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Effect of pulsed electric field treatment on enzyme kineticsand thermostability of endogenous ascorbic acid oxidasein carrots (Daucus carota cv. Nantes)

Sze Ying Leong, Indrawati Oey ⇑Department of Food Science, University of Otago, Dunedin, New Zealand

a r t i c l e i n f o

Article history:Received 31 May 2013Received in revised form 13 September 2013Accepted 16 September 2013Available online 25 September 2013

Keywords:Ascorbic acid oxidaseMichaelis–Menten enzyme kineticsThermostabilityPulsed electric fieldCarrots

a b s t r a c t

The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascor-bic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electricfield (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2 kV/cm andpulsed electrical energy between 1 and 520 kJ/kg were conducted. The enzyme kinetics and the kineticsof AAO thermal inactivation (55–70 �C) were described using Michaelis–Menten model and first orderreaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same orderof magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and400 kJ/kg, but slightly changed at pulsed electrical energy above 500 kJ/kg. However, AAO presented dif-ferent thermostability depending on the electric field strength applied. After PEF treatment at the electricfield strength between 0.2 and 0.5 kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (kvalue) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactiva-tion in carrot purée decreased, indicating that the changes in k values were less temperature dependent.It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes inenzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot puréecomposition, alteration in intracellular environment and the effective concentration of AAO released afterbeing subjected to PEF treatment.

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1. Introduction

Enzyme ascorbic acid oxidase (AAO, EC 1.10.3.3) catalyse theoxidation of L-ascorbic acid (L-AA) to produce dehydro-L-ascorbicacid (DHAA). In plants, AAO exists as either a free enzyme that isdistributed freely in the cytosol; or a membrane bound enzymelocalised at the cell wall (Hallaway, Phethean, & Taggart, 1970).Nonetheless, the role of AAO in plants is not yet fully understoodbut it has been suggested that AAO regulates the growth of plantcells and works in accordance with L-AA (Davey et al., 2000), reg-ulates the plant intracellular redox state under external and inter-nal stimuli (De Tullio, Liso, & Arrigoni, 2004) and oxygenmanagement (De Tullio, Ciraci, Liso, & Arrigoni, 2007). Recentstudy also suggests the possible role of AAO in regulating antioxi-dant capacity of vegetables during postharvest storage (Raseetha,Leong, Burritt, & Oey, 2013). Although AAO stability and catalyticactivity is not influenced by the concentration of L-AA, AAO-cata-

lysed reaction has a deleterious impact on vitamin C during foodpreparation and processing as demonstrated in previous works(Leong & Oey, 2012; Munyaka, Makule, Oey, Van Loey, & Hend-rickx, 2010a; Wawire et al., 2011). Therefore, understanding thestability and catalytic activity of AAO after food processing isimportant to prevent vitamin C degradation and maintain the anti-oxidant capacity of plant-based foods.

Pulsed electric field (PEF) processing is a non-thermal food pro-cessing technology based on the application of short pulses of highvoltage across a food product (mostly in semi-solid to liquid form)placed between two electrodes (Toepfl, Heinz, & Knorr, 2005). Thistechnology has been proven to be economically feasible to effec-tively extract compounds from plant cells at low electric fieldstrength and reduced energy consumption. For instance, juice fromsugar beet (electric field strength of 0.6 kV/cm, pulsed electricalenergy of 5 kJ/kg) (Praporscic, Ghnimi, & Vorobiev, 2005); betanine(1 kV/cm, 7 kJ/kg) (Fincan, DeVito, & Dejmek, 2004); maize germoil and phytosterol (0.6 kV/cm, 0.62 kJ/kg) (Guderjan, Töpfl,Angersbach, & Knorr, 2005) and antioxidants from fennel (0.6 kV/cm, 5 kJ/kg) (El-Belghiti, Moubarik, & Vorobiev, 2008). This isattributed to the ability of PEF to induce pore formation within cel-lular components due to the localised structural changes and the

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.09.096

⇑ Corresponding author. Address: Department of Food Science, University ofOtago, PO Box 56, Dunedin 9054, New Zealand. Tel.: +64 3 479 8735; fax: +64 3 4797567.

E-mail address: indrawati.oey@otago.ac.nz (I. Oey).

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breakdown of the cellular membrane, a process known as ‘electro-permeabilisation’ (Toepfl, Heinz, & Knorr, 2005). However, whilstextracting the targeted compounds, the loss in cell integrity alsosubsequently released other intracellular compounds located indifferent cell compartments such as endogenous enzymes withdeleterious impact on food quality. PEF-induced cell permeabilisa-tion involves permanent and irreversible membrane breakdown,occurring when the electric field strength of applied pulses exceedsthe critical electric field strength of cell membrane electroporation(Zimmermann, Pilwat, & Riemann, 1974). Below this critical elec-tric field strength, transient or reversible pore formation takesplace and cell membrane reseals after PEF treatment. Having saidthis, the breakdown of cell membrane due to PEF could provokethe release of AAO from cytosol and cell wall, and consequently ac-counts for enhancing the rate of AAO catalysis. This could facilitatethe enzymatic oxidation of L-AA. To the best of the authors’ knowl-edge, the impact of PEF treatment on AAO has not yet beeninvestigated.

The objective of this research was to study enzyme kinetics andthermostability of endogenous ascorbic acid oxidase (AAO) afterbeing treated with pulsed electric field (PEF) processing. In this re-search, endogenous AAO in carrot purée (Daucus carota cv. Nantes)was chosen as a case study. To test the release of AAO and vitaminC from solid carrot particles in the purée system as a result of PEFtreatment, a preliminary study using low to medium intensity ofpulsed electrical energy below 20 kJ/kg was conducted. Subse-quently, varying levels of pulsed electrical energy (between 1and 520 kJ/kg) at different electric field strength (between 0.2and 1.2 kV/cm) was further investigated. The enzyme kinetics ofAAO in carrot purée after being treated by PEF was studied in orderto estimate the enzymatic reaction rate and the AAO affinity to L-AA. In addition, a detailed kinetic study of AAO thermal inactiva-tion was carried out to estimate the inactivation kinetic parame-ters important to determine the thermostability of AAO after PEFtreatment.

2. Materials and methods

2.1. Chemicals and reagents

All reagents and chemicals were of analytical grade, unlessotherwise stated and bi-distilled water was used. Sodiumphosphate (NaH2PO4), ethylenediamine tetra-acetic acid (EDTA),sodium chloride (NaCl), metaphosphoric acid (MPA) and sodiumhydroxide (NaOH) were purchased from BDH Chemicals (Poole,England). Hydrochloric acid (HCl, 37%), and acetonitrile of HPLCgrade were from Merck Chemical (Darmstadt, Germany).Tris–[2-carboxyethyl]–phosphine hydrochloride (TCEP–HCl) fromSigma Aldrich (St. Louis, USA), formic acid from Riedel-de Haen(Seelze, Germany), L-ascorbic acid (L-AA) from Unilab (Auckland,New Zealand) and methanol of HPLC grade was from FisherScientific (Leicestershire, England).

2.2. Sample preparation

Fresh carrots (D. carota cv. Nantes) were purchased betweenSeptember and October 2012 from a local grower located in Branx-holme, Invercargill, New Zealand. The local grower controlled thematurity of the carrots based on the standardised commercial pro-tocol in which the carrots were usually harvested approximately18 weeks after seeding and ready for sale in the market. The carrotsused in this study were collected within 24 h after being harvestedand were screened based on similarity in colour (indicator for rip-ening), shape (indicator for variety) and size (indicator for quality).The carrots of acceptable quality – without any bruised, damaged

and infected areas were used in this study. The carrots were imme-diately stored at 4 �C for at least 24 h after arrival and kept no long-er than 2 weeks before subjected to pulsed electric field (PEF)treatment.

Approximately 1 kg of carrots was taken randomly for eachindependent PEF treatment. They were rinsed with cold tap water(8 ± 2 �C) and approximately 20 mm of both the crown and rootparts of the carrot stick were discarded. To prepare the carrotpurée, the remaining carrot sticks were grated uniformly into thincarrot slices of 2 mm thickness using Robot coupe R211 Ultra(Vincennes Cedex, France) at the speed of 1725 rpm. The gratedcarrots and bi-distilled water to the ratio of 1:1 (w/w) werehomogenised at high speed for 20 s using a 1.25 L waring blender(Watson Victor Limited, NZ) which usually resulted in an averagetotal of 1 kg carrot purée. The initial temperature of the carrotpurée before PEF treatment was standardised by preconditioningthe carrot purée at 20 �C for at least 30 min prior to the treatment.The temperature of the carrot purée was monitored using a copperthermocouple probe (1.2 mm type T, Physitemp Instruments Inc.,New Jersey, USA). From the prepared 1 kg of carrot purée, approx-imately 500 g of carrot purée was intended for PEF treatment andthe remaining purée was used as control/untreated samples. EachPEF treatment condition was independently conducted in tripli-cates. Due to batch-to-batch and day-to-day variation of carrotsthat resulted in periodic increase and decrease in AAO activityranging from 0.024 to 0.043 unit per gram of carrot purée overthe sampling period, a control sample was prepared simulta-neously with the PEF treated-sample for each replicate.

2.3. Pulsed electric field treatment

The carrot purée sample was treated using PEF equipment(ELCRACK-HVP 5, German Institute of Food Technologies, Quake-nbrück, Germany) with batch treatment configuration. The batchtreatment chamber (100 mm length � 80 mm width � 50 mmheight, 400 mL capacity) consisted of two parallel stainless steelelectrodes of 5 mm thickness separated by a distance of 80 mm.The treatment chamber was filled with approximately 250 g ofthe 500 g carrot purée and then subjected to PEF treatment. Thisstep was repeated using the remaining 250 g carrot purée fromthe same batch in order to result in approximately a total of500 g treated carrot purée at the same PEF treatment to proceedfor enzyme activity sampling. The following input operating vari-ables in ELCRACK interface programme setting were used: con-stant pulse width of 20 ls, different electric field strengthsranging from 0.2 to 1.2 kV/cm, different pulse frequencies rang-ing from 5 to 300 Hz and different pulse numbers ranging from130 to 9000. Pulse shape (square wave bipolar) was monitoredon-line with oscilloscope (Model UT2025C, Uni-Trend GroupLtd., China) during treatment. All treatments were conducted atan ambient temperature (20 ± 2 �C). In this study, the treatmentintensity was grouped based on the range of applied specificelectrical energy input: low intensity (less than 5 kJ/kg), mediumintensity (10–60 kJ/kg), high intensity (100–150 kJ/kg) and ex-treme high intensity (more than 300 kJ/kg) for different electricfield strength studied.

Table 1 summarises the programme setting of PEF input operat-ing variables applied to carrot purée which resulted in effectiveelectric field strength between 0.2 and 1.2 kV/cm and pulsed elec-trical energy between 1 and 520 kJ/kg. The pulsed electrical energy,also known as specific input energy (Wspec) applied on carrot puréeat square-wave pulse was calculated according to Zhang, Barbosa-Cánovas, and Swanson (1995) using Eq. (1).

Specific energy input; Wspec ðkJ=kgÞ ¼ V2 � ðnsÞR � W

ð1Þ

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V is the pulse peak voltage (in kV), n is the number of pulses ap-plied (dimensionless), s is the pulse width of square pulses (inmicrosecond), R is the effective load resistance (in ohm) and W isthe weight of sample (in kilogram) to be treated in the PEF treat-ment chamber. The change in electrical conductivity and tempera-ture of carrot purée prior to and after PEF treatment was measuredusing a conductivity meter (CyberScan CON 11, Eutech Instru-ments, Singapore). The pH of treated carrot purée was measuredusing pH meter (CyberScan pH 2100, Eutech Instruments, Illinois,USA). After the treatment, the samples were immediately cooleddown and kept at 4 �C or stored at �80 �C until further studies(see details below).

2.4. Preliminary study on the effect of medium intensity PEF treatmenton the activity of ascorbic acid oxidase (AAO) and vitamin C content

Carrot purée was treated below 20 kJ/kg at electric fieldstrength ranging between 0.2 and 1.2 kV/cm. After the treatment,the samples were immediately divided into two portions. One por-tion was directly cooled down and kept at 4 �C until the measure-ment of AAO activity (less than 3 h). The other portion wasimmediately transferred to cryo-vials (LabServ, Auckland, NewZealand) and stored at �80 �C for less than a month until determi-nation of vitamin C content.

2.4.1. AAO extraction and activity determinationAAO activity was measured using spectrophotometric analysis

at 265 nm as described in the previous work (Leong & Oey,2012). To extract AAO from PEF treated and untreated carrot purée,ten millilitres of the cold sodium phosphate buffer (NaH2PO4

(0.1 M, pH 6.0) containing 1 M NaCl and 0.5 mM EDTA) was addedto the carrot purée (10 g). The mixture was vortexed for 10 s andthen centrifuged at 34,700g and 4 �C for 30 min (Rotor JA-20, Beck-man Coulter J2-2M/E centrifuge, CA, USA). The supernatant wasthen kept in an ice–water bath (4 �C) for less than 2 h prior to per-forming the AAO activity assay. One unit of AAO activity was de-fined as the amount of enzyme AAO required to catalyse the

oxidation of 1 lmol of L-AA to DHAA per minute at 25 �C and pH6.0.

2.4.2. Determination of vitamin CThe frozen carrot purée was removed from �80 �C storage and

thawed in a thermostated water bath (JP Selecta Frigiterm-10,Abrera, Spain) at 25 �C for 10 min. Vitamin C extraction for carrotpurée were carried out as described by Leong and Oey (2012). Priorto injection, the pH of the sample extract was adjusted to pH 4.5with HCl (0.1 M) and then filtered through a 0.22 lm cellulose ace-tate filter (Raylab, NZ) via a three millilitre sterile syringe (BD Syr-inge, Singapore). The pH-adjusted and filtered sample was injectedinto reversed-phase PhenoSphere-NEXT C18 column (5 lm particlesize, 250 � 4.6 mm i.d.; Phenomenex NZ Limited, Milford, NewZealand) completed with a C18 column guard (4 � 3.0 mm i.d.; Phe-nomenex NZ Limited, Milford, New Zealand) in place with an Agi-lent 1200 system (MA, USA). Vitamin C (total L-AA) content wasestimated after pre-column reduction using TCEP–HCl (2.5 mM;dissolved in 5% MPA (pH 5.3) containing 1 mM EDTA) for at least9 h of incubation at 4 �C (Leong & Oey, 2012). An injection volumeof 50 lL was used to quantify vitamin C content. The elution wasundertaken isocratically using a mixture of 90% formic acid (0.1%v/v) and 10% methanol at a flow rate of 0.8 mL/min for a total elu-tion time of 30 min. L-AA was identified based on peak purity usingdiode array detector and retention time between 4.2 and 4.3 min.The quantification was performed at 245 nm and 25 �C. VitaminC content was estimated using the external L-AA standard solution(0.1 mg/ml; dissolved in 5% MPA (pH 4.0) containing 1 mM EDTA).Results were expressed as microgram of L-AA equivalents per gramof carrot purée in fresh weight (in lg L-AA/g FW).

2.5. Study on the enzyme kinetics of ascorbic acid oxidase

After PEF treatment (at different pulsed electrical energy inten-sity and electric field strength as summarised in Table 1), the carrotpurée was immediately cooled down and kept at 4 �C in an ice–water bath for AAO extraction and measurement of AAO activityas described in Section 2.4.1.

Table 1Summary of PEF treatment conditions for carrot purée and the treatment impact on the changes in electrical conductivity, pH and temperature.

Specific energy input(kJ/kg)

Electric field strength(kV/cm)

Pulsenumber

Pulse frequency(Hz)

Change in electricalconductivity (%)

Change in pH(%)

Change in temperatureA,DT (oC)

Low intensity (<5 kJ/kg)1.08 ± 0.02a 0.30 153 5 6.36 ± 2.29 1.45 ± 0.00B 0.30 ± 0.00B

2.69 ± 0.39b 0.20 1075 35 6.93 ± 0.01 0.37 ± 0.00 0.20 ± 0.004.96 ± 0.07a,b 0.60 153 5 7.66 ± 0.87 2.16 ± 0.00 0.80 ± 0.00

Medium intensity (10–60 kJ/kg)9.31 ± 0.98b 0.80 135 5 8.90 ± 0.01 0.36 ± 0.00 1.20 ± 0.0014.78 ± 1.70a,b 1.00 135 5 9.86 ± 0.01 1.07 ± 0.00 1.60 ± 0.0020.74 ± 2.02b 1.20 153 5 9.03 ± 0.01 0.72 ± 0.00 2.20 ± 0.0023.62 ± 0.86a,b 0.20 5903 200 6.27 ± 3.73 0.75 ± 0.00 1.35 ± 0.1030.13 ± 1.48b 0.50 1075 35 10.50 ± 0.01 3.36 ± 0.00 1.80 ± 0.0059.78 ± 1.68a,b 0.60 1528 50 10.14 ± 0.01 0.28 ± 0.00 3.10 ± 0.00

High intensity (100–150 kJ/kg)102.70 ± 1.48a,b 0.80 1075 35 14.05 ± 3.42 0.18 ± 0.00 6.05 ± 0.20116.93 ± 2.11a 0.30 8644 300 11.48 ± 0.01 1.32 ± 0.00 6.50 ± 0.00154.25 ± 2.59a,b 1.00 1225 40 10.05 ± 0.96 1.07 ± 0.00 10.90 ± 0.40

Extreme high intensity (>300 kJ/kg)390.25 ± 5.47a,b 0.50 8644 300 11.19 ± 0.01 1.48 ± 0.00 15.85 ± 1.07516.28 ± 3.69a,b 0.60 8644 300 28.39 ± 3.24 1.30 ± 0.00 27.90 ± 1.32

Data presented as average ± standard deviation from three independent treatments using different carrot batches (N = 3), respectively.A Initial temperature of carrot purée prior subjected to PEF treatment averaged at 20 ± 2 �C.B Standard deviation of 0.00 indicates no variation in % of pH change and DT observed among three independent treatments using different carrot batches.a To study AAO enzyme kinetics in carrot purée as described in Section 2.5, these PEF treatment conditions were employed.b To study kinetics of AAO thermal inactivation in carrot purée as described in Section 2.6, these PEF treatment conditions were employed.

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2.5.1. AAO and L-ascorbic acid reaction assayIn order to establish enzymatic oxidation of L-AA catalysed by

AAO, the reaction mixture for spectrophotometer measurementconsisted of 2500 ll of sodium phosphate buffer (NaH2PO4

(0.1 M, pH 6.0) containing 1 M NaCl and 0.5 mM EDTA), 400 ll car-rot extract and 100 ll of L-AA (different concentration of L-AAranging between 0 and 200 lM dissolved in sodium phosphatebuffer (NaH2PO4 (0.1 M, pH 5.6) containing 0.5 mM EDTA). Theblank mixture was a mixture of 2600 ll of sodium phosphate buf-fer (NaH2PO4 (0.1 M, pH 6.0) containing 1 M NaCl and 0.5 mMEDTA) and 400 ll carrot extract. The decrease in absorbance dueto L-AA oxidation by AAO was followed for a total reaction timeof 5 min at 265 nm and was recorded with the reaction kineticsprogramme module (SWIFT II program, version 2.01, AmershamBiosciences, Uppsala, Sweden).

2.5.2. Estimation of the kinetic parameters for AAO enzymatic reactionThe affinity of substrate L-AA at varying concentration

(0–200 lM) towards AAO in carrot purée followed Michaelis–Menten enzyme kinetic model (Leong & Oey, 2012). Thus, the rateof AAO enzymatic reaction was predicted by estimating kineticparameters of Vmax and KM Eq. (2) based on Michaelis–Mentenkinetic model.

Rate of enzymatic reaction;mðlmol=minÞ ¼ Vmax ½S�KM þ ½S�

ð2Þ

v is the initial reaction rate of AAO catalysing L-AA oxidation toDHAA (in lmol/min) and Vmax is the maximum reaction rateachieved at saturating substrate concentration (in lmol/min). [S]is the substrate concentration (in lM) and KM is best known asMichaelis–Menten constant that defines the substrate concentra-tion (in lM) at which the reaction rate is half of Vmax.

The kinetic parameters of Vmax and KM derived from the exper-imentally determined initial rates (v) as a function of substrateconcentration ([S]) were estimated based on non-linear regressionof Michaelis–Menten equation Eq. (2) using SAS 9.2 service pack(SAS Institute Inc., Cary, USA) for each PEF treatment to be com-pared with its corresponding untreated carrot purée. This statisti-cal approach of non-linear regression uses the whole data set toavoid generation of excessive errors. An iterative programme mod-ule was employed to optimise the kinetic parameters in order toachieve the lowest residual sum of square based on Marquardtmethod. Subsequently, the SAS output provided parameter esti-mates of Vmax and KM with a 95% confidence level at minimumresidual sum of squares, together with the standard error of theestimate. The quality of the non-linear regression analysis of thekinetic parameter estimates obtained from Eq. (2) was evaluatedbased on coefficient of determination derived from Eq. (3); ex-pressed as corrected r2.

Corrected r2 ¼ 1�ðm� 1Þ 1� SSQReg

SSQTotal

� �m� j

ð3Þ

m is the sum of observations, SSQReg is sum of squares of the regres-sion model, SSQTotal is sum of squares of total observations and j issum of parameters.

Frequently, non-linear regression estimation of enzyme kineticparameters is preferable as linear transformation of Michaelis–Menten equation will lead to bias in estimation of the Vmax andKM values since some assumptions that underlie the regression willbe violated as the error structure related to the data is disturbed(Van Boekel, 2008). Yet, the transformation of Michaelis–Mentenis necessary to help visualising the meaning of the kinetic param-eters and compare the estimated kinetic parameters using differ-ent linearised plots which includes Lineweaver–Burk Eq. (4),

Eadie–Hofstee Eq. (5), Hanes–Woolf Eq. (6) and Eadie–ScatchardEq. (7) plots.

Inverting Michealis —Menten equation :1m¼ KM

Vmax½S�þ 1

Vmaxð4Þ

Plotting m versus m=½S� : m ¼ �KMm½S� þ Vmax ð5Þ

Plotting ½S�=m versus ½S� : ½S�m¼ 1

Vmax½S� þ KM

Vmaxð6Þ

Plotting m=½S� versus m :m½S� ¼ �

1KM

mþ Vmax

KMð7Þ

These linearised plots are able to visualise the comparison be-tween untreated and PEF-treated carrot purée through estimationof Vmax and KM using linear regression analysis based on Eqs. (4)–(7) using SAS 9.2 service pack (SAS Institute Inc., Cary, USA).

2.6. Kinetic study on thermal inactivation of ascorbic acid oxidase

2.6.1. Thermal treatmentThermal inactivation of AAO was conducted sequentially after

PEF treatment (at different pulsed electrical energy intensity andelectric field strength as summarised in Table 1). Carrot purée (un-treated and PEF treated) was weighed (2.5 g) into 15 ml polyethyl-ene tubes (internal and outer diameter of 15 mm and 17 mmrespectively, BD Biosciences, California, USA), capped and equili-brated at 20 �C (Grant JB2, Cambridge, England) for at least30 min. This was done to ensure the carrot purée achieved thesame initial temperature prior to thermal treatment. The freshlyprepared carrot purée with no treatment was used as ‘control/un-treated sample’. After that, both PEF-treated and untreated carrotpurée samples (each in total 10 samples) were heated at the prede-fined temperature. In this study, five temperatures of enzyme inac-tivation were predefined based on previous work (Leong & Oey,2012), i.e. 55, 60, 62.5, 65 and 70 �C. The thermal treatment wasconducted in a thermostated water bath (Grant GD 100,Cambridge, England) for 10 different predefined time intervals tofollow the enzyme inactivation at least up to 90% of the initial en-zyme activity. The average come-up time of the samples from 20 �Cto attain the isothermal condition for each predefined temperatureduring thermal treatment was approximately 4 min, monitoredusing copper thermocouple probe (1.2 mm type T, PhysitempInstruments Inc., New Jersey, USA). In this study, the AAO inactiva-tion time in carrot purée for less than 4 min was excluded in theestimation of kinetic parameters to ensure the achievement of iso-thermal condition. To stop the heating effect, the sample tubeswere immediately cooled down in an ice–water bath (0–4 �C) forat least 20 min before performing AAO extraction and residualactivity determination.

2.6.2. Estimation of the inactivation kinetic parametersAs reported previously, thermal inactivation of AAO followed

first order reaction (n = 1) (Leong & Oey, 2012; Munyaka, Makule,Oey, Van Loey, & Hendrickx, 2010a; Wawire et al., 2011). Theinactivation rate constants (k, in min�1) for first-order inactivationunder isothermal condition at constant inactivation temperaturewere determined by plotting In(A)as a function of inactivation time(t, in min) according to Eq. (8).

lnðAÞ ¼ lnðA0Þ � kt ð8Þ

A (in unit) is the AAO activity after heating for time t at thespecified constant inactivation temperature, A0 (in unit) is theinitial AAO activity. The kinetic parameter of k value at constant

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temperature was estimated based on linear regression analysisusing SAS 9.2 service pack (SAS Institute Inc., Cary, USA).

To estimate the temperature dependency of k values, Arrheniusequation was used Eq. (9).

ln k ¼ ln kref þEa

R1

Tref� 1

T

� �ð9Þ

k is the inactivation rate constant (in min�1) at certain inactivationtemperature as estimated based on Eq. (8), while kref is the inactiva-tion rate constant (in min�1) at reference temperature (Tref = 62.5 �-C = 335.5 K); Ea is the activation energy (in kJ/mol); R is universalgas constant (0.008314 kJ/mol K) and T is actual heat treatmenttemperature (in K). In order to eliminate non-isothermal conditionduring heating, the residual enzyme activity obtained from thethermal treatment for less than 4 min was excluded from the dataanalysis. Two-step linear regression method using SAS 9.2 servicepack (SAS Institute Inc., Cary, USA) was conducted from the previ-ously derived kinetic parameter of inactivation rate constant (k)to estimate Ea and kref values, respectively.

3. Results and discussion

3.1. Effect on electrical conductivity and temperature of carrot puréeafter being subjected to pulsed electric field treatment

When carrot purée was exposed to different intensities ofpulsed electrical energy, the electrical conductivity, temperatureand pH were measured to evaluate the cell permeabilising effectof pulsed electric field (PEF) treatment. On average, the physico-chemical characteristics of the untreated carrot purée before sub-jected to PEF treatment were: electrical conductivity = 6.92 ± 0.76mSm�1 at 20 ± 2 �C and pH = 5.49 ± 0.12. As summarised inTable 1, the electrical conductivity of carrot purée generally in-creased with the increase in applied pulsed electrical energy atthe same level of electric field strength, particularly significantat 0.3, 0.6 and 0.8 kV/cm. The measurement of electrical conduc-tivity was employed in several studies as an indication for thetransport of ionic species in the cell material which was inducedby PEF because of its permeabilising effect on cell membrane(Lebovka, Bazhal, & Vorobiev, 2002). Therefore, in this study,the increase in conductivity showed that PEF enhanced transportof ionic species contributed towards modification of the intracel-lular environment of carrot purée. The increase in electricalconductivity could be related to the cellular damage. The temper-ature of carrot purée after PEF treatment increased when theapplied pulsed electrical energy was intensified. The highesttreatment temperature achieved in this study was43.70 ± 1.32 �C after PEF treatment, using pulsed electrical energyof 516.28 kJ/kg at electric field strength level of 0.6 kV/cm. Thistemperature rise is due to the transformation of energy inputdeveloped in the treatment chamber during PEF treatment, lead-ing to mild ohmic heating (Lindgren, Aronsson, Galt, & Ohlsson,2002). Minor pH-shifts (±3%, Table 1) were observed in the PEF-treated carrot purée, and thus it was reasonable to assume thatthe exposure to pulsed electrical energy of more than 100 kJ/kgdid not stimulate excessive migration of chemical species towardsthe PEF treatment electrodes as a result of electrochemicalreactions. No remarkable changes in pH were also reported inprevious works for juice processing using PEF technology(Cserhalmi, Sass-Kiss, Tóth-Markus, & Lechner, 2006; Grimi,Mamouni, Lebovka, Vorobiev, & Vaxelaire, 2011; Sanchez-Morenoet al., 2005).

3.2. Activity of ascorbic acid oxidase (AAO) and vitamin C content ofcarrot purée after being subjected to pulsed electric field treatment

In the present study, there was a pronounced increase in theresidual catalytic activity of AAO for up to 59.13 ± 3.12% when trea-ted at pulsed electrical energy below 20 kJ/kg (Fig. 1). This increasein the residual catalytic activity of AAO suggested that the perme-abilising effect of PEF resulted in the release of membrane boundAAO, hence increasing the AAO concentration available freely inthe carrot purée system. Also, the impact of PEF on AAO enzymeactivity was irreversible since the PEF-treated carrot purée re-tained similar degree of AAO activity retention after 24 h of treat-ment (data not shown). To our best knowledge, no reactivation ofAAO activity following PEF treatment has been reported in litera-ture. However, the residual catalytic activity of AAO was not signif-icantly affected (±10% deviation from the AAO activity of untreatedcarrot purée, data not shown) after being treated with pulsed elec-trical energy above 20 kJ/kg and up to 400 kJ/kg albeit exerted withvarying levels of electric field strength. The application of pulsedelectric energy of 516.28 kJ/kg inactivated enzyme AAO by61.48 ± 1.80%. This may be due to the combined thermo-electric ef-fect of PEF since the final treatment temperature was remarkablyhigh (Table 1). The finding of the current study presents foremostevidence in demonstrating the capability of employing high inten-sity PEF treatment to inactivate endogenous AAO enzyme withincarrot purée, thus providing protection against AAO-catalysed vita-min C oxidation.

It has been reported that AAO oxidises the active form of vita-min C, L-ascorbic acid (L-AA) upon matrix disruption, hence affect-ing the stability of vitamin C (Munyaka, Oey, Van Loey, &Hendrickx, 2010b). In view of this, both the permeabilising and cellcompartments disruption effects of PEF treatment would allow therelease of L-AA from its localisation within the intact plant cellssuch as apoplast, cytoplasm, vacuole and particularly those en-trapped in membrane-bound organelles such as chloroplast(approximately 10–50 mM) (Davey et al., 2000). Consequently,the free L-AA is able to participate actively in the AAO-catalysedreaction that directly relates to vitamin C oxidation. In this study,the vitamin C content in untreated carrot purée was averaged at7.43 ± 1.23 lg/g FW while all PEF-treated samples contained simi-lar amount of vitamin C but was predominantly present in the oxi-dised form, i.e. DHAA (data not shown). The complete conversionof L-AA to DHAA could be partly attributed to the activity of oxida-tive enzymes, mainly AAO. In addition, further degradation ofDHAA to other compounds such as 2,3-diketogulonic acid withno vitamin C activity was not observed for carrot purée after beingsubjected to PEF treatment. This could explain why the total vita-min C content remained constant for PEF-treated samples. In thecontext of using PEF processing technology to treat plant material,it has been reported that application of high intensity electric fieldstrength of more than 25 kV/cm exerted deleterious impact onvitamin C content (Odriozola-Serrano, Soliva-Fortuny, & Martín-Belloso, 2008; Oms-Oliu, Odriozola-Serrano, Soliva-Fortuny, &Martín-Belloso, 2009; Quitão-Teixeira, Odriozola-Serrano, Soliva-Fortuny, Mota-Ramos, & Martín-Belloso, 2009) which resulted insignificant L-AA degradation. It is evident that PEF treatment is un-able to confer protection for L-AA against oxidation and degrada-tion. To date, enzymatic oxidation of L-AA due to increasedresidual catalytic activity of AAO after the PEF treatment is oftenneglected in many previous works.

3.3. Enzyme kinetics of AAO in carrot purée after being subjected topulsed electric field treatment

In the preliminary study, application of pulsed electrical energylower than 20 kJ/kg at electric field strength between 0.2 and

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1.2 kV/cm led to an increase in the residual activity of AAO (Fig. 1),and possible changes in the intracellular environment and cellcompartment disruption which might impact the enzyme kineticsof AAO. Under the assumption that the reaction product generatedfrom the AAO-catalysed reaction, i.e. DHAA does not convert backto its substrate, i.e. L-AA, Michaelis–Menten enzyme kinetic modelwas used to estimate the kinetic parameters of AAO, i.e. Vmax andKM values. In both untreated and PEF-treated carrot purée, the rateof AAO enzyme-catalysed reaction increased with increasing L-AAconcentration until substrate saturation region was achieved. Thecurrent study found that the AAO enzyme–substrate conversionof PEF-treated carrot purée followed the same pattern as AAOactivity in untreated carrot purée and it can be described byMichaelis–Menten kinetics Eq. (2). The estimated enzyme kinetic

parameters of AAO are summarised in Table 2. The estimatedvelocity, Vmax, of the enzymatic reaction for untreated carrot puréeranged from 7.43 to 25.63 lmol/min and the estimated KM valueranged from 71.75 to 244.30 lM when the rate of enzymatic reac-tion was half of Vmax. The pronounced variation in the estimatedenzyme kinetic parameters for carrot purée before being subjectedto PEF treatment signified variation in the biological materials overthe sampling period despite the fact that all the individual carrotsticks utilised in this study were obtained from the same batchharvested at the same time from the same field. The postharveststorage and age of carrots upon sample preparation (to be pro-cessed into carrot purée) were the underlying factors that influ-enced the magnitude of estimated enzyme kinetic parameters ofAAO. Recent study on broccoli florets evidently showed fluctuationin AAO catalytic activity during postharvest storage and conse-quently affected the oxidative status of the vegetables during thesubsequent mechanical processing (Raseetha, Leong, Burritt, &Oey, 2013). Therefore, in this study, the kinetics of AAO enzymaticreaction were compared before and after being subjected to PEFtreatment using the same batch of carrot purée prepared for eachindependent treatment as demonstrated in Table 2, which is vitalto justify the impact of PEF treatment on the kinetics of AAO en-zyme–substrate conversion.

As shown in Table 2, the application of low to medium and highintensity energy levels of PEF treatment (between 1 and 400 kJ/kg)exerted negligible impact on the kinetics of AAO enzyme–substrateconversion. In addition, AAO enzyme kinetics was independent ofthe extent of electric field strength level build-up across the planttissues to induce either reversible or irreversible cell membraneelectroporation. AAO in carrot purée before and after PEF treat-ment attained similar rate of enzyme AAO-substrate conversionand affinity towards L-AA (Table 2). In other words, the estimatedenzyme kinetic parameters of Vmax and KM of the PEF-treated car-rot purée were situated in the same range as the corresponding un-treated carrot purée. Based on the estimated enzyme kineticparameters from non-transformed data using Michaelis–Mentenenzyme kinetic model Eq. (2), the depiction of linearised plotstransformed from the respective model is used to provide visualevidence to detect differences in enzyme kinetics of carrot puréebefore and after PEF treatment. The carrot purée after being treatedwith PEF treatment demonstrated similar enzyme AAO kineticswhen compared to untreated carrot purée and no significant

Fig. 1. Percentage increase in AAO activity of carrot purée after being subjected topulsed electric field treatment at different specific energy input below 20 kJ/kg. Barsand vertical error bars represent the average and standard deviation of experi-mental values from three independent treatments using different carrot batches(N = 3), respectively.

Table 2Estimated Michaelis–Menten kinetic constants for AAO in carrot purée before and after subjected to pulsed electric field treatment.

Specific energy input (kJ/kg)a Estimated kinetic parameters before PEF treatment Estimated kinetic parameters after PEF treatment

Vmax (lmol/min) KM (lM) Corrected r2 Vmax (lmol/min) KM (lM) Corrected r2

Low intensity (<5 kJ/kg)1.08 ± 0.02 25.63 ± 3.79 244.30 ± 52.69 0.999 28.96 ± 3.24 350.50 ± 52.15 0.9994.96 ± 0.07 12.37 ± 1.12 106.70 ± 18.44 0.998 14.19 ± 0.46 162.90 ± 8.60 0.999

Medium intensity (10–60 kJ/kg)14.78 ± 1.70 10.76 ± 1.04 103.80 ± 20.37 0.998 12.14 ± 0.82 140.60 ± 16.91 0.99923.62 ± 0.86 7.43 ± 0.60 81.58 ± 14.59 0.998 7.63 ± 0.52 97.08 ± 13.79 0.99959.78 ± 1.68 8.76 ± 0.80 71.75 ± 16.43 0.996 10.10 ± 0.48 120.00 ± 10.38 0.999

High intensity (100–150 kJ/kg)102.70 ± 1.48 8.89 ± 0.60 99.64 ± 13.54 0.999 9.13 ± 0.81 92.02 ± 16.63 0.998116.93 ± 2.11 7.43 ± 0.60 81.58 ± 14.59 0.998 7.50 ± 0.65 90.87 ± 16.31 0.998154.25 ± 2.59 8.89 ± 0.60 99.64 ± 13.54 0.999 9.58 ± 0.61 107.50 ± 13.10 0.999

Extreme high intensity (>300 kJ/kg)390.25 ± 5.47 8.28 ± 0.50 99.39 ± 12.44 0.999 8.94 ± 0.67 94.91 ± 14.93 0.998516.28 ± 3.69b 8.28 ± 0.50 99.39 ± 12.44 0.999 3.60 ± 0.38 106.10 ± 21.74 0.996

Data presented as estimated kinetic parameter ± standard error of the estimated parameter at 95% confidence intervals using SAS 9.2 service pack based on non-linearregression analysis using Michaelis–Menten equation Eq. (2) and the corrected r2 values calculated based on Eq. (3).

a Electric field strength used is summarised in Table 1.b Carrot purée experienced 61.48 ± 1.80% of AAO activity reduction after treated at pulsed electrical energy of 516.28 ± 3.69 kJ/kg at electric field strength of 0.6 kV/cm.

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deviation from the expected linear relationship on the basis of Eqs.(4)–(7) was observed. In addition, although an increase in residualcatalytic activity of AAO shown in Fig. 1 has suggested that the per-meabilising effect of PEF would release membrane bound AAO, PEFtreatment conferred insignificant impact on the AAO enzymekinetics based on the estimated Vmax and KM values.

The activity and stability of AAO is generally affected by tem-perature since this occurrence is evident in various vegetable tis-sues (Leong & Oey, 2012; Munyaka, Makule, Oey, Van Loey, &Hendrickx, 2010a; Wawire ., 2011). As shown in Table 1, the finaltreatment temperature for carrot purée treated with pulsed electri-cal energy ranging from 1 to 400 kJ/kg was less than 35 �C and atpulsed electrical energy of 516.28 kJ/kg, carrot purée experienceda high increase in temperature (DT = 27.90 ± 1.32 �C) and achievedthe final treatment temperature of 43.70 ± 1.32 �C. As a result, AAOresidual catalytic activity depleted by 61.48 ± 1.80% and the AAO-catalysed reaction took place at an exceptionally lower speed, asdenoted by at least a 2.3-fold reduction of Vmax from the untreatedcarrot purée (Table 2). Interestingly, the binding affinity of AAO to-wards substrate L-AA (KM value) was situated at the similar

magnitude before and after PEF treatment at this respective ap-plied pulsed electrical energy. In the context of Michaelis–Mentenenzyme kinetic model, the reduction of Vmax value after PEF treat-ment at 516.28 kJ/kg could be explained by partial enzyme inacti-vation since the maximal rate of AAO-catalysed reaction, Vmax isdirectly related to the total enzyme concentration (Van Boekel,2009). Under steady state condition, low Vmax value should leadto lower KM value which is not observed in this study. The affinityof AAO towards its substrate becomes less sensitive after PEF treat-ment at 516.28 kJ/kg. This phenomenon could be explained by thechanges in protein conformation, which is indicated by partialinactivation of enzyme molecules, or changes in intracellular envi-ronment of carrot purée after PEF treatment such as release ofinhibitors. However, further investigation on this area is stillneeded in order to confirm this hypothesis.

Moreover, while it has been claimed that AAO is a heat-sensitive enzyme which demonstrated the greatest stability attemperature between 40 and 50 �C in carrots (Leong & Oey,2012), the current study found that the combined thermo-electriceffect of PEF at the respective treatment condition (516.28 kJ/kg)

Fig. 2. Example of graphical illustrations of a first order reaction plot of AAO thermal inactivation for (a) untreated and (b) PEF-treated (0.6 kV/cm, 4.96 kJ/kg) carrot puréeand Arrhenius plot of natural logarithm of AAO thermal inactivation rate constant (k) and the reciprocal of inactivation temperature for carrot AAO for untreated and PEF-treated carrot purée at electric field strength of (c) 0.2 kV/cm and (d) 0.5 kV/cm. Symbols and error bars represent the estimated k value and standard error of the estimatedparameter at 95% confidence intervals using SAS 9.2 service pack based on Eq. (9). The lines correspond to the individual fittings of the estimated kinetic parameter of k value.

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could have been attributed to the reduction of AAO-catalysed reac-tion rather than solely on the increase in temperature that devel-oped during the treatment. Contradictory findings have beendemonstrated in the work of Van Loey, Verachtert, and Hendrickx(2001) in which the heat effect generated during PEF treatmentleads to enzyme inactivation; whereas the work of Yang, Li, andZhang (2004) shows that both PEF and heat effect are responsiblefor enzyme inactivation. In this context, constant re-evaluation ofthe design of the PEF treatment chamber configuration becomesimportant to allow uniform rate of heat dissipation to the environ-ment and homogenous distribution of electric field strength (Lind-gren, Aronsson, Galt, & Ohlsson, 2002; Meneses, Jaeger, & Knorr,2011). Nevertheless, the changes in the structures and functionsof proteins such as enzymes due to PEF have not yet been wellunderstood and remains controversially discussed. The work ofZhong et al. (2007) has reported that when employing electric fieldstrength of more than 25 kV/cm, PEF could effectively affect thesecondary structure of enzymes such as peroxidase and polyphenoloxidase. This implied that exposure to increased intensity of pulsedelectrical energy, in our case above 500 kJ/kg, possessed the poten-tial to partially inactivate endogenous AAO. Therefore, this under-pinned the importance of the effective enzyme concentrationavailable for the enzyme AAO-substrate conversion reaction whichhad influential impact on the kinetic properties of AAO, in particu-lar the estimated Vmax values. Otherwise, the overall AAO enzymekinetics after being subjected to PEF treatment (below 400 kJ/kg)remained comparable to that of untreated carrot purée regardlessof the electric field strength level applied.

3.4. Stability of AAO in carrot purée towards thermal inactivation afterbeing subjected to pulsed electric field treatment

Detailed thermal inactivation kinetics of endogenous AAO inuntreated and PEF-treated carrot purée was studied in the temper-ature range from 55 to 70 �C. In this study, it was evident that AAOactivity of PEF treated carrot purée followed the same thermalinactivation kinetics as untreated carrot purée, i.e. first order reac-tion – a linear plot was obtained between the natural logarithm of

the residual AAO activity and the inactivation time (Fig. 2a). Thefirst order kinetics of AAO inactivation coincided with other works(Leong & Oey, 2012; Munyaka, Makule, Oey, Van Loey, & Hend-rickx, 2010a; Wawire et al., 2011). The thermal inactivation kinet-ics of AAO in food matrix after being treated by PEF treatment havenot yet been investigated, and to the best of our knowledge, thisstudy is the first experiment to demonstrate that PEF treatmentdid not affect the kinetics of AAO thermal inactivation, i.e. a firstorder kinetics (Fig. 2b). The inactivation rate constants, k increasedwith increasing temperature (55–70 �C) (Table 3). The estimated kvalues varied for carrot purée treated at varying energy intensitylevels and were different from the untreated carrot purée. Thisfinding strongly suggested that AAO demonstrated different ther-mostability after being subjected to PEF treatment. The relation be-tween natural logarithm of inactivation rate constant as a functionof the reciprocal of absolute temperature could be described byArrhenius equation Eq. (9) (Fig. 2c and d). At the reference temper-ature of 62.5 �C, the estimated Ea value of AAO in untreated carrotpurée was 236.57 ± 11.46 kJ/mol (Table 3); which fell into a similarrange with intact carrots (Leong & Oey, 2012) and other vegetables(Munyaka, Makule, Oey, Van Loey, & Hendrickx, 2010a; Wawireet al., 2011).

A distinct difference for the estimated Ea and kref values wasdemonstrated for PEF-treated carrot purée at different electric fieldintensities in particular at 0.2 and 0.5 kV/cm (Fig. 2c and d). There-fore, electric field strength has been shown to play an importantrole in affecting the thermal stability of AAO which had resultedin lower estimated Ea values (i.e. less temperature dependence ofinactivation rate constants) and higher kref values (i.e. more ther-molabile) (Table 3). This phenomenon can be related to thechanges in carrot purée intracellular environment and compositiondue to permeabilising effect of PEF that may possibly affect thebehaviour of enzyme AAO towards thermal inactivation. In theelectric field strength domain of 0.2 and 0.5 kV/cm, it was apparentthat the time dependence of AAO inactivation at the reference tem-perature (i.e. kref values) increased but AAO attained lesser temper-ature dependence towards inactivation and thus significantlyhigher thermal stability (i.e. lower Ea values) when the pulsed

Table 3Estimated kinetic parameters for AAO thermal inactivation in carrot purée after subjected to different intensity of pulsed electric field treatment (Tref = 62.5 �C).

Specific energy input (kJ/kg)a Estimated inactivation rate constants, k (�10�2 min�1) Estimated kinetic parameters

55 �C 60 �C 62.5 �C 65 �C 70 �C krefb (�10�2 min�1) Ea (kJ/mol) r2

No PEF treatment 0.51 ± 0.05 1.94 ± 0.14 2.72 ± 0.19 6.36 ± 0.33 22.46 ± 1.24 2.60 ± 0.44 236.57 ± 11.46 0.993

Low intensity (<5 kJ/kg)2.69 ± 0.39 1.49 ± 0.20 1.89 ± 0.20 4.21 ± 0.43 5.36 ± 0.42 24.43 ± 2.34 4.07 ± 0.24 167.66 ± 31.84 0.9334.96 ± 0.07 1.11 ± 0.05 2.29 ± 0.13 4.11 ± 0.33 7.05 ± 0.66 24.11 ± 1.86 3.68 ± 0.09 194.87 ± 15.04 0.983

Medium intensity (10–60 kJ/kg)9.31 ± 0.98 0.98 ± 0.06 1.87 ± 0.08 3.05 ± 0.29 7.79 ± 0.66 29.37 ± 2.87 3.37 ± 0.15 218.49 ± 27.41 0.95514.78 ± 1.70 0.60 ± 0.03 1.84 ± 0.10 3.60 ± 0.36 5.81 ± 0.68 19.32 ± 1.60 2.73 ± 0.02 217.97 ± 4.26 0.99920.74 ± 2.02 0.57 ± 0.04 2.16 ± 0.11 3.14 ± 0.16 7.32 ± 0.61 42.99 ± 4.10 3.19 ± 0.11 266.11 ± 22.02 0.98023.62 ± 0.86 1.85 ± 0.12 3.22 ± 0.33 4.52 ± 0.38 8.98 ± 0.85 15.73 ± 1.38 4.53 ± 0.10 140.08 ± 13.18 0.97430.13 ± 1.48 0.57 ± 0.04 1.90 ± 0.21 5.47 ± 0.57 6.89 ± 0.82 18.22 ± 1.61 3.02 ± 0.10 220.42 ± 20.45 0.97559.78 ± 1.68 0.53 ± 0.05 1.19 ± 0.08 3.69 ± 0.47 5.21 ± 0.27 9.30 ± 0.90 2.14 ± 0.08 189.67 ± 25.60 0.948

High intensity (100–150 kJ/kg)102.70 ± 1.48 0.45 ± 0.05 2.57 ± 0.27 6.93 ± 0.91 11.61 ± 1.86 24.73 ± 2.71 3.65 ± 0.18 256.09 ± 30.49 0.959154.25 ± 2.59 0.95 ± 0.13 1.81 ± 0.16 4.18 ± 0.39 9.46 ± 0.15 16.93 ± 2.02 3.38 ± 0.12 193.82 ± 22.71 0.960

Extreme high intensity (>300 kJ/kg)390.25 ± 5.47 2.01 ± 0.01 4.27 ± 0.41 5.92 ± 0.64 7.65 ± 0.54 27.28 ± 2.98 5.47 ± 0.18 157.90 ± 17.36 0.965516.28 ± 3.69c 0.78 ± 0.05 1.13 ± 0.08 1.03 ± 0.07 7.88 ± 0.40 15.48 ± 0.90 2.64 ± 0.17 205.07 ± 39.48 0.931

Data presented as estimated kinetic parameter ± standard error of the estimated parameter at 95% confidence intervals using SAS 9.2 service pack based on linear regressionanalysis.

a Electric field strength used is summarised in Table 1.b Estimation of kref at Tref = 62.5 �C.c Carrot purée experienced 61.48 ± 1.80% of AAO activity reduction after treated at pulsed electrical energy of 516.28 ± 3.69 kJ/kg at electric field strength of 0.6 kV/cm.

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electrical energy intensified approximately ten-fold within thesame electric field strength level (Fig. 2c and d). Therefore, in thiscontext the combination of low electric field strength (below0.6 kV/cm) and pulsed electrical energy attributed to the signifi-cant changes in thermal stability of AAO to that of the untreatedcarrot purée. Amongst other PEF-treated and untreated carrotpurée, the estimated Ea value was the lowest at140.08 ± 13.18 kJ/mol for carrot purée treated with 23.62 kJ/kgand electric field strength of 0.2 kV/cm. This implies that the inac-tivation rate constants of AAO becomes less sensitive towards tem-perature change after carrot purée being treated with PEF at theaforementioned treatment condition. It could be that PEF treat-ment at low electric field strength domain below 0.6 kV/cm signif-icantly altered the carrot purée composition and intracellularenvironment that could stabilise AAO against thermal denatur-ation and inactivation. Therefore, enzyme AAO exhibits a distinctbehaviour in which the inactivation rate of AAO is less susceptibleto high temperature (i.e. less changes in the rate of AAO inactiva-tion or more thermostable).

At electric field strength domains other than 0.2 and 0.5 kV/cm,the influence of electric field strength and pulsed electrical energyon thermal stability of AAO was not apparent. Instead, AAO in car-rot purée showed comparable estimated Ea values and were insig-nificantly different with the untreated carrot purée(Ea = 236.57 ± 11.46 kJ/mol, kref = 2.60 ± 0.44 � 10�2 min�1) at theelectric field strength domains of 0.6, 1.0 and 1.2 kV/cm (Table 3).The increase in the intensity of pulsed electrical energy within thesame electric field strength domain did not alter AAO thermal sta-bility. A minor increase in the estimated kref values was observedand the Ea value (reflecting the temperature dependency of k val-ues) was situated in the same magnitude to that of untreated car-rot purée. Consequently, PEF-treated carrot purée demonstratedsimilar AAO thermal stability compared to the untreated carrotpurée. In the case of carrot purée treated at 516.28 kJ/kg, the esti-mated kinetic parameters of Ea and kref values were similar to thatof untreated carrot purée. As revealed earlier, a significant decreasein AAO residual catalytic activity of 61.48 ± 1.80% indicated thatAAO could have been partially inactivated during PEF treatmentsince the final treatment temperature was raised tremendously(Table 1). However, the estimation of temperature dependency ofk values has suggested that the degree of AAO inactivation dueto the preceding extreme high energy PEF treatment did not in-crease the sensitivity of AAO towards thermal inactivation. There-fore, it is most likely that changes in AAO thermostability were notrelated to the residual catalytic activity of AAO or the effective en-zyme AAO concentration after being subjected to PEF treatmentbut due to alteration of carrot purée composition and intracellularenvironment.

4. Conclusion

This study provides valid evidence that pulsed electric field(PEF) processing affects enzyme–substrate conversion and thermo-stability of endogenous ascorbic acid oxidase (AAO) in carrotpurée. Both electric field strength and pulsed electrical energy playan important role in affecting enzyme properties. Regardingenzyme–substrate conversion, the application of pulsed electricalenergy in the range between 1 and 400 kJ/kg did not influencethe enzyme AAO kinetics whereas pulsed electrical energy morethan 500 kJ/kg slowed down the enzymatic action of AAO. There-fore, it should be taken into account that during PEF-assistedextraction of targeted bioactive compounds, undesired enzymessuch as AAO were also unintentionally released at the same time.This would lead to degradation of untargeted bioactive compoundssuch as vitamin C which is also important to maintain the

antioxidative status of plant materials after PEF treatment. Regard-ing AAO thermostability, the changes in inactivation rate (k value)and temperature dependence of k value (Ea value) were electricfield strength dependent. It is clear that PEF treatment affects thetemperature stability of enzymes. This study suggests that PEFtechnology could offer a new potential application on enzymeproperties modification and possibly tailor the functional proper-ties of protein-rich foods. Therefore, this study calls for furtherinvestigation in this area.

Acknowledgements

The authors acknowledge University of Otago Doctoral Scholar-ship towards PhD study of Sze Ying Leong and technical assistanceof Danielle Clapperton. Authors are grateful to Jo’ann Ayers andMelinda Fiona Marzuki for proof-reading the manuscript.

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