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J. of Supercritical Fluids 75 (2013) 94– 100

Contents lists available at SciVerse ScienceDirect

The Journal of Supercritical Fluids

jou rn al h om epa ge: www.elsev ier .com/ locate /supf lu

upercritical antisolvent fractionation of lignans from the ethanolxtract of flaxseed

iuseppe Perretti, Claudia Virgili, Antonio Troilo, Ombretta Marconi,ian Franco Regnicoli ∗, Paolo Fantozzi

epartment of Economic and Food Science, via S. Costanzo n.c.n., University of Perugia, 06126 Perugia, Italy

r t i c l e i n f o

rticle history:eceived 7 November 2012eceived in revised form5 December 2012ccepted 16 December 2012

a b s t r a c t

Supercritical antisolvent fractionation (SAF) was evaluated in the fractionation and concentration of afamily of beneficial plant compounds called lignans from the ethanol extract of flaxseed. The amount oflignans obtained in different fractions was studied under varying pressures (10–30 MPa), CO2 flow rates(5–15 kg h−1) and times of treatment (60–180 min) in a three-stage fractionation column with constanttemperature fixed at 313, 323 and 333 K. The determination of lignan content was performed by HPLC cou-pled with a coulometric array detector. The effects of each individual variable as well as their interactions

eywords:upercritical carbon dioxidentisolventractionationlaxseedignans

were investigated using a full factorial design with three factors and two levels and the optimal condi-tions were calculated through response surface methodology. A statistically significant increase in lignancontent was obtained after the SAF process; from an average initial lignan content of 1.66 ± 0.13 g L−1 itwas possible to obtain a total lignan content ranging from 3.42 to 12.96 g L−1. We conclude that SAF is anappropriate technique for the isolation of lignans from flaxseed.

thanol extract

. Introduction

Flax (Linum usitatissimum L.) is a plant known for its commer-ial utility for the production of fiber and oil. The fiber is primarilyound in the long stems of flax while the oil is expressed from thearge seeds which containing 15–40% oil by weight [1]. Although theaxseed oil has important industrial applications, it also plays a role

n the food supply. As stated by Oomah [2], flaxseed oil is a promi-ent functional food due to its content of �-3 fatty acids; in fact,he essential fatty acid �-linolenic acid represents approximately2% of the total fatty acid content of flaxseed oil [2]. Flaxseeds alsoontain phenolic compounds such as lignans in very high concen-rations (>600 mg kg−1) compared to other plants and plant parts3].

Lignans are characterized by having two propyl-benzene

olecules (coniferyl alcohols) that are linked by a bond between

he 8 and 8′ positions. Lignans are biosynthesized throughhe phenylpropanoid pathway and they are stereospecifically

Abbreviations: HYDMA, idrossimataresinol; ISO, isolariciresinol; LARI, lar-ciresinol; MATA, matairesinol; PINO, pinoresinol; RF, residual fraction; RSM,esponse surface methodology; SECO, secoisolariciresinol; SDG, secoisolariciresinoliglucoside; SF, separator fraction; SAF, supercritical antisolvent fractionation;C-CO2, supercritical carbon dioxide.∗ Corresponding author. Tel.: +39 075 585 7923; fax: +39 075 585 7939.

E-mail address: gianregnicoli@libero.it (G.F. Regnicoli).

896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.supflu.2012.12.028

© 2013 Elsevier B.V. All rights reserved.

dimerized into pinoresinol (PINO). Subsequently, reduction, oxida-tion, dehydrogenation and addition reactions lead to the formationof a broad range of lignans which are present in both aglyconicand glycosylated forms. The lignans found in the highest concen-trations in flaxseed are secoisolariciresinol (SECO), matairesinol(MATA), pinoresinol (PINO), lariciresinol (LARI), idrossimataresinol(HYDMA), isolariciresinol (ISO) and secoisolariciresinol diglucoside(SDG) [4]. Lignans are stored in plants predominantly as glycosides,and they are converted by intestinal bacteria into metabolites withestrogen-like activity like equol, enterodiol and enterolactone [3].There is evidence of health benefits attributable to lignans, andenterolactone and enterodiol are considered partially responsiblefor the prevention/inhibition of human prostate, colon and skincancers as well as reduction of menopausal symptoms and post-prandial blood glucose and protection of the cardiovascular system,fertility and thyroid function [5–10].

To promote lignan consumption, SDG extracts can be incorpo-rated as ingredients into foods and drinks; in fact some patentsand applications already exist. For example, SDG can be isolatedfrom flaxseed and dried into powder by standard techniques suchas vacuum concentration, spray drying or freeze drying. These SDG-rich products can be used directly as a powder or as ingredientsin the production of foods such as soy flour or in other various

foods and drinks [11]. Typical examples include spreads, dress-ing, mayonnaise, ice creams, cream alternatives, health bars, healthdrinks, sports drinks, confectionery, bakery products, soups, cere-als, sauces, fillings and coatings [12]. Products such as these can be

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eveloped to contain 0.1–90% SDG by weight depending on serv-ng size. For products with high water content that are consumedn large amounts such as drinks and soups, the SDG content cane appropriately decreased to a range 0.001–10% by weight, forxample [11].

After the first studies of SDG by Bakke and Klosterman (1956)13], some methods for the analysis of lignans and other phenolicompounds from flaxseeds have been reported [14,15]. Literaturehows that lignans can be extracted from flaxseed and other matri-es, using dioxane, methanol [13], ethanol or enzymatic hydrolysis,ollowed by different purification systems [13–17]. The extraction

ethods vary in relation with the sample matrix and the targetompound [14]. The method of sample preparation described inhis article shows, after experimental tests, a high content of lig-ans, the use of non-toxic solvents and the use of a short timeompared to the methods shown in the literature [14].

Our laboratory has studied the application of supercritical fluidsxtraction to valorize flaxseed oil. We found extraction from groundaxseed yields 19.5 ± 0.9% oil by weight using neat SC-CO2 (super-ritical carbon dioxide), and 23.0 ± 2.2% oil by weight with the usef ethanol as a co-solvent. The quality of the extracted oils was ana-yzed by common parameters (e.g., free acidity, peroxide number)nd functional aspects (e.g., polyphenol content, lignan content).e found the oils obtained by supercritical fluid extraction were

enerally comparable to oils produced by traditional extractionechnologies, with an increase in total polyphenol extraction butithout an increase in lignan extraction [18–20]. The same authors

lready studied the behavior of flax seed oil fractionation by neatupercritical carbon dioxide under pressures from 10 to 33 MPand temperatures of 313, 323, and 333 K, even if in this specificase a low efficiency was observed (no statistical differences for

< 0.05) [18]. Additionally, we also studied the shelf life of flaxseedil and SDG content [21]. In all those experiments, the concentra-ion of lignans in flaxseed oil was very low (2–4 mg kg−1), probablyue to the polar and polymeric nature of lignans and their pres-nce as glycosides. It is known that when polar compounds haveo be concentrated and fractionated from an organic solution, theydrophobic character of CO2 can be an advantage. In this con-ext, the supercritical antisolvent fractionation (SAF) process couldmprove lignan extraction because it involves the continuous con-act between the SC-CO2 and the liquid mixture in a pressurizedessel during concentration and fractionation [22].

The aim of this study was to assess the efficiency of SAF for theoncentration of flaxseed lignans using an ethanol extraction ofignans obtained from defatted flaxseed flour.

. Materials and methods

.1. Samples and reagents

Flaxseeds (2011 harvest, Barbara variety) were purchased fromerra Bio Soc. Coop. (Urbino, Italy). The flaxseed moisture deter-ined (8.65% ± 0.05) is comprised in the common range of flaxseedoisture reported in literature [1].Standard preparations of the isolated lignans SECO, MATA, PINO,

ARI, HYDMA and ISO were purchased from Arbonova (Turku,inland). SDG was purchased from Chemos GmbH (Reenstauf,ermany). Methanol, ethanol, acetonitrile, petroleum ether, andlacial acetic acid were purchased from Sigma–Aldrich ChemiembH (Steinhein, Germany).

.2. Sample preparation

The ethanol extraction of lignans from flaxseeds was performedollowing the method of Westcott and Muir (1998) [17] modified

al Fluids 75 (2013) 94– 100 95

as follows: once removed from cold storage, 120 g of fresh seedswere ground in a laboratory grinder (Osterizer Sunbeam Model No.4153-50) at maximum speed for 30 s. Solvent defatting was per-formed over 2 h using 500 mL of petroleum ether (25:6, v:w) undermagnetic stirring at room temperature; the mixture was filteredwith a 22.09 cm2 steel mesh (0.5 mm pore size) and the resid-ual petroleum ether was evaporated for 1 h at room temperatureunder a constant stream of air. The defatted seeds, now weighingapproximately 100 g, were stirred for 2 h at room temperature in600 mL of ethanol, water and 1 M NaOH at a ratio of 4:1:1 (v:v:v).After extraction, this solution was filtered with the above describedsteel mesh, neutralized with glacial acetic acid to pH 6 and cen-trifuged at 6000 × g for 20 min to precipitate and remove watersoluble polysaccharides and proteins. For each run, the preparationof fresh ethanolic extract was repeated.

2.3. Supercritical antisolvent fractionation

The SAF experiments were conducted in triplicate, using aMuller Extract Company GmbH (Koburg, Germany) pilot plant(Fig. 1), equipped with a three-stage fractionation column (totallength 3 m, diameter 3 cm) with an internal volume of 2 L andpacked with stainless steel Raschig rings (10 mm × 10 mm); eachcolumn stage was individually thermostated. The separation sec-tion of the plant consisted of a 1 L cylindrical separator followed bytwo cyclonic separators, both set at 303 K and 6 MPa. Each exper-iment was conducted in one batch with a feed of 350 mL. Thethree-stage column temperatures were fixed at 313, 323 and 333 K,from the bottom to the top. These temperatures were chosen fol-lowing some suggestion reported in the literature of supercriticalfluids fractionation [23,24] to preserve the ethanol evaporation,to save energy and to explore the behavior at the related den-sities. This determined a density gradient in the solvent stream,with SC-CO2 densities varying along the column. The feed wasintroduced into the column from the bottom. At the end run, theresidual fraction (RF) was recovered from the bottom of the columnand measured. The separator fraction (SF) was recovered in theseparator.

2.4. Experimental design and statistical software

The effects of three variables affecting the concentration of lig-nans (time of treatment (X1), CO2 flow rate (X2) and pressure (X3))were studied following a full factorial design (23) and response sur-face methodology. Two factor levels were chosen for each variableconsidering the limits of the experimental apparatus and the orderof the runs was randomized to avoid systematic errors. The mea-sured response (Y%) was the enrichment (E%) of lignans after SAF.Table 1 shows the experimental matrix for the 23 factorial design(3 factors, each run at the two levels).

The influence of the different parameters on lignan content (per-centage ratio between the RF lignan content and the initial lignancontent) was determined using the following interactions regres-sion equation (Eq. (1)).

Y = b0 − b1X1 − b2X2 − b3X3 + b4X1X2 + b5X1X3 + b6X2X3 (1)

where Y is the enrichment (%), b0 is the constant term, b1, b2 and b3are the coefficients of the individual variables X1, X2, X3 and b4, b5,b6 are the coefficients of the pairs of interactions between the vari-

ables X1 X2, X1 X3 and X2 X3. All computations involving regressionmodels, response surface plots and ANOVA tests were performedusing the Statistic-Toolbox 7 with MATLAB (The Mathworks Inc.,Natick, USA).

96 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94– 100

Fig. 1. Fractionation pi

Table 1Experimental parameters under the full factorial design.

Exp. Time (min) CO2 flow (kg h−1) Pressure (MPa) X1 X2 X3

1 60 5 10 −1 −1 −12 60 15 10 −1 1 −13 60 5 30 −1 −1 14 60 15 30 −1 1 15 180 5 10 1 −1 −16 180 15 10 1 1 −17 180 5 30 1 −1 1

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1, time; X2, CO2 flow rate; X3, pressure.

.5. HPLC analysis of the extracts

In order to quantify the concentration of lignans obtained byAF, the fractions obtained from each experiment were analyzedy high performance liquid chromatography coupled with coulo-etric detector array (HPLC-ECD). Two Jasco PU-1580 pumps

onnected to a gradient solvent system and a Basic MarathonSpark” autosampler (Erkerode, The Netherlands) with a 100 �Loop were used. An Inertsil ODS-3 V (C18 250 mm × 4.6 mm Ø; par-icle size 5 �m) column, a CoulArray detector (ESA Inc., Chelmsford,SA) with eight electrode potentials set to 100–905 mV at incre-ents of 115 mV were used. Mobile phase A was 0.05 M KH2PO4

nd 0.05 �M sodium lauryl sulphate (SLS) and mobile phase B washase A/CH3–OH/CH3CN, 30:20:50 (v:v:v) and 0.05 �M SLS. Theobile phases were adjusted to pH 3.35 with 85% orthophosphoric

cid and were filtered with a 0.22 �m membrane filter (Millipore,

edford, USA, for aqueous solvents; MSI, USA, for organic solvents).he voltammetric data were collected and analyzed by CoulArrayoftware. In Table 2, the calibration results of the standards are

able 2alibration data.

Name RT (min) Calibration range (�g L−1) LOD (�g L−1) LOQ (�g L−1)

SDG 15.25 50–1050 26.96 28.65ISO 18.73 50–1050 21.27 27.18HYDMA 20.53 50–1050 27.87 33.13SECO 22.03 50–1050 42.58 49.35LARI 22.55 75–1050 53.57 58.74PINO 26.94 50–750 25.45 32.31MATA 30.97 50–1050 23.73 28.17

T, retention time; LOD, limit of detection; LOQ, limit of quantification.

lot plant scheme.

shown; the r2 levels are above 0.9991 for all the analytes (data notreported).

The lignans in the ethanol extract of flaxseeds were identifiedby comparison of their retention times with those of the standardsand they were quantified by using their calibration curves in thelinear range 50–1050 �g L−1.

2.6. Ethanol quantification

The ethanol contents of the extracts, RFs and SFs, were deter-mined by the OIV-MA-AS312-01A: R2009 4. C. method.

2.7. Moisture quantification

The moisture content of the flaxseed was determined by theAOAC (1984, XIV edition) 14.004 method.

3. Results and discussion

3.1. Lignan enrichment

Both total lignan content and SDG content in the RFs differed sig-nificantly (ANOVA, p < 0.05) and their values under each conditionare reported in Table 3.

The SAF process was effective in concentrating lignan content inthe RF; from an average initial lignan content of 1.66 ± 0.13 g L−1 theRF total lignan content increased to a range 3.42–12.96 g L−1, whichcorresponds to enrichment values from 201 to 753%, as reported inTable 3. Very low concentrations of lignans (0.03 ± 0.02 g L−1) weredetected in the SF. In all experiments the major lignan presentin the RF was SDG. Of the other lignans analyzed in this study,HYDMA was present at a low concentration both in the initialextract (0.04 ± 0.01 g L−1) and in the RF (0.02–0.12 g L−1). ISO wasalso detected at low levels, with an initial extract concentration of0.006 ± 0.002 g L−1 and 0.02–0.06 g L−1 in the RF. SECO, LARI, PINOand MATA were observed below the limit of quantification of theanalytical method in both the initial extract and in the RF. The spe-cific enrichment in SDG shows that there is also a potential slightfractionation of the different lignans when SDG enrichment % isdifferent than the total enrichment %. Furthermore the trace lignan

content in the SF (0.03 ± 0.02 g L−1) confirms the strong selectiveaction of SC-CO2 by extracting mainly ethanol from the column.

The mathematical models for total lignan enrichment andSDG enrichment (Eqs. (2) and (3), respectively) provide a good

G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94– 100 97

Table 3Total lignan content and SDG content (g L−1) and enrichment % (E) in the RFs.

Exp. Time (min) CO2 flow (kg h−1) Pressure (MPa) Total lignans* SDG*

(g L−1) E (%) (g L−1) E (%)

1 60 5 10 3.42ab +201ab 3.41ab +201ab

2 60 15 10 3.81ab +283ab 3.81abc +283abc

3 60 5 30 6.15abc +353abc 6.03abc +355abc

4 60 15 30 6.63bcd +388bcd 6.34bcd +392bcd

5 180 5 10 3.79ab +240ab 3.60abc +236abc

6 180 15 10 5.24abc +323abc 5.22abc +322abc

7 180 5 30 9.11cd +560cd 8.85cd +565cd

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polar moieties). On the other hand, increasing the flow rate of theshorter treatment time had a positive effect on the concentration oflignans. In fact, with 60 min of treatment at 300 bar and flow rate at

100 200 300 400 500 600 700 800

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Enrichment % (Lignans) Experimental data

Enri

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200

300

400

500

600

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* Means in the same column not sharing a common superscript letter were signi

epresentation of the real trend of the experiments.

Etot = 232.375 − 1.36041 XT − 1.3125 XP − 1.275 XF

+ 0.1027 XTP + 0.066250 XTF + 0.1575 XPF

r2 = 0.9935; RMSE = 19.6250% (2)

ESDG = 227.625 − 1.3729 XT − 1.0625 XP − 0.775 XF

+ 0.1035 XTP + 0.1425 XPF

r2 = 0.9943; RMSE = 18.3750% (3)

T, XP, and XF represent independent variables of time of treatment,ressure and CO2 flow rate, respectively while XTP, XTF, and XPF rep-esent the interactions among the three variables. The regressionoefficient is represented by r2 and RMSE is the root mean squarerror.

The graphs in Fig. 2 show the actual and predicted data disper-ion around the bisector. The equation line almost coincides withhe bisector, therefore it is completely covered and is not visible.he dispersion of the 8 experiments is linear and in agreement withhe model as confirmed by the high regression coefficient (0.9935or total lignans and 0.9943 for SDG) and by the low root meanquare error (19.6250% for total lignans and 18.3750% for SDG).

Taking into account the suitability of the regression method, its possible to describe the relationship between the different vari-bles and enrichment in lignans and SDG using response surfaceodel (RSM) methodology. The quadratic response surface (Fig. 3)

raphically reveals the results from the model. The highest levels ofignan enrichment were obtained at the highest levels of the threeariables (30 Mpa, 15 kg h−1, 180 min) confirming the evidence ofhe results reported in Table 2.

The same behavior was observed for SDG (Fig. 4).The response surfaces for pairs of variables are given in Fig. 5.The time–pressure interaction (Fig. 5a) indicates a major role for

ressure in influencing lignan enrichment. Likewise the time–CO2ow rate interaction (Fig. 5b) indicates a major role for CO2 flow rate

n determining the separation yield. Lastly, the pressure–CO2 flowate interaction (Fig. 5c) indicates another major role for pressuren the determination of the separation yield.

At the lowest pressure level, neither the stability of the sepa-ation (Fig. 5a) nor the solvent flow rate (Fig. 5c) was influenced,nd the same behavior was observed for SDG (response surfacesot reported).

In order to explore the validity of the mathematical modelor higher flow rates, times and pressures additional trials wereonducted. The experiments at 300 min of treatment, flow rate

f 15 kg h−1 and pressure of 30 or 33 MPa and also 180 min ofreatment with flow rate of 15 kg h−1 did not influence the concen-ration of lignans except for in experiment 8. This indicates a limitf the mathematical model for the higher pressure and time. This

12.96e +753e 12.87e +753e

y different (one way ANOVA; p < 0.05, n = 3).

is probably due to the changed solubility of the residual ethanolat the low concentrations in the RF, where the ethanol solubilityin water is still evident (the RF is mainly composed of water and

100 200 300 400 500 600 700 800Enrichment % (SDG ) Experimental data

Fig. 2. Regression model of actual values–predicted values for total lignans (a) andSDG (b).

98 G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94– 100

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Fig. 3. Quadratic response surface model for lignan enrichment.

4 kg h−1 it was possible to obtain higher enrichment values thanchieved in trials 1–6, as predicted by the mathematical model.

The comparisons of the different enrichments in lignansbtained with the use of similar CO2 mass (experiments 2 vs. 5 and

vs. 7) were conducted. These comparisons show a higher contentf lignans (variations not statistically significant) for the tests with

higher CO2 superficial velocity, respectively the experiments 2nd 4.

.2. Ethanol extraction

The initial content of ethanol was 66.67% of the total volumend decreased in the RF to a range 5.44–26.67%, which correspondo a decrease in value (D%) from 60 to 92% as reported in Table 4.he SF contained 100% ethanol by volume.

The ethanol content model (Eq. (4)) provided a good represen-ation of the real trends of the experimental conditions.

Dethanol = −34.7603 + 0.2530 XT + 0.9181 XP + 1.5077 XF

− 0.0021 XTP − 0.0114 XTF − 0.0014 XPF

r2 = 0.9791; RMSE = 2.000% (4)

quation (4) represents the interaction regression equation for theercent of ethanol decrease, where XT, XP, and XF are the indepen-ent variables of time of treatment, pressure and CO2 flow rate,

Fig. 4. Quadratic response surface model for SDG enrichment.

Fig. 5. Response surfaces for pairs of variables. Time–pressure (a), Time–CO2 flowrate (b), and Pressure–CO2 flow rate (c).

G. Perretti et al. / J. of Supercritical Fluids 75 (2013) 94– 100 99

Table 4Ethanol contents of the RFs in the different experiments and the decrease in ethanolcontent from the initial sample.

Exp. Time (min) CO2 flow (kg h−1) Pressure (MPa) Ethanol*

(Vol %) D (%)

1 60 5 10 26.67a 60a

2 60 15 10 18.68b 72b

3 60 5 30 13.58cd 80cd

4 60 15 30 10.95ce 84ce

5 180 5 10 10.05e 85e

6 180 15 10 16.36bd 75bd

7 180 5 30 5.44f 92f

8 180 15 30 6.75f 90f

*

n

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Fig. 7. Ethanol quadratic response surface model.

Means in the same column not sharing a common superscript letter were sig-ificantly different (one way ANOVA; p < 0.05, n = 3).

espectively. XTP, XTF, XPF represent the interactions between pairsf variables. The regression coefficient is represented by r2 andMSE is the root mean square error.

The graph of actual values versus predicted values in Fig. 6 showshe actual and predicted data dispersion around the bisector. Thequation line almost coincides with the bisector and it is thereforeompletely covered and not visible. The dispersion of the 8 experi-ents is linear and in good agreement with the model as confirmed

y the high regression coefficient (0.9791) and by the low root meanquare error (2.0000%).

As for total lignan and SDG contents, RSM methodology can alsoe applied to the change in ethanol content. The quadratic sur-ace of the model is presented in Fig. 7. The maximum decrease inthanol was obtained in experiment 7 (30 Mpa, 5 kg h−1, 180 min),n agreement with the results reported in Table 4.

The response surfaces for pairs of variables reported in Fig. 8how a different behavior for the decreasing ethanol content atifferent times of treatment.

The reduction of ethanol after 60 min of treatment (experiments–4) was greatest at a flow rate of 15 kg h−1, the same as wasbserved for lignan enrichment. However, under the condition of80 min of treatment (experiments 5–8) ethanol reduction was

−1

reatest at a flow rate of 5 kg h . The comparisons of the differ-nt ethanol reductions obtained with the use of similar CO2 massexperiments 2 vs. 5 and 4 vs. 7) were conducted. These compar-sons show a statistically significant ethanol reduction in the RF

50 60 70 80 90 10050

55

60

65

70

75

80

85

90

95

100

exp1

exp2

exp3

exp4

exp5

exp6

exp7

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Dec rea sing % (Ethanol) Experimental data

Dec

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ing %

(E

than

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Pre

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ig. 6. Regression model of actual values of experiments (black line) and predictedalues (gray line) for ethanol.

Fig. 8. Response surface models for pressure and CO2 flow rate at 60 min (a) and180 min (b) of treatment.

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igher for the tests with lower CO2 surface velocity, respectivelyhe experiments 5 and 7. This behavior is probably due to the dif-erent transport coefficients of the matter, which reflect upon theifferent balances between CO2, ethanol and water that are estab-

ished within the column in a treatment time of 180 min comparedith a treatment time of 60 min.

Alternative concentration methods to remove ethanol from thextract could be vacuum separation, distillation and membraneechniques (reverse osmosis and perevaporation), but unfortu-ately, in this case, some components could be denatured oremoved by these processes [21].

. Conclusions

SAF presents a useful technique for the concentration of lignansrom flaxseed.

The regression models and response surface models revealedhat SAF produced the greatest lignan content under the maximumevels of the variables studied (treatment time 180 min, pressure0 MPa and CO2 flow rate 15 kg h−1). Furthermore, pressure playshe greatest role, confirming that as the pressure increases, theolvent power of SC-CO2 increases too.

After the verification of the nearly complete elimination of theesidual ethanol, the characterization of the composition of the RFt the end of the process, and after the study of suitable supple-entation formulas, the purified lignans could be scaled up to

ncrease their production and could be proposed for commercialse as ingredients in functional foods.

Finally, the verification of the antioxidant activity in the RF andhe study of the residual panels as useful by-products of the flaxseedndustry can add further value to the proposed process and product.

cknowledgments

The authors thank the Italian Ministry of Agricultural Food,orestry and Fishery Policies, for their support through the grantCERSUOM”, and Dr. Valeria Sileoni for her collaboration in per-orming the statistical analyses.

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[

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