Pulsed-electric-field-assisted extraction of anthocyanins from purple-fleshed potato

Food Chemistry 136 (2013) 1330–1336

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Pulsed-electric-field-assisted extraction of anthocyanins from purple-fleshed potato Eduardo Puértolas 1, Oliver Cregenzán, Elisa Luengo, Ignacio Álvarez, Javier Raso ⇑ Tecnología 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 fields Extraction Anthocyanin Purple-fleshed potato

a b s t r a c t The influence of pulsed electric field (PEF) treatment on the anthocyanin extraction yield (AEY) from purple-fleshed potato (PFP) at different extraction times (60–480 min) and temperatures (10–40 °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 specific 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. Ó 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 flowers (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 benefits as dietary antioxidants (He & Giusti, 2010; Lachman et al., 2009; Suda et al., 2003). Pigmented potato cultivars such as purple-fleshed 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-fleshed 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: [email protected] (J. Raso). Present address: AZTI-Tecnalia, Food Research Division, Parque Tecnológico de Bizkaia, Astondo Bidea, Edificio 609, Derio, 48160 Bizkaia, Spain. 1

0308-8146/$ - see front matter Ó 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 3–4 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 fields (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; Puértolas, Luengo, Álvarez, & Raso, 2012). The process is based on the application of external electric fields that induce the electroporation of eukaryotic cell membranes, enhancing the diffusion of solutes. This permeabilisation of cell membranes can be achieved at moderate electric fields (<10 kV/cm) and low specific energies (<10 kJ/kg). An enhancement in the extraction of colorants, such as anthocyanins from grapes or red cabbage (Corrales, Toepfl, Butz, Knorr, & Tauscher, 2008; Gaschovska et al., 2010) or betanin from red beets (Chalermchat, Fincan, & Djmek, 2004; Fincan, DeVito, & Dejmek, 2004; López, Puértolas, Condón, Raso, & Álvarez, 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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In this investigation, an experimental design approach has been used to evaluate the influence of the application of PEF on the anthocyanin extraction from purple-fleshed 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-fleshed potatoes (Solanum tuberosum) variety ‘‘Vitelotte’’ were purchased at a local supermarket and stored at 4 °C until required. Before use, the entire purple-fleshed 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, Quakenbrück, 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 ¼ 1 

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

ð1Þ

where Kl, K 0l are the electrical conductivities of untreated and treated material, respectively, in a low-frequency field (1–5 kHz); Kh, K 0h are the electrical conductivities of untreated and treated material, respectively, in a high-frequency field (3–50 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 (45–105 ls), set at electric field strength ranging from 1 to 5 kV/cm were used. Specific 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 flask 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 flasks 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 quantification 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 coefficient of 30200 L cm1 mol1 and a molecular weight of

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

*

Electric field strength (kV/cm)

Treatment time (ls)

Specific 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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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 UV–vis spectra were measured simultaneously. Prior to injection, all samples were filtered through a 0.2-lm sterile syringe filter of cellulose acetate (VWR, West Chester, PA). The different anthocyanins analysed were tentatively identified according to their order of elution and their UV–vis 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 field 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 ¼ b0 þ

k k k X X X bi X i þ bii X 2i þ bij X i X j i¼1

i¼1

ð2Þ

i>j

where Y is the response variable to be modelled, Xi and Xj are independent factors, b0 is the intercept, bi the linear coefficients, bij the quadratic coefficients, bij the cross-product coefficients, 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 significantly associated (p > 0.05) with the response until a model with only a significant 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 insignificant terms (p > 0.05):

Y ¼ 1:39 þ 0:52E þ 0:02t  0:05E2  8:36  105 t2

ð3Þ

where Y is the Zp value, E is the electric field 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 coefficient (r2) of the model (0.98), the no significance in the lack of fit (p > 0.05), and the high F-values indicate that the model was significant (p < 0.0001) and could be used to predict the response. According to the F-values for the model’s parameters, the electric field strength and treatment time were the most important ones. This means that the changes in these factors have the most influence on the Zp. The presence of the square of these terms in the equation was also significant (p < 0.05), but with lower F-values. The presence of these square terms in the equation means that when the electric field strength or treatment time changed, their effect on Zp was non-linear. To illustrate the influence of the electric field 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 field 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 specific energy requirements were selected. These conditions were electric field strength of 3.4 kV/cm and a treatment time of 105 ls, which corresponded with a total specific 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 influence of electric field strength (E) and treatment time (t) on the cell disintegration index (Zp) of purple-fleshed potato (PFP).

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

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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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 acidified ethanol as solvent, or by Cevallos-Casals and Cisneros-Zevallos (2003), after sample homogenisation in 95% of acidified 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.

1

Treatment time (µs)

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 field strengths (2–4 kV/cm) and treatment time (45– 105 ls) to obtain different levels of cell disintegration index (Zp; from 0.3 to 1) on purple-fleshed 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 specific 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; Puértolas, Saldaña, Álvarez, & 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 (15–186 mg/100 g fw; Bridgers et al., 2010). However, the highest AEY obtained in the present investigation was lower than

Y ¼ 5:55 þ 0:13t þ 0:50T þ 0:08Ec  1:58  104 t 2 þ 8:77  104 tT ð4Þ Y ¼ 6:92 þ 0:14t þ 1:36T þ 0:04Ec  1:35  104 t2  0:02T 2 þ 5:16  104 tT

ð5Þ

Table 3 Anthocyanin extraction yield (AEY) (mg/100 g fw) resulting from the experimental conditions investigated for the untreated purple-fleshed potato (untreated PFP) and PEF treated purple-fleshed 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 significantly 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. Puértolas et al. / Food Chemistry 136 (2013) 1330–1336

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 significant 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 significant (p < 0.0001). The determination coefficient (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-significant lack of fit (p > 0.05) was observed. According to the F-values for the model’s parameters for the untreated and PEF-treated PFP, the most significant effect on the extraction of anthocyanins was the extraction time. This means that the changes in this factor had the most influence on the AEY. The square of extraction time was also a significant 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 influence of the extraction time is characteristic in solid–liquid 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 significant terms for AEY from both untreated and PEF-treated PFP, the more significant 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 coefficient and solubility of anthocyanins. Table 4 F- and p-values of the ANOVA analysis for models developed (Eqs. 4 and 5) to describe the influence 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-fleshed potato (untreated PFP; PEF-treated PFP).

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

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 significant 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 significantly increase the extraction yield. It has been estimated that the average diffusion coefficient 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 influence 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 efficacy 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 significant when the cells have been previously permeabilised by PEF (Lapornik, Prosek, & Wondra, 2005). A possible benefit 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)

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60

50

40

30

20 10

15

20

25

30

35

40

Temperature (ºC) Fig. 2. Influence 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-fleshed potato (untreated PFP; red colour) and PEF-treated purple-fleshed 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 figure legend, the reader is referred to the web version of this article.)

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Fig. 3. HPLC chromatograms of anthocyanin profiles of aqueous extract of purple-fleshed 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; López, Puértolas, Condón, Raso, & Álvarez, 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 purplefleshed potato treated by PEF Reverse-phase HPLC chromatogram profiles 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 five 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 López, Puértolas, Hernández-Orte, Álvarez, and Raso (2009) when comparing the anthocyanin profile 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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