57.2, 60.0, 68.3, ⇑ Corresponding author. Tel.: +90 322 3386537; fax: +90 322 3386614. E-mail address: [email protected] (E. Agcam). http://dx.doi.org/10.1016/j.foodchem.2014.05.097 0308-8146/Ó 2014 Published by Elsevier Ltd.

73.9, and 79.4 °C for 41 s processing time, respectively in Valencia orange juice (Atkins, Rouse, & Moore, 1956). However, thermal processing has negative impacts on the quality of orange juice such as losses of colour, flavor and nutritional values (Espachs-Barroso, Van Loey, Hendrickx, & Martin-Belloso, 2006). Application of pulsed electric fields (PEF) at relatively lower temperatures to inactivate foodborne and food spoilage bacteria, and enzymes, while preserving the nutritional and sensory properties are well described in the literature (Yeom, Streaker, Zhang, & Min, 2000; Giner, Gimeno, Palomes, Barbosa-Cánovas, & Martin, 2003; Barbosa-Cánovas, Tapia, & Cano, 2005; Elez-Martínez, Aguiló-Aguayo, & Martin-Belloso, 2006). Better understanding and accurate predictions of inactivation levels by PEF are necessary to achieve enzymatically stable products without over-processing (Bendicho, Estela, Giner, Barbosa-Cánovas, & Martin, 2002; Elez-Martínez et al., 2006). Several models have been developed to elucidate PEF-induced inactivation of some enzymes including the classical exponential decay model, the Hülsheger’s and Fermi’s empirical models for PME inactivation in tomato juice, the firstorder kinetic model, as well as models based on Fermi, Hülsheger or Weibull equations for pectinesterase (PE) inactivation in a commercial enzyme preparation (CEP) (Bendicho, Barbosa-Cánovas, & Martin, 2003; Elez-Martínez, Soliva-Fortuny, & Martín-Belloso, 2006; Giner et al., 2003; Giner, Grouberman, Gimeno, & Martin, 2005; Giner et al., 2000). Although information about PEF-related

E. Agcam et al. / Food Chemistry 165 (2014) 70–76

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Nomenclature A Ae Ao cp D De Ea Eae Eap Ee Ep Ee1, Ee2 I ke k0 kT

residue PME activity after pasteurization effective electric fields area in the treatment chamber (cm2) initial PME activity specific heat of the medium (J g1 K1) diameter of the chamber (cm) time required 90% reduction of PME activity at certain temperature thermal activation energy (J mol1) electrical activation energy (J mol1) activation electric fields (kV cm1 mol1) treatment electrical energy (J) electrical field strengths (volt cm1) the energies (J) for two different PEF treatments for the calculation of ze value current (ampere) reaction rate constant according to treatment energy (J1) Arrhenius constant (s1) reaction rate constant at certain temperature (s1)

inactivation and stability parametres of enzymes as well as the effects of storage on PME activity during shelf life have been reported, not enough studies were conducted for the development of mathematical models (Riener, Noci, Cronin, Morgan, & Lyng, 2009; Sampedro, Geveke, Fan, & Zhang, 2009; Vervoort et al., 2011; Yeom et al., 2000). Therefore, the objectives of this study were to quantify the effectiveness of PEF treatment conditions for PME activity during storage at 4 °C for 180 days, and to develop an inactivation kinetic model to estimate processing variables of orange juice as a function of PEF treatments. 2. Materials and methods 2.1. Orange juice Kozan-specific variety of oranges grown in the Cukurova Region (Turkey) was used in the study. Kozan Yerli orange juice had titratable acidity (TA), total soluble solids (TSS), TSS/TA and pH of 1.19% as citric acid equivalent, 11.83%, 9.94% and 3.53%, respectively. After being washed, oranges were peeled, cut into two halves, and pressed by a bench-scale automatic orange squeezing machine (CANCAN, Turkey). Orange juice was passed through 1-mm stainless steel sieves to remove seeds and coarse pulps. Juice was immediately processed by PEF and heat pasteurization units. Untreated (U) and processed samples were stored at 4 °C for 180 days and analyzed at every 60 day period. Untreated samples were fermented within 10 days, while analysis of the untreated samples was continued during storage. 2.2. PEF treatments A laboratory-scale PEF OSU-4A system (Evrendilek, Zhang, & Richter, 2004) providing square wave bipolar pulses, with 6 co-field flow treatment chambers with 0.23 cm of diametre was used for PEF treatments. The gap distance between the electrodes was 0.292 cm, and flow rate of orange juice in the treatment chambers was 0.633 mL s1. PEF processing of orange juice samples were conducted with different PEF processing parametres that each process was coded as E1 (13.8 kV cm1 electric field strength,

kt L n R Rd T T0 t t0 V ze

s rT q DT

reaction rate constant in certain voltage and current (s1) distance between electrodes (cm) number of chambers universal gas constant (J mol1 K1) resistance (ohm) temperature (K) final temperature of product after PEF treatment (K) total treatment time (s) time in minute for PME activation voltage (volt) an increase required for PEF energy to ensure a ten-fold reduction in De value. pulse width (ms) electrical conductivity of the fluid medium at processing temperature (S cm1) density of fluid medium at processing temperature (g cm3) estimated temperature increase per pair of chamber (°C)

1033.9 ls treatment time, 344.6 pulse number, 10.9 J energy), E2 (13.8 kV cm1 electric field strength, 1206.2 ls treatment time, 402.1 pulse number, 12.7 J energy), E3 (17.1 kV cm1 electric field strength, 1033.9 ls treatment time, 344.6 pulse number, 17.4 J energy), E4 (17.1 kV cm1 electric field strength, 1206.2 ls treatment time, 402.1 pulse number, 20.3 J energy), E5 (21.5 kV cm1 electric field strength, 1033.9 ls treatment time, 344.6 pulse number, 29.6 J energy), E6 (21.5 kV cm1 electric field strength, 1206.2 ls treatment time, 402.1 pulse number, 34.5 J energy), E7 (25.3 kV cm1 electric field strength, 1033.9 ls treatment time, 344.6 pulse number, 44.0 J energy) and E8 (25.3 kV cm1 electric field strength, 1206.2 ls treatment time, 402.1 pulse number, 51.3 J energy). The frequency and the pulse width of the PEF processing were 500 s1 and 3 ls, respectively (Table 1). Treatment temperature was measured during PEF processing before and after each pair of PEF treatment chamber (T1, T2, T3 and T4) by K type dual channel digital thermocouples (Fisher Scientific, Pittsburgh, PA, USA). PEF processing was conducted at 35 °C (water bath temperature); however, with increased electric field strength, the application temperature (Table 2) was raised up to 58.20 °C. After PEF processing, the orange juice samples were packed in sterile, twist-off amber coloured bottles (250 mL) inside a laminar flow hood. The samples were stored at 4 °C for 180 in darkness for further analyses. The samples were analyzed on the 0, 60, 120 and 180th day.

2.3. Heat pasteurization treatment The bench scale system designed in the Department of Food Engineering of Cukurova University (Adana, Turkey) was used for heat pasteurization applications (Agçam, Akyildiz, & Evrendilek, 2014). As a result of the preliminary experiments, heat pasteurization at 90 °C for both 10 s (HP1) and 20 s (HP2) was applied. After heat pasteurization, the orange juice samples were packed in sterile, twist-off amber coloured bottles (250 mL) inside a laminar flow hood. The samples were stored at 4 °C for 180 in darkness for further analyses. The samples were analyzed at every 0, 60, 120 and 180th days.

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Table 1 PEF processing parametres of orange juice. Symbol

Electric field strength (Ep, kV cm1)

Current (I, A)

Voltage (V, kV)

Treatment time (t, ls)

Frequency (s1)

Pulse number (n)

Pulse width (s, ls)

Energy (E, J)

E1 E2 E3 E4 E5 E6 E7 E8

13.8 13.8 17.1 17.1 21.5 21.5 25.3 25.3

2.6 2.6 3.4 3.4 4.5 4.5 5.8 5.8

4.0 4.0 5.0 5.0 6.3 6.3 7.4 7.4

1033.9 1206.2 1033.9 1206.2 1033.9 1206.2 1033.9 1206.2

500 500 500 500 500 500 500 500

344.6 402.1 344.6 402.1 344.6 402.1 344.6 402.1

3 3 3 3 3 3 3 3

10.9 12.7 17.4 20.3 29.6 34.5 44.0 51.3

Table 2 Temperature and conductivity values of PEF-treated orange juice samples. Symbol

T1 (°C)

T2 (°C)

T3 (°C)

T4 (°C)

Ta (°C)*

rT (mS cm1) **

E1 E2 E3 E4 E5 E6 E7 E8

17.5 ± 1.0 16.5 ± 1.3 16.3 ± 0.5 17.0 ± 1.2 17.6 ± 1.6 18.6 ± 0.9 18.6 ± 0.9 20.2 ± 1.3

33.8 ± 0.6 33.8 ± 1.9 36.3 ± 1.0 36.5 ± 1.3 41.2 ± 2.2 39.6 ± 0.9 39.8 ± 0.8 47.6 ± 1.3

33.8 ± 1.0 39.8 ± 1.3 41.3 ± 3.6 41.0 ± 1.2 43.3 ± 0.7 43.4 ± 1.1 43.6 ± 1.1 44.4 ± 0.6

42.5 ± 1.2 43.0 ± 1.4 48.0 ± 1.8 46.3 ± 1.0 52.0 ± 2.4 51.4 ± 1.1 51.2 ± 1.3 58.2 ± 2.2

31.9 ± 0.7 33.3 ± 1.4 35.4 ± 1.5 35.2 ± 0.9 38.5 ± 1.4 38.3 ± 0.5 38.3 ± 0.5 42.6 ± 0.9

0.45 0.45 0.45 0.45 0.46 0.46 0.46 0.46

⁄⁄⁄ * **

± values are standard deviation. Average temperature in application chambers used in calculation kinetic data. Conductivity value of orange juice at average temperature.

2.4. Conductivity measurement The conductivity of the samples was measured using a handheld conductivity metre (Sension 5 model, HACH, CO, USA) at room temperature (22–25 °C).

universal gas constant (J mol1 K1), and k0 is the Arrhenius constant (s1). PEF processing parametres can be calculated as follows:

V ¼ I:Rd

ð5Þ

Ep ¼ V=L

ð6Þ

Ee ¼ V:I:t

ð7Þ

2.5. Determination of PME activity For the measurement of PME activity, 10 mL of orange juice were mixed with 20 mL of 1% pectin-salt substrate (0.1 M NaCl) and incubated at 30 °C. The solution was adjusted to pH 7.0 with 2.0 N NaOH, and solution pH was re-adjusted to pH 7.7 with 0.05 N NaOH. After the pH reached 7.7, 0.10 mL of 0.05 N NaOH was added. The time was measured (t0 ) until the pH of solution regained 7.7 pH value. The PME activity was calculated as follows (Kimball, 1991):

ð0:05NNaOH Þð0:10 mLNaOH Þ PME ActivityðAÞ ¼ ðt0 Þð10 mLsample Þ

ð1Þ

Residual PME Activityð%Þ ¼ ðA=A0 Þ  100

ð2Þ

where V is the voltage (volt), I is the current (ampere), Rd is the resistance (ohm), Ep is the electrical fields (volt cm1), L is the distance between electrodes (cm), Ee is the treatment electrical energy (J), and t is the total treatment time (s). Arrhenius equation can be re-written for PEF conditions as follows:

lnðkt Þ ¼ lnðk0 Þ  ðV:I:tÞ=ðR:TÞ

ð8Þ

lnðkt Þ ¼ lnðk0 Þ  ðV 2 :tÞ=ðR:T:Rd Þ

ð9Þ

Eq. (10) was obtained by applying Eq. (9) as follows:

where A is the residue PME activity after pasteurization and Ao is the initial PME activity.

lnðkt Þ ¼ lnðk0 Þ  ðEae =R:TÞ:ðV:rT :DÞ=ðIÞ lnðkt Þ ¼ lnðk0 Þ  ðEap =R:TÞ:ðV:t:rT :Ae Þ

ð10Þ ð11Þ 1

2.6. Developing Arrhenius equation for PEF parametres The Arrhenius equation, which expresses relationships between temperature and reaction rate, was formulated as follows: Ea

kT ¼ k0 :e R:T

lnðkT Þ ¼ lnðk0 Þ  Ea=ðR:TÞ

ð3Þ ð4Þ

where Eae is defined as the electrical activation energy (J mol ), rT is the electrical conductivity of the fluid medium at processing temperature (S cm1), D is the diametre of the chamber (cm), Eap is the activation electric fields (kV cm1 mol1) and Ae is the effective electric fields area in the treatment chamber (cm2). In the same equation, temperature (T) is accepted as a constant for cold pasteurization such as PEF and high pressure treatments, however, temperature during the PEF treatments was not assumed to be constant in this model. Accordingly, the following Eqs. (12) and (13) were derived:

1

where kT is the reaction rate constant at certain temperature (s ), T is the temperature (K), Ea is the activation energy (J mol1), R is the

lnðkt Þ ¼ lnðk0 Þ  ðEae =RÞ:ðV:rT :DÞ=ðI:T 0 Þ

ð12Þ

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E. Agcam et al. / Food Chemistry 165 (2014) 70–76

ð13Þ

T is expressed as follows: 0

T ¼ T þ DT

ð14Þ

h i DT ¼ ðEp Þ2 :n:s:r=ð0; 01:q:cp Þ

ð15Þ

where T0 is the temperature of product after PEF treatment (K), DT is the estimated temperature increase per chamber (°C), n is the number of chamber, s is the pulse width (ls), r is the electrical conductivity (mS cm1), q is the density of the fluid medium at processing temperature (g cm3), and cp is the specific heat of the medium (J g1 K1) (Evrendilek et al., 2004). In this study, PME inactivation increased with an increase in applied energy and behaved according to the first order kinetics as expressed below:

dA=dEe ¼ ke :A

ð16Þ

lnðAÞ ¼ lnðA0 Þ  ke :Ee

ð17Þ

where ke is the reaction rate constant according to treatment energy (J1). Eq. (7) was used to find the reaction constant under different PEF conditions and placed in Eq. (17).

lnðAÞ ¼ lnðA0 Þ  ke :V:I:t

ð18Þ

kt ¼ slope:V:I or kt ¼ ke :V:I

ð19Þ

lnðAÞ ¼ lnðA0 Þ  kt :t

ð20Þ

where kt expresses the reaction rate constant in certain voltage and current in s1. The slope of the function gives us the reaction rate constant per energy and defines the relationship between treatment energy and the amount of inactivation. Eq. (21) was used to calculate De and ze values of PME. De is defined as the time required in certain PEF conditions for 90% reductions in PME activity. ze value is defined as an increase required for PEF energy to ensure a ten-fold reduction in De value. Ee1 and Ee2 are the energies (J) for two different PEF treatments for the calculation of ze value.

De ¼ 2:303=kt ;

ze ¼ ðEe2  Ee1 Þ=ðlog De1  log De2 Þ

ð21Þ

2.7. Statistical analysis The software SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for analysis of variance (ANOVA) and Duncan’s multiple comparison test in order to determine significant differences between the treatments. Each experiment was repeated at least three times. 3. Results and discussion 3.1. Effects of PEF and thermal processing on PME activation The effects of different PEF and heat treatment conditions on orange juice PME activity are shown in Fig. 1a. The lowest and highest residual PME activity of the PEF treatments were found as 6.2% and 64.9%, in the E8 and E1 treatments, respectively. The residual PME activities (%) for the other PEF treatments (E2–E7) were found as 49.3, 45.8, 34.9, 15.6, 12.5, and 10.6, respectively. PME was significantly inactivated with an increased magnitude of energy (p < 0.01). Some of the previous studies conducted with PEF inactivation of PME in orange juice revealed similar results to this study. Yeom et al. (2000) reported that 90% PME inactivation was obtained after the PEF treatment (35 kV cm1, 59 ls) in Valencia orange juice.

100 Residual acvity (%)

0

80 60 40 20 0 U

E1

E2

E3

E4

E5

E6

E7

E8

HP1

HP2

Treatments

(a)

120 y = 106.205e-0.056x R² = 0.988

100 Residual activity (%)

lnðkt Þ ¼ lnðk0 Þ  ðEap =RÞ:ðV:t:rT :Ae Þ=ðT 0 Þ

80 60 40 20 0 0

10

20

30

40

50

60

Treatment Energy (J)

(b) Fig. 1. (a) Residual PME activities of orange juice samples processed with different applications; (b) PME inactivation curve according to first order kinetics.

According to Elez-Martínez et al. (2006), 80% of PME inactivation in Navelina orange juice was detected after 35 kV cm1 of electric field strength with 1500 ls treatment time at 37.5 °C. Up to 81.4% inactivation of PME was obtained in the orange-carrot juice beverage after the PEF treatment of 25 kV cm1 electric field strength, 340 ls treatment time and 63 °C processing temperature (Rodrigo, Barbosa-Cánovas, Martinez, & Rodrigo, 2003). Min, Jin, Min, Yeom, and Zhang (2003) and Zhang, Sastry, and Yousef (1996) found 88% and 95% of PME inactivation after PEF treatment of Rohde Valance orange juice and Valencia orange juice, respectively. Although the above studies revealed a significant amount of PME inactivation, other studies reported insignificant levels of inactivation for PME activity after PEF treatment. Van Loey, Verachtert, and Hendrickx (2001) tested the sensitivity of LOX, PPO, PME, and POD, dissolved in distilled water with 10, 20 and 30 kV cm1 electric field strengths, 5 and 40 ls pulse widths, 1 and 100 Hz frequencies, and 1–1000 pulses depending on the field strength applied (1–10 pulses at 30 kV cm1, 10–100 pulses at 20 kV cm1 and 100–1000 pulses at 10 kV cm1). As a result, researchers reported that no enzymes could be inactivated more than 10% by high voltage pulses. Vervoort et al. (2011) claimed that PEF treatment was not effective enough for Valencia orange juice PME inactivation and indicated 34% inactivation at 23 kV cm1 monopolar pulses of 2 ls. The PEF application conditions are very important for the amount of PME inactivation during treatments. As the effect of PEF application depends on the batch or continuous PEF systems, geometric properties of treatment chamber, pulse shape, frequency, pulse width and temperature during treatments, various researchers found different inactivation results for the same product. The residual PME activities were detected as 6.8% and 4.2% after the heat pasteurizations at 90 °C for 10 (HP1) and 20 s (HP2) in orange juice, respectively. Based on the PME inactivation results, the E8 PEF treatment was accepted as equivalent to HP1 (90 °C,

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E. Agcam et al. / Food Chemistry 165 (2014) 70–76

8.0

y = -1.547,92x + 14,53 R² = 0,987

7.8 7.6 7.4 ln(k)

7.2 7.0

slope = -Eae/R

6.8 6.6 6.4 6.2 6.0 0.0043

0.0045

0.0047

0.0049

0.0051

0.0053

(V.σT.D)/(I.T'),(1/K)

(a) 8.0

y = 110.802,22x + 4,74 R² = 0,996

7.8 7.6

ln(k)

7.4 7.2 7.0

slope = -Eap/R

6.8 6.6 6.4 6.2 6.0 1.4E-05

1.9E-05

2.4E-05

2.9E-05

(V.σT.t.Ae)/(T'),(1/K)

(b) Fig. 2. (a) Electrical activation energy calculation curve; (b) Activation electric fields calculation curve.

10 s) heat pasteurization. Yeom et al. (2000) achieved 89% inactivation of PME in orange juice applying heat treatment at 94.6 °C for 30 s. Elez-Martínez et al. (2006) reported complete inactivation of PME in orange juice through traditional heat pasteurization (90 °C, 1 min). Similarly, 98% of PME inactivation was reported by heat treatment at 98 °C for 21 s in orange-carrot juice mixture (Rivas, Rodrigo, Barbosa-Cánovas, Martínez, & Rodrigo 2006). 3.2. PME inactivation kinetics of orange juice Faster PME inactivation was detected when PEF energy increased (Fig. 1b), and PME inactivation kinetics was fitted to first order kinetics (R2 = 0.988) in orange juice. Arrhenius equation was also used to determine the effect of temperature on inactivation rate constants. The Arrhenius equation used for the PEF treatment conditions, kinetic curves and results obtained are shown in Fig. 2a, b and Table 3. kt is the reaction rate constant in certain voltage and current, and defines the rate of inactivation of PME. In other words, if the kt value is higher, PME inactivation occurs with a faster rate. In this study, with an increase in the applied PEF processing parametres,

such as the electric fields strength, the calculated kt values increased. With applied electric fields strengths (EP) of 13.8, 17.1, 21.5 and 25.3 kV cm1, the corresponding kt values were calculated as 587.8, 937.9, 1596.7 and 2375.4 s1, respectively (Table 3). The De values were estimated as 3917.7, 2455.6, 1442.3 and 969.5 ls, respectively, due to an increase in EP. In this study, the applied PEF energy was between 10.9 and 51.3 J (Table 1), and the ze value of PME was 63.7 J (Table 3). The activation energy Ea is generally defined as the energy required per mol of the reactant for the realization of a reaction. The electrical activation energy (Eae) is expressed as the energy required per mol, and the activation electric fields (Eap) is expressed as the electric fields strength required per mol for the realization of PME inactivation under the PEF conditions. The slopes of equations in Fig. 2a and b were used for calculation Eae and Eap, respectively and, Eae and Eap were determined as 12.9 kJ mol1 and 921.2 kV cm1 mol1, respectively. Thus, it is revealed that the minimum energy required for PME inactivation is 12.9 kJ mol1 under the PEF processing conditions. Vercet, López, and Burgos (1999) determined the thermal activation energy (435 kJ mol1) of orange juice PME, and reported that the value decreased to the level of 56.9 kJ mol1 under the ultrasonic conditions. Neinaber and Shellhammer (2001) processed the orange juice under high pressure and found that the activation energy decreased with an increasing pressure. The calculated activation energies were 30.1 kJ mol1 at 400 MPa pressure and 13.5 kJ mol1 at 600 MPa. According to Polydera, Galanou, Stoforos, and Taoukis (2004), the activation energy was in the range of 177 kJ mol1 at 100 MPa and 95 kJ mol1 at 750 MPa for orange juice PME. From our results it can be concluded that comparing to the other nonthermal processes such as pressure application, PEF caused a decrease in the activation energy for PME inactivation. 3.3. Changes in PME activities of orange juice during storage The orange juice samples were stored at 4 °C for 180 days, and changes in PME residual activities during storage were given in Table 4. The PME residual activities of all the PEF-treated orange juice samples were reduced with storage. At the end of storage, the lowest PME activity was detected in the sample of E8. In addition, the greatest decline was detected in the low intensity PEF applications. E1 was a low intensity PEF-treated sample and the decline in its PME activity was determined as 32.7% after six months of storage. A decrease in the initial PME activity from E2 to E8 was measured as 23.6%, 20.8%, 9.2%, 3.9%, 1.4%, 0.4% and 1.0% after six months of storage, respectively. Although the exact mechanism to explain why the decrease in PME activity was higher under low intensity PEF than under high intensity PEF is not known, it is assumed that at low intensity electric field strength the PME molecules are not completely inactivated but substructural changes occurred. During storage these molecules cannot repair themselves thus, showing greater decline during storage. When orange juice samples processed under low intensity PEF conditions, microorganisms could not be adequately

Table 3 Kinetic data according to developed mathematical model. EP (kV cm1)

⁄

kt (s1)

De (ls)

13.8 17.1

587.8 ± 7.8 937.9 ± 12.4

3917.7 ± 48.8 2455.6 ± 30.6

21.5 25.3

1596.7 ± 21.4 2375.4 ± 31.5

1442.3 ± 17.1 969.5 ± 12.1

± values are standard deviation.

ze (J)

Eap (kV cm1mol1)

Eae (kJ mol1)

63.7 ± 0.2

921.2 ± 0.8

12.9 ± 0.0

E. Agcam et al. / Food Chemistry 165 (2014) 70–76 Table 4 Changes in PME residual activities (%) during storage. Processing U E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0. Day 100.0 ± 64.9 ± 49.3 ± 45.8 ± 34.9 ± 15.6 ± 12.5 ± 10.6 ± 6.2 ± 6.8 ± 4.2 ±

0.5aA 3.8bA 0.4cA 1.3dA 0.7eA 0.0fAB 0.3gA 0.1gA 0.2hı B 0.4hB 0.3ıB

75

4. Conclusions

60. Day

120. Day

180. Day

14.9 ± 0.9C 40.5 ± 3.6aB 38.3 ± 2.4ab B 34.9 ± 1.2bB 28.3 ± 3.4cB 18.7 ± 3.4dA 10.0 ± 1.4eA 11.1 ± 3.1eA 8.0 ± 0.1eA 9.6 ± 1.3eA 7.4 ± 0.7eA

19.7 ± 2.8B 34.5 ± 6.2aB 25.7 ± 3.8bC 23.6 ± 3.1bc C 19.8 ± 1.3cC 13.0 ± 1.5dBC 10.1 ± 2.6de A 10.4 ± 0.7de A 5.9 ± 1.1eB 9.5 ± 0.6de A 8.4 ± 0.6de A

18.0 ± 1.5BC 32.1 ± 4.6aB 25.7 ± 1.6bC 25.0 ± 2.9bC 25.6 ± 0.6bB 11.9 ± 0.7cC 11.1 ± 1.0cA 10.2 ± 1.3cA 5.2 ± 0.3dB 9.4 ± 0.4cA 8.6 ± 1.0cA

⁄

Different lower case superscript letters in the same column show the significant difference between the applications, while different upper case subscripted letters in the same row show the significant difference between storage periods. ⁄⁄ The level of significance is 0.01. ⁄⁄⁄ ± values are standard deviation.

inactivated, and the surviving microorganisms could use the remaining PME as nitrogen source to grow during storage. At high intensity electric field strength, however, a great degree of inactivation rather than injury was obtained right after the PEF treatment, and thus the decline in PME activity was smaller. These conclusions have not been made for enzyme inactivation but are a well known fact for the microbial inactivation by PEF (García, Gómez, Condón, Raso, & Pagán, 2003; Wuytack et al. 2003). The activity of PME during storage was not restored which revealed that the PEF treatments caused an irreversible inactivation of PME. Elez-Martínez et al. (2006) compared the inactivation of PME by PEF (35 kV cm1, 1000 ls, bipolar 4 ls pulses at 200 Hz) with the traditional heat pasteurization (90 °C for 1 min). PEFtreated PME was inactivated by 81.6%, and no reactivation was found during storage for 56 days. In the related literature, PEF (25 kV cm1 for 330 ls) treatment of orange-carrot blend revealed 81% inactivation in PME activity, and no reactivation of PME was found in the treated samples during the shelf-life period (Rivas et al., 2006). Yeom et al. (2000) also reported that the PME activity of orange juice significantly decreased in response to the PEF treatment (35 kV cm1 for 59 ls), and remained constant during storage at both 4 and 22 °C for 112 days. Various researchers also reported an irreversible PME inactivation due to PEF processing (AguilóAguayo, Soliva-Fortuny, & Martín-Belloso, 2008; Aguiló-Aguayo, Soliva-Fortuny, & Martín-Belloso, 2009; Sampedro et al., 2009; Salvia-Trujillo, Morales-de la Peña, Rojas-Graü, & Martín-Belloso, 2011; Vervoort et al., 2011). The PME residual activities for the HP1 and HP2 heat pasteurization treatments were detected as 6.8% and 4.2%, respectively. After six-month storage at 4 °C, the residual activities were detected as 9.4 and 8.6%, respectively. It is possible that PME reactivated during storage (p < 0.01), and thus at the end of the storage, the PME activities of HP1 and HP2 treatments increased by 2.6% and 4.4%, respectively. Similarly, Welti-Chanes, Ochoa-Velasco, and Guerrero-Beltrán (2009) also revealed that the PME activity of high pressure processed orange juice increased during storage at 4 °C for 12 days. Similar findings were also reported by other researchers (Asaka & Hayashi, 1991; Guerrero-Beltrán, Barbosa-Cánovas, & Swanson, 2004). The increased PME activity could be due to isoenzymes arising during the storage of the orange juice (Richardson & Hyslop, 1985). However, Vervoort et al. (2011) determined that the PME activity decreased in the thermally processed (at 72 °C for 20 s) orange juice samples during storage at 4 °C for 60 days. Other studies indicated that the PME activity of orange juice processed by heat pasteurization remained constant during storage (Elez-Martínez, Soliva-Fortuny, et al., 2006; Rivas et al., 2006; Salvia-Trujillo, Morales-de la Peña, & Rojas-Graü, 2011).

The PME inactivation value of orange juice samples significantly increased with an increase in the PEF treatment energy. The inactivation degrees of PME by the highest electrical field strength intensity and by the HP1 heat pasteurization treatment were 93.8% and 95.2%, respectively. An inactivation level of E8 (25.26 kV cm1, 1206.2 ls) treatment was assumed to be equivalent to the HP1 (90 °C for 10 s) heat pasteurization treatment for PME inactivation. The PEF-processed orange juice samples revealed that PME activation was either maintained or continued to decrease when low energy intensities of electric field strengths were applied at 4 °C for 180 days. In contrast, the PME activity in the heat-processed orange juice samples increased at 4 °C for 180 days, due to PME restoration during storage. The PME inactivation-related kt, De, ze, Eap and Eae values were calculated as 587.8–2375.4 s1, 969.5–3917.7 ls, 63.7 J, 921.2 kV cm1mol1 and 12.9 kJ mol1, respectively, based on the new inactivation kinetic model developed in this study. However, future studies need to be conducted to explore whether the inactivation of other enzymes can be explained by this model.

Acknowledgements The authors would like to thank the State Planning Organization (Project No: DPT 2009K 120 410), TUBITAK (Project No: 104 O 585) and Cukurova University (Project No: ZF2010BAP4 and ZF2009YL87) for financial supports.

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