Accepted Manuscript Modulation Of The Phenolic Composition And Colour Of Red Wines Subjected To Accelerated Ageing By Controlling Process Variables J.M. González-Sáiz, I. Esteban-Díez, S. Rodríguez-Tecedor, N. Pérez-delNotario, I. Arenzana-Rámila, C. Pizarro PII: DOI: Reference:

S0308-8146(14)00719-5 http://dx.doi.org/10.1016/j.foodchem.2014.05.016 FOCH 15797

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

6 January 2014 27 April 2014 6 May 2014

Please cite this article as: González-Sáiz, J.M., Esteban-Díez, I., Rodríguez-Tecedor, S., Pérez-del-Notario, N., Arenzana-Rámila, I., Pizarro, C., Modulation Of The Phenolic Composition And Colour Of Red Wines Subjected To Accelerated Ageing By Controlling Process Variables, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/ j.foodchem.2014.05.016

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MODULATION OF THE PHENOLIC COMPOSITION AND COLOUR OF RED WINES SUBJECTED TO ACCELERATED AGEING BY CONTROLLING PROCESS VARIABLES

J.M. González-Sáiz, I. Esteban-Díez, S. Rodríguez-Tecedor, N. Pérez-del-Notario, I. ArenzanaRámila, C. Pizarro* Department of Chemistry, University of La Rioja, C/ Madre de Dios 51, 26006, Logroño, La Rioja, Spain; * Corresponding author. Tel.: +34 941299626; fax: +34 941299621. E-mail address: [email protected]

Abstract The aim of the present work was to evaluate the effect of the main factors conditioning accelerated ageing processes (oxygen dose, chip dose, wood origin, toasting degree and maceration time) on the phenolic and chromatic profiles of red wines by using a multivariate strategy based on experimental design methodology. The results obtained revealed that the concentrations of monomeric anthocyanins and flavan-3-ols could be modified through the application of particular experimental conditions. This fact was particularly remarkable since changes in phenolic profile were closely linked to changes observed in chromatic parameters. The main strength of this study lies in the possibility of using its conclusions as a basis to make wines with specific colour properties based on quality criteria. To our knowledge, the influence of such a large number of alternative ageing parameters on wine phenolic composition and chromatic attributes has not been studied previously using a comprehensive experimental design methodology.

Keywords: Oak chip; Micro-oxygenation; Experimental design; Anthocyanins; Flavan-3-ols; Colour. 1

1. Introduction Maturation of red wines in oak barrels is a traditional winemaking practice aimed at enhancing wine quality. During ageing, wine undergoes important modifications, due to the release of compounds from the oak (volatile compounds, phenolic acids and tannins) and the diffusion of oxygen through wood pores. This oxidative ageing leads to high quality wines, with enhanced organoleptic characteristics (including improvement of mouthfeel complexity, reduction of astringency and aroma enrichment) and more stable colour (De Beer, Joubert, Marais, Du Toit, Fourie, & Manley, 2008). Specifically, anthocyanins are the phenolic compounds most strongly related to the chromatic characteristics of young red wines. The evolution and stabilisation of red wine colour during ageing is strongly associated with the complex reactions of anthocyanin copigmentation and condensation between anthocyanins and other phenolic compounds like flavan-3-ols (Timberlake, & Bridle, 1976; Boulton, 2001; Gómez-Cordovés, & González-SanJosé, 1995; Singleton, 1987; Bakker & Timberlake, 1997). However, traditional ageing is costly, requires long time periods, a large space in the winery, and is highly demanding in maintenance terms. These facts have prompted the development of simpler and more affordable alternative ageing systems, such as the use of oak wood pieces, commonly known as oak chips that accelerate the ageing process and reduce costs while preserving wine quality (García-Carpintero, Gómez Gallego, Sánchez-Palomo, & González Viñas, 2011). Although no restrictions have been imposed on the use of oak chips in new world winemaking countries (South Africa, Australia and Chile), their application in Europe has been shrouded in controversy. European legislation did not approve and regulate this practice until October 2006 (EC, 2006). Furthermore, it should be noted that in order to obtain wines with improved colour and aroma properties, it is also necessary to imitate the natural uptake of oxygen that occurs in barrels through the application of controlled micro-oxygenation (MO) (Gómez-Plaza & Cano-López, 2011; Ferrarini, Girardi, De Conti, & Castella,. 2001; Nevares & del Álamo, 2008, 2

Arapitsas, et al.,2012, Caillé et al., 2010). Therefore, the combined addition of controlled doses of oxygen and oak chips aims precisely to mimic the conditions that occur during traditional ageing in oak barrels. The extraction rates of wood-related compounds that may interact with anthocyanins depend on the particular characteristics of the wood used (botanical and geographical origin, toasting degree and size), chip doses and the contact time between wood pieces and wine (Del Alamo Sanza, & Nevares Domínguez, 2006; De Coninck, Jordão, Ricardo-Da-Silva, & Laureano, 2006; Cabrita, Barrocas Dias, Costa Freitas, 2011). Moreover, oxygen plays an important role in increasing colour intensity and stability through, for instance, the formation of stable pigments of monomeric anthocyanins and flavanols by direct condensation or through ethyl linkages (Timberlake, 1976; Cano-López, M.; Pardo-Minguez, López-Roca, & Gómez-Plaza, 2007; Wildenradt, & Singelton, 1974; Atanasova, Fulcrand, Cheynier, & Moutounet, 2002, Sousa, Mateus, Perez-Alonso, Santos-Buelga & De Freitas, 2005; Sousa, Mateus, Silva, GonzálezParamás, Santos-Buelga & De Freitas, 2007). In order to avoid excessive amounts of oxygen, the doses supplied have to be specifically adapted to each wood product (Del Alamo et al. 2010) Multiple approaches have evaluated the influence of alternative oenological ageing systems on wine phenolic profiles (McCord, 2003; Pérez-Magariño, Ortega-Heras, Cano-Mozo, & GonzálezSanjosé, 2009; Del Álamo, Nevares, Gallego, Fernández de Simón & Cadahía, 2010; CejudoBastante, Hermosín-Gutiérrez, & Pérez-Coello, 2011; Cejudo-Bastante, Hermosín-Gutiérrez, & Pérez-Coello, 2012; Rudnitskaya, Schmidtke, Delgadillo; Legin, & Scollary, 2009; Sartini, Arfelli, Fabiani, & Piva, 2007, García-Puente Rivas, Alcalde-Eon, Santos-Buelga, Rivas-Gonzalo & Escribano-Bailón, 2006; Gay, et al., 2010; Schmidtke et al., 2010). However, despite the importance of the main factors governing the accelerated ageing process (oxygen doses, chip doses, wood origin, toasting degree and maceration time), and the possible significant interaction effects between them, no studies have tackled this problem using a rigorous methodology to

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consider an extended number of influential factors. Only Rudnitskaya et al. (2009) have evaluated the influence of micro-oxygenation, chip maceration and vintage on wine phenolic profile using experimental design methodology. Nevertheless, the full factorial design approach proposed by the aforementioned study did not evaluate the influence of other relevant factors, such as doses of chip supplied, maceration time or wood characteristics. To our knowledge this is therefore the first paper to present an in-depth multivariate study that seeks to gain a full insight into the impact of simultaneously applying micro-oxygenation and chip maceration on the phenolic and chromatic profiles of wines, so permitting the accurate control of accelerated ageing processes in order to develop wines with specific colour profiles based on quality criteria. The multivariate approach proposed (based on combining experimental design and response surface methodologies) allowed us to evaluate the influence of the process variables that have the greatest impact on the evolution of phenolic compounds (anthocyanins and flavan-3-ols) and colour parameters during accelerated ageing with oak chips and microoxygenation, taking into account interaction effects between control variables. The main strength of this comprehensive study is that the acquired knowledge would help oenologists or winemakers to impress a specific phenolic fingerprint on their wines and to modulate colour to match quality criteria, so enabling them to produce wines according to high quality standards and consumer preferences within a Quality by Design (QbD) framework. It is important to make clear that the accelerated ageing processes were conducted in a pilot-scale system under conditions similar to those used in industrial-scale units. Given the good reproducibility of the pilot-scale experiments, this means that our findings could be transferred to industrial applications.

2. Material and methods 2.1. Chemicals and standard solutions

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Malvidin-3-O-glucoside and (+)-catechin, were purchased from Extrasynthèse (Genay, France). (‒)-Epicatechin was supplied by Aldrich Chemie (Steinheim, Germany). The purity of all the standards ranged from 97.5% to 99%. Ammonium di-hydrogen phosphate (NH4H2PO4), phosphoric acid (H3PO4) and acetonitrile were purchased from Scharlau (Barcelona, Spain). Ultrapure water was obtained from a MilliQ system (Millipore, Bedford, MA). Individual stock standard solutions of each compound were prepared in ethanol at a concentration level of 8 g L‒1. Subsequently, working solutions were prepared by diluting different amounts of each stock standard solution. Standard and work solutions were stored in darkness at 4 °C. 2.2. Wine samples and alternative ageing system The red young single-variety wine used in this study was made from the native Spanish Vitis Vinifera Tempranillo grape variety and supplied by Bodegas Riojanas S.A (Cenicero, La Rioja, Spain), whose wines are protected by the Qualified Designation of Origin Rioja. The oak pieces used in this study were commercially available products supplied by Dolmar, S.L. (Haro, La Rioja, Spain). A total of eight different types of oak chips were considered, including two different origins at four different toasting degrees. As regards their origin, American chips were from the Northern Appalachian Forests in Pennsylvania and Ohio, and the French chips came from forests in central France. The different toasting intensities considered for each type of wood were: medium (M), medium plus (M+), heavy (H) and heavy plus (H+). The wines in contact with the oak chips were aged using an SAEn 5000 microoxygenation system (PARSEC, Florence, Italy) equipped with six stainless-steel tank units (145 L capacity and 2 m in height) individually connected to remote control units that enabled temperature and oxygen doses to be controlled throughout the process. Temperature was set and kept at 18 ºC and different oxygen doses were supplied, depending on the experiment. Samples were taken from each tank after 1, 2, 3, 4 and 5 weeks and at the end of the maceration

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period (6 weeks). A control sample was also taken just before ageing started (week 0). Samples were bottled and stored at 4 ºC until analyses were performed. Fig. S1 shows a scheme for each tank. 2.3. High-performance liquid chromatography (HPLC) analysis Chromatographic analyses were performed using an Agilent 1100 series system (Agilent Technologies, Waldbronn, Germany) equipped with a high-pressure gradient pump, an autosampler, a photodiode array detector (DAD) and a fluorescence detector. The column used to perform the chromatographic separation was a Zorbax SB-C18, 250 mm × 4.6 mm ID with 5 µm particle size (Agilent), thermostatted at 20 ºC and protected by a guard column of the same material. Prior to analysis, wine samples were tempered at room temperature and filtered through a 0.45-µm nylon membrane (Aldrich Chemie ,Steinheim, Germany), discarding the first 5‒6 drops. Then 10 µl of each sample were injected in triplicate. The chromatographic method, based on the use of a ternary mobile phase gradient developed by Gómez-Alonso et al. (2007) was employed. HPLC-DAD chromatograms were recorded at 520 nm for anthocyanins. The content of several flavan-3-ols ((+)-catechin and (‒)-epicatechin) of red wines was also determined by HPLC with a fluorescence detector set to 280/320 nm excitation/emission. The identification of malvidin3-O-glucoside, (+)-catechin and (-)-epicatechin was based on the retention times of pure compounds. The other anthocyanins identified (delphinidin-3-O-glucoside, cyanidin-3-Oglucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, delphinidin-3-O-acetylglucoside, malvidin-3-O-acetylglucoside and peonidin-3-O-p-coumarylglucoside) were tentatively identified according to the elution times reported by Gomez-Alonso et al. (2007). 2.4. Data pre-processing Chromatograms were exported from Agilent Chemstation software Version B.03.02 as comma-delimited Microsoft Excel® files. Although initially whole chromatograms were stored for

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each wine sample, the regions where no peaks were detected were removed from further analysis. Thus, the working range for anthocyanins was limited to the interval 20–55 min, whereas for flavan-3-ols, the region between 19 and 32 min was retained (Figure S1). Then, chromatograms from each detector were separately processed following the same strategy according to the steps described below. Firstly, a second-order polynomial baseline correction was applied to all chromatograms over the selected ranges of retention times prior to calculations, in order to compensate for signal drifts. The rapid and versatile Interval Correction Optimised Shifting algorithm (icoshift) (Tomasi, Savorani, & Engelsen, 2011) was applied to align the signals, due to the existence of significant differences in retention times between different injections, following a two-step alignment strategy. Initially, a rough correction was performed using the whole signal range as a target signal. Although this pre-alignment approach considerably improved chromatogram alignment, it was not entirely effective when it came to correcting the large shifts found between signals over the whole working range. Thus, in a second step, the icoshift algorithm was applied again on the pre-aligned chromatograms by defining custom intervals containing the still misaligned peaks. Once the signals were satisfactorily aligned, the areas of the peaks in each chromatogram were automatically calculated using an interactive keypress-operated function called iPeak, which allows peak detection criteria to be adjusted for a specific pre-defined peak type (Gaussian in this case). After adjusting the peak detection parameters (% amplitude threshold, slope threshold, smooth width, fit width and peak density) in order to detect and integrate the target peaks, the function returned a matrix containing the position, height, width and area of each peak. In order to quantify the actual concentrations of malvidin-3-O-glucoside, (+)-catechin and (‒)-epicatechin in wine samples, calibration curves ranging from 0.5 to 300 mg L‒1 were constructed using suitable work solutions. Calibration graphs for each compound analysed had

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linear regression coefficients between 0.996 and 0.998. Moreover, the calibration graph obtained for malvidin-3-O-glucoside was used to quantify the non-commercial anthocyanins identified (Gómez-Alonso et al., 2007; Wirth et al., 2012). 2.5. Colour evaluation The chromatic parameters in the CIELAB colour space were calculated using the simplified method developed by Ayala et al. (1999) (Método Simplificado para el Color de Vinos, MSCV®), which consisted in recording the absorbance values at four specific wavelengths (450, 520, 570 and 630 nm) and using them to compute the tristimulus measurements. Thus, the main CIELAB parameters (a*, b*, L*, C* and H*) were calculated. The a* value represents the difference between green (a* < 0) and red (a* > 0), b* indicates the difference between blue (b* < 0) and yellow (b* > 0), L* describes the lightness of the colour (L* = 0 black and L* = 100 colourless), C* indicates the chroma or saturation, (C* = 0 pale colours and C* = 60 intense colours) and H* is the hue angle. 2.6. Experimental design Taking into account that the phenolic profile and, consequently, the colour of wines evolves during oxidative ageing, it is essential to simultaneously evaluate the influence that chip maceration and micro-oxygenation related factors have on the evolution of these compounds and parameters. Thus, a chemometric approach based on experimental design methodology was applied to investigate the effects of varying the main factors involved in the ageing process studied (i.e. oxygen doses, chip doses, wood origin, toasting degree and maceration time) on wine phenolic and chromatic profiles. Initially, four factors (oxygen doses, chip doses, wood origin and toasting degree) were considered to develop the hybrid design used that involved a total of 18 experiments (Table 1). The first one (E1) was an axial point with toasting degree at maximum level. The other eight experiments came from a factorial design (23) including all the factors but one (toasting degree),

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which was kept at a different discrete level (E2‒E9); six experiments came from a star design for the same variables, where the toasting degree factor was kept at a different constant level (E10‒E15), and the three remaining experiments corresponded to centre points (E16‒E18). In fact, including and replicating centre points has a double purpose in experimental design, providing a way to check for curvature in the experimental domain (and to improve the estimation of certain coefficients such as those of the squared terms in the model), and serving to estimate experimental repeatability (Lewis, Mathieu, & Phan-Tan-Luu, 1999). Finally, in order to evaluate the effect of maturation time, an extra factor labelled as “time” was included in the design, in such a way that each wine sample was additionally characterised by its corresponding ageing time. Hybrid designs are particularly recommended when performing studies with limited resources and when the experimental domain is well known. It should be stressed that this type of experimental design is highly efficient, showing a ratio between the number of parameters and the number of experiments required (R-efficiency) as near to 100% as possible. The experimental domain for each factor was defined taking into account that the aim of the combined application of oak chips and micro-oxygenation was to mimic the traditional evolution of wine in oak barrels. Thus, the doses of oxygen added had to simulate, as closely as possible, the slow and continuous diffusion of oxygen through wood pores, while the amount of chips supplied had to mimic the surface/volume ratio in barrels. Specifically, Gómez et al. (2011) suggested oxygen doses between 1 and 6 mL L−1 month−1. In the present study, oxygen doses ranged from 1 to 7 mL L−1 month−1. The oxygen doses rate ranged from 1.5 and 7.5 g L-1 in accordance with Pérez-Coello et al. (2000) and Gómez García-Carpintero et al. (2012). Moreover, the influence of wood origin was evaluated by considering two different oak wood origins (American and French) and blends of them. The percentage of American oak wood was studied from 0 to 100%. The effect of toasting was evaluated at four toasting levels: medium (M), medium plus (M+), heavy (H) and heavy plus (H+). The experimental conditions are presented in Table 1.

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Finally, the time factor was evaluated at seven different ageing stages, from week 0 (before ageing) to week 6 (end of maceration), according with previous studies (Cejudo-Bastante et al., 2011; Ortega-Heras, Pérez-Magariño, Cano-Mozo & González-San José, 2010). Thus, a total of 126 wine samples were analysed. All experiments were performed randomly to minimise the effects of uncontrolled factors that could have introduced bias into the measurements. The experimental results obtained were fitted to a polynomial quadratic equation with the following form: n

n

n

Y = b0 + ∑ bi X i + ∑∑ bij X i X j i =1

i =1 j =1

where Xi were the studied factors (X1: oxygen doses; X2: chip doses; X3: wood origin; X4: toasting degree; and X5: maceration time) and the response Y was the concentration of each phenolic compound analysed or the value of each chromatic parameter considered. The model coefficients for each response were estimated by least squares linear regression and the constructed models were analysed and validated by analysis of variance (ANOVA) using NEMRODW software.

2.7. Software Baseline correction pre-treatment was performed using the baselinew function available in PLS_Toolbox 4.2 (Eigenvector Research, 2007). Signal alignment was carried out using the icoshift tool for Matlab®, which is an open source program that can be downloaded from www.models.kvl.dk. Peak areas were calculated using the ipeak function for Matlab®, which is free

for

download

from

http://terpconnect.umd.edu/~toh/spectrum/PeakFindingandMeasurement.htm. Moreover, CIELAB parameters were calculated using MSCV® software, which is available for download at www.unirioja.es/dptos/dq/fa/colour/color.html. Finally, the experimental design and response

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surfaces were constructed and analysed using the NEMRODW statistical package (Mathieu, Nony & Phan-Tan-Luu, 2000).

3. Results and discussion 3.1. Experimental design The standard deviation and coefficients of regression for the models obtained are reported in Table S1. The proposed mathematical models were significant for all the compounds and chromatic parameters studied. Since most of the interaction coefficients were significant (Table 2), the effects of the factors could not be studied independently. Thus, in order to analyse possible synergistic or antagonistic effects, response surfaces had to be computed and visualised. The response surfaces obtained were examined separately and discussed for each family of phenolic compounds (anthocyanins and flavan-3-ols) and for CIELAB chromatic parameters. 3.2. Anthocyanins Eight monomeric anthocyanins were tentatively identified in this study: five of them were the 3-O-monoglusides characteristics of Vitis Vinifera (delphinidin-3-O-glucoside (Df-3-Gl), cyanidin-3-O-glucoside (Cn-3-Gl), petunidin-3-O-glucoside (Pt-3-Gl), peonidin-3-O-glucoside (Pn3-Gl) and malvidin-3-O-glucoside (Mv-3-Gl)); another two were anthocyanidin-acetyl-3-glucosides (delphinidin-3-O-acetylglucoside (Df-3-Gl-Ac), and malvidin-3-O-acetylglucoside (Mv-3-Gl-Ac)); and the last one was an anthocyanidin coumaryl-3-glucoside (peonidin-3-O-p-coumarylglucoside (Pn-3-Gl-Cm)). Table 3 summarises the levels of all the anthocyanins determined. In all the wine samples analysed, Mv-3-Gl was the predominant monomeric anthocyanin. When the response surfaces corresponding to the significant coefficients of these compounds were analysed, similar behaviours were observed for all of them, except for Df-3-GlAc. The response surfaces corresponding to Mv-3-Gl, the most representative anthocyanin in

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wine, were used to illustrate the most informative common behaviours (Fig. 1a-1e). The results corresponding to Df-3-Gl-Ac are examined separately at the end of this section. Most interaction effects involving toasting degree were found to be significant for the anthocyanins studied. In particular, the analysis of the interaction effect between toasting degree and maceration time (b45) showed that, in general, the concentration of anthocyanins diminished when maceration time was increased and toasting degree decreased (Fig. 1a). We also observed that the influence of time was gradually less pronounced as the toasting degree of the oak chips increased. The loss of significant influence of time occurred more quickly when heavily toasted chips were used. Thus, the largest decrease in the concentration of anthocyanins was found when medium to medium-plus toasted chips were infused into the wine, which was then aged for around four weeks (the impact of extending ageing beyond the first four weeks of treatment was very limited). There was a progressive decrease in the concentrations of monomeric anthocyanins over time, attributable to their involvement in a wide variety of complex mechanisms occurring during ageing (including self-association and copigmentation, formation of polymeric anthocyanins with flavan-3-ols and proanthocyanidins, as well as the formation of new pigments such as pyranoanthocyanins) (Atasanova, et al., 2002). On the other hand, the toasting level undoubtedly affects the wood chemical composition and consequently the amount of extractable compounds. Thus, in general, the heavier the toasting the more complex are the flavours released into the wine (Pérez-Magariño et al., 2009; Pizarro, Rodríguez-Tecedor, Esteban-Díez, Pérez-Del-Notario & González-Sáiz, 2014). At the same time, however, less tannins (structure) are extracted, resulting in wines with less ageing potential (Chira & Teissedre, 2013). The remaining significant interaction effects involving toasting degree confirmed the trends indicated for different toasting levels. The response surface for the oxygen doses–toasting degree interaction effect (b14) showed one of the most complex interactions between process variables observed in this study

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(Fig. 1b). Thus, different combinations of factor levels led to different degrees of conversion of the anthocyanins present in the wine. The use of high oxygen doses combined with medium to medium-plus toasting levels resulted in the lowest anthocyanin concentrations. Likewise, when the lightest toasting degrees were combined with low oxygen doses, a moderate conversion of anthocyanins was achieved. We also noted, that the fall in anthocyanin concentration observed when working at relatively high rates of oxygen supply and with a light toasting on the chips was more gradual as the toasting level increased, resulting in a relatively high anthocyanin content when medium to high oxygen doses were used in combination with a heavy-plus toast. Finally, the use of low oxygen doses and heavy-plus toasted chips was also associated with a relatively high conversion of monomeric anthocyanins. At this point, it should be emphasised that changes in the anthocyanin content during ageing are related to many processes competing for the same available substrates, in such a way that the final state it reaches is the result of a complex equilibrium between all the reactions and mechanisms involved, which would be very sensitive to variations in experimental conditions. Additional information is required before we can try to rationalise the trends observed for the b14 interaction effect. For this reason, this subject will be raised again when the colour changes induced by the application of the aforementioned combinations of process variables are analysed. A deeper analysis of the b14 interaction effect indicated that the interpretation of the impact of micro-oxygenation on the concentrations of monomeric anthocyanins varied slightly depending on wood origin. For blends richer in French oak for example, it was necessary to apply the highest oxygen doses to achieve the largest loss of anthocyanins (Fig. 1c). However when 100% American oak chips were used (Fig. 1d), we noted that the oxygen levels needed to reach the corresponding lowest concentration point (keeping the other factors constant) were substantially lower. The higher demand for oxygen in wines aged with French chips was surely associated with the different structure of the two types of wood (Del Álamo, et al., 2010). French

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oak is less dense and more porous than American oak, making French wood more easily impregnated. Consequently, a larger surface area available in the case of French oak would imply higher oxygen consumption. The examination of the response surface corresponding to the chip doses–wood origin interaction effect (b23) highlighted the strong influence of wood origin on the evolution pattern of anthocyanin concentration (Fig. 1e). For French oak, the levels of target compounds decreased when chip doses were increased, whereas, when American oak chips were predominant, lower levels of monomeric anthocyanins were achieved when low to medium chip doses were supplied. This difference could be justified by the difference in the extractable phenolic fraction between both types of wood. American oak wood is very poor in hydrolysable tannins (ellagitannins), while French oak wood contains significantly higher concentrations of extractable tannins (Chatonnet & Dubourdieu, 1998). In this context, the progressive increase in chip doses might have a twofold effect. On the one hand, it provided a larger effective extraction surface, which could result in a more intense extraction of phenolic compounds, and, consequently, in promoting polymerisation between anthocyanins and tannins, and on the other, it acted to strengthen the diffusion gradient between wood and wine, thus facilitating the transfer of substances into the wine. Both complementary effects were expected to be particularly pronounced in the case of French oak chips as a consequence of its richness in diffusible phenolic compounds, and were proposed as explanations for the observed trend. The response surfaces corresponding to the oxygen doses–time (b15) and chip doses– time (b25) interaction effects served to corroborate an important conclusion stated above, i.e., that from four weeks ageing onwards, there was little change in the concentrations of monomeric anthocyanins (Fig. not shown), suggesting that time was not a significant factor beyond this point. The origin-dependent behaviours of the anthocyanin content when varying oxygen and chip

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doses have already been discussed when interpreting the response surfaces for the b14 and b23 interactions effects. Finally, when analysing the only anthocyanin whose evolution during accelerated ageing was, to a certain extent, different from the rest, we observed that a reduction in Df-3-Gl-Ac concentration was enhanced when maceration time was increased continuously (without reaching a steady state), and when extreme values for oxygen doses (either low or high) were used (even though medium oxygen doses appeared to need longer ageing times to achieve comparable low concentrations). As regards chip doses and toasting degree factors, different sets of process variables led to a decrease in this compound depending on wood origin. In the case of French oak, the Df-3-Gl-Ac content was reduced by increasing the chip doses or by increasing the toasting level, in such a way that the lowest concentration detected was found when high chip doses were combined with heavy-plus toast (although quite low contents were also achieved for other combinations of these factors, e.g., high chip doses and medium to medium-plus toast, and low chip doses and heavy-plus toast). On the other hand, when American oak was used, the use of a medium toast maintained the compound at low concentrations practically across the whole range of chip doses. In spite of the fact that certain differences were found between this minor compound and the other anthocyanins as regards the effect of variation in some process variables, it should be noted that the common combination of factors promoting a decrease in the concentration of most of the anthocyanins was also related to low levels of Df-3-Gl-Ac in wine. 3.3. Flavan-3-ols The flavan-3-ols identified in the wines subjected to accelerated ageing were (+)-catechin and (‒)-epicatechin. As indicated previously, flavan-3-ols are a family of phenolic compounds highly prone to react with anthocyanins during ageing, in such a way that both classes of compounds were expected to show an intimate relationship during accelerated ageing. The

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concentration ranges detected in the present study for these compounds are summarised in Table 3. The following interaction coefficients were found to be significant for both (+)-catechin and (‒)-epicatechin: oxygen doses–wood origin (b13), chip doses–wood origin (b23), oxygen doses–toasting degree (b14), chip doses–toasting degree (b24). The evolution patterns observed when analysing the response surfaces for these interaction effects were, unsurprisingly, in agreement with those previously described for anthocyanins and, therefore, will not be discussed again in this section. In this context and taking into account the high reactivity of (+)-catechin and (‒)-epicatechin in oxidative environments, lower concentrations of these compounds were expected at higher oxygen doses (Sartini et al., 2007; Ferrarini et al., 2001). The significance of the interaction effects involving toasting degree, and the strong influence of wood origin, could be justified bearing in mind the acetaldehyde-mediated condensation reactions of flavanols with both other flavanols and anthocyanins, and the reactions between (+)-catechin and wood aldehydes to form oaklins (Sousa et al., 2005; Sousa et al., 2007). All these processes depend on the relative amounts of both the monomeric anthocyanins in the wine and the compounds released by the wood and, consequently, on both the toasting degree and the type of wood used. As regards ageing time, certain differences were encountered between (‒)-epicatechin and (+)-catechin as regards the effects found to be significant in each case. The time factor (b5) had a significant influence on (‒)-epicatechin, but there were no significant interaction effects involving this factor. Its influence on the concentration of (‒)-epicatechin could therefore be studied independently using the optimal path graph (Fig. 2a). This graph revealed that the concentration of (‒)-epicatechin decreased with time up to about the fourth week of treatment, after which no further variations were observed. We also found that only the toasting degree–time interaction effect (b45) was significant for (+)-catechin (Fig. 2b). The inspection of the corresponding response surface showed that increasing maceration time caused a decrease in

16

the concentration of (+)-catechin, although this drop in the (+)-catechin content was less marked as the toasting degree increased. We also observed that, after about four weeks’ ageing, the impact of time was almost negligible. The toasting level of the oak chips seemed to have a quite limited effect on the concentration of (+)-catechin during the first two weeks of maceration. However, from the third-fourth week of treatment, and concurrently with the decline in influence of time, the levels of (+)-catechin increased in a much clearer way when increasing toasting degree. Thus, when the two factors were combined, the lowest (+)-catechin contents were found when using medium to medium plus toasting degrees and a maceration time of around three or four weeks. These findings were in line with trends previously reported in the analysis of the evolution of the anthocyanins. 3.4. Evolution of CIELAB colour parameters The ranges for all the CIELAB parameters computed in the present study (a*, b*, L*, C* and H*) are shown in Table 3. When the response surfaces for a* and C* parameters were examined, similar patterns were observed. Hence, the most representative interaction effects found to be significant for both colour measurements would be discussed using the response surfaces obtained when using the a* parameter as a response variable (Fig. 3a-c). The fact that not only the main effect of the time factor, but all the interaction effects involving time were significant indicated a clear influence of the ageing time on the values of the a* and C* parameters, as was previously observed by Bakker and Timberlake (1997). For a more detailed illustration of this dependence, we analysed the response surface corresponding to b35 interaction for the a* parameter (Fig. 3a). This colour parameter showed an origin-dependent evolution as the ageing process progressed. Thus, the use of French oak chips appeared to lead to only a slight decrease in the a* colour value during the first two weeks of ageing, remaining stable thereafter at relatively high values. On the contrary, in the case of American oak chips, the

17

decreasing trend in the a* value over time was more marked and continued throughout the whole accelerated ageing process. The different behaviour of the two types of wood might again be due to their relative content in hydrolysable tannins, which explains why the French oak chips compensate better for potential colour losses by the formation of new pigments. The analysis of the response surface corresponding to the oxygen doses–toasting degree interaction effect (b14) for the a* parameter (Fig. 3b) and the comparison of the patterns observed with those previously outlined when evaluating the same interaction effect for anthocyanins could be used to shed light on the nature of the underlying transformations governing the observed changes. When lightly toasted oak chips were used, as the oxygen dose increased, we observed a progressive decrease in the red/green component. This behaviour could be related with our previous observations on the influence of the toasting level on wood composition. Thus, the release of tannins from medium toasted chips could favour the partial conversion of anthocyanins to form polymeric pigments, which contribute to colour stabilisation. Increasing doses of oxygen, combined with light toasting levels, probably contributed to more extensive formation of pyranoanthocyanins and ethyl-bridged compounds. All these anthocyanin derivatives formed during ageing may be associated with the progressive shift of the red-purple colour of wines to a more orangey colour. Likewise, when heavy to heavy-plus toasted chips were used in combination with high oxygen doses, the red colour of wine appeared to remain largely unchanged. In this context, it should be taken into account that oak chips which have undergone intense toasting processes would contain higher contents of compounds produced by the thermal degradation of cellulose and lignin (including aldehydes) than chips than have been lighter toasted. In this way, the competition for the available oxygen, together with the low release of hydrolysable tannins from heavy-plus toasted chips, would have surely contributed to the maintenance of appreciable concentrations of free anthocyanins in the wine, thus buffering colour changes. Nevertheless, when oxygen doses were decreased and heavy-plus toasted oak chips

18

were used, a gradual loss of the red colour component was evidenced, in such a way that relatively low a* values were observed for low to medium oxygen doses (below 4 mL L-1 month-1). In this context, the decrease in both monomeric anthocyanins and a* values, when using low oxygen doses and a heavy-plus toasting, could suggest that, under such experimental conditions, condensation reactions mediated by aldehydes (and/or reactions of anthocyanins with low molecular weight compounds to generate pyranoanthocyanins) could occur extensively, leading to the formation of new pigments responsible for the observed colour loss. It should be noted that all these complex interactions between the two factors at different levels led to the appearance of a rather wide experimental region corresponding to low values of the a* parameter (specifically, the use of heavily toasted chips at the lower range of oxygen doses, and the selection of mediumplus to heavy toasting degrees and higher oxygen doses), which could suggest the occurrence of reactions leading to the generation of pyranoanthocyanins in that “minimum” response zone. When the chip doses–toasting degree interaction effect (b24) was studied, two contrasting situations resulted in low values for the a* parameter: high chip doses combined with medium toasting degrees, and low chip doses coupled with heavy-plus toasted chips (Fig. 3c). The fact that, at high chip doses and medium toasting degrees, the wine suffered a decline in the red component could suggest that, when creating a high diffusion gradient between wood and wine, the extraction of phenolic compounds from wood was intensified, in such a way that an excess of tannins led to an orange colour which masked the red colour of anthocyanins and other polymeric pigments. The same argument discussed above to explain the decrease in the redgreen component when using heavily toasted chips (while maintaining medium doses of oxygen) also explains the correlation observed between toasting level and the a* parameter. The b* and H* colour parameters were expected to show similar evolution patterns during ageing. In fact, in most cases, the behaviours observed when analysing the respective response surfaces corresponding to the interaction effects found to be significant were similar for both

19

components. Consequently, the evolution profiles of both parameters were jointly analysed, in such a way that the response surfaces corresponding to the b* parameter were presented and discussed as representative of them both. The only striking difference observed had to do with the specific interaction effect chip doses–time (b25) for b*, and oxygen doses–time (b15) and toasting degree–time (b45) for H*. We therefore decided to discuss these interaction effects, and their respective response surfaces, separately for each parameter. As regards b*, the interaction effect between chip doses and maceration time (b25) once again highlighted the sharp difference in the observed behaviour on the basis of wood origin. When French oak was predominant, the increase of the b* component with increasing maceration time only became apparent at chip doses below 4.55 g L‒1. At higher dosages, only a progressive reduction in b* value with chip doses was produced, without any significant effect of time on the yellow-blue component (Fig. 3d). On the contrary, when the percentage of American oak was fixed at 100%, the effect of maceration time on the b* parameter was only noticeable at high chip doses (above 4.55 g L‒1), with slightly decreasing b* values as ageing progresses. However, at lower dosages, the time factor was less influential and the chip doses effect was prevalent, with higher b* values being found as chip doses increased (Fig. 3e). It should be stressed that the origin-dependent trends observed were exactly the opposite to those previously described for anthocyanins (see the discussion on the b23 interaction effect for Mv-3-Gl). This complementary behaviour could be attributed to the strong competition between the formation of polymeric pigments and pyranoanthocyanins (and their relative effects on wine colour) during wine ageing (García-Puente Rivas et al., 2005), in such a way that each process appeared to become the driving force under different conditions. On the other hand, when the b15 interaction effect was analysed for H*, it was observed that time had a greater impact when French oak was used. In fact, when the percentage of French chips was fixed at 100%, the value of the H* parameter increased as time and oxygen

20

doses increased simultaneously (Fig. 3f). Once again, the greater impact of oxygen supply with French oak wood was surely related to its particular structural properties. The inspection of the response surface corresponding to the b12 interaction effect for the b* parameter proved once again that the different oxygen consumption patterns depended on wood origin, and confirmed the already discussed dependence of change in the yellow-blue coordinate as a function of chip dosage (Fig. 3g and 3h for French and American chips, respectively). In this way, the combined effect of both factors led to a local maximum area for the b* parameter when low to medium French chip doses were used over almost the entire range of oxygen doses, whereas for American chips the lower increase in b* value found at low oxygen and high chip doses suggested reduced formation of pyranoanthocyanin-derived pigments. Finally, when analysing the b34 interaction effect, the French oak was shown to be better able to release compounds into the wine that may be involved in the formation of adducts with a reddish-orange colour (Fig. 3i). Nevertheless, for both wood origins, the increase in the yellowblue component was more marked when using heavily toasted chips, which was in accordance with toasting conditions previously reported as associated with low a* (see discussion on the b14 interaction effect for the a* parameter). To conclude the analysis, it should be noted that the lightness parameter (L*) also exhibited a strong dependence on wood origin. This fact was particularly remarkable when the oxygen doses–toasting degree (b14) and chip doses–toasting degree (b24) interaction effects were considered. For both interaction effects, the evolution of the L* parameter was analysed considering French and American origins separately. As regards b14, for French oak, the lowest L* parameters were achieved when heavy to heavy plus toasting degrees were used, although it was necessary to gradually increase the oxygen supply from low doses for H toasting to high doses for H+ in order to reach similarly low values. Likewise, a maximum response zone for L* was identified for medium toasting degrees and low oxygen doses (between 1 and 4 mL L‒1

21

month‒1) (Fig. 3j). In contrast, for American oak, the lowest levels of L* were found when medium toasted chips were used, and it was necessary to increase oxygen doses from low to high values as the toasting level increased from medium to medium plus. In addition, a response maximum was reached when combining heavy plus toasting degree and moderate oxygen doses (between 4 and 6 mL L−1 month−1) (Fig. 3k). On the other hand, the L* response surface for the chip doses– toasting degree interaction effect (b24), revealed that, for French oak, there was a decreasing trend in L* value as both toasting degree and chip doses increased (either jointly, or separately if the other factor was fixed at low levels) (Fig. 3l). However, in the case of American wood, the opposite behaviour was observed, i.e., lower L* values when decreasing toasting degree and/or chip doses (Fig. 3m). A decrease in lightness was expected to be observed as the hue angle increased. In fact, the observed trends for the L* parameter as a function of the analysed factors appeared to be in correspondence with aforementioned combinations of factors which led to low a* values. Lastly, the response surfaces corresponding to the significant interaction effects involving maceration time (b35, b45) suggested that the L* parameter, did not however show notable variations with time during the accelerated ageing processes we studied (Fig. not shown).

4. Conclusions This paper presents an in-depth multivariate study that sought to gain a full insight into the impact of micro-oxygenation and chip maceration on the phenolic and chromatic profiles of wines. Although the observed trends for the analysed compounds/parameters were relatively complex, due to the presence of significant interaction effects between process variables, and to their dependence on a complicated equilibrium among many processes occurring during ageing in competition for oxygen and substrates, certain common conclusions can be drawn. Of these the wood-origin-dependent behaviour exhibited for most considered responses is particularly noteworthy. It could be highlighted that the largest decrease in anthocyanins content was 22

observed when the wine was subjected to accelerated ageing for some four weeks, in combination with the application of high oxygen doses, medium to medium-plus toasted chips, and a chip dosage depending on wood origin (high and low chip doses for French and American oak, respectively). Slight differences in the oxygen supply needed to achieve a maximum conversion of anthocyanins were found again depending on the origin of the oak chips. The evolution patterns for flavan-3-ols during ageing, as well as those associated with changes in the a* and C* colour parameters, were largely coherent with those related to anthocyanins. However, a few particular observations could be made in the case of the red-green component: the influence of ageing time on the progressive loss of red colour was more marked for American chips; the use of high chip doses led to a decrease in the a* value for both wood origins; and several combinations of factors with heavily toasted chips also resulted in low a* measurements. As far as the b* colour parameter was concerned, the more pronounced increase in the yellowblue component with ageing time was found when a heavy toasting level was coupled with low chips doses for French oak and high chip dosages for American wood. In this case, once again different oxygen consumption rates appeared to be related to the geographical origin of the chips. Moreover, maceration time seemed to have less impact on the b* parameter when blends rich in French oak were used. Likewise, changes in lightness during accelerated ageing of wine were linked to a decrease in the red component. The knowledge acquired in this study could be very valuable in order to control accelerated ageing processes more accurately, thus enabling wineries to develop wines with specific colour profiles based on quality criteria. As far as we know, this is the first work that simultaneously examines the effects of the five main factors involved in accelerated ageing processes on the phenolic and colour profiles of wines by using such a rigorous and comprehensive approach based on experimental design.

Acknowledgments 23

The authors thank the Spanish Ministry of Science and Innovation (Project No. CTQ200803493/BQU) for its financial support, Professor R. Phan-Tan-Luu of the University of Marseille (France) for providing the software NEMROD-W and Bodegas Riojanas S.L. for their kind collaboration.

24

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Rudnitskaya, A., Schmidtke, L.M., Delgadillo, I., Legin, A., & Scollary, G. (2009). Study of the influence of micro-oxygenation and oak chip maceration on wine composition using an electronic tongue and chemical analysis. Analytica Chimica Acta, 642 (1-2), 235-245. Sartini, E., Arfelli, G., Fabiani, A., & Piva, A. (2007). Influence of chips, lees and microoxygenation during ageing on the phenolic composition of a red Sangiovese wine. Food Chemistry, 104 (4), 1599-1604. Schmidtke, L.M., Rudnitskaya, A., Saliba, A.J., Blackman, J.W., Scollary, G.R., Clark, A.C., Rutledge, D.N., Delgadillo, I., & Legin, A. (2010). Sensory, chemical, and electronic tongue assessment of micro-oxygenated wines and oak chip maceration: Assessing the commonality of analytical techniques. Journal of Agricultural and Food Chemistry, 58 (8), 5026-5033. Singleton, V.L. (1987). Oxygen with phenols and related reactions in musts, wines, and model systems: observations and practical implications. American Journal of Enology and Viticulture, 38, 69-77. Sousa, C., Mateus, N., Perez-Alonso, J., Santos-Buelga, C., & De Freitas, V. (2005). Preliminary study of oaklins, a new class of brick-red catechinpyrylium pigments resulting from the reaction between catechin and wood aldehydes. Journal of Agricultural and Food Chemistry, 53 (23), 9249-9256. Sousa, C., Mateus, N., Silva, A.M.S., González-Paramás, A.M., Santos-Buelga, C., & De Freitas, V. (2007). Structural and chromatic characterization of a new Malvidin 3-glucoside-vanillylcatechin pigment. Food Chemistry, 102 (4), 1344-1351.

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Timberlake, C.F., P. (1976). Bridle Interactions between anthocyanins, phenolic compounds, and acetaldehyde and their significance in red wines. American Journal of Enology and Viticulture, 27, 97-105. Tomasi, G., Savorani, F., & Engelsen, S.B. (2011). Icoshift: An effective tool for the alignment of chromatographic data. Journal of Chromatography A, 1218 (43), 7832-7840. Wildenradt, H.L. & Singelton, V.L. (1974). The Production of Aldehydes as a result of oxidation of polyphenolic compounds and its relation to wine ageing. American Journal of Enology and Viticulture, 25 (2), 119 – 126. Wirth, J., Caillé, S., Souquet, J.M., Samson, A., Dieval, J.B., Vidal, S., Fulcrand, H., & Cheynier, V. (2012). Impact of post-bottling oxygen exposure on the sensory characteristics and phenolic composition of Grenache rosé wines. Food Chemistry, 132 (4), 1861-1871.

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TABLE CAPTIONS Table 1. Experimental design matrix. Table 2. Estimates of the model coefficients. Table 3. Detected ranges for anthocyanins, flavan-3-ols and CIELAB parameters. Phenolic compound concentration ranges were expressed in mg L‒1.

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FIGURE CAPTIONS Fig. 1. Response surfaces obtained for the significant interactions corresponding to Mv-3-Gl: (a) b45; (b) b14; (c) b14 (% American oak fixed at 0%); (d) b14 (% American oak fixed at 100%); (e) b23. Process factors were coded as follows: 1 - oxygen doses; 2 - chip doses; 3 - wood origin; 4 toasting degree; and 5 - maceration time. When not specified, factors not varying in response surfaces were kept constant at medium levels (oxygen doses: 4 mL L−1 month−1; chip doses: 4.55 g L‒1; % American: 50%; toasting degree: M+; and time: 3 weeks). Fig. 2. (a) Optimal path graph corresponding to the time factor when using (‒)-epicatechin as a response variable, and (b) response surface obtained for the b45 interaction when using (+)catechin as a response variable. Fig. 3. Response surfaces obtained for the significant interactions corresponding to CIELAB parameters: (a) b35 for a*; (b) b14 for a*; (c) b24 for a*; (d) b25 (% American oak fixed at 0%) for b*; (e) b25 (% American oak fixed at 100%) for b*; (f) b15 for H* (% American oak fixed at 0%); (g) b12 (% American oak fixed at 0%) for b*; (h) b12 (% American oak fixed at 100%) for b*; (i) b34 for b*; (j) b14 (% American oak fixed at 0%) for L*; (k) b14 (% American fixed at 100%) for L*; (l) b24 (% American fixed at 0%) for L*; (m) b24 (% American fixed at 100%) for L*. Process factors were coded as follows: 1 - oxygen doses; 2 - chip doses; 3 - wood origin; 4 - toasting degree; and 5 - maceration time. When not specified, factors not varying in response surfaces were kept constant at medium levels (oxygen doses: 4 mL L−1 month−1, chip doses: 4.55 g L-1, % American: 50%, toasting degree: M+, and time: 3 weeks).

33

Fig. 1.

34

Fig. 2.

35

Fig. 3.

36

Table 1.

experiment name.1

O2 doses chip doses wood origin toasting degree ‒1 −1 (mL L (g L ) (% American) −1 month ) E1 4 4.55 50 100 (H+) E2 2 2.53 17 58 (H) E3 6 2.53 17 58 (H) E4 2 6.57 17 58 (H) E5 6 6.57 17 58 (H) E6 2 2.53 83 58 (H) E7 6 2.53 83 58 (H) E8 2 6.57 83 58 (H) E9 6 6.57 83 58 (H) E10 1 4.55 50 1 (M) E11 7 4.55 50 1 (M) E12 4 1.49 50 1 (M) E13 4 7.61 50 1 (M) E14 4 4.55 0 1 (M) E15 4 4.55 100 1 (M) E16 4 4.55 50 37 (M+) E17 4 4.55 50 37 (M+) E18 4 4.55 50 37 (M+) 1Seven samples were taken for each experiment from week 0 to week 6, leading to a total of 126 wine samples.

37

Table 2. Coeff.

Df-3-Gl

Cn-3-Gl

Pt-3-Gl

Pn-3-Gl

Mv-3-Gl

b0 b1 b2 b3 b4 b5 b11 b22 b33 b44 b55 b12 b13 b23 b14 b24 b34 b15 b25 b35 b45

4.177 -0.240 0.007 -0.902 0.566 -1.145 -1.152 -0.565 -0.367 0.412 0.868 0.042 -0.903 1.178 1.186 1.332 -0.053 0.080 -0.244 -0.138 0.272

0.371 -0.044 -0.005 -0.144 0.099 -0.097 -0.152 -0.137 -0.103 -0.005 0.066 0.106 -0.164 0.335 0.19 0.317 0.058 0.006 -0.066 -0.008 0.079

3.889 -0.255 0.023 -0.976 0.716 -1.121 -1.193 -0.513 -0.404 0.529 0.835 0.168 -0.872 1.366 1.253 1.418 -0.097 0.101 -0.234 -0.113 0.309

1.089 -0.071 -0.017 -0.316 0.298 -0.328 -0.351 -0.142 -0.121 0.178 0.227 0.123 -0.341 0.473 0.346 0.407 -0.056 0.02 -0.07 -0.035 0.113

16.99 -1.159 0.053 -4.802 4.016 -4.615 -5.326 -2.153 -1.643 2.078 3.012 1.325 -4.321 6.648 5.359 6.608 -1.132 0.708 -1.072 -0.453 1.509

Df-3-GlAc 0.664 -0.006 -0.029 0.085 -0.072 -0.048 -0.055 -0.053 -0.058 -0.083 -0.003 -0.001 0.001 0.024 0.003 0.055 0.163 -0.009 -0.008 -0.043 0.038

Mv-3-GlAc 0.618 -0.034 0.005 -0.168 0.139 -0.167 -0.168 -0.062 -0.051 0.079 0.116 0.047 -0.157 0.197 0.188 0.221 -0.053 0.035 -0.036 -0.019 0.046

Pn-3-GlCm 0.618 -0.034 0.005 -0.168 0.139 -0.167 -0.168 -0.062 -0.051 0.079 0.116 0.047 -0.157 0.197 0.188 0.221 -0.053 0.035 -0.036 -0.019 0.046

(+)Catechin 5.268 -0.066 -0.192 -0.458 0.454 -0.785 -0.874 -0.175 -0.442 -0.018 0.566 -0.081 -0.528 0.890 0.611 1.086 0.110 0.108 -0.069 -0.080 0.158

(-) Epicatechin 1.261 -0.030 -0.098 -0.130 0.129 -0.217 -0.204 -0.066 -0.115 0.071 0.143 0.000 -0.127 0.295 0.130 0.215 0.038 0.016 -0.022 -0.011 0.038

a*

b*

C*

49.848 -0.264 -0.598 -0.202 -0.311 -0.622 0.535 -0.008 0.179 -0.389 0.055 -0.460 0.414 0.300 1.24 1.987 0.202 -0.621 -0.274 -0.621 1.067

12.700 -0.877 0.021 -1.382 0.911 0.126 -0.276 -1.189 -0.397 -2.784 -0.285 0.561 -1.598 2.389 1.001 2.685 1.122 0.208 -0.299 -0.216 0.134

51.434 -0.434 -0.481 -0.497 -0.170 -0.568 0.329 -0.060 0.005 -0.854 0.012 -0.315 0.080 0.798 1.45 2.184 0.385 -0.568 -0.249 -0.66 1.003

H*

L*

14.236 -0.924 0.169 -1.400 1.178 0.183 -0.527 -1.397 -0.463 -2.629 -0.169 0.727 -1.933 2.535 0.740 2.491 1.140 0.442 -0.216 -0.239 -0.375

Bold numbers indicate significant effects (5%)

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48.802 -0.275 0.128 0.385 0.229 -0.323 -1.465 -1.162 -0.961 -2.155 -0.167 1.033 -1.357 2.78 1.911 3.640 4.583 -0.045 -0.187 0.92 -0.473

Table 3. Compound/ Parameter Anthocyanins Df-3-Gl Cn-3-Gl Pt-3-Gl Pn-3-Gl Mv-3-Gl Df-3-Gl-Ac Mv-3-Gl-Ac Pn-3-Gl-Cm Flavan-3-ols (+)-Catechin (‒) Epicatechin Chromatic parameters a* b* C* H* L*

Range (mg L‒1) 10.4–32.4 0.90–3.85 8.64–30.8 2.67–9.87 31.4–133 3.13–4.53 2.26–5.70 0.90–9.10 (mg L‒1) 25.7–36.9 19.0–22.5 47.28–51.64 7.55–14.54 48.12–52.95 9.67–16.56 40.41–52.08

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Highlights

The impact of alternative ageing on phenolic and chromatic profiles was examined. > The effect of five essential process factors was evaluated by a rigorous methodology. > Multivariate study allowed evaluating interaction effects between process factors.> Noteworthy wood-origindependent behaviour exhibited for most considered responses. > Wineries could develop wines with specific colour profiles based on quality criteria.

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