3.1. Materials
Winery wastes. Winery wastes from Cooperativa Vitivinícola do Ribeiro (Ribadavia, Ourense, Spain), years 2007–2008, were collected and processed to separate the solid and liquid fractions. The obtained liquid phase was centrifuged to remove suspended solids, diluted with tap water and stored at 4 °C until use. The total phenolic and sugar content in the diluted liquid phase were 1.8 g (expressed as gallic acid equivalents)/L and 6.0 g/L, respectively.
Resins. The food grade resins used were an acrylic polymer, Amberlite XAD7HP, two resins with a formaldehyde-phenol polycondensed matrix, Amberlite XAD761 and Amberlite XAD1180, and three PS-DVB resins, Amberlite XAD2, Amberlite XAD4 and Amberlite XAD16, supplied by Sigma Chemical Corporation. PS-DVB copolymers with different hydrophobicity, Sepabeads SP700, Sepabeads SP207, Sepabeads SP825, Sepabeads SP850 and Diaion HP20, a resin with a polymethacrylate estructure, Diaion HP2MG and a chemically modified PS-DVB polymer, Sepabeads SP70, were kindly supplied by Resindion S.R.L. (Mitsubishi Chemical Corporation). The physicochemical characteristics of these resins are summarized in
Table 8.
Table 8.
Physicochemical characteristics of the commercial resins used for the recovery of phenolic compounds from winery wastes.
Table 8.
Physicochemical characteristics of the commercial resins used for the recovery of phenolic compounds from winery wastes.
Resin Name | Structure | Surface Area (m2/g) | Pore Radius (Å) | Porosity (mL/g) | Particle Size (mm) | Density (g/mL) |
---|
Amberlite | | | | | | |
XAD2 | PS-DVB | 330 | 90 | 0.65 | 0.25–0.84 | 1.02 |
XAD4 | PS-DVB | 725 | 40 | 0.98 | 0.25–0.84 | 1.02 |
XAD7HP | Acrylic ester | 450 | 90 | 1.14 | 0.25–0.84 | 1.05 |
XAD16 | PS-DVB | 800 | 100 | 1.82 | 0.25–0.84 | 1.02 |
XAD761 | Phenol-formaldehyde | 300 | 600 | 0.43 | 0.56–0.76 | 1.11 |
XAD1180 | Phenol-formaldehyde | 600 | 300 | 1.68 | 0.35–0.60 | 1.01 |
Diaion | | | | | | |
HP20 | PS-DVB | 600 | 260 | 1.3 | 0.25–0.60 | 1.01 |
HP2MG | Polymethacrylate | 470 | 170 | 1.2 | 0.25–0.60 | 1.09 |
Sepabeads | | | | | | |
SP70 | Chemically modified PS-DVB (Br-PS-DVB) | 800 | 70 | 1.6 | 0.25–0.85 | 1.01 |
SP207 | PS-DVB | 630 | 120 | 1.1 | 0.25–0.60 | 1.18 |
SP700 | PS-DVB | 1200 | 90 | 2.3 | 0.25–0.70 | 1.01 |
SP825 | PS-DVB | 1000 | 57 | 1.4 | 0.30–0.50 | 1.01 |
SP850 | PS-DVB | 1000 | 38 | 1.2 | 0.30–0.80 | 1.01 |
The resins were activated by contact with sufficient methanol to cover the resin bed by 2.5–5 cm (Sigma-Aldrich, Madrid, Spain). Resins and methanol were blended gently by shaking one minute and then the suspension was stirred at 175 rpm and 25 °C during 15 min. Before use resins were rinsed with deionized water at a liquid to solid ratio of 5 (g/g). The moisture content of the resins was determined by drying the beads in an oven at 100 °C up to constant weight, and adsorption experiments were carried out utilizing known amounts of resins.
Absorption. The centrifuged winery liquid wastes were contacted in batch mode with weighed quantities of hydrated resins in sealed Erlenmeyer flasks at 25 °C in an orbital shaker at 175 rpm. The concentration of phenolics adsorbed at time t onto a mass unit of resin (qt, mg/g) was measured as gallic acid equivalents and calculated by the equation:
where C0 and Ct are the concentrations of phenol in the aqueous solution (mg/L) at the initial stage and at t time, respectively, V is the volume of the solution added into the flask (L), and W is the weight of the wet resin (g). Experiments were performed in triplicate.
The kinetic assays were performed in 25 mL Erlenmeyer flasks, with 5 mL of the liquid phase separated from winery wastes and further diluted and 3 g of resins at an initial pH 4.0, at 25 °C for up to 3 h. The content of each flask was filtered trough a 0.45 μm membrane filter and the liquid phase was analyzed. The pseudo-first-order rate equation of Lagergren is generally described by equation (2), and assumes that the rate of solute uptake is proportional to the gradient in saturation concentration.
where qt and qe are the amount of phenol adsorbed (mg/g) at contact time t (min) and at equilibrium, k1 is the pseudo-first-order rate constant (min−1). Integration and linearization leads to:
The pseudo-second order kinetic model is represented by:
where t is the contact time, qt and qe are the concentrations of phenolics adsorbed (expressed as mg/g) at the considered time and at the equilibrium respectively, k2 is the pseudo-second order kinetic parameter. Integration and linearization results in:
Washing. Distilled water was used to remove unadsorbed compounds susceptible of reducing purity of the extracts desorbed in further stages. Washing was performed in two stages with distilled water at a water:resin ratio of 3 (g:g) in an orbital shaker (175 rpm) at 25 °C for 20 min.
Desorption. Ethanol/water mixtures were selected on the basis of availability, suitability for food uses, and the reported cleanup capability. Optimization of the desorption conditions to produce a purified antioxidant extract was addressed by applying a central composite factorial design. The independent variables were temperature (°C), and the ethanol content (%, v/v), coded as:
The dependent variables or objective functions were expressed according to the general expression of a second order polynomial equation (equation 8). The phenolic desorption yield (Y1, %), the sugars desorption yield (Y2, %), the total phenolic content (Y3, g GAE/g extract), the total sugar content (Y4, g D-glucose/g extract) and the radical scavenging activity (Y5, mM Trolox) were expressed as a function of linear, interaction, and second-order terms involving the normalized, dimensionless variables T and E:
where Yi(i = 1–5) are the dependent variables, T and E are the dimensionless, normalized, independent variables, and a0, aT, aE, aTE, aTT and aEE are regression coefficientes calculated from experimental data.
During elution the resin, saturated at conditions previously selected, was contacted with the ethanol:water solution at a moist resin to ethanol ratio 1:3 (g:mL) (resin moisture is approximately 65%). The desorbed extract was analyzed for phenolic and sugar content and for radical scavenging activity. The resin regeneration procedure consisted on leaving the resin overnight in 1 M NaOH and further washing with deionized water.
3.2. Analytical Methods
The total phenolic content was determined by the Folin-Ciocalteu assay [
20], and expressed as Gallic Acid Equivalents (GAE). The total sugar content was determined by the Antrone method [
21], and expressed as Glucose Equivalents.
The phenolic compounds were analysed in an Agilent HPLC 1100 equipped with a Waters Spherisorb ODS-2 column (5 μm, 250 mm × 4.6 mm) and DAD detector, operating at 30 °C with a flow rate of 1 mL/min. Gradient elution using solvent A (acetonitrile/water/formic acid, 10:85:5) and solvent B (acetonitrile/water/formic acid, 90:5:5) was performed: 0 min, 100% A, 0% B; 40 min, 85% A, 15% B; 45 min, 0% A, 100% B; 60 min, 100% A, 0% B. Quantification was performed from calibration curves obtained with standard compounds diluted in methanol.
Samples were conditioned for gas chromatography-mass spectrometry analysis (GC-MS). About 0.25 mL of 3-octanol (10 ppm) was added as internal standard into 25 mL of a diluted extract solution (0.5 g extract/L). This mixture was extracted with dichloromethane (DCM). The organic phase was transferred to a graduate glass tube and concentrated under nitrogen. GC-MS analysis was carried out in splitless mode in a Hewlett-Packard 5890-II gas chromatograph coupled to a mass spectrometer HP-5970 using He as carrier gas. Separation was performed using a 60 m × 0.25 mm × 0.25 μm film thickness HP-Innowax capillary column. The temperature was maintained at 45 °C for 1 min, increased to 230 °C at 3 °C/min, and then held for 30 min. Mass spectrometer was in EI mode (electron energy 70 eV; source temperature 250 °C), and data acquisition was made in scanning mode from 30 to 300 amu/s and 1.9 spectra/s. Compounds were identified by comparison of the retention time and mass spectra with library data of mass spectra (Wiley 7n) and authentic compounds. Quantification was performed by the internal standard method (using 3-octanol as standard).
The degree of polymerization of the procyanidins was estimated by RP-HPLC analysis of the depolymerized fractions present in the reaction media after the thiolysis at 65 °C of the desorbed product diluted in methanol [
18]. RP-HPLC analysis were carried out in a Smart (Amersham-Pharmacia Biotech, Uppsala, Sweden) equipment fitted with a C18 Hypersil ODS column (Supelco). Elution was carried out at a flow rate of 1 mL/min of solvent A (0.1% aqueous TFA) and solvent B (0.082% TFA in water/CH
3CN (1:4)). The gradient, expressed as concentration of B varied as follows: 0–30 min from 12% to 30%, 30–40 min from 30% to 100%, 40–45 min from 100% to 12%.
The antioxidant activity was evaluated as the radical scavenging using the TEAC (Trolox Equivalent Antioxidant Capacity) assay. A 7 mM ABTS [2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate)] stock solution was reacted with 2.45 mM potassium persulfate and kept in the dark at room temperature for 12–16 h before use. The formed ABTS•+ solution was diluted with phosphate buffer saline (PBS) (pH 7.4) to an absorbance of 0.700 at 734 nm and equilibrated at 30 °C. One mL of diluted ABTS•+ solution was mixed with 10 μL of antioxidant compounds or Trolox standards in ethanol or PBS and the absorbance was read up to 6 min, using appropriate solvent blanks. The percentage inhibition of absorbance at 734 nm was calculated as a function of the concentration of extracts and Trolox.