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

Original article
Effect of pulsed electric field processing on carotenoid
extractability of carrot pure
e

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.

It was previously reported that the release and absorp-

Introduction
Carotenoids, the main tetraterpenoid organic pig-
ments, naturally exist in the chloroplasts and chro-
moplasts of plants. Although over 650 specific
carotenoids have been identified and isolated from nat-
ural sources, only about 60 are common in the human
diet, and about 20 can be identified in human plasma
and tissues (Dutta et al., 2005). Carrots are a globally
important vegetable crop, providing a good source of
carotenoids. The principal carotenoids of carrots
are 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 that
provide benefits to human health, such as strengthen-
ing the immune system, decreasing the risk of develop-
ing certain types of cancer, reducing the risk of
cardiovascular diseases and preventing macular degen-
eration and cataracts (Al-Delaimy et al., 2005; Maiani
et al., 2009).
The physical state and location of carotenoids in
plants strongly affect accessibility during digestion,
which is subsequently related to release and absorption.
*Correspondent: Fax: +64 3 479 7567;
e-mail: indrawati.oey@otago.ac.nz
tion of carotenoids present in raw fruit and vegetables
are highly inefficient and could be as low as 3% (Hedr
en
et al., 2002). In plants, carotenoids could occur (i) as part
of the photosynthetic apparatus where they are associ-
ated closely with proteins in chromoplasts and trapped
within 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). The
unique location and different forms of carotenoids
strongly impact upon the adsorption of these compounds
from different food matrices.
Carrot pomace is a rich source of antioxidants, die-
tary fibre and natural carotenoids, which have been
shown to exhibit health-promoting effects. During car-
rot juice manufacture, around 80% of carotenoids
may be retained in the carrot pomace (Kumar & Ku-
mar, 2012), possibly due to carotenoids being depos-
ited in crystalline form in the chromoplasts, and
carotene crystals being stabilised by other components
such as protein or residual membranes (Reiter et al.,
2003). Due to this crystalline nature, the stability of
carotenoids in carrot pomace is high. Food processing,
such as mechanical homogenisation or heat treatment,
disintegrates carrot tissue and consequently improves
the bioavailability of carotenoids (Van Buggenhout

doi:10.1111/ijfs.12510
© 2014 Institute of Food Science and Technology

et al., 2010). Cooking of plant materials, such as heat-
ing to break down plant cell structure, has been shown
to enhance carotenoid bioavailability (Hedr
en et al.,
2002). Depending on the severity of the heat treat-
ment, degradation of carotenoids could occur after
being released from the cell membranes.
Pulsed electric field (PEF) processing is one of the
nonthermal emerging technologies that affects cell
membrane permeability. The focus of PEF applica-
tions has been on permeabilisation of cell membranes
to enhance the extraction of bioactive compounds
from the inner part of cells (Donsı et al., 2010), mainly
from the cytosol, such as anthocyanin from grapes,
purple-fleshed potato (Corrales et al., 2008; Gac-
hovska et al., 2010; Pu
ertolas et al., 2013), betanine
from red beetroot (L
opez et al., 2009) and phenolics
from Cabernet Franc grapes (El Darra et al., 2013). In
this process, the efficacy of electropermeabilisation
strongly depends on the PEF parameters. Electric field
strength is an important parameter that controls the
efficiency of cellular tissue electroplasmolysis. It was
previously reported that for low electric field strengths
(≤100–200 V cm
1), the total time of electric current
application should be long for electroplasmolysis of
cellular tissue. This causes the destruction of cellular
membranes due to ohmic heating, with consequent
damage of thermosensitive elements and deterioration
of product quality. In contrast, using high electric field
strengths (>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) has
been suggested to obtain optimum permeabilisation
with minimum energy consumption (Jemai & Voro-
biev, 2002). In addition to electric field strength, pulse
frequency is another meaningful parameter in the elec-
troporation process. Theoretical modelling shows that
the degree of electroporation decreases with increasing
frequency (Bilska et al., 2000). The PEF-induced
breakage in plant tissue at different frequencies was
studied by Asavasanti et al. (2011), and the results
revealed that a larger number of cells are irreversibly
permeabilised at low frequency.
The next step after release of carotenoids from the
inner part of the cells is to extract these compounds
by organic solvents; however, the use of conventional
carotenoid extraction methods requires a large amount
of organic solvents, which are costly, environmentally
hazardous, and requires expensive disposal procedures
(Mustafa et al., 2012). The oil solubilisation character-
istics of carotenoids have led to studies on recovery of
these components using vegetable oils. The direct
enrichment of vegetable oil with carotenoids improves
the human nutritional quality aspects. Studies con-
ducted so far have mainly focused on the extraction of
carotenoids from animal tissues using vegetable oils
(No & Meyers, 1992; Sachindra & Mahendrakar,
© 2014 Institute of Food Science and Technology
Carrot pomace with higher carotenoid extractability S. Roohinejad et al. 2121
2005); little is known about the extraction of carote-
noids from plant materials (Benakmoum et al., 2008).
Long-chain fatty acids and triglycerides were mostly
used in those studies. Carotenoid extraction yield was
found to be higher when oils with a higher degree of
polyunsaturated fatty acids (e.g. sunflower oil) were
used during the extraction process (No & Meyers,
1992; Sachindra & Mahendrakar, 2005; Benakmoum
et al., 2008).
Up to the present time, most studies have investi-
gated the aqueous phase (juice) rather than the solid
phase (pomace) after PEF treatment. This technology
has been shown to be an effective pretreatment
for juice extraction (El-belghiti & Vorobiev, 2005;
Vorobiev & Lebovka, 2006; Grimi et al., 2007). The
issue of whether electroporation due to PEF treatment
is also effective at releasing membrane-bound com-
pounds such as carotenoids, which are not freely situ-
ated in the cytosol but strongly trapped in cell
organelles such as the chromoplast or chloroplast, is
not 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 PEF
processing. The purpose of the present work was to
investigate the effect of moderate electric field
strengths 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 carotenoid
extractability of carrot pomace after PEF processing.
Material and methods
Reagents and chemicals
Calcium chloride, ethanol (95%), methanol (HPLC
grade) and acetone were purchased from Biolab (Scores-
by, Vic., Australia), 2-diphenyl-1-picrylhydrazyl and
butylated hydroxytoluene from Sigma-Aldrich (St.
Louis, MO, USA) and n-hexane from J.T. Baker (Phil-
lipsburg, NJ, USA). Commercially available vegetable
oils 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 December
2011 were purchased from a supplier of locally grown
products (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 storage
and rinsed with tap water (approximately 8 °C) to
International Journal of Food Science and Technology 2014
2122 Carrot pomace with higher carotenoid extractability S. Roohinejad et al.
remove any soil residues and debris. Both the top and
bottom parts of the carrot root (approximately
20 mm) were discarded. After washing, the carrots
were sliced into 0.8-mm-thick discs using an automatic
slicer (Robot Coupe R211 Ultra Model, Montceau-en-
Bourgogne, France) to have similar size of carrot
pieces and minimise size variation. To make one batch
of carrot pur
ee, the carrot discs (500–600 g) were pro-
cessed into a pur
ee by mixing with distilled water (1:1
ratio) and blending using a Waring blender (Watson
Victor Limited, Wellington, New Zealand) at mixing
speed of 18 000 r.p.m. and room temperature
(18–20 °C) for 20 s resulting in 1–1.2 kg of carrot
pur
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 5
PEF unit (Quakenbr€ uck, Germany) with batch treat-
ment configuration. For each PEF treatment, 100 g
of preconditioned carrot pur
ee was placed inside the
PEF chamber of dimensions 80 9 100 9 50 mm
with a sample capacity of 400 mL, consisting of two
stainless steel parallel-plate electrodes with a gap
of 80 mm. Each PEF combination of treatments
(100 g each; conditions shown in Table S1) was
independently carried out in triplicate using three
independent carrot pur
ee batches and compared with
untreated samples (control) from each of the corre-
sponding batches. The temperature and conductivity
of the carrot pur
ee were measured prior to, and after
PEF treatment, using an electrical conductivity meter
(CyberScan CON 11; Eutech Instruments, Singapore,
Singapore). The weight of the sample placed in the
chamber was recorded, and the specific energy con-
sumption (Wspec) during the PEF treatment was
calculated using eqn (1):
W ¼ UINs=m
where U and I represent, respectively, the voltage
across the electrodes in volts (V) and the amplitude of
the electric current strength in amperes (A), and N is
the number of pulses, s is the duration of pulse in s,
and m is the total mass of carrot pur
ee in the PEF
treatment chamber in grams. All treatments were
applied as follows: (i) different moderate electric field
strengths (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 constant
electric field strength of 1 kV cm
1 and pulse width of
20 ls. The processing time was calculated by multiply-
ing the number of pulses and effective pulse duration
as presented in Tables 1 and 2. All samples were trea-
ted using bipolar square pulses.
International Journal of Food Science and Technology 2014
After PEF treatment, the treated carrot pur
ee was
immediately 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 as
controls. Each entire sample treated in the PEF cham-
ber was transferred to 250 mL polyallomer centrifuge
bottle (Beckman, Fullerton, CA, USA) and centrifuged
twice at 15 300 g for 30 min at 4 °C, and the solid
phase (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):
m
Y¼  100 ð2Þ
mi
where m is the weight of juice or pomace, and mi is
the initial weight of carrot pur
ee before separation.
Carotenoid contents in both juice and pomace were
measured immediately after separation. Carrot juice
and freeze-dried pomace were stored at
80 °C for
subsequent measurement. The schematic diagram of
the 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 carrot
pomace or 5 g carrot juice) were homogenised with
50 mL extraction solvent (50% hexane, 25% acetone,
25% ethanol, containing 0.1% butylated hydroxytolu-
ene (v/v) and 5 g CaCl22H2O) to achieve a distinct
separation of the water and organic layers. The mix-
ture was stirred for 20 min at 4 °C and further washed
by adding 15 mL of reagent grade water, followed by
stirring for 10 min at 4 °C. The mixture was trans-
ferred into a separation funnel. The water layer was
ð1Þ discarded, and the organic phase was collected and
brought to 50 mL with extraction solvent and stored
in dark vials to minimise light degradation until subse-
quent analysis. The absorbance of carotenoids was
measured using a UV/visible spectrophotometer
(Ultraspec 3300 Pro; Amersham Biosciences, Uppsala,
Sweden) at the specific absorption wavelength and
extinction coefficient of the carotenoids of interest
against 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 ð3Þ
where A is the absorbance at kmax, V is the total vol-
ume of extract and e1%
1cm is the extinction coefficient of
carotenoids (a-carotene: 2860; b-carotene: 2620 L
© 2014 Institute of Food Science and Technology

mol
1 cm
1. Concentration of carotenoids was
expressed as lg per g of wet weight of sample.
The dry matter and water content of all treated
samples were determined gravimetrically. Treated and
untreated carrot pomace (5 g each) were dried in a
convection oven at 80 °C for at least 48 h or until a
constant weight was achieved. The carotenoid content
of pomace was calculated on a dry matter basis
because samples treated by PEF contained different
water contents depending on the specific carrot batches
used.
Extraction of carotenoids from carrot pomace with
vegetable oils
Carotenoids of PEF-treated and untreated carrot pom-
ace were extracted using different vegetable oils and
compared 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 of
five 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 °C
with constant shaking at 300 r.p.m. for 24 h in the
dark. The samples were placed in amber bottles and
flushed for 1 min with nitrogen before shaking. The
suspension were then transferred to 50 mL polyallo-
mer centrifuge bottle (Beckman) and centrifuged at
60 800 g for 15 min, and the clear supernatant oil was
decanted. The carotenoid content in oils was deter-
mined using the same procedure as previously
described.
Statistical analysis
The results were expressed as mean  standard devia-
tion for three independent samples. The data obtained
from the same carrot batch were subjected to one-way
analysis of variance (ANOVA) followed by Student’s
paired t-test. Significant differences among mean val-
ues were determined by Tukey’s test significance
defined at P < 0.05. Statistical analysis was carried out
using Minitab software (version 16; Minitab Inc., State
College, PA, USA).
Results and discussion
Effect of electric field strength on conductivity and juice
yield
Different techniques have been used to assess cell
membrane permeability or integrity, such as micros-
copy, determination of liquid release, conductivity
measurement of the secreted liquid and electrical
impedance measurement of cellular tissues (Donsı
et al., 2010). In the current study, electrical
© 2014 Institute of Food Science and Technology
Carrot pomace with higher carotenoid extractability S. Roohinejad et al. 2123
conductivity was used as an indicator of the intactness
and permeability of cell membranes. Table S1 summa-
rises the change in conductivity, load resistance and
temperature 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 frequency
of 5 Hz and bipolar pulse width of 20 ls for total
pulse time of 3 ms. Increasing the electric field strength
at 5 Hz up to 0.6 kV cm
1 significantly (P < 0.05)
increased the change in conductivity and decreased the
load resistance of carrot pur
ee. Further elevation of
electric field strength above 0.6 kV cm
1 did not result
in any significant increase in conductivity, indicating
that maximum cell rupture was obtained at this elec-
tric field strength. Electric field strengths between 0.6
and 1 kV cm
1 showed no significant difference in
their effect on conductivity (P > 0.05; Table S1). In
addition, at 5 Hz and electric field strengths of 0.3 kV
cm
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 treatment
was still below 25 °C. PEF treatment at moderate elec-
tric field strengths (0.5 and 1 kV cm
1 for 10
4 to
10
2 s) can damage the cell membrane of plant tissue
with little temperature increase (Fincan & Dejmek,
2002; Lebovka et al., 2002). Therefore, PEF can be
used to minimise the degradation of heat-, light- and
oxygen-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 strengths
on ion leakage in onions and found that at constant
frequency, increasing the electric field strength up to
0.5 kV cm
1 increased damage efficiently, but there
was no significant impact on the efficiency beyond this
level of field strength. It was also previously reported
that the critical level of electric field strength required
for cell rupture in various plant tissues ranges from
0.2 to 0.5 kV cm
1, depending on the type of tissue
(Bazhal et al., 2003). These authors suggested that
additional energy beyond than that required for plant
cell rupture resulted in a progressive increase in
energy consumption but with no further increase in
cell disintegration. Therefore, there is an optimal elec-
tric field strength corresponding to a minimum
amount of total energy consumption to achieve suffi-
cient cell rupture. The present study confirmed this
phenomenon and found no significant increase in cell
permeability at electric field strengths higher than
0.6 kV cm
1.
Previous studies have also found that electric field
strengths between 0.5 and 1 kV cm
1 and treatment
times of 10
4 to 10
2 s led to high degree of cell dam-
age for the majority of plant tissues (Lebovka et al.,
International Journal of Food Science and Technology 2014
2124 Carrot pomace with higher carotenoid extractability S. Roohinejad et al.
2002). The average electrical conductivity of tissue
increases with the degree of its damage and is directly
proportional to the number of permeabilised cells
(Lebovka et al., 2002). A slight membrane breakdown
of potato, apple and fish tissues was reported between
0.015 and 0.2 kV cm
1, whereas significant membrane
breakdown was observed when the electric field
strength was increased from 0.4 to 0.8 kV cm
1
(Angersbach et al., 2000). When an external electric
field is applied to a cell, a transmembrane potential is
induced. Strong polarisation due to the electric field
causes an increase in cell membrane conductance and
permeability (Coster, 1965).
In addition to conductivity, the impact of electric
field strength on mass transfer was investigated. Only
47% of the juice was obtained from non-PEF-treated
carrot pur
ee after centrifugation (Fig. S2a). This
amount of juice was mainly extracted from the
mechanically treated cells due to pur
ee preparation.
When samples were treated with a high electric field
strength, the observed decrease in the moisture con-
tent of the pomace (Table S1) was likely due to the
cell membranes being ruptured, leading to an increase
in permeability of the cell walls and an increase in
release of intracellular compounds. There was no dif-
ference in juice yield for any of the electric field levels
applied; however, there was a significant difference
between the control and treatments of 0.6 kV cm
1
and above (Fig. S2a). The quantity of extracted juice
increased at an energy level of 2.6 kJ kg
1 (imparted
by the 0.6 kV cm
1 treatment). Above this energy
level, any further enhancement of juice yield recovery
was not observed. Therefore, this energy could be suf-
ficient to give maximal electropermeabilisation effects
to carrot cells.
Most reports have shown that PEF treatment
enhances juice yield (Bazhal & Vorobiev, 2000; Es-
htiaghi & Knorr, 2002); however, a few studies did not
report any significant increase (McLellan et al., 1991).
The results of this study are in good agreement with
those reported by Geulen et al. (1994) and El-belghiti
& Vorobiev (2005) who found that juice yield
increased with an increase in PEF energy level. The
difference in the optimum energy level found in this
study, 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 juice
yield increased by 30% (carrot pieces of 3 mm) and
50% (carrot pieces of 1.5 mm) in untreated carrot
mash to about 70% after PEF treatment at 2.6 kV
cm
1 (Geulen et al., 1994). The difference between
juice extractions in different studies may be attributed
to differences in separation techniques or initial mois-
ture content of the samples.
International Journal of Food Science and Technology 2014
Effect of pulsed electric field frequency on conductivity
and yield
Increasing the electrical field strength, pulse duration
or number of pulses can enhance both the degree of
membrane rupture and increase the density of pores in
the membrane and cell wall (Ersus & Barrett, 2010);
however, little is known about the effect of pulse
frequency on plant cell membrane electroporation
(Vorobiev & Lebovka, 2009). Table S1 shows that
increasing PEF frequency at constant electric field
strength (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, an
increase in juice yield was achieved by applying PEF
treatment whereas increasing the frequency did not
contribute to a further increase in juice yield, which
consequently maintained the same moisture content of
pomace (Table S1). It has been reported that even at
high and low pulse duration, the effect of increasing
PEF frequency on ion leakage is not significant (Ersus
& Barrett, 2010). These authors suggested that when
pulses were applied with a high frequency of repeti-
tion, the delay between two consecutive pulses was too
short for membrane charging, therefore did not cause
significant cell membrane rupture.
Effect of electric field strength and frequency on
distribution of carotenoids in carrot pur
ee
The effect of electric field strength on carotenoid con-
tent in juice and pomace was further examined to
determine whether the conductivity increase was due
to carotenoids being released, or leaching of other
minerals or solid substances into the juice. Increasing
the electric field strength at constant frequency for a
fixed treatment time significantly (P < 0.05) increased
the concentration of a- and b-carotenes (lg g
1) in
carrot pomace compared with untreated pur
ee
(Fig. S2b). The lack of an increase in carotenoid
extraction when the field strength was increased from
0.6 to 1 kV cm
1 showed that this electric field
strength (0.6 kV cm
1) was enough to achieve the
maximum cell disintegration.
Increasing the electric field strength at constant fre-
quency for a fixed treatment time had no significant
impact on the concentration of carotenoids in carrot
juice (Fig. S2b). In other words, the majority of the
carotenoids released from carrot cells remained in the
carrot pomace, and the amount of carotenoids released
to the juice was negligible. This might be due to the
reason 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
© 2014 Institute of Food Science and Technology

with Grimi et al.(2007) who reported that approxi-
mately, the same content of carotenoids was obtained
in 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 different
behaviour compared with the effect of increasing the
field strength. At a fixed electric field strength of 1 kV
cm
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 pomace
and juice (Fig. S3b). This study shows that at high
electric field strength, frequencies above 10 Hz are not
capable of increasing the partitioning of carotenoids
from pomace to juice. It should be taken into consid-
eration that the carrots came from different batches
with some variation in the initial carotenoid content.
Therefore, the effect of PEF treatment on carotenoid
extractability was compared with the untreated sam-
ples from the same batch. There is a critical frequency
of ~1 Hz below which permeabilisation of onion tissue
was reported to be significantly increased (Asavasanti
et al., 2012). These authors reported a correlation
between PEF frequency and the subsequent speed of
intracellular convective motion, that is, cytoplasmic
streaming. In the current study, no improvement in
the extraction of carotenoids was found when the
frequency was increased from 10 to 75 Hz.
Extraction of carotenoids with different oils
Vegetable oils are the most common type of plant oils
and are extensively used in food products. A few
studies 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 are
limited studies on extraction of carotenoids from plant
materials using vegetable oils (Benakmoum et al.,
2008). When direct carotenoid extraction was carried
out using untreated and PEF-treated carrot pomace at
electric field strength of 0.6 kV cm
1 and frequency of
5 Hz, the yield of carotenoids extracted from PEF-
treated carrot pomace was significantly (P < 0.05)
higher than untreated samples in all vegetable oils, but
not for samples treated at frequency of 50 Hz
(Table S2).
The low net yield of extracted carotenoids after PEF
treatment at 50 Hz can be explained by the inefficient
processing parameters used to release the majority of
carotenoids from the carrot cells. The findings of the
current study are consistent with those of Lebovka et al.
(2000) who found that changing the pulse frequency
above 10 Hz had no effect on electroporation efficacy.
Loghavi et al. (2008) suggested that lower pulse
© 2014 Institute of Food Science and Technology
Carrot pomace with higher carotenoid extractability S. Roohinejad et al. 2125
frequencies may cause more damage to the cell because
there is more time for charging the cell membranes
between pulses, thereby facilitating pore formation. In
another study, Lebovka et al. (2001) proposed a model
for the effect of PEF on plant tissues in terms of what
they called a ‘correlated percolation phenomenon’
where membrane resealing and moisture transport after
PEF are taken into consideration. These authors
reported 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 degradation
are also released together with the carotenoids. As a
result of carotenoid antioxidant activity, they are eas-
ily degraded by exposure to hydroperoxides. During
PEF treatment at 50 Hz, naturally occurring enzymes
(such as lipoxygenase) can catalyse the hydroperoxida-
tion of polyunsaturated fatty acids, such as linoleic
acids, producing conjugate hydroperoxides. Radicals
from the intermediate steps of this reaction may be
responsible 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 organelle
membranes (Verchot-Lubicz & Goldstein, 2010). Asa-
vasanti et al. (2011) reported a strong correlation
between the persistence of cytoplasmic streaming and
overall damage to the tissue as a function of frequency
in onion tissue. The high net yield of extracted carote-
noids after PEF treatment at 5 Hz in the current study
might 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 cell
membranes (Asavasanti et al., 2012).
The results of current study revealed that the most
improvement in carotenoid extractability from the
carrot pomace treated at electric field strengths of
0.6 kV cm
1 and frequency of 5 Hz was obtained using
sunflower oil and soya bean oil, and the least using pea-
nut oil. Although the oils used had long-chain fatty
acids, the significant difference in the solubility of carot-
enoids in the sunflower oil/soya bean oil and peanut oil
might be attributed to the diversity in the composition
of their fatty acids. Sunflower oil contains 11% satu-
rated, 20% monounsaturated and 69% polyunsaturated
fatty acids, whereas peanut oil contains 17% saturated,
46% monounsaturated and 32% polyunsaturated fatty
acids. Higher carotenoid extractability of sunflower oil
and soya bean oil might be due to their higher propor-
tion of polyunsaturated fatty acids. This finding is in
agreement with previous studies reporting that sun-
flower oil gives higher carotenoid yield than the other
vegetable oils (e.g. groundnut oil, coconut oil and rice
International Journal of Food Science and Technology 2014
2126 Carrot pomace with higher carotenoid extractability S. Roohinejad et al.
bran oil) studied (Sachindra & Mahendrakar, 2005).
Extraction of carotenoids with vegetable oils from PEF-
treated carrot pomace has commercial potential, such as
the manufacture of emulsions with carotenoid-saturated
vegetable oil. Further study needs to be done to opti-
mise the conditions of carotenoid extraction using sun-
flower oil with different extraction conditions such as
oil-to-PEF-treated carrot pomace ratio, extraction time
and 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 carotenoid
extractability. Electroporation due to PEF treatment
can be used to improve the extractability of carote-
noids in carrot pomace with limited loss of carotenoids
into the juice during extraction. It should be taken
into account that the PEF processing parameters
should be optimised to achieve the greatest carotenoid
extraction yield. Using moderate field strengths up to
1 kV cm
1 at 5 Hz increased the release of carotenoids
from carrot pomace. Increasing PEF frequency above
10 Hz at a constant field strength of 1 kV cm
1 did
not show any further increase in carotenoid extract-
ability, which was also confirmed by direct extraction
of carotenoids with vegetable oils from carrot pomace.
Sunflower and soya bean oils had the highest level of
carotenoid extractability for carrot pomace treated at
0.6 kV cm
1 and 5 Hz, but no significant difference
was observed between the vegetable oils for extraction
of carrot pomace treated at 50 Hz. This has important
implications for developing carotenoid-enriched
vegetable oils from agro-industrial carrot waste as a
value-added technology to be used in the food and
pharmaceutical industries. This study suggests that
PEF-treated carrot pomace, a rich source of dietary
fibre, and higher bioavailable carotenoids with excel-
lent functional properties could be beneficial as a func-
tional food to be used in commercial baby food or
other food industries.
Acknowledgments
The authors acknowledge the awarding of a University
of Otago PhD Scholarship for Shahin Roohinejad and
the technical assistance of Ian Ross, Nerida Downes
and Michelle Petrie from the Department of Food
Science, University of Otago.
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Supporting Information
Additional Supporting Information may be found in
the 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).
International Journal of Food Science and Technology 2014