effect of pulsed electric field processing on carotenoid extractability of carrot purée

Original article

Effect of pulsed electric field processing on carotenoid

extractability of carrot pur�ee

Shahin Roohinejad,1 David W. Everett1,2 & Indrawati Oey1,*

1 Department of Food Science, University of Otago, PO Box 56, Dunedin 9054, New Zealand

2 Riddet Institute, Private Bag 11 222, Palmerston North 4442, New Zealand

(Received 9 September 2013; Accepted in revised form 8 January 2014)

Summary The purpose of this research was to study the effect of electric field strength (0.1–1 kV cm�1) and fre-

quency (5–75 Hz) during pulsed electric field (PEF) processing on the extractability of carotenoids in car-

rots, which was examined using different vegetable oils. Increasing electric field strengths up to 1 kV cm�1

at 5 Hz significantly (P < 0.05) increased the extraction of carotenoids from carrot pomace. Increasing

frequency above 10 Hz at 1 kV cm�1 did not improve the carotenoids extraction. The yield of carotenoids

extracted was dependent on the vegetable oils. Sunflower and soya bean oils had the highest carotenoid

extractability, and peanut oil was the lowest for carrot pomace treated at 0.6 kV cm�1 and 5 Hz, but no

significant difference was observed among vegetable oils for carrot pomace treated at 50 Hz. This study

suggests that PEF can improve the carotenoids extractability of carrots depending on the electric field

strength and frequency used.

Keywords Carotenoids, carrot, extractability, pomace, pulsed electric field.

Introduction

Carotenoids, the main tetraterpenoid organic pig-ments, naturally exist in the chloroplasts and chro-moplasts of plants. Although over 650 specificcarotenoids have been identified and isolated from nat-ural sources, only about 60 are common in the humandiet, and about 20 can be identified in human plasmaand tissues (Dutta et al., 2005). Carrots are a globallyimportant vegetable crop, providing a good source ofcarotenoids. The principal carotenoids of carrotsare b-carotene (60–80%) followed by a-carotene(10–40%), lutein (1–5%) and the other minor carote-noids (0.1–1%) (Bandyopadhyay et al., 2007). Carote-noids possess free radical-scavenging capacity thatprovide benefits to human health, such as strengthen-ing the immune system, decreasing the risk of develop-ing certain types of cancer, reducing the risk ofcardiovascular diseases and preventing macular degen-eration and cataracts (Al-Delaimy et al., 2005; Maianiet al., 2009).

The physical state and location of carotenoids inplants strongly affect accessibility during digestion,which is subsequently related to release and absorption.

It was previously reported that the release and absorp-tion of carotenoids present in raw fruit and vegetablesare highly inefficient and could be as low as 3% (Hedr�enet al., 2002). In plants, carotenoids could occur (i) as partof the photosynthetic apparatus where they are associ-ated closely with proteins in chromoplasts and trappedwithin the cell structures, (ii) as membrane-bound semi-crystalline structures within the chromoplasts or chloro-plast, or (iii) dissolved in oil droplets in chromoplasts(Hornero-M�endez & M�ınguez-Mosquera, 2007). Theunique location and different forms of carotenoidsstrongly impact upon the adsorption of these compoundsfrom different foodmatrices.Carrot pomace is a rich source of antioxidants, die-

tary fibre and natural carotenoids, which have beenshown to exhibit health-promoting effects. During car-rot juice manufacture, around 80% of carotenoidsmay be retained in the carrot pomace (Kumar & Ku-mar, 2012), possibly due to carotenoids being depos-ited in crystalline form in the chromoplasts, andcarotene crystals being stabilised by other componentssuch as protein or residual membranes (Reiter et al.,2003). Due to this crystalline nature, the stability ofcarotenoids in carrot pomace is high. Food processing,such as mechanical homogenisation or heat treatment,disintegrates carrot tissue and consequently improvesthe bioavailability of carotenoids (Van Buggenhout

*Correspondent: Fax: +64 3 479 7567;

e-mail: [email protected]

International Journal of Food Science and Technology 2014, 49, 2120–2127

doi:10.1111/ijfs.12510

© 2014 Institute of Food Science and Technology

2120

et al., 2010). Cooking of plant materials, such as heat-ing to break down plant cell structure, has been shownto enhance carotenoid bioavailability (Hedr�en et al.,2002). Depending on the severity of the heat treat-ment, degradation of carotenoids could occur afterbeing released from the cell membranes.

Pulsed electric field (PEF) processing is one of thenonthermal emerging technologies that affects cellmembrane permeability. The focus of PEF applica-tions has been on permeabilisation of cell membranesto enhance the extraction of bioactive compoundsfrom the inner part of cells (Dons�ı et al., 2010), mainlyfrom the cytosol, such as anthocyanin from grapes,purple-fleshed potato (Corrales et al., 2008; Gac-hovska et al., 2010; Pu�ertolas et al., 2013), betaninefrom red beetroot (L�opez et al., 2009) and phenolicsfrom Cabernet Franc grapes (El Darra et al., 2013). Inthis process, the efficacy of electropermeabilisationstrongly depends on the PEF parameters. Electric fieldstrength is an important parameter that controls theefficiency of cellular tissue electroplasmolysis. It waspreviously reported that for low electric field strengths(≤100–200 V cm�1), the total time of electric currentapplication should be long for electroplasmolysis ofcellular tissue. This causes the destruction of cellularmembranes due to ohmic heating, with consequentdamage of thermosensitive elements and deteriorationof product quality. In contrast, using high electric fieldstrengths (>1000 V cm�1) will increase energy con-sumption (Bazhal et al., 2003). Therefore, using mod-erate electric field strengths (300–1000 V cm�1) hasbeen suggested to obtain optimum permeabilisationwith minimum energy consumption (Jemai & Voro-biev, 2002). In addition to electric field strength, pulsefrequency is another meaningful parameter in the elec-troporation process. Theoretical modelling shows thatthe degree of electroporation decreases with increasingfrequency (Bilska et al., 2000). The PEF-inducedbreakage in plant tissue at different frequencies wasstudied by Asavasanti et al. (2011), and the resultsrevealed that a larger number of cells are irreversiblypermeabilised at low frequency.

The next step after release of carotenoids from theinner part of the cells is to extract these compoundsby organic solvents; however, the use of conventionalcarotenoid extraction methods requires a large amountof organic solvents, which are costly, environmentallyhazardous, and requires expensive disposal procedures(Mustafa et al., 2012). The oil solubilisation character-istics of carotenoids have led to studies on recovery ofthese components using vegetable oils. The directenrichment of vegetable oil with carotenoids improvesthe human nutritional quality aspects. Studies con-ducted so far have mainly focused on the extraction ofcarotenoids from animal tissues using vegetable oils(No & Meyers, 1992; Sachindra & Mahendrakar,

2005); little is known about the extraction of carote-noids from plant materials (Benakmoum et al., 2008).Long-chain fatty acids and triglycerides were mostlyused in those studies. Carotenoid extraction yield wasfound to be higher when oils with a higher degree ofpolyunsaturated fatty acids (e.g. sunflower oil) wereused during the extraction process (No & Meyers,1992; Sachindra & Mahendrakar, 2005; Benakmoumet al., 2008).Up to the present time, most studies have investi-

gated the aqueous phase (juice) rather than the solidphase (pomace) after PEF treatment. This technologyhas been shown to be an effective pretreatmentfor juice extraction (El-belghiti & Vorobiev, 2005;Vorobiev & Lebovka, 2006; Grimi et al., 2007). Theissue of whether electroporation due to PEF treatmentis also effective at releasing membrane-bound com-pounds such as carotenoids, which are not freely situ-ated in the cytosol but strongly trapped in cellorganelles such as the chromoplast or chloroplast, isnot settled. Therefore, this study was designed to eval-uate the effects of PEF on the distribution of carote-noids in carrot pur�ee, juice and pomace after PEFprocessing. The purpose of the present work was toinvestigate the effect of moderate electric fieldstrengths and frequency on the extractability of carote-noids (a-carotene, b-carotene) in carrot pur�ee. Differ-ent vegetable oils were also used to test the carotenoidextractability of carrot pomace after PEF processing.

Material and methods

Reagents and chemicals

Calcium chloride, ethanol (95%), methanol (HPLCgrade) and acetone were purchased from Biolab (Scores-by, Vic., Australia), 2-diphenyl-1-picrylhydrazyl andbutylated hydroxytoluene from Sigma-Aldrich (St.Louis, MO, USA) and n-hexane from J.T. Baker (Phil-lipsburg, NJ, USA). Commercially available vegetableoils used for this experiment, including canola oil,sunflower oil, peanut oil, rice bran oil and soya bean oil,were purchased from New Zealand markets.

Sample preparation

The same variety of fresh carrots (Daucus carota ssp.Nantes) harvested between November and December2011 were purchased from a supplier of locally grownproducts (Kaan’s Catering Supplies Ltd., Dunedin,New Zealand), within 48 h after harvest. Upon arrival,samples were immediately stored at 4 °C under sub-dued light for <4 weeks during the period of process-ing and analysis. On the day of each experiment,carrots (2 kg) were randomly removed from storageand rinsed with tap water (approximately 8 °C) to

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remove any soil residues and debris. Both the top andbottom parts of the carrot root (approximately20 mm) were discarded. After washing, the carrotswere sliced into 0.8-mm-thick discs using an automaticslicer (Robot Coupe R211 Ultra Model, Montceau-en-Bourgogne, France) to have similar size of carrotpieces and minimise size variation. To make one batchof carrot pur�ee, the carrot discs (500–600 g) were pro-cessed into a pur�ee by mixing with distilled water (1:1ratio) and blending using a Waring blender (WatsonVictor Limited, Wellington, New Zealand) at mixingspeed of 18 000 r.p.m. and room temperature(18–20 °C) for 20 s resulting in 1–1.2 kg of carrotpur�ee. Afterwards, the resulting pur�ee was precondi-tioned at 20 °C before PEF treatment. The whole sam-ple preparation was carried out under subdued light.

Pulsed electric field treatment

Carrot pur�ee was treated using an ELRACK-HPV 5PEF unit (Quakenbr€uck, Germany) with batch treat-ment configuration. For each PEF treatment, 100 gof preconditioned carrot pur�ee was placed inside thePEF chamber of dimensions 80 9 100 9 50 mmwith a sample capacity of 400 mL, consisting of twostainless steel parallel-plate electrodes with a gapof 80 mm. Each PEF combination of treatments(100 g each; conditions shown in Table S1) wasindependently carried out in triplicate using threeindependent carrot pur�ee batches and compared withuntreated samples (control) from each of the corre-sponding batches. The temperature and conductivityof the carrot pur�ee were measured prior to, and afterPEF treatment, using an electrical conductivity meter(CyberScan CON 11; Eutech Instruments, Singapore,Singapore). The weight of the sample placed in thechamber was recorded, and the specific energy con-sumption (Wspec) during the PEF treatment wascalculated using eqn (1):

W ¼ UINs=m ð1Þwhere U and I represent, respectively, the voltageacross the electrodes in volts (V) and the amplitude ofthe electric current strength in amperes (A), and N isthe number of pulses, s is the duration of pulse in s,and m is the total mass of carrot pur�ee in the PEFtreatment chamber in grams. All treatments wereapplied as follows: (i) different moderate electric fieldstrengths (0.1, 0.3, 0.6, 0.8 or 1 kV cm�1) with con-stant frequency of 5 Hz, pulse width of 20 ls and (ii)different frequency (10, 30, 50 or 75 Hz) with constantelectric field strength of 1 kV cm�1 and pulse width of20 ls. The processing time was calculated by multiply-ing the number of pulses and effective pulse durationas presented in Tables 1 and 2. All samples were trea-ted using bipolar square pulses.

After PEF treatment, the treated carrot pur�ee wasimmediately cooled down and placed in the dark chil-ler (4 °C for <3 h) until subsequent carotenoid analy-sis, to minimise oxidation. For each PEF combination,carrot samples without PEF treatments were used ascontrols. Each entire sample treated in the PEF cham-ber was transferred to 250 mL polyallomer centrifugebottle (Beckman, Fullerton, CA, USA) and centrifugedtwice at 15 300 g for 30 min at 4 °C, and the solidphase (pomace) was separated from the liquid phase(juice) by filtration (LabServ LBS0001.90 filter paper;ThermoFisher Scientific, Auckland, New Zealand).The weight of separated juice and pomace was mea-sured. The percentage of juice and pomace yield (Y)was calculated using eqn (2):

Y ¼ m

mi� 100 ð2Þ

where m is the weight of juice or pomace, and mi isthe initial weight of carrot pur�ee before separation.Carotenoid contents in both juice and pomace weremeasured immediately after separation. Carrot juiceand freeze-dried pomace were stored at �80 °C forsubsequent measurement. The schematic diagram ofthe experimental procedure is shown in Fig. S1.

Determination of carotenoid content

Carotenoids were extracted from carrot samples (Sa-dler et al., 1990) under dark conditions to protect car-otenoids from light degradation. Samples (5 g carrotpomace or 5 g carrot juice) were homogenised with50 mL extraction solvent (50% hexane, 25% acetone,25% ethanol, containing 0.1% butylated hydroxytolu-ene (v/v) and 5 g CaCl2�2H2O) to achieve a distinctseparation of the water and organic layers. The mix-ture was stirred for 20 min at 4 °C and further washedby adding 15 mL of reagent grade water, followed bystirring for 10 min at 4 °C. The mixture was trans-ferred into a separation funnel. The water layer wasdiscarded, and the organic phase was collected andbrought to 50 mL with extraction solvent and storedin dark vials to minimise light degradation until subse-quent analysis. The absorbance of carotenoids wasmeasured using a UV/visible spectrophotometer(Ultraspec 3300 Pro; Amersham Biosciences, Uppsala,Sweden) at the specific absorption wavelength andextinction coefficient of the carotenoids of interestagainst the extraction solvent blank (Hart & Scott,1995). The concentration of carotenoids (C) was calcu-lated using eqn (3) and based on Lambert Beer theory:

C ¼ A� V� 106=e1%1cm � 100�W ð3Þwhere A is the absorbance at kmax, V is the total vol-ume of extract and e1%1cm is the extinction coefficient ofcarotenoids (a-carotene: 2860; b-carotene: 2620 L

© 2014 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014

Carrot pomace with higher carotenoid extractability S. Roohinejad et al.2122

mol�1 cm�1. Concentration of carotenoids wasexpressed as lg per g of wet weight of sample.

The dry matter and water content of all treatedsamples were determined gravimetrically. Treated anduntreated carrot pomace (5 g each) were dried in aconvection oven at 80 °C for at least 48 h or until aconstant weight was achieved. The carotenoid contentof pomace was calculated on a dry matter basisbecause samples treated by PEF contained differentwater contents depending on the specific carrot batchesused.

Extraction of carotenoids from carrot pomace withvegetable oils

Carotenoids of PEF-treated and untreated carrot pom-ace were extracted using different vegetable oils andcompared with hexane. Freeze-dried PEF-treated (e.g.0.6 kV cm�1 and 5 Hz, or 0.6 kV cm�1 and 50 Hz)carrot pomace (1 g) was accurately added to 30 g offive different vegetable oils, namely soya bean oil,canola oil, sunflower oil, rice bran oil and peanut oil.The extraction was carried out via incubation at 30 °Cwith constant shaking at 300 r.p.m. for 24 h in thedark. The samples were placed in amber bottles andflushed for 1 min with nitrogen before shaking. Thesuspension were then transferred to 50 mL polyallo-mer centrifuge bottle (Beckman) and centrifuged at60 800 g for 15 min, and the clear supernatant oil wasdecanted. The carotenoid content in oils was deter-mined using the same procedure as previouslydescribed.

Statistical analysis

The results were expressed as mean � standard devia-tion for three independent samples. The data obtainedfrom the same carrot batch were subjected to one-wayanalysis of variance (ANOVA) followed by Student’spaired t-test. Significant differences among mean val-ues were determined by Tukey’s test significancedefined at P < 0.05. Statistical analysis was carried outusing Minitab software (version 16; Minitab Inc., StateCollege, PA, USA).

Results and discussion

Effect of electric field strength on conductivity and juiceyield

Different techniques have been used to assess cellmembrane permeability or integrity, such as micros-copy, determination of liquid release, conductivitymeasurement of the secreted liquid and electricalimpedance measurement of cellular tissues (Dons�ıet al., 2010). In the current study, electrical

conductivity was used as an indicator of the intactnessand permeability of cell membranes. Table S1 summa-rises the change in conductivity, load resistance andtemperature increase in carrot pur�ee after PEF treat-ment at different electric field strengths (0.1, 0.3, 0.6,0.8, 1 kV cm�1) combined with a constant frequencyof 5 Hz and bipolar pulse width of 20 ls for totalpulse time of 3 ms. Increasing the electric field strengthat 5 Hz up to 0.6 kV cm�1 significantly (P < 0.05)increased the change in conductivity and decreased theload resistance of carrot pur�ee. Further elevation ofelectric field strength above 0.6 kV cm�1 did not resultin any significant increase in conductivity, indicatingthat maximum cell rupture was obtained at this elec-tric field strength. Electric field strengths between 0.6and 1 kV cm�1 showed no significant difference intheir effect on conductivity (P > 0.05; Table S1). Inaddition, at 5 Hz and electric field strengths of 0.3 kVcm�1 and above, PEF-treated samples had significantly(P < 0.05) lower moisture content than the control(samples without PEF treatment). Increasing the elec-tric field strength led to an increase in temperature,but the final temperature of the sample after treatmentwas still below 25 °C. PEF treatment at moderate elec-tric field strengths (0.5 and 1 kV cm�1 for 10�4 to10�2 s) can damage the cell membrane of plant tissuewith little temperature increase (Fincan & Dejmek,2002; Lebovka et al., 2002). Therefore, PEF can beused to minimise the degradation of heat-, light- andoxygen-sensitive compounds such as carotenoids(Torregrosa et al., 2005).These results are in agreement with Ersus & Barrett

(2010) who studied the effect of electric field strengthson ion leakage in onions and found that at constantfrequency, increasing the electric field strength up to0.5 kV cm�1 increased damage efficiently, but therewas no significant impact on the efficiency beyond thislevel of field strength. It was also previously reportedthat the critical level of electric field strength requiredfor cell rupture in various plant tissues ranges from0.2 to 0.5 kV cm�1, depending on the type of tissue(Bazhal et al., 2003). These authors suggested thatadditional energy beyond than that required for plantcell rupture resulted in a progressive increase inenergy consumption but with no further increase incell disintegration. Therefore, there is an optimal elec-tric field strength corresponding to a minimumamount of total energy consumption to achieve suffi-cient cell rupture. The present study confirmed thisphenomenon and found no significant increase in cellpermeability at electric field strengths higher than0.6 kV cm�1.Previous studies have also found that electric field

strengths between 0.5 and 1 kV cm�1 and treatmenttimes of 10�4 to 10�2 s led to high degree of cell dam-age for the majority of plant tissues (Lebovka et al.,



2002). The average electrical conductivity of tissueincreases with the degree of its damage and is directlyproportional to the number of permeabilised cells(Lebovka et al., 2002). A slight membrane breakdownof potato, apple and fish tissues was reported between0.015 and 0.2 kV cm�1, whereas significant membranebreakdown was observed when the electric fieldstrength was increased from 0.4 to 0.8 kV cm�1

(Angersbach et al., 2000). When an external electricfield is applied to a cell, a transmembrane potential isinduced. Strong polarisation due to the electric fieldcauses an increase in cell membrane conductance andpermeability (Coster, 1965).

In addition to conductivity, the impact of electricfield strength on mass transfer was investigated. Only47% of the juice was obtained from non-PEF-treatedcarrot pur�ee after centrifugation (Fig. S2a). Thisamount of juice was mainly extracted from themechanically treated cells due to pur�ee preparation.When samples were treated with a high electric fieldstrength, the observed decrease in the moisture con-tent of the pomace (Table S1) was likely due to thecell membranes being ruptured, leading to an increasein permeability of the cell walls and an increase inrelease of intracellular compounds. There was no dif-ference in juice yield for any of the electric field levelsapplied; however, there was a significant differencebetween the control and treatments of 0.6 kV cm�1

and above (Fig. S2a). The quantity of extracted juiceincreased at an energy level of 2.6 kJ kg�1 (impartedby the 0.6 kV cm�1 treatment). Above this energylevel, any further enhancement of juice yield recoverywas not observed. Therefore, this energy could be suf-ficient to give maximal electropermeabilisation effectsto carrot cells.

Most reports have shown that PEF treatmentenhances juice yield (Bazhal & Vorobiev, 2000; Es-htiaghi & Knorr, 2002); however, a few studies did notreport any significant increase (McLellan et al., 1991).The results of this study are in good agreement withthose reported by Geulen et al. (1994) and El-belghiti& Vorobiev (2005) who found that juice yieldincreased with an increase in PEF energy level. Thedifference in the optimum energy level found in thisstudy, compared with that reported by El-belghiti &Vorobiev (2005) of 9 kJ kg�1, could be due to the dif-ference in the size and shape of the carrot pieces used(carrot pur�ees compared with disc and sliced carrots).Moreover, it was previously reported that the juiceyield increased by 30% (carrot pieces of 3 mm) and50% (carrot pieces of 1.5 mm) in untreated carrotmash to about 70% after PEF treatment at 2.6 kVcm�1 (Geulen et al., 1994). The difference betweenjuice extractions in different studies may be attributedto differences in separation techniques or initial mois-ture content of the samples.

Effect of pulsed electric field frequency on conductivityand yield

Increasing the electrical field strength, pulse durationor number of pulses can enhance both the degree ofmembrane rupture and increase the density of pores inthe membrane and cell wall (Ersus & Barrett, 2010);however, little is known about the effect of pulsefrequency on plant cell membrane electroporation(Vorobiev & Lebovka, 2009). Table S1 shows thatincreasing PEF frequency at constant electric fieldstrength (1 kV cm�1) did not significantly (P > 0.05)affect the changes in conductivity, load resistance, tem-perature increase and moisture content (%).The effect of PEF frequency on the percentage of

juice yield is reported in Fig. S3a. In all cases, anincrease in juice yield was achieved by applying PEFtreatment whereas increasing the frequency did notcontribute to a further increase in juice yield, whichconsequently maintained the same moisture content ofpomace (Table S1). It has been reported that even athigh and low pulse duration, the effect of increasingPEF frequency on ion leakage is not significant (Ersus& Barrett, 2010). These authors suggested that whenpulses were applied with a high frequency of repeti-tion, the delay between two consecutive pulses was tooshort for membrane charging, therefore did not causesignificant cell membrane rupture.

Effect of electric field strength and frequency ondistribution of carotenoids in carrot pur�ee

The effect of electric field strength on carotenoid con-tent in juice and pomace was further examined todetermine whether the conductivity increase was dueto carotenoids being released, or leaching of otherminerals or solid substances into the juice. Increasingthe electric field strength at constant frequency for afixed treatment time significantly (P < 0.05) increasedthe concentration of a- and b-carotenes (lg g�1) incarrot pomace compared with untreated pur�ee(Fig. S2b). The lack of an increase in carotenoidextraction when the field strength was increased from0.6 to 1 kV cm�1 showed that this electric fieldstrength (0.6 kV cm�1) was enough to achieve themaximum cell disintegration.Increasing the electric field strength at constant fre-

quency for a fixed treatment time had no significantimpact on the concentration of carotenoids in carrotjuice (Fig. S2b). In other words, the majority of thecarotenoids released from carrot cells remained in thecarrot pomace, and the amount of carotenoids releasedto the juice was negligible. This might be due to thereason that carotenoids are almost insoluble in water,which limits their leaching into the juice during pro-cessing. The results of current study are in agreement



with Grimi et al.(2007) who reported that approxi-mately, the same content of carotenoids was obtainedin juice solutions, either with or without the applica-tion of PEF, using moderate electric field strengths(0.25–1 kV cm�1).

The effect of increasing PEF frequency on the con-tent of carotenoids in carrot pur�ee showed a differentbehaviour compared with the effect of increasing thefield strength. At a fixed electric field strength of 1 kVcm�1 and pulse width of 20 ls, increasing the PEF fre-quency did not show any significant effect (P > 0.05)on the content of carotenoids in both carrot pomaceand juice (Fig. S3b). This study shows that at highelectric field strength, frequencies above 10 Hz are notcapable of increasing the partitioning of carotenoidsfrom pomace to juice. It should be taken into consid-eration that the carrots came from different batcheswith some variation in the initial carotenoid content.Therefore, the effect of PEF treatment on carotenoidextractability was compared with the untreated sam-ples from the same batch. There is a critical frequencyof ~1 Hz below which permeabilisation of onion tissuewas reported to be significantly increased (Asavasantiet al., 2012). These authors reported a correlationbetween PEF frequency and the subsequent speed ofintracellular convective motion, that is, cytoplasmicstreaming. In the current study, no improvement inthe extraction of carotenoids was found when thefrequency was increased from 10 to 75 Hz.

Extraction of carotenoids with different oils

Vegetable oils are the most common type of plant oilsand are extensively used in food products. A fewstudies have been carried out on the extraction of car-otenoids from animal wastes such as from shrimp (Sa-chindra & Mahendrakar, 2005) and crawfish (No &Meyers, 1992) using vegetable oils; however, there arelimited studies on extraction of carotenoids from plantmaterials using vegetable oils (Benakmoum et al.,2008). When direct carotenoid extraction was carriedout using untreated and PEF-treated carrot pomace atelectric field strength of 0.6 kV cm�1 and frequency of5 Hz, the yield of carotenoids extracted from PEF-treated carrot pomace was significantly (P < 0.05)higher than untreated samples in all vegetable oils, butnot for samples treated at frequency of 50 Hz(Table S2).

The low net yield of extracted carotenoids after PEFtreatment at 50 Hz can be explained by the inefficientprocessing parameters used to release the majority ofcarotenoids from the carrot cells. The findings of thecurrent study are consistent with those of Lebovka et al.(2000) who found that changing the pulse frequencyabove 10 Hz had no effect on electroporation efficacy.Loghavi et al. (2008) suggested that lower pulse

frequencies may cause more damage to the cell becausethere is more time for charging the cell membranesbetween pulses, thereby facilitating pore formation. Inanother study, Lebovka et al. (2001) proposed a modelfor the effect of PEF on plant tissues in terms of whatthey called a ‘correlated percolation phenomenon’where membrane resealing and moisture transport afterPEF are taken into consideration. These authorsreported that, for high PEF frequencies, the pulse repe-tition time may not be long enough for pores to expand;therefore, relatively less tissue is damaged.It should be considered that oxidative enzymes and

other compounds involved in carotenoid degradationare also released together with the carotenoids. As aresult of carotenoid antioxidant activity, they are eas-ily degraded by exposure to hydroperoxides. DuringPEF treatment at 50 Hz, naturally occurring enzymes(such as lipoxygenase) can catalyse the hydroperoxida-tion of polyunsaturated fatty acids, such as linoleicacids, producing conjugate hydroperoxides. Radicalsfrom the intermediate steps of this reaction may beresponsible for oxidative degradation of carotenoids(Yang et al., 2013).Cytoplasmic streaming is an important transport

process in plant cells involving a convective phenome-non and plays a significant role in metabolism and dis-tribution of molecules and proteins across organellemembranes (Verchot-Lubicz & Goldstein, 2010). Asa-vasanti et al. (2011) reported a strong correlationbetween the persistence of cytoplasmic streaming andoverall damage to the tissue as a function of frequencyin onion tissue. The high net yield of extracted carote-noids after PEF treatment at 5 Hz in the current studymight be due to the persistence of cytoplasmic stream-ing during low PEF frequency, which plays an impor-tant role in accelerating the physical damage of cellmembranes (Asavasanti et al., 2012).The results of current study revealed that the most

improvement in carotenoid extractability from thecarrot pomace treated at electric field strengths of0.6 kV cm�1 and frequency of 5 Hz was obtained usingsunflower oil and soya bean oil, and the least using pea-nut oil. Although the oils used had long-chain fattyacids, the significant difference in the solubility of carot-enoids in the sunflower oil/soya bean oil and peanut oilmight be attributed to the diversity in the compositionof their fatty acids. Sunflower oil contains 11% satu-rated, 20% monounsaturated and 69% polyunsaturatedfatty acids, whereas peanut oil contains 17% saturated,46% monounsaturated and 32% polyunsaturated fattyacids. Higher carotenoid extractability of sunflower oiland soya bean oil might be due to their higher propor-tion of polyunsaturated fatty acids. This finding is inagreement with previous studies reporting that sun-flower oil gives higher carotenoid yield than the othervegetable oils (e.g. groundnut oil, coconut oil and rice



bran oil) studied (Sachindra & Mahendrakar, 2005).Extraction of carotenoids with vegetable oils from PEF-treated carrot pomace has commercial potential, such asthe manufacture of emulsions with carotenoid-saturatedvegetable oil. Further study needs to be done to opti-mise the conditions of carotenoid extraction using sun-flower oil with different extraction conditions such asoil-to-PEF-treated carrot pomace ratio, extraction timeand extraction temperature.

Conclusions

This study shows the feasibility of using PEF treat-ment to develop functional natural food ingredients,for example carrot pomace with improved carotenoidextractability. Electroporation due to PEF treatmentcan be used to improve the extractability of carote-noids in carrot pomace with limited loss of carotenoidsinto the juice during extraction. It should be takeninto account that the PEF processing parametersshould be optimised to achieve the greatest carotenoidextraction yield. Using moderate field strengths up to1 kV cm�1 at 5 Hz increased the release of carotenoidsfrom carrot pomace. Increasing PEF frequency above10 Hz at a constant field strength of 1 kV cm�1 didnot show any further increase in carotenoid extract-ability, which was also confirmed by direct extractionof carotenoids with vegetable oils from carrot pomace.Sunflower and soya bean oils had the highest level ofcarotenoid extractability for carrot pomace treated at0.6 kV cm�1 and 5 Hz, but no significant differencewas observed between the vegetable oils for extractionof carrot pomace treated at 50 Hz. This has importantimplications for developing carotenoid-enrichedvegetable oils from agro-industrial carrot waste as avalue-added technology to be used in the food andpharmaceutical industries. This study suggests thatPEF-treated carrot pomace, a rich source of dietaryfibre, and higher bioavailable carotenoids with excel-lent functional properties could be beneficial as a func-tional food to be used in commercial baby food orother food industries.

Acknowledgments

The authors acknowledge the awarding of a Universityof Otago PhD Scholarship for Shahin Roohinejad andthe technical assistance of Ian Ross, Nerida Downesand Michelle Petrie from the Department of FoodScience, University of Otago.

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Supporting Information

Additional Supporting Information may be found inthe online version of this article:Table S1. Effect of pulsed electric field on change in

conductivity of carrot pur�ee.Table S2. Carotenoid extraction from freeze-dried

PEF-treated and untreated carrot pomace using hex-ane and different vegetable oils (n = 3).Figure S1. The schematic diagram of experimental

procedure.Figure S2. Effect of electric field strength (combined

with frequency of 5 Hz) on the juice and pomace yield(a) and carotenoids content (b).Figure S3. Effect of pulsed electric field (PEF) fre-

quency (1 kV cm�1) on the juice and pomace yield (a)and carotenoids content (b).



effect of pulsed electric field processing on carotenoid extractability of carrot purée

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