Comparative Study of the Enological Potential of Different Winemaking

Apr 30, 2014 - Different white winemaking byproducts (pomace, skins, seeds, and stems) were compared as natural sources of phenolic compounds having ...
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Comparative Study of the Enological Potential of Different Winemaking Byproducts: Implications in the Antioxidant Activity and Color Expression of Red Wine Anthocyanins in a Model Solution M. José Jara-Palacios,† Belén Gordillo,† M. Lourdes González-Miret,† Dolores Hernanz,‡ M. Luisa Escudero-Gilete,† and Francisco J. Heredia*,† Laboratorio de Color y Calidad de Alimentos, Á rea de Nutrición y Bromatologı ́a, and ‡Departamento of Quı ́mica Analı ́tica, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain

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ABSTRACT: Different white winemaking byproducts (pomace, skins, seeds, and stems) were compared as natural sources of phenolic compounds having biological and sensory properties of enological interest. Antioxidant and copigmentation effects of these byproducts were studied in a wine-like model solution. RRLC-DAD was used to establish differences in the phenolic composition, and the ABTS method was used to compare the antioxidant activities. Spectrophotometric and colorimetric analyses were performed to assess the magnitude of copigmentation and the changes induced in the color expression of red wine anthocyanins. Antioxidant and copigmentation properties significantly varied depending on the type of byproduct, which was related to their qualitative and quantitative phenolic composition. Seeds and pomace showed the highest antioxidant potential, whereas skins and pomace led to the strongest and visually perceptible color effects on red wine anthocyanins by multiple copigmentation (darker, more saturated, and vivid bluish colors). Results open the possibility of technological applications for the wine industry based on reusing winemaking byproducts to improve the biological value and color characteristics of red wines. KEYWORDS: winemaking byproducts, phenolic compounds, antioxidant activity, multiple copigmentation, anthocyanin color

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INTRODUCTION Nowadays, an efficient management of byproducts derived from the elaboration and processing of agricultural products is a necessary requirement for a sustainable food industry.1 Focusing on the wine industry, research has consistently demonstrated the important environmental impact of the liquid and solids residues obtained from grape vinification such as wine lees, pomaces, stems, or wastewater sludge and the technical and economic difficulties in their elimination or transformation.2,3 Problems associated with the management of winemaking byproducts are related to their high organic loading, which makes difficult their biological degradation. On the other hand, as winemaking is a seasonal activity, an intensive accumulation of residues is generated during a short period every year (grape harvesting), especially in high production regions. Under these circumstances, the European Union is becoming exigent about the preservation of water, soil, and biodiversity and seriously promotes to wine producers regions looking for new initiatives that permit a more sustainable management and exploitation of their winemaking byproducts (Council Regulation (EC) no. 491/2009). Traditionally, winemaking byproducts have been sent to distilleries for obtaining ethanol or to be used as fertilizers or biomass. However, these activities are usually carried out by external companies representing economic costs for the wine industry.2 As a consequence, there is increasing interest in finding alternative solutions for the exploitation and valorization of those byproducts, which would involve economic, social, and environmental advantages4−7 Over the past decade, the chemical composition of winemaking byproducts has been extensively investigated, and they © 2014 American Chemical Society

were confirmed to represent low-cost sources of many bioactive compounds, being even richer than other types of agri-food wastes.8 Especially important in this respect are phenolic compounds, which have potential industrial applications (pharmaceutical, cosmetic, nutritional, or agricultural) due to their strong antioxidant, anti-inflammatory, antimicrobial, or biostimulant effects.9−12 These compounds have also interesting applications in the field of enology not only because they are responsible for the antioxidant properties of wines but also because they play a crucial role in organoleptic characteristics such as color, aroma, or taste.13 Particularly in red wines, it is wellknown that colorless phenolics are involved in the chemical stabilization of anthocyanin pigments by means of noncovalent interactions through intermolecular copigmentation reactions.14 Studies carried out in model solution and focused on the application of objective color measurements have demonstrated that copigmentation causes the stabilization of the colored forms of the anthocyanins and consequently enhances their color.15,16 Thus, copigmentation is considered a relevant interaction because obtaining wines with stable and attractive colors is a major focus for quality control purposes. Among grape components, colorless phenols including flavonoids and some phenolic acids appeared as good anthocyanin copigments, which are abundant compounds in winemaking byproducts. Moreover, these compounds can act as effective oxidation substrates, which partially avoid undesirable color changes due to browning/ Special Issue: International Workshop on Anthocyanins (IWA2013) Received: November 13, 2013 Accepted: April 30, 2014 Published: April 30, 2014 6975

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they were used as a crude mixture of colorless phenolics (copigments) of wine anthocyanins in copigmentation experiments. Total Phenolic Content. The spectrophotometric determination of the total phenolic content was performed with a Hewlett-Packard UV− vis HP8453 spectrophotometer (Palo Alto, CA, USA), using 10 mm path length glass cells and distilled water as reference. The total phenolic content of the samples was determined using the Folin−Ciocalteu method.20 Briefly, 0.25 mL of sample, 1.25 mL of Folin−Ciocalteu reagent, and 3.75 mL of a solution of sodium carbonate at 20% were mixed, and distilled water was added to make up a total volume of 25 mL. The solution was homogenized and left to stand for 120 min for the reaction to take place and stabilize. Absorbance was measured at 765 nm. Gallic acid was employed as a calibration standard, and results were expressed as gallic acid equivalents (mg GAE/L). Phenolic Composition Analysis. Rapid-resolution liquid chromatography (RRLC) was performed on an Agilent 1260 system equipped with a diode array detector. Samples were filtered through a 0.45 μm pore size membrane filter, and 30 μL of sample was injected in a C18 Poroshell 120 column (2.7 μm particle size, 5 cm × 4.6 mm; Agilent, Palo Alto, CA, USA) maintained at 25 °C. Water/formic acid (99:1, v/ v) as solvent A and acetonitrile as solvent B were used, setting the flow rate at 1.5 mL/min. The linear gradient elution was 0 min, 100% A; 5 min, 95% A and 5% B; 20 min, 50% A and 50% B; 22 min, 100% A; 25 min, 100% A. The wavelengths of detection were 280 nm (flavanols and benzoic acids), 320 nm (hydroxycinnamic acids and their tartaric esters), and 370 nm (flavonols). Phenolic compounds were identified by their retention time, UV−vis spectra, and mass spectra data in an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany) equipped with an ESI source and a triple-quadrupole ion trap mass analyzer, as described by Jara-Palacios et al.21 The identification of phenolic compounds was achieved by comparison of the retention times and mass spectra with those of the available pure standards and our data library. The external calibration method was used for quantification by comparing the areas with standards of gallic, protocatechuic, caffeic, and p-coumaric acids, catechin, epicatechin, procyanidin B1, quercetin, and kaempferol. Caftaric and coutaric acids were quantified using the calibration curves of caffeic and p-coumaric acids, respectively. Procyanidin dimers B2, B3, and B4, procyanidin B2−3-O-gallate, trimers, and tetramer were quantified with the calibration curve of procyanidin B1. Quercetin and isorhamnetin derivatives were quantified as quercetin and kaempferol drivatives as kaempferol. Total phenolic acids, total flavanols, total flavanol oligomers, and total flavonols were also calculated by the sum of individual phenolic acids, flavanols, flavanol oligomers, and flavonols identified, respectively. The samples were analyzed in triplicate and the results expressed as milligrams per liter. Antioxidant Activity. The antioxidant activity was measured in vitro on the basis of the ability to scavenge the ABTS•+ radical.22 The ABTS•+ radical was produced by the oxidation of 7 mM ABTS with potassium persulfate (2.45 mM) in water. The mixture was kept in the dark at room temperature for 16 h before using it, and then the ABTS•+ solution was diluted with phosphate-buffered saline (PBS) at pH 7.4 to give an absorbance of 0.70 ± 0.02 at 734 nm. Then, 50 μL of each sample was mixed with 2 mL of the ABTS•+ diluted solution and vortexed for 10 s, and the absorbance was measured at 734 nm after 4 min of reaction at 30 °C. Results were obtained by interpolating the absorbance of samples on a calibration curve obtained with Trolox (30−1000 μM). Three independent experiments in triplicate were performed for each sample, and the results were expressed as Trolox equivalent antioxidant activity (TEAC; μmol of Trolox equivalent (TE) with the same antioxidant activity of 1 L of sample). Copigmentation Experiments. Copigmentation experiments were carried out in a wine-like medium using a crude anthocyanin solution prepared from Syrah red grapes and different crude phenolic solutions (colorless copigments) obtained from white winemaking byproducts. The crude anthocyanin solution was obtained by macerating 1 g of homogeneous lyophilized powder of Syrah skins in 20 mL of wine-like medium (the same as previously described) for 12 h with occasional

oxidation. In fact, it has been recently reported that the addition of dehydrated waste grape skins during winemaking increased the concentration of some flavonoids in wines, preventing color loss during storage.17,18 Despite their potential use in enology, winemaking byproducts have received little attention in comparison to other wine additives (wood chips, enzymes, enological tannins, etc.), probably due to the difficulties related to the technical and legal concerns of its application. However, their application as natural wine additives could represent a sustainable alternative to maximize the exploitation of this valuable agricultural waste as well as to improve the quality of wines, making them more competitive. In this sense, further investigations are needed to advance the knowledge of the contribution of these agricultural byproducts to wine color and color stability. Therefore, the main objective of this study was to compare the potential of different white winemaking byproducts (pomace, seeds, skins, and stems) as natural sources of antioxidants and copigments, in a model solution. The information reported in this work could be useful for high production winemaking areas.

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MATERIALS AND METHODS

Standards and Reagents. Gallic acid, protocatechuic, caffeic, and caftaric acids, (+)-catechin (C), (−)-epicatechin (EC), quercetin, kaempferol, myricetin, sodium carbonate, potassium persulfate, and tartaric acid were purchased from Sigma-Aldrich (Madrid, Spain), and malvidin-3-glucoside was from Extrasynthese (Genay, France). Procyanidin dimer B1 standard was isolated in the laboratory by semipreparative HPLC.19 2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Fluka (Madrid, Spain), and HPLC grade acetonitrile was from Carlo Erba (Rodano, Italy). Folin reagent, ethanol, and formic acid were obtained from Panreac (Barcelona, Spain). Winemaking Byproducts and Sample Preparation. White winemaking byproducts from Zalema grapes (Vitis vinifera sp.) used in this study were pomace (PM), skins (SK), seeds (SD), and stems (ST). They were obtained from a winery located in Condado de Huelva Designation of Origin (southwestern Spain). The Zalema cultivar was selected because it is a high production variety rich in phenolic compounds that represents the main and more extensively white grape cultivated in the zone. Pomace is the main organic winemaking byproduct generated from grape vinification, which is constituted by a mixture of skins and pulp rests, seeds, and stems. Three kilograms of pomace was collected the day of harvest after Zalema grapes were pressed for winemaking. To be also individually used in the experiments, particles of stems, seeds, and skins were manually separated from the pomace. All winemaking byproducts were stored at −20 °C and further freeze-dried (lyophilizer Cryodos-80, Telstar Varian DS 102) until extracted. The moisture contents of byproducts were PM, 50%; ST, 40%; SD, 30%; and SK, 70% SK. The extraction of the nonanthocyanin phenolic compounds from each winemaking byproduct was carried out in a wine-like medium containing 5 g/L tartaric acid in 12% ethanol, buffered with 1 M NaOH to pH 3.6, and ionic strength adjusted to 0.2 M by the addition of NaCl. For this purpose, 2 g of the homogeneous lyophilized powder of samples (PM, SD, SK, and ST) was individually macerated in 15 mL of wine-like medium for 12 h at room temperature (18−20 °C), with occasional agitation and sonication. The supernatants were centrifuged (4190g, 10 min) to separate out the liquid fraction containing the phenolic compounds extracted, which was filtered through 0.45 μm MilliporeAP20 filters (Bedford, MA, USA). The crude phenolic solution obtained from each winemaking byproduct was analyzed for its phenolic composition (spectrophotometric and chromatographic analysis) and antioxidant activity. Also, 6976

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Table 1. Mean Values and Standard Deviations (n = 3) of the Phenolic Composition, Total Phenolic Content, and Antioxidant Activity for the Crude Phenolic Solution from Winemaking Byproductsa pomace

skins

seeds

stems

total phenolic contentb (mg/L) total phenolic acidsc (mg GAE/L) total flavanolsc total oligomersc total flavonolsc antioxidant activity (μmol TE/L)

1138.42 ± 0.19a 45.93 ± 0.43a 92.32 ± 1.23a 77.26 ± 0.37a 3.31 ± 0.00a 463.29 ± 41.78a

1049.01 ± 3.78b 28.16 ± 0.46b 50.79 ± 2.65b 40.91 ± 2.57b 5.61 ± 0.01b 297.53 ± 15.87b

2575.21 ± 5.59c 30.56 ± 0.55c 141.13 ± 0.76c 98.97 ± 0.53c 0.56 ± 0.01c 888.73 ± 21.78c

1000.56 ± 6.72b 31.34 ± 0.10d 50.54 ± 0.07b 37.89 ± 0.11b 1.46 ± 0.07d 305.57 ± 15.42b

benzoic acids gallic acid protocatechuic acid

36.13 ± 0.35a 0.98 ± 0.19a

22.33 ± 0.39b 0.41 ± 0.03b

30.18 ± 0.04c ndd

20.12 ± 0.07d 0.15 ± 0.01c

hydroxycinnamic acids caftaric acid caffeic acid cis-coutaric acid trans-coutaric acid

6.40 ± 0.01a 1.46 ± 0.01a 0.26 ± 0.02a 0.70 ± 0.01a

3.44 ± 0.06b 1.32 ± 0.01b 0.28 ± 0.01a 0.38 ± 0.02b

nd nd 0.15 ± 0.01b 0.23 ± 0.02c

8.67 ± 0.01c 1.92 ± 0.02c 0.17 ± 0.02b 0.31 ± 0.01d

flavanols (+)-catechin (C) (−)-epicatechin (EC) procyanidin B1 procyanidin B2 procyanidin B3 procyanidin B4 trimer C−C−EC trimer C1 tetramer procyanidin B2−3-O-gallate

7.86 ± 0.98a 7.20 ± 0.25a 25.03 ± 0.07a 4.94 ± 0.81a 5.22 ± 0.07a 8.05 ± 0.33a 4.72 ± 0.53a 4.77 ± 0.50a 5.80 ± 0.59a 18.73 ± 0.24a

6.32 ± 0.04b 3.55 ± 0.04b 17.37 ± 1.20b 4.08 ± 0.03a 3.27 ± 0.5b 4.52 ± 0.03b 2.72 ± 0.05b 1.66 ± 0.03b 3.52 ± 0.29b 3.78 ± 0.95b

22.03 ± 0.07c 20.10 ± 0.37c 20.51 ± 0.03c 7.08 ± 0.07b 4.68 ± 0.05a 11.17 ± 0.07c 5.43 ± 0.05c 10.80 ± 0.04c 9.08 ± 0.06c 30.22 ± 0.26c

8.90 ± 0.03d 3.74 ± 0.01d 17.43 ± 0.03d 1.46 ± 0.02c 2.52 ± 0.04b 4.16 ± 0.02d 5.50 ± 0.02c 0.98 ± 0.03d 1.33 ± 0.02d 4.52 ± 0.09d

flavonols quercetin 3-O-rutinoside quercetin 3-O-glucuronide quercetin 3-O-glucoside kaempferol hexoside kaempferol 3-O-glucoside isorhamnetin 3-O-glucoside quercetin kaempferol

0.17 ± 0.01a 0.38 ± 0.01a 2.07 ± 0.03a 0.20 ± 0.01a 0.18 ± 0.03a 0.15 ± 0.01a 0.16 ± 0.01a nd

0.19 ± 0.01a 0.57 ± 0.01b 3.48 ± 0.01b 0.27 ± 0.01a 0.65 ± 0.01b 0.19 ± 0.01a 0.26 ± 0.02b tr

0.17 ± 0.02a 0.15 ± 0.01c 0.24 ± 0.04c nd nd nd nd nd

0.21 ± 0.07a 0.20 ± 0.01d 0.86 ± 0.01d tre 0.19 ± 0.01a nd tr tr

a

Different letters in the same row indicate significant differences (p < 0.05) by Tukey test. bAs Folin−Ciocalteu method. cSum of individual phenolic acids, flavanols, oligomers, and flavonols identified, respectively. dNot detected. eTraces. The CIELAB parameters (L*, a*, b*, C*ab, and hab) were calculated from the absorption spectra by using the original software CromaLab,24 following the recommendations of the Commission International de L’Eclariage:25 the 10° Standard Observer and Standard Illuminant D65. Color difference (ΔE*ab) was determined by applying the CIE76 color difference formula. It was calculated as the Euclidean distance between two points in the three-dimensional CIELAB space defined by L*, a*, and b*: ΔE*ab = [(ΔL*)2 + (Δa*)2 + (Δb*)2 ]1/2. It is assumed that color differences (ΔE*ab) >3−5 units can be perceived by an average observer.26 Copigmentation Measurements. The spectrophotometric determination of the magnitude of the copigmentation was made by comparing the absorbance at 520 nm of the crude anthocyanin solution (A0) and the absorbance at 520 nm of the same solution containing different crude phenolic mixtures from white winemaking byproducts (Ac), at each concentration level and expressed as the percentage [(Ac − A0)/A0] × 100.27 The color variation due to copigmentation was also evaluated by tristimulus colorimetry according to the methodology described in Gordillo et al.,16 which offers an objective measurement of color because

agitation and sonication. Then, it was centrifuged (4190g, 10 min) and the supernatant filtered through 0.45 μm Millipore-AP20 filters (Bedford, MA, USA). The phenolic composition (anthocyanin pigments and other colorless monomeric phenols) of the crude anthocynin solution was analyzed by HPLC following the method described in Gordillo et al.23 Copigmented solutions were prepared by adding separately each crude phenolic solution from winemaking byproducts (PM, ST, SD, and SK) to the crude anthocyanin solution at seven levels (50, 100, 200, 300, 400, 500, and 600 mg/L). The final anthocyanin concentration was the same in all cases (200 mg/L). All of the solutions (2 mL) were prepared in triplicate and equilibrated to reach equilibrium for 2 h and stored closed in darkness at 25 °C, after which their absorption spectra were recorded. Colorimetric Measurement. The absorption spectra (380−770 nm) of all solutions were recorded at constant intervals (Δλ = 2 nm) with a Hewlett-Packard UV−vis HP8453 spectrophotometer (Palo Alto, CA, USA), using 2 mm path length glass cells and distilled water as a reference. 6977

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Figure 1. RRLC chromatograms recorded at 280, 320, and 370 nm of crude phenolic solution from seeds (blue), skins (red), stems (green), and pomace (magenta). Peaks: (a) 1, gallic acid; 2, protocatechuic acid; 3, procyanidin B1; 4, procyanidin B3; 5, catechin; 6, trimer C−C−EC; 7, tetramer; 8, procyanidin B4; 9, trimer C1; 10, procyanidin B2; 11, epicatechin; 12, procyanidin B2-O-gallate; (b) 1, caftaric acid; 2, cis-coutaric acid; 3, trans-coutaric acid; 4, caffeic acid; (c) 1, quercetin 3-O-rutinoside; 2, quercetin 3-O-glucuronide; 3, quercetin 3-O-glucoside; 4, quercetin pentose; 5, kaempferol hexoside; 6, kaempferol 3-O-glucoside; 7, isorhamnetin 3-O-glucoside; 8, quercetin; 9, kaempferol. it is based on the consideration of the whole visible spectrum and allows the real assessment of color to be obtained. Following this methodology, diverse color difference formulas were applied in the CIELAB color space by using the scalar (L*, a*, b*) and cylindrical (L*, C*ab, hab) CIELAB color coordinates of samples. This provides a better evaluation of the quantitative and qualitative color implications of the copigmentation and their incidence on visual perception. The new colorimetric variables were determined as follows. The total color of each sample was assessed as the CIELAB color difference (ΔE*ab) applied between its color (L*, a*, and b*) with respect to distilled water (L* = 100, a* = 0, b* = 0), as shown in eq 1. It represents a quantitative color attribute.

weight of the three color attributes that make up the total color difference. relative contribution (%) of lightness: %ΔL * (c − 0))2 ] × 100 = [(ΔLc − 0)2 /(ΔEab

relative contribution (%) of chroma: %ΔC * (c − 0))2 ] × 100 = [(ΔCc − 0)2 /(ΔEab

(4)

relative contribution (%) of hue: %Δh * (c − 0))2 ] × 100 = [(Δhc − 0)2 /(ΔEab

* = [(L* − 0)2 + (a* − 0)2 + (b* − 0)2 ]1/2 total color: ΔEab (1)

(5)

Δhc−0 being deduced as follows:

The total color difference induced by copigmentation was assessed as the CIELAB color difference (ΔE*ab) applied between the color of the crude anthocyanin solution (L*0, a*0, and b*0) and the color of the same solution copigmented with each crude phenolic mixture from white winemaking byproducts (L*c, a*c, and b*c), as shown in eq 2:

* (c − 0))2 − ((ΔLc − 0)2 + (ΔCc − 0))2 ]1/2 Δhc − 0 = [(ΔEab Statistical Analysis. All statistical analyses were performed using Statistica v.8.0 software.28 Univariate analysis of variance (Tukey test) was applied to establish differences for the phenolic composition, antioxidant activity, and copigmentation effects among the crude phenolic solutions from winemaking byproducts (GP, SD, SK, and ST). Moreover, correlations between the phenolic composition (main phenolic groups or individual phenolic compounds), and the antioxidant activity was studied by linear and multiple regressions. In all cases (differences or correlations), the statistically significant level was considered at p < 0.05.

* − 0) total color difference: ΔEab(c = [(Lc* − L0*)2 + (ac* − a0*)2 + (bc* − b0*)2 ]1/2

(3)

(2)

The relative contribution (%) of lightness, chroma, and hue to each total color difference induced by copigmentation was calculated by means of the color formulas shown in eqs 3, 4, and 5. They represent the 6978

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RESULTS AND DISCUSSION Phenolic Composition. A total of 24 phenolic compounds were identified and quantified in the crude phenolic solutions Table 2. Concentration and Distribution of Individual Phenolic Compounds Identified by HPLC in the Crude Anthocyanin Solution Prepared from Syrah Grape Skins

total anthocyaninsa total phenolic acidsa total flavanolsa anthocyanins delphinidin-3-glucoside cyanidin-3-glucoside petunidin-3-glucoside peonidin-3-glucoside malvidin-3-glucoside petunidin-3-acetyl-glucoside peonidin-3-acetyl-glucoside malvidin-3-acetyl-glucoside petunidin-3-p-coumaroylglucoside peonidin-3-p-coumaroyl -glucoside malvidin-3-p-coumaroyl -glucoside hydroxycinnamic acids caftaric acid cis-coutaric acid trans-coutaric acid flavanols flavonols myricetin-3-O-glucuronide myricetin-3-O-glucoside quercetin-3-O-glucuronide quercetin-3-O-glucoside laricitrin-3-O-glucoside kaempferol-3-O-glucoside isorhamnetin-3-O-glucoside syringetin-3-O-glucoside

concentration (mg/L ± SD, n = 3)

relative proportion (%)

222.65 ± 10.03 1.97 ± 0.02 7.63 ± 0.97

95.8 0.9 3.3

3.12 ± 0.09 3.12 ± 0.14 12.42 ± 1.25 11.79 ± 1.62 98.32 ± 2.49 5.35 ± 0.71 8.09 ± 1.13 39.47 ± 1.90 2.9 ± 0.21

4.2 1.3 5.3 5.1 42.3 2.3 3.6 16.9 1.3

6.34 ± 0.36

2.7

25.13 ± 2.1

10.8

0.25 ± 0.01 0.76 ± 0.01 0.93 ± 2.1

0.1 0.4 0.4

Figure 2. (a) Magnitude of copigmentation and (b) total color of the crude anthocyanin solution containing increasing concentrations of crude phenolic solutions from Zalema winemaking byproducts (SK, skins, PM, pomace; ST, stems; SD, seeds); mean ± SD, n = 3. Different letters in the same byproduct indicate significant differences (p < 0.05).

tr

0.96 ± 0.01 0.89 ± 0.01 0.97 ± 0.03 2.95 ± 0.02 0.18 ± 0.01 0.14 ± 0.01 0.92 ± 0.01 0.59 ± 0.01

0.4 0.4 0.4 1.3