Recovery and concentration of antioxidants from winery wastes

Molecules 2012, 17, 3008-3024; doi:10.3390/molecules17033008 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Recovery and Concentration of Antioxidants from Winery Wastes María Luisa Soto 1, Enma Conde 1,2, Noelia González-López 1,2, María Jesús Conde 1, Andrés Moure 1,2, Jorge Sineiro 3, Elena Falqué 4, Herminia Domínguez 1,2,*, María José Núñez 3 and Juan Carlos Parajó 1,2 1 Departamento de Enxeñaría Química, Universidade de Vigo (Campus Ourense), Edificio Politécnico, As Lagoas, Ourense 32004, Spain 2 CITI, Tecnópole, San Cibrao das Viñas, Ourense 32901, Spain 3 Escola Técnica Superior de Enxeñaría, Universidade de Santiago de Compostela, Avda. Lope Gómez de Marzoa sn, Santiago de Compostela 15782, Spain 4 Departamento de Química Analítica, Universidade de Vigo (Campus Ourense), Edificio Politécnico, As Lagoas, Ourense 32004, Spain * Author to whom correspondence should be addressed; E-Mail: herm[email protected]; Tel.: +34-988-387-082; Fax: +34-988-387-001. Received: 21 December 2011; in revised form: 10 February 2012 / Accepted: 27 February 2012 / Published: 9 March 2012 Abstract: Grape and wine byproducts have been extensively studied for the recovery of phenolic compounds with antioxidant activity and a variety of biological actions. The selective recovery and concentration of the phenolic compounds from the liquid phase separated from further diluted winery wastes has been proposed. Adsorption onto non ionic polymeric resins and further desorption with ethanolic solutions was studied. Several commercial food grade resins were screened with the aim of selecting the most suited for the practical recovery of phenolic compounds with radical scavenging activity. Under the optimized desorption conditions (using Sepabeads SP207 or Diaion HP20 as adsorbents and eluting with 96% ethanol at 50 °C) a powdered yellow-light brown product with 50% phenolic content, expressed as gallic acid equivalents, was obtained. The radical scavenging capacity of one gram of product was equivalent to 2–3 g of Trolox. Keywords: winery wastes; phenolics; adsorption; resins; radical scavengers OPEN ACCESS Molecules 2012, 17 3009 1. Introduction Winemaking is a seasonal activity of environmental and economic relevance in the producing countries. In some industries the final residue is the grape pomace generated in the pressing stage, but in the wine industries that produce spirits the wet distilled grape pomace is the final residue of the plant. The compounds from distilled pomace are more active than those obtained from the pressing pomace and are highly thermostable [1]. A simple alternative to recover antioxidants from distilled grape pomace, consists of the utilization of the liquid phase accompanying the pomace; this liquid presents a radical scavenging capacity comparable to synthetic antioxidants, but the phenolic purity is low (15%, dry basis) [2]. The phenolic compounds from the liquid phase accompanying the distilled grape pomace could be successfully adsorbed onto activated charcoal, but they could not be eluted [3]. However, the reversible adsorption of these grape phenolics onto resins was observed [4]. In that work, the liquid phase found in the distilled grape pomace was concentrated in nanofiltration membranes and further refined by adsorption onto polymeric resins and elution with ethanol. The direct adsorption onto commercial resins of the phenolic components from the winery wastes leaving the distillation stage has not been tried. Adsorption using nonpolar macroporous polymers presents a series of advantages, including the wide range of structures and properties available, high adsorption capacity and selectivity, good performance to recover and to separate bioactive compounds, chemical stability, relatively low cost and easy regeneration. Increasing applications of resins are found in scientific literature for the recovery and non-thermal concentration and fractionation of the crude phenolic extracts from products and byproducts of the food industry, i.e., citrus peel and molasses [5], apple and grape pomace [6–9] or for solvent extracts from autohydrolysis liquors of grape pomace [10]. Among the phenolic compounds identified in winery products are catechins (catechin, epicatechin), flavonols (quercetin, kaempferol, myricetin), benzoic acids (gallic, protocatechuic, 4-hydroxybenzoic, syringic, gentisic) and cinnamic acids (p-coumaric) [7]. These dietary phenolics present high antioxidant capacity, confer protection against cronic and degenerative diseases [11], are metabolizable [12] and stable at high temperatures [1]. Based on these properties, the concentrated phenolic product from wineries could be of interest for food, pharmaceutical and cosmetic applications. Such products could be proposed as agents protecting from oxidation during storage and as bioactive components in the formulation of functional foods. The aim of the present work was to select commercial food grade resins for the efficient adsorption of phenolic compounds with antioxidant activity present in winery wastes. Separation of solid and liquid phases from winery wastes and suitable dilution of the latter was accomplished before addressing the selective adsorption and desorption stages. Kinetic studies were carried out to compare the performance of the different resins and to establish the time required to reach equilibrium. For the resins selected on the basis of their adsorption capacity, the experimental desorption conditions were optimized to obtain concentrated extracts selectively enriched in the active components. Molecules 2012, 17 3010 2. Results and Discussion 2.1. Adsorption In order to select the most effective adsorbents a comparative batch adsorption experiment was performed. The total phenolics and the ABTS [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate)] radical scavengers adsorbed were measured by q, as mg of gallic acid equivalent (GAE)/g resin and as mg Trolox equivalents (TE)/g resin, respectively. The kinetic data corresponding to the time course of the adsorption of phenolics and ABTS radical scavengers onto the evaluated resins are shown in Figure 1a,b. Figure 1. Adsorption kinetics of (a) phenolic compounds (expressed as gallic acid equivalents, GAE) and (b) ABTS radical scavengers (expressed as Trolox equivalents, TE) from winery wastes. The symbols correspond to experimental data and the lines to the calculated trend according to the selected models. 0 0.5 1.0 1.5 2.0 2.5 3.0 0 0.1 0.2 0.3 0.4 0.5 0.6 1.0 1.5 2.0 2.5 3.0 3.5 q(mg GAE/g resin) Time (h) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 0 0.5 1.0 1.5 2.0 2.5 3.0 0 0.1 0.2 0.3 0.4 0.5 0.6 1.0 1.5 2.0 2.5 3.0 3.5 q(mg GAE/g resin) Time (h) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 a 0 0.5 1.0 1.5 2.0 2.5 3.0 0 0.1 0.2 0.3 0.4 0.5 0.6 1.0 1.5 2.0 2.5 3.0 3.5 q(mg GAE/g resin) Time (h) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 0 0.5 1.0 1.5 2.0 2.5 3.0 0 0.1 0.2 0.3 0.4 0.5 0.6 1.0 1.5 2.0 2.5 3.0 3.5 q(mg GAE/g resin) Time (h) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 a 0 2 4 6 8 10 024681012 14 16 18 20 Time (min) q (mg TE/g resin) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 0 2 4 6 8 10 024681012 14 16 18 20 Time (min) q (mg TE/g resin) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 b 0 2 4 6 8 10 024681012 14 16 18 20 Time (min) q (mg TE/g resin) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 0 2 4 6 8 10 024681012 14 16 18 20 Time (min) q (mg TE/g resin) Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 Amberlite XAD16HP Amberlite XAD1180 Amberlite XAD2 Amberlite XAD4 Amberlite XAD761 Amberlite XAD7HP Diaion HP2MG Diaion HP20 Sepabeads SP70 Sepabeads SP207 Sepabeads SP825 Sepabeads SP850 Sepabeads SP700 b The adsorption yields ranged from 67 to 90%, with increasing values for Diaion HP2MG (68.7) and HP20 (75.2), Amberlite XAD2 (69.2), XAD7HP (70.4), XAD1180 (75.3), XAD761 (79.2), XAD16HP (80.2), XAD4 (81.8), and Sepabeads SP70 (83.6), SP850 (84.1), SP825 (85.6), SP207 (88.1) and SP700 (89.5). The time to reach the steady state was relatively short, and for further experiments was fixed at 0.5 h; Amberlite XAD2 and XAD4 required 3 h. These values were shorter than those needed to retain phenolics from winery wastes onto activated carbon [3]. The adsorption of ABTS radical scavengers was accomplished in shorted times than the adsorption of phenolics. In 2 min 91% of the Molecules 2012, 17 3011 active compounds were adsorbed onto SP207, SP70, SP825 and SP850, between 64 and 78% onto HP20, HP2MG, XAD 7HP, XAD 16HP, XAD761 and XAD1180, whereas 20 min were required to achieve these yields onto XAD2. Operating with XAD4 a different behaviour was observed, reaching 75% adsorption yield after 20 min. The experimental data in Figure 1 were fitted to the pseudo first order model and the pseudo second order model. For all the tested resins, the R2 values (0.8544–0.9855) observed with pseudo first order models were lower than those (0.9929–0.9999) found for the pseudo second order model. According to these values, summarized in Table 1, the sorption kinetics followed a pseudo second order model, also reported for other phenolics and resins [10,13,14]. Table 1. Regression coefficients for pseudo first and pseudo second order models for the adsorption of (a) total phenolics (as mg gallic acid equivalents/g resin) and (b) ABTS radical scavenging compounds (as mg Trolox equivalents/g resin) present in winery wastes. a Pseudo First Order Model Pseudo Second Order Model k1 (min−1) qe calc (mg/g) R2 k 2 (g/mg·min) qe calc (mg/g) R2 Amberlite XAD2 2.74 0.80 0.8544 0.025 1.98 0.9998 XAD4 2.25 1.54 0.9376 0.011 2.18 0.9929 XAD7HP 22.5 0.49 0.9716 0.190 1.98 0.9998 XAD16HP 29.0 0.89 0.9826 0.118 2.33 0.9997 XAD761 17.9 0.70 0.9612 0.103 2.29 0.9997 XAD1180 22.8 0.47 0.9597 0.197 2.18 0.9999 Diaion HP20 24.5 0.56 0.9614 0.170 2.19 0.9999 HP2MG 26.8 0.38 0.9716 0.281 1.93 0.9999 Sepabeads SP70 16.2 0.52 0.8779 0.139 2.43 0.9998 SP207 22.3 0.65 0.9855 0.139 2.57 0.9998 SP700 26.1 0.46 0.9681 0.240 2.60 0.9999 SP825 28.6 0.55 0.9558 0.220 2.39 0.9999 SP850 20.1 0.72 0.9630 0.112 2.45 0.9998 b Pseudo First Order Model Pseudo Second Order Model k1 (min−1) qe calc (mg/g) R2 k2 (g/mg·min) qe calc (mg/g) R2 Amberlite XAD2 5.54 3.81 0.9475 9.59 7.91 0.9998 XAD4 9.11 7.97 0.9916 2.42 8.51 0.9983 XAD7HP 30.3 1.49 0.9169 105 6.90 0.9996 XAD16HP 21.2 2.77 0.9478 29.3 8.26 0.9997 XAD761 8.63 1.41 0.5970 40.1 7.90 0.9992 XAD1180 13.5 1.80 0.9828 38.7 8.04 0.9996 Diaion HP20 15.7 1.33 0.7960 50.9 8.09 0.9999 HP2MG 15.7 0.80 0.7926 105 6.90 0.9999 Sepabeads SP70 17.1 1.23 0.6610 62.5 8.94 0.9999 SP207 43.8 2.65 0.9722 61.1 9.05 0.9999 SP700 36.0 1.84 0.9579 60.9 9.06 0.9999 SP825 28.4 1.32 0.9453 125 8.92 0.9999 SP850 19.4 1.80 0.8165 40.6 9.06 0.9999 Molecules 2012, 17 3012 2.2. Desorption The efficient desorption of the adsorbed compounds using ethanolic solutions was proposed. Despite the fact that methanol could provide higher elution yields than ethanol [6] a food grade solvent was preferred. The process was optimized by means of a central composite design of experiments, since a systematic variation of parameters has been recommended for the recovery of purified plant extracts enriched in certain target compounds [7]. The matrix with the real values of the independent variables temperature and ethanol concentration, and the correspondent coded variables (T, E) is shown in Table 2. The experimental values for the objective functions obtained for the resins selected on the basis of their potential for selectively retaining the target compounds present in the diluted stream from winery wastes are shown in Tables 2–4. The maximal phenolics desorption yields were 67, 62 and 51%, respectively, for HP20, SP207 and XAD16HP, whereas the sugar desorption yields were 22, 31 and 19%, respectively. As a general trend, desorption of phenolic compounds is favoured by higher ethanol concentrations than desorption of sugars. The effect of temperature on the desorption yields depends on the resin and does not present a definite trend. Similar recovery yields have been reported during the alkaline elution of resins used for the simultaneous removal of phenolic compounds and polar anions from citrus peel juice and molasses [5] and for ethanol elution of phenolics from apple juice [15]. The range of phenolic content of the desorbed products was 44–51% for HP20, 35–51% for SP207 and between 30–47% for XAD16 HP. The range of sugar content of the desorbed extracts was 24–29%, for HP20, 22–31%, for SP207 and 16–26% for XAD16HP. The highest phenolic purity for HP20 was obtained for experiment 7, operating at 25 °C and eluted with 88% ethanol. An expected correlation between the phenolic content of the desorbed extracts and the radical scavenging capacity was observed. The ABTS radical scavenging capacity of the desorbed product ranged from 8.9 to 13.0 mM Trolox for HP20, from 11.2 to 13.3 mM Trolox for SP207 and from 7.6 to 14.0 mM Trolox for XAD16HP. In order to compare the efficieny of the desorbed product, these values can be referred to the activity of one gram of the dry desorbed product. These activities ranged from 1.94 to 2.63 grams of Trolox per gram of desorbed product for HP20, from 1.97 to 2.66 g/g for SP207 and from 1.5 to 3.1 g/g for XAD16HP. The ABTS radical scavenging capacity of one gram of BHT (butylated hydroxytoluene) was equivalent to 1.80 g Trolox, one gram of BHA (butylated hydroxyanisole) was comparable to 2.06 g Trolox and gallic acid to 4.93 g Trolox [10]. The effects of the independent variables were evaluated by a Student t-test and the significance of the models by F-test. The coefficients for the linear, quadratic and interaction effects of T and E on the objective functions are shown in Table 5 for the selected resins. Molecules 2012, 17 3013 Table 2. Coded and real variables of a central composite design for two factors and experimental and calculated values of the objective functions during operation with Diaion HP20. Coded Variables Real Variables Objective Functions Exp. T E T (°C) EtOH (%) Y1ex p Y 1calc Y2ex p Y 2calc Y3ex p Y 3calc Y4ex p Y 4calc Y5ex p Y 5calc 1 −1 −1 25 48 62.0 59.6 20.9 21.3 0.444 0.439 0.242 0.255 11.4 11.1 2 1 −1 45 48 63.2 62.2 21.6 22.1 0.475 0.467 0.263 0.270 8.90 9.60 3 −1.4142 0 20.858 68 62.2 64.2 22.3 22.3 0.475 0.479 0.276 0.269 10.3 11.2 4 1.4142 0 49.142 68 63.8 63.8 23.5 23.3 0.469 0.476 0.279 0.281 11.3 10.8 5 0 −1.4142 35 40 56.7 58.7 21.6 20.9 0.434 0.441 0.268 0.255 10.7 10.3 6 0 1.4142 35 96 63. 6 63.6 21.7 22.1 0.497 0.500 0.275 0.281 12.4 13.1 7 −1 1 25 88 66.9 65.9 22.4 22.2 0.514 0.511 0.279 0.279 13.0 11.9 8 1 1 45 88 62.3 62.7 23.0 22.8 0.483 0.478 0.289 0.282 12.8 12.8 9 0 0 35 68 64.1 64.5 21.8 22.2 0.475 0.469 0.262 0.261 10.9 11.1 10 0 0 35 68 64.5 64.5 22.2 22.2 0.474 0.469 0.264 0.261 11.6 11.1 11 0 0 35 68 64.5 64.5 22.6 22.2 0.457 0.469 0.260 0.261 11.1 11.1 12 0 0 35 68 64.6 64.5 22.2 22.2 0.460 0.469 0.256 0.261 10.9 11.1 13 0 0 35 68 64.6 64.5 22.1 22.2 0.479 0.469 0.265 0.261 11.0 11.1 Y1: Phenolic desorption yield (%); Y2: Sugars desorption yield (%); Y3: Total phenolic content (g GAE/g extract); Y4: Total sugar content (g D-glucose/g extract); Y5: Radical scavenging activity (mM Trolox). Molecules 2012, 17 3014 Table 3. Experimental and calculated values of the objective functions during desorption from Sepabeads SP207 resin. Exp. Y1ex p Y 1calc Y 2ex p Y 2calc Y 3ex p Y 3calc Y 4ex p Y 4calc Y 5ex p Y 5calc 1 52.7 51.7 23.1 26.1 0.471 0.438 0.291 0.301 12.2 12.2 2 58.7 56.8 25.1 26. 8 0.454 0.423 0.272 0.275 11.7 11.2 3 55.9 56.3 24.6 22.6 0.475 0.475 0.284 0.265 12.6 12.5 4 59.5 61.2 24.6 24.5 0.488 0.482 0.268 0.260 11.4 12.0 5 49.8 51.4 31.2 28.4 0.349 0.396 0.308 0.304 11.2 11.4 6 58.3 58.9 23.2 23.9 0.511 0.458 0.285 0.262 12.4 12.7 7 58.9 58.7 21.7 22.2 0.425 0.462 0.225 0.249 12.5 12.4 8 61.6 60.5 25.1 24.2 0.446 0.487 0.251 0.267 13.3 12.7 9 59.5 57.6 25.8 24.8 0.400 0.400 0.244 0.240 11.6 11.7 10 58.3 57.6 24.4 24.8 0.423 0.400 0.254 0.240 11.2 11.7 11 58.2 57.6 24.6 24.8 0.401 0.400 0.234 0.240 11.8 11.7 12 55.9 57.6 25.2 24.8 0.372 0.400 0.236 0.240 12.0 11.7 13 56.1 57.6 23.9 24.8 0.405 0.400 0.233 0.240 11.9 11.7 Y1: Phenolic desorption yield (%); Y2: Sugars desorption yield (%); Y3: Total phenolic content (g GAE/g extract); Y4: Total sugar content (g D-glucose/g extract); Y5: Radical scavenging activity (mM Trolox). Table 4. Experimental and calculated values of the objective functions during desorption from Amberlite XAD16HP resin. Exp. Y1ex p Y 1calc Y 2ex p Y 2calc Y 3ex p Y 3calc Y 4ex p Y 4calc Y 5ex p Y 5calc 1 41.7 42.1 16.6 16.6 0.350 0.387 0.220 0.245 7.98 7.65 2 46.1 46.0 16.5 17.2 0.307 0.328 0.176 0.199 8.87 8.87 3 49.3 47.8 18.5 18.1 0.413 0.392 0.249 0.238 8.43 8.70 4 50.5 49.8 19.1 18.1 0.397 0.399 0.241 0.235 9.22 9.02 5 39.5 39.8 16.1 15.6 0.356 0.319 0.233 0.202 7.63 7.85 6 45.1 42.7 15.5 14.7 0.297 0.315 0.165 0.177 8.92 8.77 7 44.3 46.6 16.0 16.6 0.323 0.321 0.188 0.183 9.36 9.29 8 43.7 45.5 15.1 16.1 0.408 0.390 0.234 0.225 8.25 8.52 9 51.5 50.4 18.4 17.8 0.410 0.437 0.235 0.247 13.3 13.3 10 50.9 50.4 17.8 17.8 0.419 0.437 0.235 0.247 14.0 13.3 11 49.0 50.4 16.5 17.8 0.469 0.437 0.254 0.247 12.9 13.3 12 50.5 50.4 18.8 17.8 0.432 0.437 0.259 0.247 13.1 13.3 13 50.0 50.4 17.5 17.8 0.452 0.437 0.235 0.247 13.4 13.3 Y1: Phenolic desorption yield (%); Y2: Sugars desorption yield (%); Y3: Total phenolic content (g GAE/g extract); Y4: Total sugar content (g D-glucose/g extract); Y5: Radical scavenging activity (mM Trolox). Molecules 2012, 17 3015 Table 5. Regression coefficients and statistical parameters for the objective functions. Y1: Phenolic Desorption Yield (%) Y2: Sugars Desorption Yield (%) Y3: Total phenolic Content (g GAE/g extract) Y4: Total Sugar Content (g D-glucose/g extract) Y5: Radical Scavenging Activity (mM Trolox) coefficient probability coefficient probability coefficient probability coefficient probability coefficient probability Diaion HP20 a0 64.5 3.80 × 10−12 22.2 1.64 × 10−12 0.469 1.32 × 10−12 0.261 5.22 × 10−11 11.1 5.14 × 10−9 aT −0.142 0.799 0.362 0.063 −0.001 0.751 0.004 0.212 −0.158 0.561 aE 1.70 0.016 0.393 0.048 0.021 0.000 0.009 0.023 0.989 0.007 aTE −1.42 0.103 −0.043 0.857 −0.015 0.015 −0.003 0.547 0.587 0.154 aTT −0.228 0.704 0.298 0.133 0.004 0.292 0.007 0.082 −0.057 0.844 aEE −1.66 0.023 −0.333 0.099 0.001 0.797 0.004 0.325 0.302 0.314 R2 0.758 0.721 0.878 0.688 0.728 Error 1.519 0.463 0.009 0.009 0.734 F 4.39 3.62 10.1 3.09 3.75 Sepabeads SP207 a0 57.6 2.19 × 10−11 24.8 1.86 × 10−8 0.400 9.74 × 10−8 0.240 1.14 × 10−8 11.7 1.03 × 10−10 aT 1.72 0.027 0.672 0.367 0.002 0.864 −0.002 0.767 −0.184 0.277 aE 2.65 0.004 −1.60 0.056 0.022 0.169 −0.015 0.051 0.447 0.024 aTE −0.819 0.379 0.348 0.735 0.009 0.647 0.011 0.248 0.347 0.161 aTT 0.576 0.413 −0.621 0.433 0.039 0.039 0.011 0.143 0.266 0.157 aEE −1.25 0.101 0.673 0.397 0.013 0.420 0.022 0.015 0.166 0.355 R2 0.821 0.535 0.572 0.730 0.684 Error 1.74 1.97 0.0405 0.0178 0.442 F 6.41 1.61 1.87 3.79 3.03 Amberlite XAD16HP a0 50.4 4.94 × 10−11 17.8 1.47 × 10−9 0.437 8.12 × 10−9 0.247 2.70 × 10−8 13.3 1.92 × 10−11 aT 0.694 0.290 0.021 0.953 0.002 0.837 −0.001 0.879 0.114 0.443 aE 1.02 0.137 −0.289 0.433 −0.001 0.919 −0.009 0.266 0.323 0.0545 aTE −1.27 0.182 −0.283 0.583 0.032 0.075 0.022 0.069 −0.500 0.039 aTT −0.787 0.265 0.173 0.656 −0.020 0.125 −0.006 0.510 −2.24 1.45 10−6 aEE −4.58 0.0002 −1.32 0.009 −0.059 0.001 −0.029 0.008 −2.52 6.62 10−7 R2 0.889 0.672 0.819 0.736 0.985 Error 1.71 0.983 0.031 0.021 0.396 F 11.2 2.87 6.35 3.90 91.6 Molecules 2012, 17 3016 The quadratic effect of the ethanol concentration in the eluting solvent was only significant on desorption yields whereas the linear effect of this variable was significant on all objective functions during operation with HP20. With SP207, the linear effect of the ethanol concentration was significant on all functions, except on the phenolic content of the product. Operating with XAD16HP the quadratic effect of the ethanol concentration was the most significant, followed by the interaction effect between temperature and ethanol, particularly on the phenolic and sugar content of the extracts and on the radical scavenging capacity. A comparison between the experimental and calculated objective functions for randomly selected operational conditions confirmed the good prediction ability of the models for all resins (Table 6). The response surface models and the contour plots defined with the variables significant at 90% level are shown in Figure 2 for the studied objective functions. Table 6. Comparison between experimental and calculated values of the objective functions at the optimal conditions. Real Variables Coded Variables Objective Functions Resin T (°C) EtOH (%) T E Y 1exp Y1calc Y2exp Y2calc Y3exp Y2calc Y 4exp Y 2calc Y 5exp Y5calc HP20 50 96 1.5 1.4142 56.8 59.8 22.9 23.2 0.473 0.475 0.266 0.297 13.3 14.0 SP207 50 96 1.5 1.4142 60.0 61.0 21.6 24.2 0.521 0.570 0.291 0.318 12.7 13.7 XAD16HP 45 96 1 1.4142 39.3 40.8 16.3 14.5 0.272 0.343 0.163 0.202 8.39 5.93 Y1: Phenolic desorption yield (%); Y2: Sugars desorption yield (%); Y3: Total phenolic content (g GAE/g extract); Y4: Total sugar content (g D-glucose/g extract); Y5: Radical scavenging activity (mM Trolox). Figure 2. 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