Polyphenolic compounds as chemical markers of wine ageing in contact with cherry, chestnut, false acacia, ash and oak wood

Food Chemistry 143 (2014) 66–76

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Polyphenolic compounds as chemical markers of wine ageing in contact with cherry, chestnut, false acacia, ash and oak wood B. Fernández de Simón a, M. Sanz b, E. Cadahía a,⇑, J. Martínez c, E. Esteruelas b, A.M. Muñoz b a

Centro de Investigación Forestal, CIFOR-INIA, Apdo. 8111, 28080 Madrid, Spain I+D+I Industrial Tonelera Navarra S.L. (INTONA), 31522 Monteagudo, Navarra, Spain c ICVV, Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja, Universidad de La Rioja y CSIC), 26006 Logroño, Spain b

a r t i c l e

i n f o

Article history: Received 3 April 2013 Received in revised form 1 July 2013 Accepted 19 July 2013 Available online 27 July 2013 Keywords: Wine Barrels Non-oak woods Polyphenols LC-DAD-ESI/MS

a b s t r a c t The nonanthocyanic phenolic composition of four red wines, one white, and one rosé aged using barrels and chips of cherry, chestnut, false acacia, ash and oak wood was studied by LC-DAD-ESI/MS, to identify the phenolic compounds that woods other than oak contribute to wines, and if some of them can be used as chemical markers of ageing with them. A total of 68 nonanthocyanic phenolic compounds were identified, 15 found only in wines aged with acacia wood, 6 with cherry wood, and 1 with chestnut wood. Thus, the nonanthocyanic phenolic profile could be a useful tool to identify wines aged in contact with these woods. In addition, some differences in the nonanthocyanic phenolic composition of wines were detected related to both the levels of compounds provided by each wood species and the different evolution of flavonols and flavanols in wines during ageing in barrels or in contact with chips. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction In recent years, some papers about the chemical composition of non-oak heartwoods have been shown in scientific literature, with a view to their use in cooperage, although only oak and chestnut are approved by OIV to wine ageing (De Rosso, Cancian, Panighel, Dalla Bedona & Flamini, 2009; Fernández de Simón, Esteruelas, Muñoz, Cadahía, & Sanz, 2009; Flamini, Dalla Bedona, Cancian, Panighel, & De Rosso, 2007; Rodriguez, Suarez, Diñero, Del Valle, & Piccinelli, 2010; Sanz et al., 2010a, 2010b, 2011, 2012a, 2012b). Thus, heartwood from species as false acacia (Robinia pseudoacacia), chestnut (Castanea sativa), and cherry (Prunus avium), and more rarely, ash (Fraxinus excelsior and F. vulgaris), mulberry (Morus alba and M. nigra), beech (Fagus sylvatica), alder (Alnus glutinosa) and some local woods are being considered as possible sources of wood for the production of wines and their derived products, like spirits, and especially vinegars, in order to give them a special personality. The most studied compounds were polyphenols, pointing out important chemical differences in relation to oak wood that should be taken into account when considering its use in cooperage. The oak heartwood shows high levels of monomer ellagitannins, such as castalagin, roburin E, vescalagin, and grandinin, and low molecular weight (LMW) phenolic compounds, ellagic and gallic acids, besides lignin derivatives, especially vanillin ⇑ Corresponding author. Tel.: +34 913476789; fax: +34 913476767. E-mail address: [email protected] (E. Cadahía). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.096

that can vary greatly depending on the species and geographical origin of the wood as well as the processing that undergoes in cooperage (Cadahía, Muñoz, Fernández de Simón, & García-Vallejo, 2001; Chatonnet, Boidron, & Pons, 1989). Oak heartwood does not contain other kinds of phenolic compounds, for example flavonoids. Chestnut heartwood shows the most similar polyphenolic profile to oak, although its LMW phenolic and tannic contents are higher, highlighting the presence of gallotannins and the high levels of gallic acid (Canas, Leandro, Spranger, & Belchior, 2000; Sanz et al., 2010b). The other studied woods show many qualitative and quantitative differences in their polyphenolic profile, including condensed tannins as both type procyanidin and prorobinetin, other flavonoids (flavanonols, flavanones, chalcones, aurones, flavonols and flavones), secoiridoids, phenylethanoids, dilignols and oligolignols. In all woods, the toasting at cooperage results in a progressive increase in lignin constituents with regard to intensity, and at the same time, in a degradation of most other polyphenols, leading to a minor differentiation among species. However, both before and after toasting the polyphenolic profile can be used to identify the species of wood (Sanz et al., 2012b). Although most of woods used in cooperage, as well as in chips and barrels, are toasted at different intensities, and so the differences in the polyphenolic profile of a wine aged with different woods could be very small, the useful phenolic markers to discriminate among wood species could also allow us to differentiate the wines aged with them. Little information about the effects of

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non-oak woods on the characteristics of wines, vinegars, and other beverages, compared to oak, has been presented in literature. Someone have pointed out a different evolution of the phenolic and volatile composition, and organoleptic properties in beverages aged in barrels or in contact with chips made of different woods (Caldeira, Anjos, Portal, Belchior, & Canas, 2010; Cerezo, Espartero, Winterhalter, García-Parrilla, & Troncoso, 2009; Cerezo et al., 2008; Chinnici, Natali, Sonni, Bellachioma, & Riponi, 2011; De Rosso, Panighel, Dalla Vedova, Stella & Flamini, 2009; Kozlovic, Jeromel, Maslov, Pollnitz, & Orlic, 2010; Sanz et al., 2012c). In some cases, phenolic markers that allow discriminate the wood used for ageing have been identified. This is the case of red wines and vinegars aged in acacia barrels, in which compounds like dihydrorobinetin, robinetin and other flavonoids were detected, but were not detected when beverages ageing in contact with oak wood (Cerezo et al., 2009; Sanz et al., 2012c). Some authors highlight that wines or vinegars aged in non-oak barrels had better organoleptic characteristics (Chinnici et al., 2011; Hillmann, Mattes, Brockhoff, Dunkel Meyerhof, & Hofmann, 2012; Kozlovic et al., 2010). However the physical–mechanical properties of wood barrel, like porosity that influence the gas exchange during ageing, can in some cases promote a fast polyphenol oxidation (Chinnici et al., 2011; De Rosso et al., 2009; Torija et al., 2009). That effect could be minimised using non-oak wood alternative to barrel products like powder, shavings, chips, cubes, or staves, as cheaper substitute techniques. The polyphenolic profile variability of beverages, and their evolution during ageing, can make the analysis of markers found in the wood more complex. In wines, this variability can be attributed to several factors, including some aspects of the raw material (grape variety, climatologic conditions, agronomic characteristics, degree of grape ripening) and winemaking process (time of maceration and fermentation in contact with the grape skins and seeds, pressing, fining, etc.) (Castillo-Muñoz, Gómez, García, & Hermosín, 2007; Monagas, Suárez, Gómez-Cordovés, & Bartolomé, 2005). These differences remain throughout ageing, even though a clear evolution in the concentrations of most phenolic compounds happens. In this context, it is important to have tools that reveal the botanical origin of wood used to wine ageing, as well as if woods other than oak have been used. The main goal of this work is to know the phenolic compounds that wood other than oak contributes to the wines, and if some of them can be used as chemical markers of ageing with non-oak woods. For this aim, medium toasting chips and 225 L barrels were made with cherry, chestnut, acacia, ash and oak wood, and the polyphenolic composition of four red wines (two 100% cv. Syrah, one 100% cv. Grenache, and one 100% cv. Tempranillo), one white wine (100% cv. white Grenache), and one rosé wine (100% cv. Grenache) aged with them was studied by LC-DAD-ESI/MS.

2. Materials and methods 2.1. Woods and wines Cherry (P. avium), chestnut (C. sativa), acacia (R. pseudoacacia), ash (F. excelsior), and oak (French and Spanish -Navarra- Q. petraea and American Q. alba) heartwood were provided as staves for making barrels by Tonelería Intona, SL (Navarra, Spain). The wood was naturally seasoned for 24 months until 15% humidity. Barrels were made following a traditional process, at medium intensity level of toasting, over a wood fire (185 °C for 45 min). The barrel heads were not toasted. All barrels were with a capacity of 225 L and staves of 28 mm thickness. Moreover, some staves of each were cut after seasoning at chip size (1  0.5 cm, approximately), and toasted at a medium intensity level (200 °C for 35 min), in an

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industrial-scale convection oven, with special oven trays for chips. All the wood was manufactured (seasoned and toasted) by the same cooperage. Two red wines from D.O. Cataluña and Somontano (Spain) were produced on an industrial scale in 2009, from cv. Syrah (100%) grapes, according to traditional methods. They were put into the cherry, chestnut, acacia, European ash, and French oak barrels (4 of each wood) in November 2009, and were kept during 6 (short ageing) and 12 months (long ageing) respectively, and finally bottled. During this storage time in barrels, wine samples from each barrel were taken, after ageing 2, 4, 6, 9 and 12 months. All wines were analysed in duplicate, so 320 samples were analysed. Other red wine from D.O. Navarra was produced on an industrial scale in 2010, from cv. Grenache (100%) grapes, according to traditional methods. It was put into the cherry, chestnut, acacia, European ash, French, American and Spanish oak barrels (4 of each wood) in January 2010, and was kept during 12 months, taken wine samples from each barrel after ageing 6 and 12 months. The same wine was in contact with chips of the same woods during 2 months in stainless steel 50 L tanks, with two quantities of chips: 150 and 75 g for each tank. Four thanks of each wood and each dosage were used. Other three wines were aged in contact with chips of the same woods during two months in stainless steel 50 L tanks. Two were from D.O. Navarra and produced on an industrial scale in 2010, according to traditional methods: one white wine from cv. White Grenache (100%) grapes and one rosé wine from cv. Grenache (100%) grapes. The dosage of chips was 75 g for each tank. Lastly, a dosage of 300 g of chips for each tank was used with a red wine from D.O. Rioja (100% Tempranillo grapes), produced on an industrial scale in 2010 according to traditional methods. All of the tanks with wood were micro-oxygenated using an Eco2 device (Oenodev, France) and ceramic diffusers with a dosage rate from 1.5 to 2 mL/L/month. All wines were analysed in duplicate at the end of ageing (448 samples). 2.2. Chemicals Reference compounds were obtained from commercial sources with purity higher 98%: caffeic acid, 2,4-dihydroxybenzoic (b-resorcylic) aldehyde, methyl gallate, ethyl gallate, gallic acid and protocatechualdehyde (Fluka Chimie AG, Buchs, Switzerland), 2,4dihydroxybenzoic acid (b-resorcylic), methyl vanillate, and methyl syringate (Aldrich Chimie, Neu-Ulm, Germany), ellagic acid and aromadendrin (Apin, Oxon, UK), (+)-catechin, (-)-epicatechin, quercetin, protocatechuic acid, syringic acid, benzoic acid, and taxifolin (Sigma Chemical, St. Louis, MO), eriodictyol (Roth, Kalsruhe, Germany), dihydrorobinetin, fustin, robinetin, isorhamnetin, vanillic acid, ferulic acid, procyanidin B1 and B2, naringenin, isosakuranetin, butein, prunin, kaempferol, p-coumaric acid, p-hydroxybenzoic acid, tyrosol, tryptofol, myricetin, quercetin-3-glucoside, quercetin-3-galactoside, and resveratrol (Extrasynthèse, Genay, France), and robtein (Transmit, Marburg, Germany). Methanol, diethyl ether, ethyl acetate, anhydrous sodium sulphate, and acetic acid were purchased from Panreac (Barcelona, Spain). Acetonitrile HPLC grade was from Scharlab (Barcelona, Spain) and formic acid and ammonium acetate MS spectroscopy from Fluka Chimie AG (Buchs, Switzerland) 2.3. Extraction of phenolic compounds The wine samples from each barrel or tank were analysed separately, following the previously described method by Cadahía (Cadahía, Fernández de Simón, Sanz, Poveda, & Colio, 2009). Samples, concentrated to 25% of their initial volume, were extracted with diethyl ether and ethyl acetate. The organic fractions were combined and evaporated to dryness, and the residue re-dissolved

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in MeOH-H2O (1:1) to be analysed by HPLC-DAD and LC-DAD/ESIMS analyses.

range test. Multivariate canonical discriminant analysis was also carried out using the program SAS (version 9.1; SAS Institute, Cary, NC).

2.4. HPLC/DAD analysis 3. Results and discussion Quantification of phenolics was performed by LC-DAD using an Agilent 1100 L liquid chromatography system equipped with a diode array detector (DAD), and managed by a Chemstation for LC 3D systems Rev B.03.02 (Agilent Technologies, Palo Alto, CA, USA). The column was a 200 mm  4 mm i.d., 5 lm, Hypersil ODS C18, maintained at 30 °C and protected with a 4 mm  4 mm i.d. guard column of the same material (Agilent Technologies). The HPLC profiles were monitored at 280, 340 and 310 nm, and the UV/ Vis spectra were recorded from 190 to 400 nm. The volume injected was 20 lL. The elution conditions used for wine extracts were as follows: flow rate, 1 mL min 1; temperature, 30 °C; solvent A = water/acetic acid (98:2 v:v), solvent B = water/acetonitrile/acetic acid (78/20/2 v:v:v); gradient profile, 0–55 min, 100–20% A, 55– 70 min, 20–10% A, 70–80 min, 10–5% A, 80–90 min 5–0% A, 90– 100 min 0% A. Detection was performed by scanning from 190 to 400 nm. The quantification of compounds was carried out by the external standard method, using detection at 280, 310 and 340 nm depending on their UV spectra. The concentration of each substance was measured by comparing it with calibrations made with the pure compound analysed under the same conditions and linear regression coefficients between 0.9990 and 0.9999 were obtained. In general, more than one linear regression was carried out for each compound, at different concentration levels. Calibration of a similar compound was used when the pure reference standard was not available. Thus, the hydroxycinnamic derivatives were quantified with the corresponding free acid calibration, and peaks 46 and 50 with ferulic acid. Flavonols were quantified as quercetin-3-glucoside (peaks 53, 59, 60 and 64) or myricetin (peaks 43 and 44), resveratrol derivatives with aglycone calibration, peaks 1, 20, 26, 33, 34, 36 and 41 with dihydrorobinetin calibration, and peak 56 with that of robinetin. Unidentified flavanols (peaks 12, 14, 22 and 35) were quantified as procyanidin B2, in agreement with their UV/Vis profile (99.8% matching those of commercial standard), peak 49 as (+)-catechin, and peak 37 as ellagic acid. The samples were analysed in duplicate. 2.5. LC-DAD/ESI-MS analysis Chromatographic separations were performed on an Agilent series 1100 (Palo Alto, CA) chromatography system equipped with a diode array detector and a quadrupole mass spectrometer (Agilent series 1100 MSD) with an electrospray interface. The binary mobile phase consisted of solvents A (2% acetic acid in HPLC grade water) and B (HPLC grade acetonitrile). The column, gradient, the volume injection, and the temperature of the analytical column were the same as that referred to above for the HPLC analysis. The flow rate was fixed at 0.7 mL min 1 during the entire chromatographic process. The DAD was set at 255, 280, and 340 nm to monitor the UV/Vis absorption. The UV/Vis spectra were recorded from 190 to 650 nm. ESI parameters were as follows: drying gas (N2) flow, 10 L min 1, temperature, 350 °C, a nebulizer pressure 55 psi (380 Pa); and capillary voltage, 4000 V. Mass spectra were acquired using electrospray ionisation in the negative mode at the voltage gradient: m/z 0–200, 80 V fragmentation voltage; m/z 200–3000, 200 V fragmentation voltage, and recorded for the range of m/z 100–3000. 2.6. Statistical analysis The obtained data were analysed carrying out univariate analysis using ANOVA, applying the Student Newman-Keuls multiple

3.1. Identification of phenolic compounds Different phenolic profiles of wines were obtained in relation to wood kind used during ageing and wine origin (Fig. 1). A wide variety of nonanthocyanic phenolic compounds were found, including nonflavonoids and flavonoids. Most of them have been previously described in different varietal wines aged in oak barrels (CastilloMuñoz et al., 2007; Hernández, Estrella, Dueñas, Fernández de Simón, & Cadahía, 2007; Monagas et al., 2005), but some were only detected in wines aged in contact with cherry, chestnut or acacia wood. Hydroxybenzoic and hydroxycinnamic aldehydes were not detected, possibly due to their tendency to react with wine glycols formatting acetal derivatives during the extraction with organic solvents, and successive concentrations carried out previously to the HPLC analysis (Spillman, Pollnitz, Liacopoulos, Skouroumounis, & Sefton, 1997). In fact, some of them were identified and quantified when these same wines were extracted in a different way and analysed by GC–MS (Unpublished results). Among the detected compounds, 35 were identified by comparing their retention times and UV/vis and mass spectra with those of the commercial standards, as indicate in Table 1 and 2 with a superscript after compound name. Moreover, some hydroxycinnamic derivatives were tentatively identified, such as cis and trans caftaric acids, cis and trans coutaric acids, trans fertaric acid, and two hexose ester of p-coumaric acid (peaks 3, 5, 6, 11, 16, 23 and 28), in accordance to data in literature (Castillo-Muñoz et al., 2007; Monagas et al., 2005). An unidentified structure of hydroxycinnamic compound was also assigned to peak 50, which showed an UV/vis spectrum very similar to p-coumaric acid (kmax at 306 nm), and the same two fragment ions that the hexose esters (m/z 163 and 145), but very high retention time (51.6 min) and different molecular ion (m/z 315). Glutathionyl adducts derived from hydroxycinnamic acids were not detected. Peaks 12, 14, 22 and 35 showed the typical UV profile of proanthocyanidins/catechin-like compounds with an absorbance maximum between 277 and 280 nm, and molecular ions at m/z 577 or m/z 865, characteristics of B-type procyanidin dimers or trimers, respectively (Dueñas, Sun, Hernández, Estrella, & Spranger, 2003). Peak 49 was consistent to a flavan-3-ol gallate (kmax at 277, [M–H] at m/z 441, fragment ions at m/z 289 and 169) (Monagas et al., 2005), but the assignment of catechin or epicatechin was not possible by means of the MS analysis. Flavonol glycoside was the possible structure of peaks 43, 44, 53, 59, 60 and 64, on basis of their UV/vis spectra, showing maxima absorbance around 350 nm, and mass spectra data showing the [M–H] ion and the fragment corresponding to the loss of sugar moiety. Among them, the structures of two myricetin derivatives (3-glucuronide ([M– H] at m/z 493, and [M–H-176] at m/z 317) and 3-glucoside ([M–H] at m/z 479, and [M–H-162] at m/z 317)), quercetin-3glucuronide ([M–H] at m/z 477, and [M–H-176] at m/z 301), laricitrin-3-glucoside ([M–H] at m/z 497, and [M–H-162] at m/ z 331), isorhamnetin-3-glucoside ([M–H] at m/z 477, [M–H162] at m/z 315, and ([M–H-162-CH2] at m/z 300), and syringetin-3-galactoside ([M–H] at m/z 507, [M–H-162] at m/z 345] were assigned taking into account spectral and literature data (Castillo-Muñoz et al., 2007; Monagas et al., 2005). Looking at stilbenes, peaks 45 and 58 were identified as trans and cis-resveratrol glucosides since in addition to the molecular ion (m/z 389), a fragment ion corresponding to resveratrol after loss of the glucose moiety was observed in their MS spectra (Monagas et al., 2005).

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Fig. 1. LC-DAD chromatograms at 280 nm of red wines (100% Syrah, DO Somontano) aged with cherry (A), chestnut (B), acacia (C), ash (D), and oak (E) wood barrels during 12 months. Peak numbers shown in Tables 1 and 2.

In addition, peak 37 showed an UV/vis spectrum very similar to ellagic acid (kmax at 256 and 364 nm) and two fragment ions due to the loss of CO2 from the deprotonated molecule, and to the ellagic acid fragment (m/z at 469, 425 and 301). The structure of valoneic acid dilactone was assigned to this peak following data from chestnut wood (Sanz et al., 2010b). Finally, a structure of dihydroflavonol was assigned to peaks 20 (UV/Vis spectrum showing kmax at 290 nm and a shoulder at 316 nm, [MS: m/z (%): 319 (4), 301 (100), 193 (100)]), 26 (kmax at 280 nm, shoulder at 316 nm, [MS: m/z (%): 303 (10), 285 (100), 163 (22), 135 (20)]), and 33 (kmax at 280 nm, shoulder at 316 nm, [MS: m/z (%): 317 (100), 299 (100), 284 (23), 274(10), 135 (14)]), dihydroflavone to peaks 34 (kmax at 278 nm, shoulder at 314 nm, [MS: m/z (%): 287 (90), 177 (4), 151 (100), 135 (62)]), and 36 (kmax at 278 nm, shoulder at 312 nm, [MS: m/z (%): 271 (57), 135 (100), 91 (11)]), flavonoid compound to peak 41 (kmax at 280 nm, shoulder at 315 nm, [MS: m/z (%): 289 (40), 227 (30), 151 (100)]), hydroxycinnamic compound to

peak 46 (kmax at 320 nm, shoulder at 290 nm, [MS: m/z (%): 195 (97), 163 (21), 135 (100), 91 (48)]), and aurone to peak 56 (kmax at 262 and 400 nm, [MS: m/z (%): 285 (100), 149 (60), 135 (6), 109 (3)]), following the same assignations that in acacia wood, remaining unidentified the peak 1 (kmax at 280 nm and a shoulder at 316 nm in UV/vis spectrum, and [MS: m/z (%): 181 (45), 137 (47), 109 (100)]) (Sanz et al., 2011). 3.2. Polyphenolic compounds as markers of wood species used in ageing wines Some of these compounds were only generated in wines through the wood barrels used, and so, they can only be detected in wines after their contact during ageing. In Table 1 we show their levels at the end of ageing in all studied wines. All red wines ageing in cherry wood barrels showed flavonoid compounds extracted from toasted (barrel staves) and untoasted

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Table 1 Quantitative evaluation (mg L Peak

1

) from HPLC-DAD chromatograms of phenolic markers in wines aged in acacia, cherry or chestnut wood barrels or chips.

Compound

Barrel Syrah 12 M

Wines 38 42 48 61 66 68

aged with cherry wood Taxifolina Prunina Aromadendrina Eriodictyola Naringenina Isosakuranetina

5.65b 1.36a 10.74a 1.13a 9.80a 5.25a

Wines 2 52 25 37

aged with chestnut wood Gallic acida,b Ellagic acida,b Ethyl gallatea,b Valoneic acid dilactone

69.6a 24.3a 23.2a 4.01a

Wines 1 13 17 18 20 26 31 33

aged with acacia wood Unknown compound 2,4-Dihydroxybenzoic acida 2,4-Dihydroxybenzaldehydea Dihydrorobinetina Pentahydroxydihydroflavonol Tetrahydroxydihydroflavonol Fustina Trihidroxymethoxy dihydroflavonol Robtin Butin Unidentified flavonoid Hydroxycinnamic compound Robinetina Tetrahydroxyaurone Buteina

34 36 41 46 47 56 63

Chips Syrah 6M 3.64c 0.76bc 5.56c 0.63b 5.57c 3.98b

Grenache 12 M

Tempranillo 6 g/L

4.59c 0.93b 6.85b 0.76b 6.41b 4.23b

6.22a 0.97b 7.82b 0.65b 7.07b 6.25a

43.9d 20.4b 11.7b 1.69c

64.8b 22.8a 22.1a 2.71b

38.9e 20.4b 4.01c 0.62d

5.99a 2.68a 29.13a 114.8b 0.94c 7.46b 4.39a 3.71b

1.21c 2.19b 16.48b 79.24c 1.75b 5.69c 4.33a 2.78b

5.41a 2.56a 14.32b 147.0a 2.41a 12.73a 3.95a 5.61a

1.71b 1.05c 6.24c 18.82d 0.38c 2.96d 1.47b 0.61c

2.59b 4.11b 2.21b 1.21a 42.38a 3.39a 3.22a

1.49c 3.41c 0.98d 0.85ab 30.01b 3.28a 2.63b

4.52a 7.55a 1.77bc 0.33c 31.96b 3.73a 1.37cd

F-values

1.03cd 2.03d 4.01a 0.83ab 9.87c 2.14b 1.86c

Grenache 3 g/L

Grenache 1.5 g/L

3.67c 0.71bc 3.12d 0.59b 3.70c 2.19c

2.41d 0.54 cd 1.43e 0.50b 2.47d 1.79c

White Grenache 1.5 g/L 2.69d 0.31de 2.90de 0.26c 3.07d 1.48c

Rosé Grenache 1.5 g/L 2.30d 0.41de 2.71de 0.24c 2.88d 2.01c

65.86⁄⁄⁄ 35.47⁄⁄⁄ 81.08⁄⁄⁄ 33.48⁄⁄⁄ 290.17⁄⁄⁄ 35.43⁄⁄⁄

46.2d 17.8ab 9.28b 0.46d

7.56f 4.87c 0.98c 0.27d

10.1f 6.55c 1.24c 0.32d

468.02⁄⁄⁄ 24.75⁄⁄⁄ 47.45⁄⁄⁄ 36.50⁄⁄⁄

1.41b 0.49d 2.45d 8.54e 0.25d 1.41de 0.74c 0.16c

0.37c 0.27d 1.29d 4.16f 0.13d 0.49e 0.41c 0.09c

0.31c 0.24d 1.06d 3.47f 0.09d 0.47e 0.49c 0.14c

0.22c 0.43d 1.43d 3.64f 0.25d 0.51e 0.34c 0.17c

655.30⁄⁄⁄ 42.04⁄⁄⁄ 189.44⁄⁄⁄ 45.84⁄⁄⁄ 28.53⁄⁄⁄ 48.24⁄⁄⁄ 12.57⁄⁄⁄ 36.58⁄⁄⁄

0.41d 1.19e 1.27cd 0.53bc 5.59c 1.99b 1.08de

0.17d 0.94e 0.85d 0.19c 3.28c 1.60b 0.52e

0.24d 0.43e 0.21e 0.08c 2.71c 0.32c 0.13f

0.31d 0.43e 0.45d 0.36c 2.26c 0.51c 0.17f

63.06⁄⁄⁄ 125.07⁄⁄⁄ 103.88⁄⁄⁄ 14.80⁄⁄⁄ 29.12⁄⁄⁄ 31.15⁄⁄⁄ 44.88⁄⁄⁄

50.1c 22.9a 10.3b 0.74d

⁄ ⁄⁄

, and ⁄⁄⁄ indicate significance at p < 0.01, 0.001, and 0.0001, respectively. Compounds identified using commercial standard. b Compounds not selected as markers, but showing high significant differences. Averages were calculated for four wines analysed in duplicate. The variation coefficients were <3%. Different letters in the same row denote a statistical difference with 95% confidence level (Student Newman-Keuls multiple range test). ,

a

(barrel heads) cherry wood that were not present in red wines from remainder wood barrels. Also in red, rosé and white wines aged in contact with cherry wood chips we can found these same flavonoids. Among them, it highlights aromadendrin, naringenin, taxifolin and isosakuranetin because their higher levels, but also prunin and eriodictyol were detected, and during all ageing time (Fig. 1, Table 1). So, these compounds could be proposed as possible phenolic markers of ageing in contact with cherry wood for authenticity purposes. It is the first time that have been identified in wines aged in contact with cherry wood, in spite of recent studies carried out about the phenolic composition of red Italian wines and red wine vinegars, aged in cherry barrels (Cerezo et al., 2008; Chinnici et al., 2011; De Rosso et al., 2009). These five flavonoids have been detected in untoasted cherry wood, showing concentrations between 7500 and 1200 lg g 1 of wood. There are extremely sensitive to heat, and their concentrations decrease more than 85% when wood is toasted at medium level (similar to toasting used in our work), not being detected at higher intensity (Sanz et al., 2010a, 2010b, 2012b). Therefore, the barrels with untoasted head should provide greater amount of these compounds than chips. As it can be seen in Table 1, besides the type of ageing (barrel or chips), grape variety, length of stay in barrel, and of course, dose of chips, may determine the level of these compounds in aged wines. Thus, after 12 months of ageing, the red wine 100% Syrah showed higher values than red wine 100% Grenache, with statistical significant differences; the two Syrah wines after 12 and 6 months of ageing also showed statistical significant differences, as well as among the dose of chips, since in the three wines aged with 1.5 gL 1 of cherry chips, only the eriodictyol concentrations showed significant differences, while we found significant

differences for taxifolin, aromadendrin and naringenin between 1.5 and 3 gL 1, and in addition for isosakuranetin between 3 and 6 gL 1. In an early paper, we also described some nonflavonoid compounds as chemical markers of cherry wood, related to chestnut, acacia and ash woods: methyl syringate, benzoic acid, methyl vanillate, p-hydroxybenzoic acid, 3,4,5-trimethoxylphenol and pcoumaric acid, as well as the presence of condensed tannins in untoasted cherry wood, specially (+)-catechin (Sanz et al., 2012b). Among them, only p-hydroxybenzoic and p-coumaric acids, and (+)-catechin were detected in aged wines, probably because the interference of other compounds in the HPLC analysis, since when the volatile and semi volatile compounds of these wines were analysed by GC–MS, all them were found (unpublished results). These three compounds cannot be considered as phenolic markers of ageing in contact with cherry wood since they were detected in all wines, before and after ageing. In Table 2, the levels of no-marker phenolic compounds in final wines from Syrah are shown, and in Table 3 the F-value and p from ANOVA of these data from all aged red wines. It can be seen that p-hydroxybenzoic acid and (+)-catechin showed statistically significant higher concentrations in wines aged in contact with cherry wood, but no differences were found for p-coumaric acid, probably because its concentration increases during ageing by hydrolysis of its derivatives, and it is involved in the condensation reactions which occur during ageing. The higher levels of (+)-catechin in these wines disagree with the lowest levels found by De Rosso et al. (2009) in wines aged in cherry barrels compared to those aged in oak, acacia, chestnut and mulberry barrels, that the authors relate to higher oxidation levels in cherry barrels.

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B. Fernández de Simón et al. / Food Chemistry 143 (2014) 66–76 Table 2 Quantitative evaluation at the end of ageing (mg L Peak

Compound

1

) of peaks in HPLC-DAD chromatograms of Syrah red wines aged in cherry, chestnut, acacia, ash, and oak wood barrels.

D.O. Cataluña aged 6 months

D.O. Somontano aged 12 months

Cherry

Chestnut

Acacia

Ash

Oak

Cherry

Chestnut

Acacia

Ash

Oak

Hydroxybenzoic acids 2 Gallic acida 4 Protocatechuic acida 8 p-Hydroxybenzoic acida 19 Vanillic acida 27 Syringic acida 52 Ellagic acida

21.38d 1.02a 0.82a 3.59b 5.16a 5.94c

43.91a 0.24b 0.32b 3.25b 3.95bc 20.41a

33.09b 0.9a 0.33b nq c 3.32c 4.54c

27.77c 0.82a 0.38b 4.88a 4.09b 5.54c

30.46bc 0.73a 0.41b 3.62b 3.56bc 11.61b

34.35b 2.17a 1.74a 2.63b 4.32a 7.44b

69.64a 1.48b 0.46b 3.22b 4.41a 24.4a

35.34b 1.06b 0.41b nq d 3.07a 4.55c

34.77b 1.62b 0.41b 4.73a 3.56b 5.11c

34.05b 1.11b 0.52b 1.57c 2.55d 7.88b

Hydroxycinnamic acids 21 trans-Caffeic acida 29 cis p-Coumaric acida 32 trans p-Coumaric acida 39 Ferulic acida

22.66b 4.01a 46.68a 0.56c

15.62d 1.27b 9.45c 1.02b

20.21c 4.31a 40.14b 1.41a

21.28bc 4.2a 41.89b 1.29a

24.82a 4.25a 47.13a 1.07b

11.47a 4.96a 16.32a nd

3.94c 5.46a 8.67c nd

3.72c 2.85b 8.17c nd

8.55b 4.91a 11.54b nd

1.39d 4.6a 4.01d nd

Hydroxycinnamic derivatives 23 trans p-Coumaric hexose 28 trans p-Coumaric hexose 3 cis-Caftaric acid 5 trans-Caftaric acid 6 cis-Coutaric acid 11 trans-Coutaric acid 16 trans-Feftaric acid 50 Hydroxycinnamic comp.

5.67a 1.71a nd c 0.07c 0.03c 0.04c 0.16b 0.25b

1.84c 0.57b 0.02b 0.54b 0.11a 0.36a 0.19b 0.24b

3.39b 1.85a 0.04a 0.71a 0.06b 0.17b 0.91a 0.92a

6.11b 1.91a 0.02b 0.41b 0.03c 0.13b 0.05c 0.26b

5.73a 2.01a nd c 0.07c nd d nd c nd c 0.31b

7.02bc 4.54ab 0.25 7.38d 0.25c 1.36d 1.21 1.27c

8.35ab 3.46b 0.31 14.24b 0.48a 2.44b 1.47 1.43b

9.41a 5.17a 0.27 12.34c 0.42b 2.26c 1.23 2.25a

6.96bc 3.92ab 0.28 7.96d 0.27c 1.46d 1.22 1.21c

5.59c 1.57c 0.34 15.35a 0.51a 2.71a 1.09 1.25c

Flavanols 49 15 30 12 14 22 26 35

4.52b 47.7a 10.81a 3.13a 2.18a 5.59a 11.14 5.66a

4.36bc 38.88b 9.81ab 2.02bc 1.59b 1.42b 5.78 2.63b

nq d 35.34c 9.32b 1.71c 2.41a 2.03b 4.92 5.07a

4.06c 37.5c 9.17b 2.16bc 1.58b 3.48b 7.54 2.52b

5.12a 40.13b 10.35ab 2.32b 2.01a 3.31b 9.11 3.05b

2.58a 28.39a 2.68 0.63 0.59b 5.11a 6.27a 0.46b

2.25a 24.28ab 3.78 0.71 0.66b 1.61b 5.61a 1.12a

nq b 20.38b 3.59 0.44 1.67a nq c nq c 1.14a

1.33a 20.52b 3.02 0.31 0.65b 2.34b 2.96b 0.44b

2.25a 20.6b 3.35 0.41 0.35b 1.88b 3.15b 0.45b

Flavonols 43 Myricetin-3-glucuronide 44 Myricetin-3-glucoside 51 Quercetin-3-galactosidea 53 Quercetin-3-glucoronide 55 Quercetin-3-glucosidea 59 Laricitrin-3-glucoside 57 Myricetina 60 Isorhamnetin-3-glucoside 64 Syringetin-3-galactoside 65 Quercetina 67 Kaempferola

0.81a 5.11 9.66 0.84b 3.48b 13.70 1.91b 7.82a nd c 27.82a 1.05

0.35b 4.28 7.57 1.39a 4.38a 11.96 1.53bc 6.11b 0.54b 23.99c 1.02

0.72a 4.66 8.01 0.74b 3.14bc 12.62 1.81b 6.28b 2.49a 23.72c 0.67

0.59ab 4.62 8.63 0.72b 2.97c 13.07 2.55a 7.73a 0.82b 23.41c 0.56

0.62ab 4.69 8.86 0.57b 2.93c 13.42 1.37c 6.59b 2.37a 26.24b 0.56

0.76b 7.25 10.43 3.51 2.81 8.29 2.48c 2.97b 0.67a 7.72b 0.84

0.91b 7.35 10.65 3.56 2.67 8.05 3.58b 4.54a 0.88a 8.85a 0.74

1.89a 8.28 10.57 3.57 2.7 8.48 4.41a 3.92a 0.79a 7.84b 0.57

0.65b 6.88 10.31 2.46 2.57 7.48 3.11bc 4.36a 0.56a 8.24ab 0.56

0.79b 8.54 10.47 3.58 2.77 4.91 3.45b 3.75a nd b 8.61a 0.78

Stilbenes 45 54 58 62

1.72 1.35 2.66a 0.66b

1.45 1.18 1.97c 0.95a

2.35 1.77 1.49d 0.27c

1.61 1.38 2.32b 0.61b

2.01 1.57 1.74 cd 0.24c

0.79 0.67a 0.72b 0.29

0.62 0.35c 0.91a 0.22

0.67 0.51b 0.7ab 0.21

0.79 0.62ab 0.67b 0.28

0.74 0.53b 0.39c 0.08

0.82b 1.31a 1.32a 8.99b 0.21a

0.73c 1.14b 1.22a 11.73a 0.17a

0.84b 0.58c 1.04b 7.95b 0.19a

1.01a 0.91b 1.32a 8.11b 0.2a

0.84b 1.38a 1.22a 9.16b nd b

0.86b 0.41b 0.85a 6.17b 0.095c

1.29a 0.29b 1.02a 23.18a 0.21a

1.11a 0.75a 0.62b 7.61b nd d

1.33a 0.46b 0.94a 7.02b 0.12b

1.14a 0.22b 0.81a 6.01b nd d

(Epi)catechin gallate (+)-Catechina (-)-Epicatechina Procyanidin dimer Procyandin trimer Procyanidin dimer Procyanidin B2a Procyanidin dimer

trans-Resveratrol gluc. trans-Resveratrola cis-Resveratrol glucoside cis-Resveratrola

Other compounds 10 Tyrosola 40 Tryptofola 9 Methyl gallatea 25 Ethyl gallatea 7 Protocatechuic aldehydea

a Compounds identified using commercial standard. nd = not detected. nq = not quantified because the interference by other peaks. Averages were calculated for four wines analysed in duplicate. The variation coefficients were less than 3%. Different letters in the same row of each ageing time denote a statistical difference with 95% confidence level (Student Newman-Keuls multiple range test). ⁄, ⁄⁄, and ⁄⁄⁄ indicate significance at p < 0.01, 0.001, and 0.0001, respectively.

Valoneic acid dilactone could be proposed as possible phenolic marker of ageing wines in contact with chestnut wood for authenticity purposes. It was only able to be detected in these wines, and during all ageing time (Fig. 1, Table 1). As far as we know, it is the first time that has been identified in wines aged in contact with chestnut wood, in spite of recent studies carried out about the phenolic composition of red Italian and Spanish wines, and red wine vinegars, aged in chestnut barrels or with chestnut chips (Alañón et al., 2013; Cerezo et al., 2008; De Rosso et al., 2009; Gambuti, Capuano, Lisanti, Strollo, & Moio, 2010). This compound was

identified in untoasted and light toasted chestnut wood besides a lot of gallotannins (galloyl and hexahydroxydiphenoyl derivatives) and ellagitannins. The hydrolysable tannins are extremely sensitive to heating in cooperage, and their concentrations decrease 70% at light toast, and more than 95% when wood is toasted at medium level (similar to toasting used in our work), not being detected at higher intensity (Sanz et al., 2010b). They would be extracted from untoasted wood of barrel heads, but they have not been detected in our aged wines, since once in the wine, ellagitannins are slowly but continuously transformed through

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B. Fernández de Simón et al. / Food Chemistry 143 (2014) 66–76

Table 3 F-values and p from the analysis of variance (ANOVA) of quantitative evaluation of phenolic compounds in red wines aged in contact with cherry, chestnut, acacia, ash, and oak wood. Peak

Compound

Speciea

ageing modeb

Hydroxybenzoic acids 2 4 8 19 27 52

Gallic acid Protocatechuic acid p-Hydroxybenzoic acid Vanillic acid Syringic acid Ellagic acid

82.28***(c a bc c b) 11.28***(b c bc c a) 171.27***(a b b b b) 146.26***(b b c a b) 15.15***(a b c b c) 181.57***(c a c c b)

Specie x ageing mode

9.80*(a b) 70.17***(b a) 10.18*(a b) 1.37 20.50***(a b) 1.19

42.57*** 13.48*** 81.23*** 64.70*** 11.67*** 86.68***

Hydroxycinnamic acids 21 trans-Caffeic acid 29 cis p-Coumaric acid 32 trans p-Coumaric acid 39 Ferulic acid

13.77***(a c bc b bc) 2.14 6.28***(a b a a a) 5.64**(b b a b a)

1.21 17.40***(a b) 4.92 11.28**(b a)

7.54*** 2.83* 4.07*** 3.99***

Hydroxycinnamic derivatives 23 trans p-Coumaric hexose 28 trans p-Coumaric hexose 3 cis-Caftaric acid 5 trans-Caftaric acid 6 cis-Coutaric acid 11 trans-Coutaric acid 16 trans-Feftaric acid 50 Hydroxycinnamic compound

2.62 1.53 9.91***(b b b b a) 13.06***(b b b b a) 8.82***(c a ab bc a) 6.37***(c a ab bc ab) 8.58***(b b b b a) 7.74***(b b a b c)

4.22 54.65***(b a) 33.63***(b a) 47.94***(b a) 0.38 17.56***(a b) 23.08*** (a b) 98.36***(b a)

1.90 7.55*** 7.83*** 11.19*** 4.62*** 6.46*** 11.87*** 14.94***

Flavanols 49 15 30 12 14 22 26 35

(Epi)catechin gallate (+)-Catechin (-)-Epicatechin Procyanidin dimer Procyandin trimer Procyanidin dimer Procyanidin B2 Procyanidin dimer

24.62***(b b a b b) 7.68***(a b b b b) 2.69 2.75 6.01***(ab ab a b c) 18.43***(a b b a b) 4.86*(a ab b b ab) 2.61

87.32***(a 40.57***(a 121.86***(a 73.15***(a 132.27***(a 46.12***(a 59.99***(a 21.68***(a

Flavonols 43 44 51 53 55 59 57 60 64 65 67

Myricetin-3-glucuronide Myricetin-3-glucoside Quercetin-3-galactoside Quercetin-3-glucoronide Quercetin-3-glucoside Laricitrin-3-glucoside Myricetin Isorhamnetin-3-glucoside Syringetin-3-galactoside Quercetin Kaempferol

13.71***(c c a c b) 9.04***(a a a a b) 0.54 6.83***(a a ab a b) 5.13***(a a a a b) 0.98 8.37***(a a a a b) 7.69***(a a a a b) 8.15***(c ab a ab b) 1.21 5.56***(a a ab ab b)

1.50 145.55***(a 96.29***(a 112.05***(a 246.17***(a 0.03 296.13***(a 137.24***(a 0.04 97.94***(a 267.34***(a

Stilbenes 45 54 58 62

trans-Resveratrol glucoside trans-Resveratrol cis-Resveratrol glucoside cis-Resveratrol

Other compounds 10 40 9 25 7

Tyrosol Tryptofol Methyl gallate Ethyl gallate Protocatechuic aldehyde

1.14 2.30 2.50 1.01 14.03***(bc b bc a c) 1.66 2.88 52.02***(b a b b b) 2.46

b) b) b) b) b) b) b) b)

23.86*** 8.75*** 14.38*** 9.24*** 20.83*** 15.85*** 10.83*** 7.59***

b) b)

7.53*** 21.12*** 12.04*** 15.40*** 29.37*** 0.46 40.71*** 25.40*** 4.67*** 10.84*** 33.25***

41.02***(a b) 0.02 50.07***(a b) 2.86

5.72*** 1.49 7.12*** 1.29

41.02***(a b) 3.07 81.40***(b a) 25.87***(a b) 0.23

10.87*** 1.93 12.48*** 42.48*** 1.54

b) b) b) b) b) b)

a

Letters between parentheses show the differences among species, in the order cherry, chestnut, acacia, ash and oak. Barrel, chip. Different letters denote a statistical difference with 95% confidence level (Student Newman-Keuls multiple range test), with a being the highest concentration. * Indicate significance at p < 0.01, 0.001, and 0.0001, respectively. ** Indicate significance at p < 0.01, 0.001, and 0.0001, respectively. *** Indicate significance at p < 0.01, 0.001, and 0.0001, respectively. b

condensation, hydrolysis, and oxidation reactions, giving rise to the formation of other compounds, as their ethyl derivatives and the flavano-ellagitannins (Jourdes, Michel, Saucier, Quideau, & Teissedre, 2011), and a similar behaviour could be expected from the gallotannins. Consequently, their levels in aged wines will be much lower than those of other polyphenols, and a fractionation step was always required previously to their detection and quantification (García-Estévez, Escribano-Bailón, Rivas-Gonzalo, & AlcaldeEon, 2010). The levels of valoneic acid dilactone detected in these

wines showed statistically significant differences related to the type of ageing (barrel or chips), grape variety, length of stay in barrel, but not to dose of chips (Table 1), probably because its concentration in medium toasted chips was very low. Literature data show a greater ratio of gallic and ellagic acids extraction from chestnut than for oak and, as a consequence, the formation of gallic ethyl ester is more likely in beverages aged in contact with chestnut wood (Canas, Leandro, Spranger, & Belchior, 1999; Cerezo et al., 2008). These three compounds show

B. Fernández de Simón et al. / Food Chemistry 143 (2014) 66–76

statistically significant differences between red wines aged in contact with chestnut wood and red wines from the remainder woods (Tables 2 and 3)., although they cannot be regarded as phenolic markers of ageing in contact with chestnut since they can be detected in almost all wines, especially in those aged in oak. However, because of their high statistical significance, and the absence of other potential markers besides valoneic acid dilactone, we have considering interesting to include them also in Table 1. All red, rosé and white wines aged in contact with acacia wood showed 15 nonanthocyanic phenolic compounds that were not present in wines from the remainder woods (Fig. 1, Table 1). Dihydrorobinetin, robinetin and 2,4-dihydroxybenzaldehyde showed the highest concentrations, in this order, in all these wines. In addition, four dihydroflavonols (fustin, tetrahydroxy-, trihydroxymethoxy-, and pentahydroxy- dihydroflavonol), two dihydroflavones (robtin and butin), one chalcone (butein), one aurone (tetrahydroxyaurone), 2,4-dihydroxybenzoic acid, and three compounds not fully identified (peaks 1, 41 and 46) were also detected at appreciable levels in wines from acacia and during all ageing process. All of them had been previously found in untoasted and medium toasted acacia wood, but not in the remainder woods (Sanz et al., 2012b). In an early paper about these Syrah wines (Sanz et al., 2012c), we have proposed the most abundant of them as possible phenolic markers of ageing in acacia barrels, and the results obtained in this work confirm this statement, although significant differences have been found in their concentrations related to type of ageing (barrel or chips), grape variety, length of stay in barrel, and of course, dose of chips (Table 1). So, after 12 months of ageing in barrels, Grenache wine extracted significant higher quantities of dihydrorobinetin, pentahydroxy- dihydroflavonol, tetrahydroxydihydroflavonol, trihidroxymethoxydihydroflavonol, robtin, and butin, and lower of 2,4-dihydroxybenzaldehyde, the hydroxycinnamic compound, robinetin, and butein, than Syrah wine, confirming that every wine has a different ability to extract the compounds from wood barrel, as it happens with the other woods, and data in literature (Fernández de Simón et al., 2008). Concerning to type of ageing, differences in flavonoid levels are related to the contribution of those more sensitive to heat from barrel head pieces (untoasted wood). Thus, dihydrorobinetin was 35 times higher in Grenache wines aged 12 months in acacia barrel than in the same wine aged 2 months with 1.5 gL 1 of medium toasted acacia chips, as it happens in vinegars aged with untoasted and toasted acacia wood (Cerezo et al., 2009). However, the differences found in the levels of b-resorcilyc aldehyde and acid are not related to their higher levels in toasted wood, since they were lower in wines aged with chips. Thus, other factors such as solubility or chemical reactivity could influence the final concentrations of these compounds in wines. Lastly, the polyphenolic profile of all wines aged in contact with ash wood has not shown specific polyphenols provided by this wood, untoasted or toasted, although compounds such as secoiridoids, phenylethanoid glycosides and lignols were previously identified in this wood (Sanz et al., 2012a). However, their levels in untoasted wood were not high, and the effect of temperature was already very important when light toasting was applied, being detected only four of these compounds after medium toast, with concentrations lower than 160 lg/g of wood. Therefore, we cannot propose any polyphenolic compound as marker of wine ageing in contact with ash wood. However, the concentrations of two compounds, vanillic acid and tyrosol, showed statistically significant differences among red wines aged in contact with ash wood and the remainder woods (Table 3). Indeed, considering the differences in the LMW polyphenol composition of all these woods, toasted and untoasted, compounds such as tyrosol, vanillic acid, vanillin, syringaldehyde, and coniferaldehyde showed statistically significant higher concentrations in ash wood (Sanz et al., 2012b). Since

73

hydroxybenzoic and hydroxycinnamic aldehydes were not detected using this extraction method, only the higher contribution of this wood on tyrosol and vanillic acid has been detected. 3.3. Other nonanthocyanic polyphenolic compounds in aged red wines Before ageing, each wine showed a specific nonanthocyanic polyphenolic profile with quantitative differences among them. For example, the Syrah wine from D.O. Cataluña was richer in hydroxycinnamic acids, flavanols, flavonol aglycones, and stilbenes, while the Syrah wine from D.O. Somontano was richer in hydroxycinnamic derivatives and some flavonol glycosides (unpublished data). This variability can be attributed to several factors, including some aspect of the raw material and winemaking process, and it is especially important among red, rosé and white wines. The differences remain throughout ageing, even though a clear evolution in the concentrations of most phenolic compounds happens. This evolution may be conditioned in part by the characteristics of the wood barrel used during ageing. So, we have studied if the statistical differences among the levels of these compounds in aged red wines may be relate to the characteristics of wood, whether its species, or how to use it, barrels or chips. Rosé and white aged wines were not included in this part of study because of their differences in the polyphenolic profiles before ageing. Most of studied compounds showed statistically significant differences related to wood species used during ageing, regardless ageing mode (barrel or chips) (Table 3). It have highlighted the Fvalues of ellagic, p-hydroxybenzoic, vanillic and gallic acids, and ethyl gallate, all them related to polyphenolic characteristics of woods. The evolution of compounds with high oxidation facility, such as hydroxycinnamic acids and their derivatives, flavanols and flavonols, also lead, in some cases, to statistically significant differences at the end of ageing related to wood species, and probably in relation to their physical–mechanical properties like porosity, which influence the gas exchange during ageing, and therefore oxidative condensation and copigmentation procedures (De Rosso et al., 2009). Therefore, it appears that there is not clear relationship between type of wood and oxidative metabolism in wine, since polyphenolic composition before ageing exerts great influence, as it can be seen in the results of these compounds shown for Syrah wines in Table 2, and in literature (Gambuti et al., 2010). According to our results, when the same wine is aged in cherry, chestnut, acacia, ash, and oak barrels, the evolution of their polyphenols shows differences related to wood species, but a defined behaviour of a wine related to wood specie cannot be established. Thus, for example, in the case of long ageing Somontano Syrah wines, the decrease of trans-caftaric and trans-coutaric acids was more intense for wines aged in cherry and ash barrels, than in oak barrels, showing also a higher increase of the free acids. However, in Cataluña Syrah wines, after 6 months of ageing the levels of hydroxycinnamoyltartaric acids were very low in all wines, and the lowest levels of free acids were found in wines aged in chestnut barrels. In Italian red wines after 9 months of ageing in acacia, cherry, chestnut, mulberry and oak barrels no significant changes in hydroxycinnamoyltartaric acids were detected (De Rosso et al., 2009). In order to have an overall view of the influence that botanical species of wood has on the nonanthocyanic phenolic composition of wines, regardless the marker compounds, we carried out a multivariate analysis (CDA) of data, grouping the wines in accordance only with wood species, not taking into account the ageing mode. The graphic representation of the samples, in the space defined by the two main canonical functions obtained, shows a distribution in four groups (Fig. 2-top). Canonical function 1 (Can 1) allow to distinguish wines aged with chestnut wood (B) from the other four species. The more correlated variables were ellagic and gallic acids,

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B. Fernández de Simón et al. / Food Chemistry 143 (2014) 66–76

Fig. 2. Canonical discriminant analysis of phenolic compounds in red wines aged in barrels or with chips from different woods, taking into account only kind of wood (top), or kind of wood together with kind of ageing (bottom). A = cherry; B = chestnut; C = acacia; D = ash; E = oak. In bottom figure, capital letter = barrels; small letter = chips. n = 328. Top: 70.76% of dispersion (47.64% Can1 and 23.12% Can2); Canonical correlation of 0.9692 and 0.9394, for Can 1 and Can 2, respectively. Bottom: 70.61% of dispersion (50.69% Can1 and 19.92% Can2); Canonical correlation of 0.9902 and 0.9755, for Can 1 and Can 2, respectively.

and ethyl gallate, with negative coefficients, and p-hydroxybenzoic, and trans-caffeic acids with positive, all them higher than 0.55 according to total canonical structure. The presence of gallic and ellagic acids and ethyl gallate at important levels in wines aged in contact to chestnut wood was therefore the most remarkable difference related to wood specie. These high levels of ellagic and gallic acids and ethyl gallate could have an effect on the organoleptic characteristics of beverages since they have been shown to be related to a puckering astringent mouth feel, and bitterness and astringency produced by red wine at taste thresholds lower than the concentrations detected in some beverages after their contact

with chestnut wood. Wines from ash (D) and oak (E) are fully overlapped, both along Can 1 and Can 2, and wines from acacia (C) and cherry (A) are separated among them throughout Can 2, being vanillic, p-hydroxybenzoic and syringic acids the more correlated variables, with positive coefficients. It appears therefore that differences in the amounts of compounds provided by each wood to wine during ageing have greater impact in their nonanthocyanic composition than differences on the evolution of the compounds with high oxidation facility, i.e. on the oxidative condensation and copigmentation processes related to the gas exchange during ageing.

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Comparing red wines aged in barrels and in contact with chips, regardless wood specie (Table 3), the evolution of flavanols and mainly flavonols, and also some hydroxycinnamoyl tartaric acids, allows us to differentiate the wines according to ageing system, since both flavonols and flavanols were significantly higher in wines from barrels, showing very high F-values, specially flavonols. Although the fall of flavanols and flavonols concentrations during wine ageing has been fully reported in the literature, differences in final concentration of these compounds related to oak barrel or chips use were not found. These differences between two ageing systems are probably related to the enhancement of sorption effects of these compounds that could be produced because of the major and irregular surface of contact of chips, such as it has been demonstrated for polyphenolic compounds and aroma components (Barrera-García et al., 2007). Similar results were obtained in Spanish Tempranillo wines aged with chestnut chips and barrels (Alañon et al., 2013), showing major sorption effect for flavonol aglycones and the easily hydrolysable quercetin-3-glucoside. When ANOVA was applied to these data taking into account the two factors, wood specie and ageing mode, the highest F-values were obtained for ellagic, p-hydroxybenzoic, vanillic, and gallic acids, and ethyl gallate, all related to wood specie, followed by myricetin, kaempferol and quercetin-3-glucoside, related to ageing mode. In their CDA graphic representation (Fig. 2, bottom), Can 1 allows to distinguish wines aged in contact with chips (small letters) from those aged in barrels (capital letters), and among these last, those aged in acacia barrels (C). The more correlated variables to canonical function 1 were myricetin, kaempferol and quercetin3-glucoside with positive coefficients (as all other flavanols and flavonols), confirming their relation to ageing mode. The distances among samples throughout Can 2 leads us to distinguish wines aged in contact with chestnut wood (B and b) of everyone else, regardless ageing mode. Also wines aged in cherry barrels (A) are almost completely separated from those aged in other wood barrels, but again wines aged in ash (D) and oak (E) barrels are fully overlapped, such as those aged in contact with no chestnut chips. Ellagic and gallic acids, and ethyl gallate, with negative coefficients, and p-hydroxybenzoic and caffeic acids, with positive, were the more correlated variables to Can 2. It therefore appears that there are two factors that cause differences in the nonanthocyanic phenolic composition of wines: the different amounts of certain compounds provided by each wood to wine during ageing could help in the classification of wines according to ageing wood, and the differences on flavonols and flavanols evolution in wines aged in barrels or in contact with chips according to ageing mode. However, the few differences found in some nonanthocyanic polyphenols in wines aged in contact with oak or with ash were very little significant in the overall studied wine and lead to the no differentiation of these wines.

4. Conclusions The nonanthocyanic phenolic profile could be a useful tool to identify wines aged in contact with cherry, chestnut, and acacia wood, either in barrels or with chips. Thus, aromadendrin, naringenin, taxifolin, isosakuranetin, eriodictyol, and prunin, all them contributed by cherry wood, valoneic acid dilactone contributed by chestnut wood, and dihydrorobinetin, robinetin, 2,4-dihydroxybenzaldehyde, tetrahydroxydihydroflavonol, fustin, butin, trihydroxymethoxydihydroflavonol, 2,4-dihydroxybenzoic acid, tetrahydroxyaurone, butein, and three compounds not fully identified, released from acacia wood, could be used as phenolic markers of ageing with them, for authenticity purposes in different wines, from different grape variety, duration of ageing, and chip dosage.

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Some other differences in nonanthocyanic phenolic composition of aged wines have been detected related to both, wood species and ageing method, such as quantitative differences in certain compounds provided by each wood, or different evolution of flavonols and flavanols in wines aged in barrels or in contact with chips. However, profile of wines aged with oak or ash lead to no significant differentiation. In addition to this study, further studies on organoleptic characteristics, volatile composition, colour features, and monomeric and derived pigments would be necessary, for a better understanding of the influence that no oak woods can have on the quality of aged wines. Acknowledgements This study was financed by Ministerio de Ciencia e Innovación (Project INIA-FEDER RTA2009-0046) from Spain, as well as Tonelería Intona, SL, and Navarra Government (Project: ‘‘Caracterización de maderas alternativas al roble en tonelería para uso alimentario’’). Miriam Sanz received a contract from the Spanish Government through the Torres Quevedo program. The authors gratefully thank Bodegas Torres (Cataluña, Spain) and Bodegas Enate (Huesca, Spain) who kindly provided wine samples. References Alañón, M. E., Schumacher, R., Castro-Vázquez, L., Díaz-Maroto, M. C., HermosínGutiérrez, I., & Pérez-Coello, M. S. (2013). Enological potential of chestnut wood for ageing tempranillo wines Prat II: Phenolic compounds and chromatic characteristics. Food Research International, 51, 536–543. Barrera-García, V. D., Gougeon, R. D., Di Majo, D., De Aguirre, C., Boiley, A., & Chassagne, D. (2007). Different sorption behaviour for wine polyphenols in contact with oak wood. Journal of Agricultural and Food Chemistry, 55, 7021–7027. Cadahía, E., Fernández de Simón, B., Sanz, M., Poveda, P., & Colio, J. (2009). Chemical and chromatic characteristics of tempranillo, cabernet sauvignon, and merlot wines from DO Navarra aged in Spanish and French oak barrels. Food Chemistry, 115, 639–649. Cadahía, E., Muñoz, L., Fernández de Simón, B., & García-Vallejo, C. (2001). Changes in low molecular weight phenolic compounds in Spanish, French and American oakwoods during natural seasoning and toasting. Journal of Agricultural and Food Chemistry, 49, 1790–1798. Caldeira, I., Anjos, O., Portal, V., Belchior, A. P., & Canas, S. (2010). Sensory and chemical modifications of wine brandy aged with chestnut and oak wood fragments in comparison to wooden barrels. Analytica Chimica Acta, 660, 43–52. Canas, S., Leandro, M. C., Spranger, M. I., & Belchior, A. P. (1999). Low molecular weight organic compounds of chestnut wood (Castanea sativa L.) and corresponding aged brandies. Journal of Agricultural and Food Chemistry, 47, 5023–5030. Canas, S., Leandro, M. C., Spranger, I., & Belchior, A. P. (2000). Influence of botanical species and geographical origin on the content of low molecular weight phenolic compounds of woods used in Portuguese cooperage. Holzforchung, 54, 255–261. Castillo-Muñoz, N., Gómez, S., García, E., & Hermosín, I. (2007). Flavonol profiles of Vitis vinifera L. red grapes and their single-cultivar wines. Journal of Agricultural and Food Chemistry, 55, 990–1002. Cerezo, A. B., Espartero, J. L., Winterhalter, P., García-Parrilla, C., & Troncoso, A. M. (2009). (+)-Dihydrorobinetin: A marker of vinegar ageing in acacia (Robinia pseudoacacia) wood. Journal of Agricultural and Food Chemistry, 57, 9551–9554. Cerezo, A. B., Tesfaye, W., Torija, M. J., Mateo, E., García-Parrilla, C., & Troncoso, A. M. (2008). The phenolic composition of red wine vinegar produced in barrels made from different woods. Food Chemistry, 109, 606–615. Chatonnet, P., Boidron, J. N., & Pons, M. (1989). Incidence du traitement thermique du bois de chêne sur sa composition chimique. 2ª partie: Évolution de certain composésen function de l’intensité de brûlage. Connaissance Vigne and Vin, 23, 223–250. Chinnici, F., Natali, N., Sonni, F., Bellachioma, A., & Riponi, C. (2011). Compartive changes in colour features and pigment composition of red wines aged in oak and cherry wood casks. Journal of Agricultural and Food Chemistry, 59, 6575–6582. De Rosso, M., Cancian, D., Panighel, A., Dalla Bedona, A., & Flamini, R. (2009a). Chemical compounds released from five different woods used to make barrels for ageing wines and spirits: Volatile compounds and polyphenols. Wood Science and Technology, 43, 375–385. De Rosso, M., Panighel, A., Dalla Bedona, A., Stella, L., & Flamini, R. (2009b). Changes in chemical composition of a red wine aged in acacia, cherry, chestnut, mulberry, and oak wood barrels. Journal of Agricultural and Food Chemistry, 57, 1915–1920.

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Dueñas, M., Sun, B., Hernández, T., Estrella, I., & Spranger, M. I. (2003). Proanthocyanidin composition in the seed coat of lentils (Lens culinaria). Journal of Agricultural and Food Chemistry, 51, 7999–8004. Fernández de Simón, B., Cadahia, E., Sanz, M., Poveda, P., Perez-Magariño, S., OrtegaHeras, M., et al. (2008). Volatile compounds and sensorial characterization of wines from four Spanish denominations of origin, aged in Spanish rebollo (Quercus pyrenaica Willd.) oak wood barrels. Journal of Agricultural and Food Chemistry, 56, 9046–9055. Fernández de Simón, B., Esteruelas, E., Muñoz, A. M., Cadahía, E., & Sanz, M. (2009). Volatile compounds in acacia, chestnut, cherry, ash, and oak woods, with a view to their use in cooperage. Journal of Agricultural and Food Chemistry, 57, 3217–3227. Flamini, R., Dalla Bedona, A., Cancian, D., Panighel, A., & De Rosso, M. (2007). GC/MSpositive ion chemical ionisation and MS/MS study of volatile benzene compounds in five different woods used in barrel making. Journal of Mass Spectrometry, 42, 641–646. Gambuti, A., Capuano, R., Lisanti, M. T., Strollo, D., & Moio, L. (2010). Effect of ageing in new oak, one-year-used oak, chestnut barrels and bottle on colour, phenolics and gustative profile of three monovarietal red wines. European Food Research and Technology, 231, 455–465. García-Estévez, I., Escribano-Bailón, M. T., Rivas-Gonzalo, J. C., & Alcalde-Eon, C. (2010). Development of a fractionation method for the detection and identification of oak ellagitannins in red wines. Analytica Chimica Acta, 660, 171–176. Hernández, T., Estrella, I., Dueñas, M., Fernández de Simón, B., & Cadahía, E. (2007). Influence of wood origin in the polyphenolic composition of a Spanish red wine ageing in bottle, after storage in barrels of Spanish, French and American oak wood. European Food Research and Technology, 224, 695–705. Hillmann, H., Mattes, J., Brockhoff, A., Dunkel Meyerhof, W., & Hofmann, T. (2012). Sensomics analysis of taste compounds in balsamic vinegar and discovery of 5acetoxymethyl-2-furaldehyde as a novel sweet taste modulator. Journal of Agricultural and Food Chemistry, 60, 9974–9990. Jourdes, M., Michel, J., Saucier, C., Quideau, S., & Teissedre, P. L. (2011). Identification, amounts, and kinetics of extraction of C-glucosidic ellagitannins during wine ageing in oak barrels or in stainless steel tanks with oak chips. Analytical and Bioanalytical Chemistry, 401, 1531–1539. Kozlovic, G., Jeromel, A., Maslov, L., Pollnitz, A., & Orlic, S. (2010). Use of acacia barrique barrels- Influence on the quality of Malvazika from Istria wines. Food Chemistry, 120, 698–702.

Monagas, M., Suárez, R., Gómez-Cordovés, C., & Bartolomé, B. (2005). Simultaneous determination of nonanthocyanin phenolic compounds in red wines by HPLCDAD/ESI-MS. American Journal of Enology and Viticulture, 56, 139–147. Rodriguez, S., Suarez, B., Diñero, Y., Del Valle, P., & Piccinelli, A. (2010). Alternative woods for ageing distillates – An insight into their phenolic profiles and antioxidant activities. Food Science Biotechnology, 19, 1129–1134. Sanz, M., Cadahía, E., Esteruelas, E., Muñoz, A. M., Fernández de Simón, B., Hernández, T., et al. (2010a). Phenolic compounds in cherry (Prunus avium) heartwood with a view to their use in cooperage. Journal of Agricultural and Food Chemistry, 58, 4907–4914. Sanz, M., Cadahía, E., Esteruelas, E., Muñoz, A. M., Fernández de Simón, B., Hernández, T., et al. (2010b). Phenolic compounds in chestnut (Castanea sativa Mill.) heartwood. Effect of toasting at cooperage. Journal of Agricultural and Food Chemistry, 56, 9631–9640. Sanz, M., Fernández de Simón, B., Cadahía, E., Esteruelas, E., Muñoz, A. M., et al. (2012a). LC-DAD/ESI-MS/MS study of phenolic compounds in ash (Fraxinus excelsior L. and F. americana L.) heartwood. Effect of toasting intensity at cooperage. Journal of Mass Spectrometry, 47, 905–918. Sanz, M., Fernández de Simón, B., Cadahía, E., Esteruelas, E., Muñoz, A. M., et al. (2012b). Polyphenolic profile as a useful tool to identify the wood used in wine ageing. Analytica Chimica Acta, 732, 33–45. Sanz, M., Fernández de Simón, B., Esteruelas, E., Muñoz, A. M., Cadahía, E., et al. (2011). Effect of toasting intensity at cooperage on phenolic compounds in acacia (Robinia pseudoacacia) heartwood. Journal of Agricultural and Food Chemistry, 59, 3135–3145. Sanz, M., Fernández de Simón, B., Esteruelas, E., Muñoz, A. M., Cadahía, E., et al. (2012c). Polyphenols in red wine aged in acacia (Robinia pseudoacacia) and oak (Quercus petraea) wood barrels. Analytica Chimica Acta, 732, 83–90. Spillman, P. J., Pollnitz, A. P., Liacopoulos, D., Skouroumounis, G. K., & Sefton, M. A. (1997). Accumulation of vanillin during barrel-aging of white, red, and model wines. Journal of Agricultural and Food Chemistry, 45, 2584–2589. Torija, M. J., Mateo, E., Vegas, C. A., Jara, C., González, A., Poblet, M., et al. (2009). Effect of wood type and thickness on acetification kinetics in traditional vinegar production. International Journal of Wine Research, 1, 155–160.