Valorization of oilseed residues: Extraction of polyphenols from flaxseed hulls by pulsed electric fields

Industrial Crops and Products 52 (2014) 347–353

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Valorization of oilseed residues: Extraction of polyphenols from flaxseed hulls by pulsed electric fields N. Boussetta a,∗ , E. Soichi a , J.-L. Lanoisellé a,b , E. Vorobiev a a Université de Technologie de Compiègne, Unité Transformations Intégrées de la Matière Renouvelable, EA 4297, Centre de Recherches de Royallieu, BP 20529, 60205 Compiègne Cedex, France b Laboratoire d’Ingénierie des Matériaux de Bretagne, EA 4250 Université de Bretagne Sud, IUT de Lorient, Antenne de Pontivy, Allée des pommiers, 56300 Pontivy, France

a r t i c l e

i n f o

Article history: Received 15 July 2013 Received in revised form 23 October 2013 Accepted 29 October 2013 Keywords: Polyphenols Acidic hydrolysis Alkaline hydrolysis Ethanol Extraction

a b s t r a c t This work aims at obtaining extracts with high level of polyphenols from flaxseed hulls treated by pulsed electric fields (PEF). The effect of the different operating parameters was studied on the extraction of polyphenols such as the PEF treatment duration, the PEF electric field strength, the solvent composition (ethanol, acid or base content) and the rehydration duration of the product. Results have shown that a PEF treatment allowed the extraction of up to 80% of polyphenols when applied at 20 kV/cm for 10 ms. For lower PEF electric field strength, the extraction efficiency was smaller. The rehydration of the product before PEF application improved the treatment efficiency. The highest polyphenols increase (≈37%) was observed when the product was rehydrated for 40 min before PEF application. The addition of ethanol, citric acid and sodium hydroxide has increased the extraction of polyphenols. The highest polyphenols yield was reached with a solvent containing 20% of ethanol and 0.3 mol/L hydroxide sodium. The alkaline hydrolysis was more effective than the acidic hydrolysis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The re-use of agricultural residues have been received much attention over the last few years due to the increasingly shortage of natural resources and the need for environmental protection. Many investigations have been aimed at converting the waste materials into food ingredients, bio-fuels, and other value-added applications (Makris et al., 2007; Pan et al., 2012; Piwowarska and Gonzalez-Alvarez, 2012). In particular, flax, Linum usitatissimum, is mainly produced for fiber and oil. Recently, flaxseed has attracted attention from the scientific community due to its favorable chemical composition (Muir and Westcott, 2003). Flaxseed appears to be a key raw material in the nutraceuticals and functional foods industry, as it is an important source of omega-3 fatty acids, soluble fiber (mucilage) and polyphenols (lignans) (Oomah, 2001). The polyphenols of flaxseed have been shown to reduce the levels of LDL-cholesterol in blood, the risk of diabetes, and hormone related cancer. They have antioxidant activity, cardioprotective

∗ Corresponding author at: Université de Technologie de Compiègne, Unité Transformations Intégrées de la Matière Renouvelable, Centre de Recherches de Royallieu, B.P. 20529-60205, Compiègne Cedex, France. Tel.: +33 3 44 23 44 41; fax: +33 3 44 97 15 91. E-mail address: [email protected] (N. Boussetta). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.10.048

effect, and improve renal function in lupus nephritis patients (Muir and Westcott, 2003; Oomah, 2001; Prasad, 2000). The flaxseed polyphenols are mainly found in the seed coat of seed, where they are ester-linked to 3-hydroxy-3-methyl glutaryl (HMG) residues, and possibly bound to other compounds. These polyphenols can be released from such structures by hydrolytic cleavage of the ester bonds (Ford et al., 2001). The traditional polyphenols extraction methods involve a grinding pretreatment and a sequential or a simultaneous alcoholic solid–liquid extraction and alkaline treatment (Eliasson et al., 2003). These methods are time and energy consuming; they can take a few hours. Moreover, they involve extensive subsequent solid–liquid separation and purification steps. Recently there has been an increasing demand for new extraction techniques that are environmentally friendly, faster, and more efficient than the traditional extraction methods. Among these techniques, microwaves (Bagherian et al., 2011), high pressure (Corrales et al., 2008), and pulsed electric field (PEF) (Grimi et al., 2011; Loginova et al., 2011; Boussetta et al., 2012; Shynkaryk et al., 2009) have shown their efficiencies for the extraction of biomolecules from different plants. In particular, PEF is a non-thermal technique providing electrical pulses of a few microseconds. The PEF action is mainly localized on a microscopic scale. Pores are formed in cell membranes thus accelerating the release of intracellular compounds. For example, the impact of PEF

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Fig. 1. Experimental set-up.

on the recovery of oils from oilseeds and the impact on high fatty plant cells were studied (Guderjan et al., 2007). In this work the application of pulsed electric fields on the recovery of oil and functional food ingredients as antioxidants, tocopherols, polyphenols and phytosterols as well as oil quality parameters from hulled and non-hulled rapeseed were investigated. In summary, PEF have been applied on various fresh fruits and vegetables. However, there are only a few studies concerning the treatment of oilseed but nothing concerning the seed hulls. The main objective of this study is to optimize the PEF-assisted extraction of polyphenols from flaxseed hulls. The effect of pretreatment (electric field intensity, input treatment energy) and diffusion (solvent, pH) operating parameters will be investigated. This study aimed at optimizing the combination of factors in order to reach the highest extraction yield of polyphenols. 2. Materials and methods 2.1. Biological material Flaxseed (L. usitatissimum, cultivar Baladin) hulls (seed teguments) were provided by Lasalle Beauvais (Beauvais, France). The dry matter content of dried seeds is 96.3%. 2.2. Polyphenols extraction 2.2.1. Rehydration of flaxseeds hulls The flaxseeds hulls (10 g) were mixed with a mixture of water and ethanol (250 mL). Various concentrations of ethanol (from 0% to 50%, v/v) in water were supplemented with 0.05–0.3 mol/L sodium hydroxide for alkaline extraction or with 0.05–0.3 mol/L citric acid for acidic extraction. The suspension was introduced in a beaker under agitation at 150 rpm for up to 60 min. The temperature of the mixture was 20 ◦ C. After rehydration, the suspension of flaxseeds hulls was treated by PEF. 2.2.2. Pulsed electric fields treatment The PEF apparatus consisted of a pulsed high voltage power supply (Tomsk Polytechnic University, Tomsk, Russia) and a batch 1-L treatment chamber with stainless electrodes as previously

described (Boussetta et al., 2012). The electrodes of the treatment chamber were two parallel disks. As two plane electrodes are used for the treatment, the electric field should be homogeneous. The electrode area was 95 cm2 . The distance between the electrodes can be varied from 1 to 10 cm. The circuit configuration and the electrodes shape generated exponential decay pulses. The PEF pulse length was about ti = 10 ␮s. The test set-up and the pulse shape are described in Fig. 1. Flaxseed hulls (10 g) suspended in the extraction solvent (250 mL) at 20 ◦ C were introduced between the electrodes. The extraction solvent was the same as that used during the rehydration step. The high voltage pulse generator provided 40 kV to 10 kA pulses. These data are the maximum capability of the generator. The distance between electrodes was varied so that the corresponding electric field strengths E were 10, 15 and 20 kV/cm. Note that the change in distance between electrodes has no effect on the pulse shape. The PEF treatment consisted of applying up to nPEF = 1000 pulses with a frequency f of 0.33 Hz. This pulse frequency was imposed by the generator. Thus the time of PEF application tPEF = nPEF ·ti was varied from 1 to 10 ms. In this study, the number of pulse was counted and the pulse length was defined as the time required for a given pulse to decay from its peak voltage to 37% of the peak voltage. The voltage (Ross VD45-8.3-A-K-A, Ross Engineering Corp., Campbell, USA) and current (Pearson 3972, Pearson Electronics Inc., Palo Alto, USA) sensors were connected with a 108 MHz sampling system via an oscilloscope (Tektronix TDS1002, Oregon, USA). The software HPVEE 4.01 (Hewlett-Packard, Palo Alto, USA) was used for acquisition of data. From the measured voltage V(t) and current I(t), the instantaneous power P(t) = VI is calculated numerically, and the total dissipated energy per pulse Wp is obtained by integration of the power P for the whole duration T of the pulse (Eq. (1)).

 Wp =

T

V (t)I(t)dt

(1)

0

2.2.3. Grinding Flaxseeds hulls were crushed for 40 s in a laboratory coffee grinder (SEB, Ecully, France) (180 W). The total energy input

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is 720 kJ/kg. Powdered samples were sieved to select particles (50.0 ± 0.1 g) smaller than 1000 ␮m and were immediately used for diffusion experiments. 2.2.4. Diffusion After the PEF treatment, the suspension of flaxseed hulls was introduced in a cylindrical glass beaker of 600 mL. A round incubator shaker (Aerotron, Infors Sarl, Paris, France) was used for the diffusion experiments. The rotary shaking frequency was fixed at 150 rpm. The temperature inside the chamber was fixed at 20 ◦ C. The extraction was performed for 120 min. For control and grinding extractions, flaxseeds hulls were introduced in the beaker with a mixture of water and ethanol in the same conditions as for PEF-treated samples. To avoid any evaporation and degradation of polyphenols under the impact of air or light, the beaker was closed and covered during the extraction process. 2.3. Analysis

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2.4. Polyphenols extraction index Zp The polyphenols extraction index Zp is the normalized polyphenols content of the flaxseed hulls extracts. It is defined as follows:

Zp =

P(t) − Pmin (t) Pmax (t) − Pmin (t)

(2)

where Pmin (t) and Pmax (t) are the minimum and the maximum polyphenols content, respectively and P(t) is the polyphenols content from PEF treated samples at time t. The minimum polyphenols content was obtained from control diffusion (without pretreatment). The maximum polyphenols content was defined from grinding assisted extraction. 2.5. Effective diffusivity of polyphenols Total polyphenols extraction can be described by Fick’s second law (Eq. (3)): 2

2.3.1. pH determination The pH of the solution was determined by using a pHmeter (CONSOR C931, Bioblock Scientific, France) at 20 ◦ C. 2.3.2. Total soluble matter content The concentration of total soluble matter (or total solutes) was measured by a digital refractometer (Atago, USA) at room temperature. The results are expressed in Brix (g of dry matter DM/100 g solution). 2.3.3. Moisture content The moisture content was determined after drying the flaxseeds hulls in an air current-heated oven at 105 ◦ C to constant weight. Product weights before and after drying were determined. Results were expressed on dry matter (DM) basis. The analyses were performed in triplicate and standard deviation was calculated. 2.3.4. Total polyphenols content Total polyphenols amount was assayed colorimetrically by means of the Folin–Ciocalteu method based on oxidation/reduction reactions of phenols (Singleton et al., 1999). A volume of 0.2 mL of diluted extract and 1 mL of ten-fold diluted Folin–Ciocalteu reagent (Sigma–Aldrich, St-Quentin Fallavier, France) were mixed. Then, 0.8 mL of Na2 CO3 (75 g/L) (VWR, Fontenay-sous-Bois, France) was added. The sample was incubated for 10 min at 50 ◦ C and then cooled at room temperature. For the control sample, 0.2 mL of phosphate citrate buffer (0.2 mol/L of Na2 HPO4 , 0.1 mol/L of citric acid) at pH 4 was taken. The absorbance was measured at 750 nm by the UV/Vis spectrophotometer (Libra S32, Biochrom, Lagny-sur-Marne, France). Gallic acid (Sigma–Aldrich, St-Quentin Fallavier, France) was used for the calibration curve. Results were expressed as g GAE/100 g dry matter (DM). The analyses were performed in triplicate and standard deviation was calculated. 2.3.5. Centrifugation analysis The multisample analytical centrifuge (LUMiSizer, LUM GmbH, Berlin, Germany), used in this study, employs the STEP technology (Space- and Time-resolved Extinction Profiles Technology), which allows to measure the intensity of the transmitted near infrared light (%) as a function of time and position over the entire sample length simultaneously, without scanning. The volume of the sample was fixed at 2 mL. The centrifugation speed was fixed at 4000 rpm. The temperature was fixed at 20 ◦ C.

∂C ∂ C =D ∂t ∂x2

(3)

where C is the polyphenols concentration (g/L), t is the time (s), x is the thickness of the particle (m) and D is the diffusion coefficient (m2 /s). Flaxseed hulls particles were considered as thin slabs of uniform thickness 2l. It was supposed that stirring was sufficiently intensive and so the resistance to the external mass transfer was neglected. The solution of Fick’s second law for a stirred solution of limited volume was given by Eq. (4) (Crank, 1975):

 2˛(1 + ˛) Mt =1− exp M∞ 1 + ˛ + ˛2 q2n ∞

n



Dq2 t − 2n l

 (4)

where Mt (g/100 g) and M∞ (g/100 g) are respectively the total amount of polyphenols in the slabs at the time t and after infinite time of diffusion. ˛ is the mass ratio of the liquid and flaxseed hulls. qn are the non-zero positive roots of the tan qn = −˛qn . In these experimental conditions, ˛ was equal to 25. This summation series converges rapidly for large values of time t. Therefore, only the first five leading terms were taken into account for D estimation. 2.6. Statistical analysis Each experiment was repeated at least three times. Means and standard deviations of data were calculated. The error bars in all figures correspond to the standard errors. 3. Results and discussion The effect of PEF energy input and treatment duration on the polyphenols extraction index Zp (Eq. (1)) is presented in Fig. 2. The polyphenols extraction increases with the PEF treatment duration. It reaches a maximum for the longest PEF treatment (10 ms). In these conditions the total polyphenols content is 2.7 times higher compared to a control extraction. The PEF effect is usually attributed to the damage of cell membranes facilitating the release of intracellular compounds (Lebovka et al., 2001, 2002). Some damage of cell walls cannot be excluded under the high PEF of 10–20 kV/cm. Grinding causes mechanical rupture of cells and suspended hulls. As a result, extraction is more intensive after the grinding. The polyphenols extraction index Zp = 0.6 is obtained for the PEF energy input of 300 kJ/kg (Fig. 2). It means that 60% of polyphenols are extracted compared to the grinding assisted extraction. Similar total energy input was required to reach high

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Fig. 4. Effect of the electric field intensity on total polyphenols yields and total solutes yields (tPEF = 4 ms, aqueous treatment, total real time of PEF application ≈20 min). Fig. 2. Effect of the total PEF energy input on the polyphenols extraction index Zp . Inset: effect of the PEF treatment duration and the grinding on the polyphenols extraction (PEF: 20 kV/cm, solvent: water).

level of polyphenols extraction from grape seeds (Boussetta et al., 2012). Fig. 3 presents the light transmission (extract clarity) versus centrifugation time for PEF-assisted diffusion (after 2, 4, 6 ms of PEF treatment), grinding assisted diffusion and control extraction. The extracts obtained at the end of the diffusion process are used for sedimentation experiments. Extracts from grinding have the lowest clarities (the extracts are turbid), control and PEF-treated samples have rather the same clarities. The sedimentation of the dispersed particles during centrifugation is rather slow for grinded samples whereas it is much faster for control and PEF-treated extracts. The particles present in the extracts are mainly residues from treated linseed hulls. The benefits of applying PEF versus grinding for polyphenols extraction would be the selective release of biocompounds (Figs. 2 and 3). Grinding results in a solution with a high turbidity

Fig. 3. Extracts clarity (transmission, %) versus centrifugation time for PEF and grinding assisted diffusions and control extraction. The extracts were obtained after 60 min of diffusion at 50 ◦ C. Transmission data were obtained from the LUMisizer centrifuge.

that contains smaller particles size than that obtained after PEF extraction. The subsequent purification of the extracts is thus more difficult. For example, the solid liquid separation by centrifugation is longer from ground samples (Fig. 3). For the next experiments, a PEF treatment of 300 kJ/kg will be applied as it seems to offer a good compromise between a reduced energy input and a high level of polyphenols content (2.3 times higher than a control). The effect of the PEF electric fields strength on polyphenols and total solutes yields was studied (Fig. 4). The control extraction (0 kV/cm) was performed for 20 min (the same duration as the PEF treatment) and without mixing (0 rpm). The total polyphenols content is 4 times greater with a PEF treatment at 20 kV/cm compared to a control experiment (0 kV/cm). When decreasing the PEF intensity at 10 and 15 kV/cm, the treatment efficiency on polyphenols extraction is smaller. The same tendency is observed for the total solutes extraction. Note that the level of polyphenols in the flaxseed hulls extracts represents approximately 0.6–0.7% of the total solutes content. When applied on fresh fruits and vegetables, PEF intensity is usually quite low (0.5–2 kV/cm) (Grimi et al., 2011). In the case of oilseeds germ (maize, olives and soybeans), low values of electric field strength in the range of 0.6–1.3 kV/cm can be applied for enhanced and gentle recovery of functional food ingredients (oil, isoflavonoids, phytosterols) through induction of stress reactions (Guderjan et al., 2005). For the extraction of oil from hulled and non hulled rapeseed, maximum permeabilisation of the cell membrane was achieved for hulled rapeseed 55% at 7.0 kV/cm and 120 pulses (84 kJ/kg) and 17% at 5 kV/cm and 60 pulses (42 kJ/kg) for non-hulled rapeseed (Guderjan et al., 2007). However, flaxseeds hulls are a lignocellulosic product with a low level of water (3.7%). It has been shown that the required PEF intensity is higher (20 kV/cm) for products with a high dry matter content like dried grape seeds (Boussetta et al., 2012). On the other hand, products rich in cellulose and lignins are also treated with a high PEF intensity of 20 kV/cm (Grimi et al., 2011). The presence of water in the product would enhance the current and voltage flows through the product thus resulting in a more efficient treatment (Lebovka et al., 2002). Water is reported to be much more easily polarized by electric fields than are most solutes. Water molecules dissociate into ions at high field strength. The presence of polar groups in interfaces or in the interior of macromolecules has a destabilizing effect, reducing association constant and unfolding free energies. It results in morphological changes such as increased spacing between the plasmic membrane and the cell wall, leakage of cytoplasmic materials and disruption of organelles (Benz et al.,

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Table 1 Effect of the flaxseed hulls moisture on the extraction efficiency of PEF (P: polyphenols content increase with PEF treatment compared to a control (untreated) diffusion at 150 rpm). Rehydration duration (min)

Moisture (%)

P (%)

0 20 40 60

3.7 69.5 72.2 72.3

30.9 31.2 37.8 38.1

± ± ± ±

0.1 0.5 0.8 0.5

1979; Barsotti et al., 1999). All these phenomena may contribute to solutes loss (in particular, polyphenols compounds) during pretreatment. The influence of the flaxseeds hulls rehydration before PEF treatment was studied (Table 1). Electrical experiments were performed at 20 kV/cm with a total specific energy input of 300 kJ/kg (tPEF = 4 ms). Flaxseeds hulls were rehydrated for 0, 20, 40 or 60 min before PEF application. Hulls contain initially about 3.7% of moisture. This product has a strong ability to absorb water until reaching a saturation level (72.3% of moisture) observed after 40 min of contact with the solvent. The rehydration of the product before PEF improves the treatment efficiency. The highest polyphenols increase (≈37%) is observed when the product is rehydrated at least 40 min. Another study shows that before PEF treatment rapeseed was also soaked in tap water for a few minutes up to a moisture content of 50–60% to receive an optimal conductivity (Guderjan et al., 2007). The electropermeabilisation induced by PEF is facilitated in polar liquids. The addition of water is thus beneficial; as the water is absorbed, the volume of the air inside the hulls decreases. When strong electric fields are applied in a product containing air, microdischarges can occur inside the air bubbles. The air has a lower dielectric breakdown strength than the liquid media. Its presence leads to inhomogeneities of the electric field distribution (isolation effect). The lower dielectric permittivity of air causes an enhancement of the electric field within the bubbles increasing the chance for a dielectric breakdown and arcing. When modeling the electric field distribution within bubbles present in the treatment chamber, it was shown that the field strength in the boundary region of a bubble is very low, possibly leading to under-processing, in particular between several bubbles (Gongora-Nieto et al., 2003). Therefore, the rehydration of dried products seems to be the most suitable solution. The effect of the ethanol content on polyphenols extraction was studied (Fig. 5). The flaxseeds hulls were first rehydrated for 40 min, PEF treated and extracted by a diffusion step for 60 min. The addition of 50% (w/w) ethanol to the solvent resulted in a clearly increased extraction of polyphenols. The polyphenols content was up to 3 times higher after only 20 min of extraction compared to experiments with 0% ethanol. For all cases, the application of PEF increases the extraction yields. Immediately after PEF (at 60 min), the polyphenols content increase compared to a control extraction (no PEF) was 38%, 21% and 12% respectively for 0%, 20% and 50% ethanol. At the end of the extraction, the polyphenols content was increased compared to a control extraction (no PEF) by 37%, 24%, 18% respectively for extracts with 0%, 20% and 50% ethanol. When samples contain higher polyphenols content, the increase in extraction is rather smaller. Indeed, the higher final polyphenols yield (≈314 mg/g DM) is reached for PEF assisted extraction with 50% ethanol. Flaxseed polyphenols are mainly composed of lignans (about 1% of flaxseed meal), phenolics acids (ferulic and p-coumaric acids) and flavonoids. Most of these compounds are largely present as glycosides that make them more polar. Mixtures of methanol and water are the solvents most commonly employed for the extraction of these molecules. However, the use of ethanol instead of methanol is preferable as it is a generally recognized as safe solvent and allows reaching high extraction yields of polyphenols. On the other hand,

Fig. 5. Effect of the ethanol content on the polyphenols extraction for untreated and PEF-treated samples (PEF: 300 kJ/kg, 20 kV/cm, rehydration duration: 40 min, solvent: water/ethanol, diffusion agitation: 150 rpm).

the highest concentration of phenolic acids, lignans and flavonoids is in the aleurone layer and in the seed coat (Pandey and Rizvi, 2009). The ethanol may increase the permeabilisation of the cell membranes of the seed coat and the aleurone layer by the solubilization of their phospholipids compounds. Studies have shown that the presence of even small amount of ethanol (0.4%, v/v) caused cell membrane integrity lost, especially for long time contact from plant tissues (Wang et al., 2011). The release of polyphenols would thus be facilitated. For the following experiments, extraction will be performed in the presence of 20% ethanol. This ethanol content is the maximum recommended for potential PEF application at the industrial scale. The effect of different citric acid concentrations (0.05–0.3 mol/L) on the extraction yields of polyphenols was studied (Fig. 6). The PEF treatment was applied after 40 min of rehydration. It has been observed that concentrations above 0.3 mol/L (pH < 2.55) have a negative effect on the extraction of polyphenols (results not shown). The pH values of the solvent containing 0, 0.05, 0.1 and 0.3 mol/L acid are respectively 4.03, 2.92, 2.74 and 2.55. Results show that the extraction of polyphenols is improved as the concentration of acid increases. After 40 min of extraction, the addition of

Fig. 6. Effect of the citric acid molarity on the polyphenols extraction for untreated and PEF-treated samples (PEF: 300 kJ/kg, 20 kV/cm, rehydration duration: 40 min, solvent: 20% ethanol, diffusion agitation: 150 rpm).

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Fig. 7. Effect of the sodium hydroxide molarity on the polyphenols extraction for untreated and PEF-treated samples (PEF: 300 kJ/kg, 20 kV/cm, rehydration duration: 40 min, solvent: 20% ethanol, diffusion agitation: 150 rpm).

0.3 mol/L acid allows extracting 24% more polyphenols than extraction without acid. In the presence of PEF, the extraction is still improved. At the end of the PEF assisted extraction, the polyphenols content is increased by 24%, 38%, 31% and 26% compared to control extraction (no PEF) respectively for extracts with 0, 0.05, 0.1 and 0.3 mol/L. However, the rather same level of polyphenols (≈249 mg GAE/g DM) is extracted for PEF assisted extraction in the presence of 0, 0.05 and 0.1 mol/L acid. The highest polyphenols content is reached for PEF assisted extraction in the presence of 0.3 mol/L acid. The higher extraction yields in the presence of acid may be related to the extraction of bound polyphenols. Studies have shown that acidic extraction allows the release of bound polyphenols (Hahn et al., 1984). Another study (Kozlowska et al., 1983) showed that the majority (59%) of phenolic acids in flaxseed are ester-bound. They identified free and bound phenolic acids in flaxseed. Free and bound phenolic acids are extracted in methanol and in boiling 2 mol/L HCl, respectively (Hahn et al., 1984). In the case of sorghum, free phenolic acids are found in the outer layers of the kernel (pericarp, testa, and aleurone), whereas the bound phenolic acids are associated with the cell walls (Hahn et al., 1984). The addition of acid also allows the recovery of the aglycone form of lignan, but this method could be destructive if too long heating period or too high acid concentration are used (Kraushofer and Sontag, 2002; Li et al., 2008). The effect of different sodium hydroxide concentrations (0.05–0.3 mol/L) on the extraction yields of polyphenols was studied (Fig. 7). All experiments were carried out in the presence of 20% ethanol. The pH values of the solvent containing 0, 0.05, 0.1 and 0.3 mol/L sodium hydroxide are respectively 4.03, 12.65, 12.91 and 13.33. The PEF treatment was applied after 40 min of rehydration. Even a low concentration of sodium hydroxide (0.05 mol/L) improves drastically the yields of polyphenols at the very beginning of the extraction. In the absence of PEF, the presence of 0.05 mol/L hydroxide sodium increases up to 3.8 times the polyphenols yields compared to an extraction with 0 mol/L. Compared to control experiments (no PEF), the application of PEF results on a supplementary polyphenols release of up to 25%, 18%, 20% and 9% respectively in the presence of 0, 0.05, 0.1 and 0.3 mol/L. For higher concentration (0.3 mol/L), the effect of PEF is less marked as the initial content of polyphenols in the extract is higher. The highest polyphenols content (1033 mg GAE/g DM) is obtained in the presence of 0.3 mol/L sodium hydroxide for PEF assisted extraction. Note that compared to the acidic extraction (Fig. 6), the alkaline

Fig. 8. Effect of the ethanol content (a), the acid molarity (b) and the sodium hydroxide molarity (c) on the diffusion coefficient D for untreated and PEF-treated samples. (PEF: 300 kJ/kg, 20 kV/cm, rehydration duration: 40 min, solvent: 20% ethanol, diffusion agitation: 150 rpm).

extraction allows reaching higher yields of polyphenols. Acidic extraction has been reported in the literature to be less efficient than alkaline extraction for polyphenols extraction (Li et al., 2008). Sodium hydroxide is commonly employed at concentrations from 0.1 mol/L (Hano et al., 2006) to 1 mol/L (Degenhardt et al., 2002) for lignan hydrolysis. Release of the bound lignans from their esterified complex has been generally achieved by alcoholic solid–liquid extraction and alkaline treatment (Eliasson et al., 2003; Renouard et al., 2010). The hydroxide sodium also allows the release of bound acid phenolics. Alkaline hydrolysis released 730 mg phenolic acids/kg defatted flaxseed flour which represents 89% of total phenolic acids (Kozlowska et al., 1983). The mean total flavonoids content in flaxseed, measured by absorbance in 80% methanolic extract at 404 nm, has been reported to be 50–80 mg/100 g defatted flaxseed flour (Oomah et al., 1996). Other authors found 200 mg/100 g in flaxseed hulls (Struijs et al., 2007). On the other hand, sodium hydroxide has been largely used for alkaline lysis in the field of proteomics and particularly in the isolation of plasmid DNA (Birnboim and Doly, 1979). The effect of the ethanol content, the acidic molarity and the basic molarity on the diffusion coefficient was presented in Fig. 8. Values vary from 0.47 × 10−12 to 7.05 × 10−11 m2 /s. For all experiments, the diffusion coefficient was always higher for PEF assisted extraction than control extraction. When changing the ethanol content, the diffusion coefficient was the highest for an ethanol content of 20% (Fig. 8a). Values obtained for 0% and 50% ethanol were quite similar. Soluble phenolic compounds are generally extracted using water, methanol, ethanol or acetone. The presence of attached sugars tends to render the phenolic compounds more water soluble and combinations of the above solvent with water are thus better solvents for glycosides. In particular, the glycoside form of lignans show enhanced solubility compared to the corresponding aglycones. Soluble phenolic compounds are mainly distributed in the cell vacuoles, while most lignan, flavonoids, and insoluble polyphenols deposit in the cell wall to combine the hydrogen bond, hydrophobic bond with proteins and polysaccharides (Dixon and Paiva, 1995). Water and low concentration of ethanol can access to cells, but high concentration of ethanol can prevent the dissolution of polyphenols and then influencing the extraction rate. With

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high concentration of ethanol, more liposoluble materials were extracted which led to the increase of interference factors, making the purification progress more difficult. The effective diffusion coefficient was showed to be in the range of 0.065–0.130 m2 /s in water and in the range of 0.01–0.076 m2 /s in pure ethanol (Guerrero et al., 2008). However, the addition of acid resulted in a small decrease in the diffusion coefficient for both control and PEF treated samples (Fig. 8b). On the contrary, the presence of sodium hydroxide significantly increased the diffusion coefficient (Fig. 8c). There exists an optimal sodium hydroxide molarity. Indeed, the maximal diffusion coefficient was reached for 0.05 mol/L sodium hydroxide. The basic hydrolysis is more effective than the acidic hydrolysis. It can be assumed that cell structure is more easily damaged by sodium hydroxide thus increasing the release of intracellular compounds. On the other hand, the resulting phenolics hydrolysate from basic experiments are still in their glycone form and are consequently hydro-soluble. Note that experiments for acidic and basic hydrolysis were performed in the presence of 20% ethanol. However, the acidic hydrolysis generally resulted in the formation of aglycone form of polyphenols that are more liposoluble. 4. Conclusions This study shows that the PEF pretreatment allows improving the extraction of polyphenols from flaxseed hulls. The optimal parameters that result on the higher extraction yield are a rehydration of hulls for 40 min followed by a PEF treatment energy input of 300 kJ/kg at 20 kV/cm and a subsequent diffusion step in 20% ethanol and 0.3 mol/L sodium hydroxide. Further studies are needed to determine the effect of such a PEF assisted extraction on other flaxseed hulls biomolecules like proteins and mucilage. The PEF pretreatment should be compared to other electrotechnologies. References Bagherian, H., Zokaee Ashtiani, F., Fouladitajar, A., Mohtashamy, M., 2011. Comparisons between conventional, microwave- and ultrasound-assisted methods for extraction of pectin from grapefruit. Chem. Eng. Process.: Process Intensificat. 50 (11–12), 1237–1243. Barsotti, L., Merle, P., Cheftel, J.C., 1999. Food processing by electric fields: physical aspects. Food Rev. Int. 15 (2), 163–180. Benz, R., Beckers, F., Zimmermann, U., 1979. Reversible electrical breakdown of lipid bilayer membranes: a charge-pulse relaxation study. J. Membr. Biol. 48, 181–204. Birnboim, H.C., Doly, J., 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523. Boussetta, N., Vorobiev, E., Le, L.H., Cordin-Falcimaigne, A., Lanoisellé, J.L., 2012. Application of electrical treatments in alcoholic solvent for polyphenols extraction from grape seeds. LWT: Food Sci. Technol. 46 (1), 127–134. Corrales, M., Toepfl, S., Butz, P., Knorr, D., Tauscher, B., 2008. Extraction of anthocyanins from grape by-products assisted by ultrasonics, high hydrostatic pressure or pulsed electric fields: a comparison. Innov. Food Sci. Emerg. Technol. 9 (1), 85–91. Crank, J., 1975. The Mathematics of Diffusion. Oxford University Press, London, UK. Degenhardt, A., Habben, S., Winterhalter, P., 2002. Isolation of the lignan secoisolariciresinol diglucoside from flaxseed (Linum usitatissimum L.) by high-speed counter-current chromatography. J. Chromatogr. A 943 (2), 299–302. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell. 7, 1085–1097. Eliasson, C., Kamal-Eldin, A., Andersson, R., Aman, P., 2003. High-performance liquid chromatographic analysis of seoicolariciresinol diglucoside and hydroxycinnamic acid glucosides in flaxseed by alkaline extraction. J. Chromatogr. A 1012 (2), 151–159. Ford, J.D., Huang, K.S., Wang, H.B., Davin, L.B., Lewis, N.G., 2001. Biosynthetic pathway to the cancer chemopreventive secoisolariciresinol

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