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Food Chemistry 136 (2013) 13301336

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Pulsed-electric-eld-assisted extraction of anthocyanins from purple-eshed potato


Eduardo Purtolas 1, Oliver Cregenzn, Elisa Luengo, Ignacio lvarez, Javier Raso
Tecnologa de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, c/Miguel Servet 177, 50013 Zaragoza, Spain

a r t i c l e

i n f o

Article history:
Received 23 May 2012
Received in revised form 13 September 2012
Accepted 20 September 2012
Available online 29 September 2012
Keywords:
Pulsed electric elds
Extraction
Anthocyanin
Purple-eshed potato

a b s t r a c t
The inuence of pulsed electric eld (PEF) treatment on the anthocyanin extraction yield (AEY) from purple-eshed potato (PFP) at different extraction times (60480 min) and temperatures (1040 C) using
water and ethanol (48% and 96%) as solvents has been investigated. Response surface methodology
was used to determine optimal PEF treatment and optimise anthocyanin extraction. A PEF treatment
of 3.4 kV/cm and 105 ls (35 pulses of 3 ls) resulted in the highest cell disintegration index (Zp = 1) at
the lowest specic energy requirements (8.92 kJ/kg). This PEF treatment increased the AEY, the effect
being higher at lower extraction temperature with water as solvent. After 480 min at 40 C, the AEY
obtained for the untreated sample using 96% ethanol as the solvent (63.9 mg/100 g fw) was similar to
that obtained in the PEF-treated sample using water (65.8 mg/100 g fw). Therefore, PEF was possible with
water, a more environmental-friendly solvent than ethanol, without decreasing the AEY from PFP.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Colour is recognised as a major factor affecting food acceptance.
Enhancing food colour during processing by adding colorants is a
common practice in the food industry to make food more appealing for consumers. In recent years, there has been an increasing
interest in the food industry toward replacing synthetic colorants
by natural ones, as a consequence of the doubts about the safety
of synthetic colorants and the consumer demand for natural products (Sowbhagya & Chitra, 2010). Anthocyanins, a group of phenolic compounds, are some of the most extensive natural pigments,
responsible for the red, purple and blue colours of fruits, vegetables
and owers (Mazza & Miniati, 1993). Interest in anthocyanin pigments has increased recently, due to the range of colours of their
molecules, with their possible applications as natural dyes and also
potential health benets as dietary antioxidants (He & Giusti,
2010; Lachman et al., 2009; Suda et al., 2003).
Pigmented potato cultivars such as purple-eshed potatoes
(PFPs) are a rich source of anthocyanins. It has been reported that
the concentration of anthocyanins in PFP is similar to the highest
anthocyanin production crops, such as blueberries, blackberries,
cranberries or grapes (Bridgers, Chinn, & Truong, 2010). Due to
the fact that they are a low-cost crop, purple-eshed potatoes
may be a potential source of natural anthocyanin pigments for
industry (Jansen & Flamme, 2006).

Corresponding author. Tel.: +34 976762675; fax: +34 976761590.


E-mail address: jraso@unizar.es (J. Raso).
Present address: AZTI-Tecnalia, Food Research Division, Parque Tecnolgico de
Bizkaia, Astondo Bidea, Edicio 609, Derio, 48160 Bizkaia, Spain.
1

0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.09.080

Studies conducted on the extraction of anthocyanins from different plants have showed that extraction yield depends on different factors, such as solvent type and concentration or time and
temperature of extraction (Cacace & Mazza, 2003). Anthocyanins
are soluble in water; however, extraction methods generally used
for obtaining anthocyanin pigments from plants are usually based
on the use of other polar solvents, such as methanol or ethanol. For
example, Bridgers et al. (2010) observed that ethanol and methanol
extracts of PFP had approximately 34 times higher values of
anthocyanins compared to water extracts. However, the use of
these kinds of solvents increases the cost of the process and may
cause important environmental problems. Consequently, improving the anthocyanin extraction with water by the use of pretreatment steps, such as application of pulsed electric elds (PEFs)
is an interesting approach.
PEF is an emerging technology that has gained increasing interest in recent years for improving mass transfer operations in the
food industry (Donsi, Ferrari, & Pataro, 2010; Knorr et al., 2011;
Purtolas, Luengo, lvarez, & Raso, 2012). The process is based
on the application of external electric elds that induce the electroporation of eukaryotic cell membranes, enhancing the diffusion of
solutes. This permeabilisation of cell membranes can be achieved
at moderate electric elds (<10 kV/cm) and low specic energies
(<10 kJ/kg).
An enhancement in the extraction of colorants, such as anthocyanins from grapes or red cabbage (Corrales, Toep, Butz, Knorr, &
Tauscher, 2008; Gaschovska et al., 2010) or betanin from red beets
(Chalermchat, Fincan, & Djmek, 2004; Fincan, DeVito, & Dejmek,
2004; Lpez, Purtolas, Condn, Raso, & lvarez, 2009), by application of a PEF treatment has been reported. However, these studies
only evaluated the aqueous extraction of these compounds.

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E. Purtolas et al. / Food Chemistry 136 (2013) 13301336

In this investigation, an experimental design approach has been


used to evaluate the inuence of the application of PEF on the
anthocyanin extraction from purple-eshed potato at different
temperatures, using water and ethanol as solvents. The last objective of the study was to demonstrate if the cell permeabilisation by
PEF could reduce extraction time and decrease or eliminate the use
of organic solvents, such as ethanol, without affecting anthocyanin
extraction yield.

2. Materials and methods


2.1. Raw materials
Purple-eshed potatoes (Solanum tuberosum) variety Vitelotte
were purchased at a local supermarket and stored at 4 C until required. Before use, the entire purple-eshed potatoes (PFPs) were
washed with cold water and then peeled. Two types of samples
were used in this study: for determination of the cell disintegration
index, disc samples of 2 cm in diameter and 2 cm in thickness were
used; for the extraction experiments, potatoes were diced into
pieces of approximately 1 cm3.

2.2. PEF treatment

2.3. Cell disintegration index


Cell disintegration index (Zp) was used to identify the PEF treatment conditions for the pre-treatment of the PFP cells before the
anthocyanin extraction. This index characterises the proportion
of permeabilised cells based on the frequency dependence of conductivity of intact and permeabilised plant tissues (Angersbach,
Heinz, & Knorr, 1999).
The cell disintegration index analysis was carried out using
impedance measurement equipment (DIL, Quakenbrck, Germany). For the experiments, cylindrical pieces (2 cm in length;
2 cm2 of surface) of untreated or PEF-treated PFP were placed in
the measuring cell of the equipment. Zp was calculated using the
following equation:

Zp 1 

 
K h K 0h  K 0l

; 0 6 Zp 6 1
K 0h K h  K l

where Kl, K 0l are the electrical conductivities of untreated and treated material, respectively, in a low-frequency eld (15 kHz); Kh, K 0h
are the electrical conductivities of untreated and treated material,
respectively, in a high-frequency eld (350 MHz). The Zp varies between 0 for intact tissues and 1 for a tissue with all the cells
permeabilised.
2.4. Anthocyanin extraction

PEF equipment used in this investigation was supplied by ScandiNova (Modulator PG; ScandiNova, Uppsala, Sweden). The apparatus generates square waveform pulses of a width of 3 ls with a
frequency up to 300 Hz. The maximum output voltage and current
were 30 kV and 200 A, respectively. The equipment consists of a direct current power supply which converts the 3-phase line voltage
to a regulated DC voltage. It charges up 6 IGBT switching modules
(high-power solid-state switches) to a primary voltage of around
1000 V. An external trigger pulse gates all the modules and controls its discharge to a primary pulsed signal of around 1000 V. Finally, a pulse transformer converts this primary 1000 V pulse to
the desired high-voltage pulse.
The treatment chamber consisted of a cylindrical methacrylate
tube closed with two polished stainless steel cylinders. The gap between the electrodes was 2 cm. The diameter of the treatment
chamber was 2 cm for the determination of the cell disintegration
index, and 3.4 cm for the extraction experiments.
Actual voltage and current intensity applied were measured
with a high-voltage probe (P6015A; Tektronix, Wilsonville, OR)
and a current probe (Stangenes Industries Inc., Palo Alto, CA),
respectively, connected to an oscilloscope (TDS 220, Tektronix).
PEF treatments ranging from 5 to 35 pulses of 3 ls (45105 ls),
set at electric eld strength ranging from 1 to 5 kV/cm were used.
Specic energy of these treatments ranged from 0.54 to 13.50 kJ/kg
(Table 1). A pulse frequency of 1 Hz was used.

Twelve pieces (14 0.5 g) of the untreated and PEF-treated PFP


were put in a 250 ml Erlenmeyer ask that contained 140 ml of
solvent. Solvents used were distilled water, 48% ethanol and 96%
ethanol tempered at different temperatures (10, 25, and 40 C).
In all cases, 1% of hydrochloric acid was added to obtain a pH in
the extraction solvent of around 1, which is in the pH range of
maximum anthocyanin colour stability, preventing the degradation of those compounds (Brigita, Mirko, & Alenka, 2005). During
extraction, all asks were incubated at the appropriate temperature in a water bath. Samples of 1 ml of the extraction solvent were
taken at different extraction times. The samples were centrifuged
for 5 min at 6000g and the supernatant was collected for the determination of the anthocyanins yield.
2.5. Anthocyanin quantication
Monomeric anthocyanin content was determined using the
spectrophotometric method proposed by Francis (1989). A UV500
spectrophotometer (Unicam Limited, Cambridge, UK) and 1-cm
path length cells were used for spectral measurements at
535 nm. Pigment content was calculated as malvidin 3-p-coumaroyl-rutinoside-5-glucoside; that is the major anthocyanin of the
PFP Vitelotte cultivar (Lachman et al., 2012), using an extinction
coefcient of 30200 L cm1 mol1 and a molecular weight of

Table 1
Cell disintegration index (Zp) of purple-eshed potato (PFP) after application of PEF treatments at different electric eld strengths and treatment times. Specic energy for the
different treatments conditions has been also included.

Electric eld strength (kV/cm)

Treatment time (ls)

Specic energy (kJ/kg)

Zp*

1
2
2
3
3
3
4
4
5

75
45
105
15
75
145
45
105
75

0.54
1.26
3.02
0.97
4.86
8.75
5.20
12.1
13.5

0.19 0.03
0.12 0.02
0.53 0.11
0.00 0.00
0.83 0.04
1.00 0.01
0.80 0.02
0.98 0.03
1.00 0.01

Each value represents mean standard deviation.

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E. Purtolas et al. / Food Chemistry 136 (2013) 13301336

718 g mol1 (Giusti & Wrolstad, 2001). Anthocyanin extraction


yield (AEY) was expressed as mg of anthocyanins extracted by
100 g of fresh weight (fw) of PFP.

The CCD and the corresponding analysis of the data were carried out using the software package Design-Expert 6.0.6 (Stat-Ease
Inc., Minneapolis, MN).

2.6. HPLC analysis of anthocyanins

3. Results and discussion

Before HPLC analysis, an aliquot of the corresponding extract


(4 mL) was passed through a C-18 cartridge of 6 mL capacity and
500 mg sorbent weight (Biotage, Lund, Sweden), previously activated with methanol followed by 0.01% aqueous HCl.
Anthocyanins were retained on the cartridge, while residual
sugars and organic acids were eluted with 5 mL of 0.01% aqueous
HCl. Anthocyanins were recovered with 4 mL of methanol containing 0.01% HCl.
HPLC/DAD analyses were performed on a ProStar high-performance liquid chromatograph (Varian Inc., Walnut Creek, CA)
equipped with ProStar 240 ternary pump, a ProStar 410 autosampler
and a ProStar 335 photodiode array detector. The system was controlled with Star chromatography workstation v.6.41 (Varian). A reversed-phase column Microsorb-MV 100-5 C18 (25  0.46 cm;
5 lm particle size) with a precolumn (5  0.46 cm; 5 lm particle
size) of the same material was used. The temperature of the column
and precolumn was maintained at 40 C.
An elution gradient consisting of water/acetonitrile/formic acid
87/3/10 (v:v:v) (solvent A) and water/acetonitrile/formic acid 40/
50/10 (v:v:v) (solvent B) was used as follows: 0 min 6% B, 20 min
20% B, 35 min 40% B, 45 min 90% B and 55 min 6% B. Flow rate
through the column was 0.5 mL/min, sample injection 20 lL,
absorbance detection wavelength 520 nm, and UVvis spectra
were measured simultaneously. Prior to injection, all samples were
ltered through a 0.2-lm sterile syringe lter of cellulose acetate
(VWR, West Chester, PA).
The different anthocyanins analysed were tentatively identied
according to their order of elution and their UVvis spectral characteristics published in the literature (Ieri, Innocenti, Andrenelli,
Vecchio, & Mulinacci, 2011; Lachman et al., 2009; Mulinacci
et al., 2008).

3.1. Optimisation of PEF treatment conditions

2.7. Experimental design


Response surface methodology (RSM) was used to determine
optimal PEF treatment conditions for the pre-treatment of the
PFP cells before the anthocyanin extraction and to optimise anthocyanin extraction at different temperatures using water and ethanol as solvents (Montgomery, 2004). A central composite design
(CCD) was constructed to investigate the effects of electric eld
strength (from 2 to 4 kV/cm; a = 2) and treatment time (from 45
to 105 ls; a = 2) on Zp. CCD was also used to investigate the effects
of extraction time (from 60 to 480 min; a = 1), solvent concentration (from 0% to 96% ethanol; a = 1) and extraction temperature
(from 10 to 40 C; a = 1) on the AEY for untreated and PEF-treated
PFP. Each point of the CCD was carried out in duplicate.
The data obtained were modelled with the following second-order polynomial equation:

Y b0

k
k
k
X
X
X
bi X i
bii X 2i
bij X i X j
i1

i1

i>j

where Y is the response variable to be modelled, Xi and Xj are independent factors, b0 is the intercept, bi the linear coefcients, bij the
quadratic coefcients, bij the cross-product coefcients, and k the
total number of independent factors. A backward regression procedure was used to determine the parameters of the models. This procedure systematically removed the effects that were not
signicantly associated (p > 0.05) with the response until a model
with only a signicant effect was obtained.

Between the different methods proposed for assessing cell


membrane permeabilisation, such as microscopic observations,
measurement of the liquid release or evaluation of the conductivity
of the exuded liquid, the determination of Zp via electrical impedance measurements has been one of the most used to select the
optimum PEF treatment conditions (Angersbach et al., 1999; De
Vito, Ferrari, Lebovka, Shynkaryk, & Vorobiev, 2008; Lebovka, Bazhal, & Vorobiev, 2002). This method provides a precise and rapid
measurement of the degree of permeabilisation in a short time.
The application of a multiple regression analysis to the Zp values
obtained after treatment conditions (Table 1) resulted in the following quadratic model, after neglecting the statistically insignicant terms (p > 0.05):

Y 1:39 0:52E 0:02t  0:05E2  8:36  105 t2

where Y is the Zp value, E is the electric eld strength (kV/cm), and t


is the PEF treatment time (ls). Table 2 shows the results of the ANOVA for the quadratic model developed (Eq. 3). The determination
coefcient (r2) of the model (0.98), the no signicance in the lack
of t (p > 0.05), and the high F-values indicate that the model was
signicant (p < 0.0001) and could be used to predict the response.
According to the F-values for the models parameters, the electric eld strength and treatment time were the most important
ones. This means that the changes in these factors have the most
inuence on the Zp. The presence of the square of these terms in
the equation was also signicant (p < 0.05), but with lower
F-values. The presence of these square terms in the equation
means that when the electric eld strength or treatment time
changed, their effect on Zp was non-linear.
To illustrate the inuence of the electric eld strength and
treatment time on Zp of PFP, a contour plot was generated that
shows combinations of these two parameters to obtain different
degrees of cell permeabilisation (Fig. 1). The maximum permeabilisation (Zp = 1) was achieved at electric eld strengths and treatment times varying from 3.4 to 4 kV/cm and from 105 to 87 ls,
respectively. In order to carry out the extraction experiments, the
conditions of this range that corresponded to the minimum specic energy requirements were selected. These conditions were
electric eld strength of 3.4 kV/cm and a treatment time of
105 ls, which corresponded with a total specic energy requirement of 8.92 kJ/kg. El-Belghiti and Vorobiev (2005) reported that

Table 2
F- and p-values of the ANOVA analysis for the quadratic model (Eq. 3) developed to
describe the inuence of electric eld strength (E) and treatment time (t) on the cell
disintegration index (Zp) of purple-eshed potato (PFP).

E
t
E2
t2
Model
Lack of t
r2
Adjusted-r2
Signal-to-noise ratio
Variation coefcient
p < 0.05 is signicant.

F-value

p-Value

103.80
137.08
9.71
20.72
65.80
0.66
0.98
0.97
22.55
16.25

<0.0001
<0.0001
0.0067
0.0003
<0.0001
>0.05

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E. Purtolas et al. / Food Chemistry 136 (2013) 13301336

105.00

the 180 mg anthocyanin/100 g fw found by Bridgers et al. (2010),


using an extraction temperature of 80 C and 70% of acidied ethanol as solvent, or by Cevallos-Casals and Cisneros-Zevallos (2003),
after sample homogenisation in 95% of acidied ethanol and then
extraction at 4 C. However, other authors have observed lower
AEY. For example, Brown, Culley, Yang, Durst, and Wrolstad
(2005) reported AEY ranging from 15 to 38 mg/100 g fw when
the extraction was performed with 70% of acetone and PFP was
previously frozen in liquid nitrogen and ground to a powder. This
large variability could be due to the diverse extraction techniques,
methods of analysis and the different techniques, method of analysis and expression of results used by the different authors. Furthermore, cultivar, crop season, growing conditions, or degree of
ripeness are other factors that strongly affect the anthocyanin content of PFP (Lachman et al., 2009, 2012).
The permeabilisation of the cell membranes of PFP by application of a PEF treatment resulted in an improvement in the AEY at
extraction temperatures of 10 and 25 C when water or 48% ethanol were used as solvents (Table 3). At 40 C, an increment of the
AEY in the PEF-treated sample was only observed when water
was used as solvent. On the other hand, when 96% ethanol was
used as solvent an improvement in extraction by PEF was only observed at 10 C.

Treatment time (s)

90.00

0.9
0.8

75.00

0.7

0.6
60.00

0.5
0.4
0.3

45.00
2.00

2.50

3.00

3.50

4.00

Electric field strength (kV/cm)


Fig. 1. Combination of electric eld strengths (24 kV/cm) and treatment time (45
105 ls) to obtain different levels of cell disintegration index (Zp; from 0.3 to 1) on
purple-eshed potato (PFP) tissue.

3.3. Response surface modelling of AEY as a function of process


parameters

the permeabilisation of the majority of the cells of a carrot tissue


required a specic energy of 9 kJ/kg, which is of the same order
as that required in this study for permeabilisation of the PFP cells.

In order to determine and quantify the potential advantages of


the application of a PEF treatment for anthocyanin extraction from
PFP, RSM was used. This approach enables the evaluation of the effect of several factors and their interactions on response variables.
This technique has been successfully used by different authors for
optimisation of the extraction of anthocyanins from PFP and fruits
such as blackcurrants or grapes (Cacace & Mazza, 2003; Fan, Han,
Gu, & Chen, 2008; Purtolas, Saldaa, lvarez, & Raso, 2011). The
application of a multiple regression analysis to the independent
and response variables shown in Table 2 resulted in the following
second order polynomial equations for untreated PFP (Eq. 4) and
PEF-treated PFP (Eq. 5):

3.2. Anthocyanin extraction


The anthocyanin extraction yield (AEY) resulting from the
experimental conditions investigated for the control (untreated
PFP) and the sample treated by PEF (PEF-treated PFP) is shown in
Table 3. Due to the thermal susceptibility of these compounds,
the maximum extraction temperature used in this study was
40 C. Several authors have reported a sharp decrease in anthocyanin concentrations at extraction temperatures higher than 45 C
(Cacace & Mazza, 2003).
The AEY varied from 8.1 to 63.9 mg/100 g fw in the untreated
PFP and from 14.1 to 67.9 mg/100 g fw in the PEF-treated PFP.
These contents are within the range of values reported in the literature (15186 mg/100 g fw; Bridgers et al., 2010). However, the
highest AEY obtained in the present investigation was lower than

Y 5:55 0:13t 0:50T 0:08Ec  1:58  104 t 2 8:77  104 tT


4
Y 6:92 0:14t 1:36T 0:04Ec  1:35  104 t2
 0:02T 2 5:16  104 tT

Table 3
Anthocyanin extraction yield (AEY) (mg/100 g fw) resulting from the experimental conditions investigated for the untreated purple-eshed potato (untreated PFP) and PEF
treated purple-eshed potato (PEF-treated PFP) (3.4 kV/cm; 105 ls).
Extraction time (min)
60
60
60
60
60
270
270
270
270
270
480
480
480
480
480

Temperature (C)
10
10
25
40
40
10
25
25
25
40
10
10
25
40
40

Ethanol (%)
0
96
48
0
96
48
0
48
96
48
0
96
48
0
96

Each value represents mean standard deviation.


Values followed by different small letter are signicantly different according to t-test (p < 0.05).

AEY untreated
a

8.1 1.04
15.2 0.28a
19.2 0.78a
25.2 0.35a
30.6 0.65a
29.8 0.39a
36.8 1.,53a
39.1 1.55a
49.2 2.68a
52.5 2.23a
28.9 1.22a
41.9 0.90a
50.5 0.14a
61.5 1.44a
63.9 1.68a

AEY PEF-treated
14.1 2.23b
17.1 0.05b
25.1 1.87b
29.9 0.38b
30.7 2.42a
35.9 0.77b
46.8 0.74b
50.5 0.86b
51.3 2.43a
55.6 1.85a
41.8 0.73b
49.5 3.06b
62.5 0.85b
65.8 1.11b
67.9 2.62a

E. Purtolas et al. / Food Chemistry 136 (2013) 13301336

where Y is the AEY (mg/100 g fw), t is the extraction time (min), T


the extraction temperature (C), and Ec the ethanol concentration
(%).
Table 4 shows the results of the analysis of variance for the signicant terms of the models. The statistical analysis indicated that
both models were adequate to estimate AEY as a function of the
three independent factors investigated. The model F-values were
125.46 and 229.09, for untreated and PEF-treated PFP, respectively,
indicating that both models were signicant (p < 0.0001). The
determination coefcient (r2) for each model was higher than
0.98, which means that less than 2% of the total response variation
remained unexplained by the models obtained. The adjusted-r2
values that corrected the r2 according to the number of responses
and terms in the model were very similar to r2 for both equations.
Finally, for both models, a non-signicant lack of t (p > 0.05) was
observed.
According to the F-values for the models parameters for the untreated and PEF-treated PFP, the most signicant effect on the
extraction of anthocyanins was the extraction time. This means
that the changes in this factor had the most inuence on the
AEY. The square of extraction time was also a signicant term in
both equations. The presence of these square terms in the equation
means that when the extraction time changed, its effect on oil yield
extraction was non-linear. The negative effect of the square terms
for both equations indicates an optimum value for extraction time;
above this value, the increment of the treatment time will not substantially increase the extraction yield. This inuence of the extraction time is characteristic in solidliquid extraction of intracellular
compounds from plant materials using solvents. In this process,
the concentration of the compound within the plant tissue tends
to develop an equilibrium with the concentration that will be dissolved into the solvent, the rate of extraction being slower with a
lower concentration gradient (Cacace & Mazza, 2003; Hojnik,
Skerget, & Knez, 2008).
The linear terms of the temperature and the ethanol concentration were also signicant terms for AEY from both untreated and
PEF-treated PFP, the more signicant effect being the temperature.
The increase of the AEY with the presence of ethanol in the extraction medium and with the increment of the temperature is consistent with mass transfer principles. The presence of ethanol
favoured extraction by enhancing solubility and diffusivity. Anthocyanin solubility is higher in ethanol than in water; on the other
hand, ethanol affects cell permeability by acting on the phospholipid bilayer of biological membranes (Bridgers et al., 2010;
Goldstein & Chin, 1981). Temperature also favours extraction by
increasing both diffusion coefcient and solubility of anthocyanins.
Table 4
F- and p-values of the ANOVA analysis for models developed (Eqs. 4 and 5) to describe
the inuence of extraction time (t), extraction temperature (T) and ethanol
concentration (Ec) on the cell anthocyanin extraction yield (AEY) (mg/100 g fw) of
untreated and PEF-treated (3.4 kV/cm; 105 ls) purple-eshed potato (untreated PFP;
PEF-treated PFP).

t
T
Ec
t2
T2
tT
Model
r2
Adjusted-r2
Signal-to-noise ratio
Variation coefcient
p < 0.05 is signicant.

Untreated PFP

PEF-treated PFP

F-value

p-Value

F-value

p-Value

364.16
199.35
26.86
26.85

10.10
125.46
0.99
0.98
38.38
6.68

<0.0001
<0.0001
0.0006
0.0006

0.0112
<0.0001

990.63
284.97
11.15
33.98
15.41
7.19
229.09
0.99
0.99
47.86
3.99

<0.0001
<0.0001
0.0102
0.0004
0.0044
0.0279
<0.0001

The main difference between Eqs. (4 and 5) was that the square
of temperature was also a signicant term for anthocyanin extraction in PEF-treated PFP. The negative effect of this square term
indicates that in the range of temperatures investigated when
the PFP is treated by PEF above a maximum value, the increment
of temperature will not signicantly increase the extraction yield.
It has been estimated that the average diffusion coefcient of a
small solute in a membrane is often about a million times lower
than that in the adjacent aqueous solutions (Nobel, 1999). Therefore, the inuence of the temperature observed in the PEF-treated
PFP could be a consequence of the fact that the effect of the temperatures in anthocyanin diffusivity is lower when the cell membranes are permeabilised by the application of the PEF treatment.
In order to illustrate the advantages of the PEF permeabilisation
of the PFP before extraction in terms of increasing AEY or reducing
temperature and concentration of ethanol, Fig. 2 was plotted using
the corresponding regression models for untreated and PEF-treated
PFP (Eqs. 4 and 5). Fig. 2 shows that AEY increased with treatment
temperature and ethanol concentration in both untreated and PEFtreated samples. Independently of the extraction temperature, the
use of ethanol as solvent was more effective for the untreated PFP
than for the PEF-treated PFP. For example, for the untreated PFP at
25 C, the AEY using 96% ethanol was 20% higher than using water
as solvent, but only 5% for the PEF-treated PFP. This decrease of the
efcacy of ethanol as solvent as compared with water when samples were treated by PEF could be due to the high solubility of
anthocyanins in ethanol and the fact that the improvement of
extraction due to the increment of the diffusivity as a consequence
of the ethanolic denaturation of the phospholipid bilayer is less
signicant when the cells have been previously permeabilised by
PEF (Lapornik, Prosek, & Wondra, 2005).
A possible benet of the PEF permeabilisation of the PFP before
extraction is the possibility of decreasing the extraction temperature without affecting the AEY. For example, for a constant AEY
of 60 mg/100 g fw, the application of a PEF treatment permitted
the reduction of the extraction temperature from 40 to 25 C using
water as solvent and from 31 to 20 C when the solvent was 96%

80

70

AEY (mg /100 g fw)

1334

60

50

40

30

20
10

15

20

25

30

35

40

Temperature (C)
Fig. 2. Inuence of extraction temperature (C) and ethanol concentration (0%, solid
line; 48%, segmented line; 96%, dotted line) on anthocyanin extraction yield (AEY;
mg/100 g fw) for both untreated purple-eshed potato (untreated PFP; red colour)
and PEF-treated purple-eshed potato (PEF-treated PFP; green colour) (3.4 kV/cm;
105 ls) after an extraction time of 480 min. (For interpretation of the references to
colour in this gure legend, the reader is referred to the web version of this article.)

E. Purtolas et al. / Food Chemistry 136 (2013) 13301336

1335

Fig. 3. HPLC chromatograms of anthocyanin proles of aqueous extract of purple-eshed potato (PFP) at 520 nm for (A) control and (B) PEF treated sample (3.5 kV/cm102 ls). 1, malvidin 3-O-rutinoside-5-O-glucoside; 2, petunidin 3-O-caffeoyl-rutinoside-5-O-glucoside; 3, delphinidin 3-O-p-coumaroyl-rutinoside-5-O-glucoside; 4,
petunidin 3-O-p-coumaroyl-rutinoside-5-O-glucoside; 5, petunidin 3-O-feruloyl-rutinoside-5-O-glucoside; 6, malvidin 3-O-p-coumaroyl-rutinoside-5-O-glucoside; 7,
malvidin 3-O-feruloyl-rutinoside-5-O-glucoside.

ethanol. The potential of reducing extraction temperature by permeabilisation of cells by PEF has been observed by other authors
in the extraction of other compounds such as sugar from sugar
beets (Loginova, Vorobiev, Bals, & Lebovka, 2011; Lpez, Purtolas,
Condn, Raso, & lvarez, 2009). Finally, it is observed that in the
range of extraction temperatures between 35 and 40 C, where
the AEY was the highest for all solvents, the quantity of anthocyanins extracted from the untreated sample using 96% ethanol as solvent was similar to those extracted in the PEF-treated PFP using
water or ethanol. Therefore, in this range of temperatures, the
application of a PEF treatment to the PFP before extraction could
permit the use of water, a more environmentally friendly solvent
than ethanol, without decreasing the amount of anthocyanins
recovered from PEP.
3.4. HPLC characterisation of anthocyanins extracted from purpleeshed potato treated by PEF
Reverse-phase HPLC chromatogram proles detected at 520 nm
for untreated and PEF-treated PFP are presented in Fig. 3A and B.
HPLC chromatograms were similar to those obtained by Hillebrand
et al. (2011) for the same PFP cultivar. The PFP cultivar Vitelotte
contains ve minor anthocyanins and two major anthocyanins that
correspond to petunidin 3-p-coumaroylrutinoside-5-glucoside and
malvidin 3-p-coumaroyl-rutinoside-5-glucoside. The results of the
HPLC analyses showed that the application of a PEF treatment to
the PFP before extraction did not affect the extraction of a determined anthocyanin. The only difference observed was that the
peak areas were approximately 20% higher in the chromatograms
corresponding to the PEF-treated PFP. Similar results were obtained by Lpez, Purtolas, Hernndez-Orte, lvarez, and Raso
(2009) when comparing the anthocyanin prole of a control wine
with a wine obtained from grapes treated by PEF.
4. Conclusions
In this investigation, it has been demonstrated that independently of the extraction temperature or solvent used for extraction
of anthocyanins from PFP, the extraction yield was always higher

when the tissues were permeabilised previously by application of


a PEF treatment. Considering the low energetic cost of the treatment (8.9 kJ/kg) required for permeabilisation of the PFP cells,
PEF is a promising technique not only to improve AEY but also to
reduce the extraction temperature, and to reduce or eliminate
the use of organic solvents in the recovery of anthocyanins from
PFP without decreasing extraction yield.

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