Application of LC-MS and tristimulus colorimetry to assess the ageing aptitude of Syrah wine in the Condado de Huelva D.O. (Spain), a typical warm climate region

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Analytica Chimica Acta on June 2012, available at: https://doi.org/10.1016/j.aca.2012.03.051 .” 1 *Manuscript Click here to view linked References 1 Application of LC-MS and Tristimulus Colorimetry to asses the ageing 2 aptitude of Syrah wine in the Condado de Huelva D.O. (Spain), a typical 3 warm climate region 4 5 B. Gordilloa, F.J. Rodriguez-Pulidoa, N. Mateusb, M.L.Escudero-Giletea, M.L. 6 González-Mireta, F.J. Heredia a,*, V. de Freitasb. 7 8 a Food Colour & Quality Lab. Dept. Nutrition & Food Science. Facultad de 9 Farmacia. Universidad de Sevilla. 41012-Sevilla, Spain 10 b Centro de Investigação em Química, Departamento de Química, 11 Faculdade de Ciências. Universidade do Porto. 4169-007-Porto, Portugal 12 13 Abbreviated running title: 14 Pigment composition and colour quality of aged red wines 15 16 17 * Corresponding author: 18 Francisco J. Heredia 19 Food Colour & Quality Lab., Dept. Nutrition & Food Science. Facultad de 20 Farmacia. Universidad de Sevilla. 41012-Sevilla, Spain 21 Tel.: +34 954556495 Fax: +34 954557017 22 e-mail: [email protected] 23 2 24 Abstract 25 The study of the evolutions of different wine pigment families, 26 copigmentation/polymerisation processes and colour characteristics during the 27 first year of ageing in oak barrel has allowed the assessment of the ageing 28 aptitude of Syrah wines a warm climate region. 29 A total of 32 anthocyanic pigments were identified, including 14 major 30 compounds from grape and 18 minor derivatives formed during the vinification. 31 The anthocyanin profile changed toward more chemical complexity, being 32 vitisin-like pyranoanthocyanins the predominant minor pigments during the first 33 months of ageing. As wine became older, a progressive increase on the content 34 of 4-vinylcatechin, 4-vinylphenol and 4-vinylcatechol compounds took place. 35 Results showed that copigmentation occurred during the whole process of 36 ageing inducing visual perceptible colour effects. Simultaneously to the 37 copigmentation decrease, the degree of polymerisation increased during 38 ageing, being maximum at 9 months old wines (77%). The colour of wines 39 evolved progressively in a positive way from 3 to 9 months of ageing, becoming 40 darker and with more vivid colour. However, from 9 to 12 month of ageing, the 41 chemical structure of wines was negatively affected resulting in lighter, with 42 more red-orange hues and less vivid colours. The inclusion of the chemical and 43 colorimetric information on the PCA model allows us to reach very good 44 discriminations among the Syrah wines with different wood contact period. 45 46 Keywords: ageing process; copigmentation; detailed pigment composition; 47 quality colour; oak wood. 48 3 49 1. Introduction 50 The climatic conditions of a given geographical area is one of the most 51 important factors influencing the overall wine style of a region since determine 52 the types and the quality of grapes that can be grown [1]. 53 In warm climate regions, red grapes have not been traditionally cultivated 54 because the high night temperatures and the severe light exposures during the 55 ripening affects negatively the anthocyanic biosynthesis and its accumulation in 56 the fruit skin, giving rise to poor coloured grapes [2]. As a consequence, red 57 wines elaborated from them usually show important problems with colour quality 58 and stability during winemaking maturation, especially when they are subjected 59 to ageing processes. 60 However, due to the market demand, the interest for red wine 61 vinifications in warm regions is becoming more and more increasing in the last 62 63 restricted wine-producing zone in south-western Spain with the typical 64 climatological conditions of warm climate, new red grape varieties are being 65 introduced in order to diversify the wine choice of the DO and to expand their 66 potential market [3]. This D.O. includes approximately 6000 hectares of neutral 67 or slightly alkaline soil, having a typical Mediterranean climate with a clear 68 Atlantic influence: gentle winters and springs and long and warm summers 69 (average temperature 18ºC, minimum over 10ºC in winter and over 40 ºC in 70 summer), relative humidity ranging between 60% and 80%, and mean rainfall 71 around 700 mm year -1. 72 Vitis vinifera cv. Syrah grape is the main and more extensively grown in the 73 zone. It was selected because has been described as an excellent and robust 4 74 grape, well-adapted to warm climate, easy to cultivate and little vulnerable to 75 diseases. 76 In fact, the elaboration of young Syrah wines with better phenolic 77 78 been already possible thanks to important investments in new technologies 79 such as the application of low maceration temperatures prior to fermentation, 80 which resulted in a more effective extraction of the anthocyanin pigments and 81 improved the colour stabilization during the earlier stages of wine maturation [4, 82 5]. Notwithstanding, as was previously stated, to produce high quality wines 83 from Syrah grapes besides to preserve the levels of phenols in the winemaking, 84 perform a long fermentation and watch carefully the temperature, summiting 85 them to ageing processes is also recommended since they are especially fit for 86 ageing in oak barrels [6]. Taking into account the absence of previous studies 87 about the ageing potential of this variety during longer ageing processes in this 88 region, further investigations are still necessary. 89 Nowadays, is well-known that the expression of the colour in red wines 90 depends not only from the anthocyanin concentration originally extracted from 91 grapes but also on physicochemical phenomena of copigmentation that led to 92 the formation of new derived pigment which stabilize wine colour [7]. 93 Recently, different groups of anthocyanin-derived pigments with 94 particular colour characteristics have been identified in red wines, such as 95 pyranoanthocyanins, direct flavanol anthocyanin condensation products and 96 acetaldehyde-mediated flavanol anthocyanin condensation products [8]. 97 Although some of these pigments have only been detected in very small 98 quantities in wines, they present unique spectroscopic features that may 5 99 somehow contribute together to the overall colour of aged red wines by means 100 of additive, synergistic, or suppressive effects [9]. A common colorimetric 101 characteristic of all these derived pigments is the greater resistance to 102 degradation reactions that could further enhance their sensorial importance. 103 Different mechanisms have been proposed and confirmed the formation 104 of this compounds [10,11] being some of them favoured by the presence of 105 oxygen [12]. For this reason, although wine ageing in oak barrels implies a high 106 cost outlay for the wineries, it is commonly used because the positive effects 107 108 109 as increasing colour stability, spontaneous clarification and wine complexity 110 [13]. 111 Therefore, the main objective of this paper is to study more in depth the 112 detailed anthocyanic composition of the aged Syrah wines elaborated in the 113 114 their role in the wine colour quality according to the ageing time. For these 115 purposes, not only the occurrence of the main anthocyanins was studied, but 116 also those of the anthocyanin-derived pigments in order to establish the 117 contribution of each pigment family at each moment of wine ageing. As the 118 identification of minor pigments has proven difficult, especially because their 119 levels are much lower than those of original anthocyanins, high performance 120 liquid chromatography diode array detection coupled to mass spectrometry 121 (HPLC DAD MS) has been used in pigment identification. 122 Regarding colour assessment, Tristimulus Colorimetry, based on objective and 123 direct spectral measurements, represent a useful methodology for colour 6 124 specifications in red wines by means of the quantitative and qualitative 125 colorimetric variables, which also provide high precision and accuracy to follow 126 the integral colorimetric effect induced by copigmentation processes [14]. 127 Moreover, applying principal component analysis, the usefulness of the pigment 128 composition and colorimetric variables in the differentiation of the wines as a 129 function of the ageing time was also assessed. 130 131 2. Materials and methods 132 2.1. Wine samples and ageing process 133 A red wine made from grapes Vitis vinifera var. Syrah (2007 vintage) 134 south-western 135 Spain, was used for the experiment. The wine was elaborated by 136 - 137 high concentrations of phenolic compounds, particularly anthocyanin pigments. 138 It consisted of two stages: a first stage of 11 days of pre-fermentative cold 139 maceration (between 5-8ºC), followed by 15 days of traditional maceration 140 (between 20-25ºC). The cold-maceration process was carried out by using an 141 industrial refrigeration system (HITSA-TOPAIR, mod RAE-101, Madrid, Spain) 142 for the recirculation of refrigerant liquid through cooling water jackets to keep 143 low temperatures. At the end of the stabilization process in stainless steel tanks 144 during 5 months, the wine was distributed into three new American oak barrels 145 (225 L, medium toast), where it was maintained for a 12 months. Samples were 146 analyzed at 3, 6, 9, and 12 months of ageing, so a total of 12 wines (200 mL) 147 148 were submitted to study. 7 149 2.2. HPLC DAD MS analysis 150 The HPLC used was an ELITE LaChrom fitted with a L-2130 quaternary 151 pump, a L-2200 autosampler and a L-2455 DAD. The stationary phase was a 152 MERK RP 153 L and the mobile phase was composed by two solvents: solvent A, a 10% 154 (v/v) aqueous formic acid solution, solvent B, formic acid: acetonitrile: water 155 (1:3:6) (v/v). The elution conditions were: flow rate set at 1 mL min-1, starting 156 with a linear gradient from 20% to 66% of B in 50 min, a second linear gradient 157 from 66% to 100% of B for 10 min followed by an isocratic elution of 100% of B 158 for another 10 min. The column was washed with 100% of acetonitrile for a 7 159 min period proceeded by a re-equilibration step of 15 min of isocratic 20% of B. 160 The UV vis absorbance spectra were recorded from 250 to 700 nm and the 161 peaks recorded at 280 and 520 nm. 162 MS analyses were performed using a Finnigan LCQ MS detector 163 (Thermoquest) equipped with an API source, using an electrospray ionization 164 (ESI) interface. The HPLC system was connected to the probe of the mass 165 spectrometer via the UV cell outlet. Both the sheath and the auxiliary gas was 166 nitrogen. The sheath gas flow was 1.2 Lmin and the auxiliary gas flow 6 167 Lmin . The source voltage and the capillary voltage were 4.50 kV and 26 V, 168 respectively, and the capillary temperature 195 ºC. Spectra were recorded in 169 positive ion mode between m/z 150 and 2000. The mass spectrometer was 170 programmed to do a series of consecutive scans: a full mass and a MS2 scan 171 of the most abundant ion in the full mass. The normalized energy of collision 172 was 45%. 8 173 The quantification of the anthocyanins compounds was made by 174 comparing the areas and the retention times with the malvidin 3-glucoside 175 standard, and anthocyanin concentration was expressed as mgL-1. On the basis 176 of their chemical structure, the overall pigment profile was divided into four 177 major pigment families: a) Monomeric anthocyanins, grouped as Anthocyanidin- 178 3-O-gluc (GL), acetylated anthocyanins (AC) and p-coumaroyl anthocyanins 179 (COUM), b) pyranoanthocyanins (PY), c) acetaldehyde-mediated flavanol 180 anthocyanin products (ACD), and d) direct flavanol anthocyanin condensation 181 products (DC). The proportion of the different pigment families estimated by 182 summing the content of each member found and the total pigment content 183 estimated as the sum of the all compounds identified (SUM_TA) were also 184 calculated. 185 2.3. Copigmented and polymerized anthocyanins determination 186 The contribution of copigmented anthocyanins (%CA) and polymeric 187 pigments (%PP) to the total wine colour at pH 3.6 were determined following the 188 method proposed by Boulton [15]. Wine samples were firstly adjusted to pH 3.6. 189 The colorimetric effect of the copigmentation phenomenon was also evaluated 190 by Tristimulus Colorimetry as reported by Gordillo et al. [14]. 191 Colorimetric measurements 192 The whole visible spectrum of the wines (380 770 nm) was recorded at 193 = 2 nm) with a Hewlett-Packard UV-vis HP8452 194 spectrophotometer (Palo Alto, CA), using 2 mm pathlength glass cells and 195 distilled water as a reference. The CIELAB parameters (L*, a*, b*, C*ab and hab) 196 were determined by using the original software CromaLab [16], following the 197 Commission Internationale : the 10° Standard 15 345 Among the newly formed red wine pigments, whereas some groups of 346 compounds showed similar behaviour, others seemed to be inversely affected, 347 which resulted in a lower decrease than monomeric anthocyanins. The total 348 amounts of the minor anthocyanins diminished from 34.9±1.3 mgL-1 to 22.6±0.3 349 mgL-1 during the whole ageing process (35%); but from 6 months only a slightly 350 decrease was globally produced. The minor pigments content of the aged Syrah 351 wines studied were similar to those described in aged Monastrell wines by 352 Cano-López [13], but it was significant lower than those reported in aged 353 Tempranillo/Graciano wines [29]. These differences can be explained by a dual 354 effect: the warm climate (the case of Monastrell and Syrah wines), which can 355 exert a negative influence on the phenolic content and copigmentation process 356 [30], and the grape variety, since it is well known that Graciano has high 357 phenolic content which improve the copigmentation reactions. 358 During the first stage of ageing (from 3 to 6 months), together with 359 monomeric anthocyanins, the predominant minor anthocyanins corresponded to 360 the vitisin-like pyranoanthocyanins (A and B type), contributing for 60-69% to 361 the total derived pigments. Their presence in wine is related to the secondary 362 metabolic products (pyruvate and acetaldehyde) mainly released by yeast 363 during alcoholic fermentation, and because of their slow degradation rate, they 364 could become the main derived pigments in young and early aged wines [31]. 365 During ageing, since the formation of vitisins is not strongly favoured, the 366 proportion of both kinds of compounds naturally diminished with time. However, 367 although the concentration of the B-type vitisins was higher at the beginning of 368 the ageing process, A-type vitisins were the most abundant member of this sub- 369 family as wine became older. Specifically, the remaining amount for the B-type 16 370 vitisins at the end of the ageing process was only of 10% (1.4±0.02 mgL-1) while 371 for the A-type vitisins was of 40% (5.8±0.2 mgL-1); indicating that the last ones 372 were also more resistant towards the conditions undergone during ageing. This 373 is probably due to the fact that, unlike acetaldehyde, pyruvic acid cannot be 374 used as an intermediate in polymerisation processes [24]. The occurrence of 375 these anthocyanin derivatives is of special interest in red wines typical from 376 warm climates because these relatively small molecules remain in solution, in 377 contrast to the classical flavanol anthocyanin polymeric pigments that tend to 378 precipitate. Oppositely, in the last three months of ageing (from 9 and 12 month) 379 a progressive increase on the content of the lower polar pyranoanthocyanins 380 took place, being the predominant derived pigments in 12 months wines 381 (63.8%). The occurrence of 4-vinylcatechin and 4-vinylphenol was confirmed at 382 every moment of ageing showing similar behaviour (Table 1). Their 383 concentration tended to increase from 0.2 to 0.9 and from 5.3 to 7.4 mgL-1, 384 respectively, being the increases significant for both compounds. The 385 occurrence of 4-vinylcatechol adduct was detected only from 9 months, 386 reaching the highest levels in the last stage of ageing in the barrel (6.2±0.1 387 mgL-1). The difference found about the occurrence of those type of 388 pyranoanthocyanins is explained because the reaction between 4-vinylcatechol 389 and Mv 3-gl to produce Pinotin A is reported to proceed rather slowly and 390 requires years of storage to complete [32], while the formation of the Mv 3gl-4- 391 vinylphenol or vinylcatechin adducts take place during fermentation [10]. 392 Regarding acetaldehyde mediated derived anthocyanins; the main 393 member of this family (Mv 3-glc-ethyl-cat) was present during the whole process 394 of ageing. Nevertheless, after 9 months, the global content decreased 17 395 significantly from 4.4±1.2 to 0.7±0.01 mgL-1, which might indicate a lower 396 resistance against the ageing process of this pigment family. 397 Finally, it is worth mentioning the lower importance of direct condensation 398 adducts over the pigment profile of aged Syrah wines during the first and the 399 last stages of ageing, where practically were quantified as very low 400 concentrations. The highest contents of this sub-family of pigments were found 401 at intermediate stages of ageing (from 6 to 9 months), reaching concentrations 402 around 3.0±0.3 mgL-1. Despite the lower amounts of this anthocyanin-derived 403 pigments comparatively to pyranoantocyanins or ethyl mediated adducts, it was 404 shown that they can induce significant modification in the colour of the genuine 405 anthocyanins at concentrations similar to those that can exist in red wines [33]. 406 Thus, they could also be considered as compounds with sensorial impact in 407 Syrah aged wines. 408 To summarize, as wine became older, the proportion of the original grape 409 anthocyanins (monomeric anthocyanins) tended to decrease from 92% to 70% 410 (Figure 3). On contrast, the anthocyanin-derived pigments played an increasing 411 role (from 8% to 30%), being mainly due to compounds providing the wine with 412 orange hues (pyranoanthocyanins). This gradual chemical transformation is in 413 accordance with the positive maturation effects attributed to barrels [34]. As 414 reported in literature, most of the anthocyanin derived pigments have a greater 415 colour expression and stability than monomeric anthocyanins at wine pH [8]. 416 Thus, despite being minor pigments, is expected they can contribute 417 418 419 substantially to the overall colour of the aged Syrah wines. 18 420 3.3. Effect of ageing time on copigmentation, polymerisation and wine colour 421 Although copigmentation is a phenomenon mainly described in young 422 red wines, the results showed that it occurred in the Syrah wines during the 423 whole process of ageing in oak barrels (Figure 4), which was in accordance with 424 the results of some authors [35,36]. Notwithstanding, the general values 425 obtained were lower than those reported for young red wines, were 426 copigmented anthocyanins can even reach up to 50% of total colour [7]. The 427 highest value of the magnitude of copigmentation was found in the three 428 months samples (28.4%±0.5), but as the ageing advanced, the degree of 429 copigmentation significantly decreased reaching a minimum value of 9.14%±2.6 430 in 12 months samples. 431 Tristimulus Colorimetry was also applied to asses the colorimetric effect 432 of copigmentation on the total colour of the aged Syrah wines as reported in 433 [14]. Thus, the wine colour with and without copigmentation effect was 434 calculated at each stage of ageing, and was represented graphically in the 435 CIELAB (a*b*) diagram (Figure 5). The CIELAB differences ( E*ab, L, C*ab, 436 and hab) for every pair of wines were also calculated to follow the trend of the 437 changes. As observed, the different location of the copigmented and no 438 copigmented wines in the (a*b*) diagram confirms that not only the magnitude 439 of copigmentation was influenced by the ageing process but also the colour 440 changes induced by this phenomenon. 441 At shorter ageing times (3 and 6 months), copigmentation induced the 442 greatest amplitude of the colour effect ( E*ab=11.1±0.35 and 8.24±0.37, 443 respectively) contributing to the total colour mainly with quantitative changes, 444 which consisted in strong increases of chroma and decreases of lightness 19 445 ( Cab=+8.6±0.2 and +5.4±0.1; L=-7.0±0.3 and -6.2±0.3, respectively). On 446 contrast, at longer ageing times (9 and 12 months), although the colour effects 447 were significantly lower ( E*ab=6.35±0.8 and 3.9±0.7, respectively) the most 448 important contribution of the copigmentation was qualitative, leading to a more 449 bluish hues ( hab=-4.9 ±1.1, respectively). In any case, all the colour differences 450 calculated were higher than 3.0 CIELAB u. (visually perceptible by the human 451 eye) [37], which confirms the relevance of this physicochemical phenomenon on 452 the quality colour of aged wines, mainly at earlier stages. 453 Simultaneously to the copigmentation evolution, the degree of 454 anthocyanin polymerisation increased during ageing, being maximum at 9 455 months old wines (%PP=77.0%±3.6) (Figure 4). This significant increase 456 coincided with the observed losses of monomeric anthcoyanins and gradual 457 formation of new derived pigments, which reflects the complex pigment 458 transformation described in the previous section. However, after 9 months of 459 ageing, a slightly decrease of the polymerisation occurred (%PP=70.7%±3.6), 460 evidencing that extended ageing times affected negatively the chemical stability 461 of pigments in the Syrah wines studied. 462 Regarding the colour evolution, the changes on the colorimetric 463 parameters are shown in Figure 6. The results showed that the evolution of the 464 anthocyanins and the copigmentation/polymerisation processes resulted in 465 important changes both on quantitative (L* and C*ab) and qualitative (hab) 466 psychophysical components of the colour. As expected, the ageing time 467 influenced significantly the trend of the changes. 468 From 3 to 9 months of ageing, the quantitative attributes of the wine 469 colour evolved progressively in a positive way. Specifically, the lightness of the 20 470 samples decreased by 6% (from 80.4±0.6 to 75.6±0.1 CIELAB u.), while 471 chroma increased by 13% (from 21.4±0.4 to 24.7±0.4 CIELAB u.), that is, the 472 wines became darker, with a more vivid colour. This colorimetric evolution 473 indicates that no colour fall was produced in this period of time. Thus, it can be 474 said that the significant loss of pigment and %copigmentation observed was 475 due more to their transformation into derived pigments than to degradation, 476 which also was confirmed by the increase in the %PP. However, from 9 to 12 477 month of ageing, the lightness and chroma of samples were negatively affected. 478 As observed, the lightness increased by 4% (from 75.6±0.1 to 78.7±0.5 CIELAB 479 u.) and the chroma decreased by 13% (from 24.6±0.4 to 21.3±0.4 CIELAB u.), 480 that is, wines became lighter and with less vivid colour. Thus, despite not being 481 especially significant the loss of anthocyanins in the last period of ageing, it 482 seems to be mainly responsible for the colour loss since the polymerisation 483 process tended also to decrease. It is well-known that the partial elimination of 484 new polymeric pigments by precipitation is a usual chemical transformation of 485 more matured red wines, which additionally can be intensified in warm climate 486 regions. The colour difference ( E*ab) calculated between the colour of the 9 487 and 12 months samples revealed that this colour variation was of 5.1±0.8 488 CIELAB u., thus, visually detectable by the human eye. 489 As far as qualitative attribute of colour (hab), it was remarkably affected, 490 showing a clear tendency during the whole ageing process. It increased with 491 increasing ageing time from 0.9º±0.8 to 11.3º±0.1, that is, from redness toward 492 more red-orange hues, which is expected according to the increasing role of 493 anthocyanin derived pigments over the total pigment content. 21 494 3.4. Differentiation of aged Syrah wines based on the pigment and colorimetric 495 characteristics 496 By comparing the four groups of wines (3, 6, 9 and 12 months) on the 497 basis of their pigment composition and colorimetric characteristics, a PCA 498 analysis was performed in order to examine which of these variables are more 499 involved in the differentiation of aged Syrah wines from Condado de Huelva 500 D.O. region, with different wood contact period. Figure 7 shows the distribution 501 of the samples along the first two principal components, where an accurate 502 differentiation and classification of wines into each of their respective groups 503 was obtained. The two principal components, with eigenvalues>1, explained 504 91.3% of the variability in the original data. 505 The first factor, which explained 72.5% of the variance, clearly separates 506 Syrah wines aged for 3 and 6 months, grouped in the negative area of PC1, 507 from the respective ones aged for 9 and 12 months, located separately in the 508 positive area of the graphic. Results showed that several compounds were 509 useful for the differentiation between shorter and longer aged Syrah wines, 510 including not only monomeric but also derived anthocyanic pigments. In 511 particular, significant negative correlations were obtained for Glucosides, 512 Acetates, Coumarates, Vitisin A dvs., Vitisin B dvs., and acetaldehyde mediated 513 derived anthocyanins, which generally are more characteristics of earlier 514 maturation stages in red wines. On contrast, significant positive correlations 515 were obtained for minor anthocyanic compounds Mv 3-glc 4-vinylcatechol, Mv 516 3-glc-4-vinyl-catechin and Mv 3-glc 4-vinylphenol, usually associate with more 517 extended ageing periods. Regarding the colour variables, shorter aged wines 518 were significant correlated with lighter colour and higher levels of 22 519 copigmentation (negative correlations), while longer aged wines with re-orange 520 hues and higher degree of polymerisation (positive correlations). 521 On the other hand, the second factor which explained 18.8% of the 522 variance, showed that 3 and 12 aged wines could be also differentiated from 6 523 and 9 aged wines on the basis of the direct condensation adducts content and 524 chroma, both of them being significant positive correlated. This meant that 525 intermediate ageing periods were characterised by more intense colours and 526 527 higher content of direct condensation anthocyanin adducts. 528 4. Conclusions 529 This work provide a better knowledge of the ageing aptitude of Syrah wines 530 elaborated in warm climate based on the evolution of its detailed pigment 531 composition and colorimetric behaviour. HPLC DAD MS analysis permitted the 532 identification of several major and minor anthocyanic pigments. Although the 533 total pigment content decreased during the whole ageing process, as wines 534 became older, the relative amounts of compounds which give higher chemical 535 stability and orange hue increased and that of compounds less stable that gives 536 blue hues decreased. With respect to the colorimetric characteristics, the results 537 showed that wines summited to shorted or intermediate ageing times (between 538 3 to 9 months) exhibited most desirable global characteristics as required 539 nowadays to produce high quality wines, that is, darker and more redness vivid 540 colour. On contrast, those submitted to longer ageing times (between 9 to 12 541 542 543 months) finally showed lighter and less vivid colour with more red-orange hues. 23 544 Acknowledgement 545 Authors are indebted to Consejería de Innovación, Ciencia y Empresa (Junta de 546 Andalucía, Spain) for the financial support (Project P07-AGR-0289) and The 547 Ministry of Science and Innovation of Spain the financial support: Projects 548 AGL2008-05569-C02-01 and AGL2008-05569-C02-02. The authors thank 549 Vinicola del Condado winery (Bollullos Par del Condado, Huelva, Spain) for 550 technical assistance during vinifications and Dra. Zélia Azevedo for the LC-MS 551 552 553 analysis. 24 554 References 555 [1] G.V. Jones, M. A. White, O.R. Cooper, and K. Storchmann, Climatic 556 Change., 73 (2001) 319-343. 557 [2] K. Mori, S. Sugaya, and H. Gemma, Sci. Hortic., 105 (2005) 319-330. 558 [3] D. Hernanz, V. Gallo, A.F. Recamales, A.J. Meléndez-Martínez, M.L. 559 González-Miret, and F.J. 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Pyroanthocyanins Figure 2 Click here to download Figure: Gordillo_SyrahAgeing_Figure2.doc Figure 2. (a) 300 250 200 150 100 50 0 Monoglucosides Acetate dvs. Coumaric dvs. Free Anthocyanins (b) 16 14 12 10 8 6 4 2 0 Vitisin A dvs. Vitisin B dvs. Vynilcatechol Vynilcatechin Vynilphenol Direct conden. dvs. Acetaldehyde mediated dvs. Anthocyanin derived pigments Different letters for each group of pigments among successive ageing stages mean significant differences (p < 0.05). a 3 months 6 months 9 months 12 months b a c c b a b c c c c a 3 months 6 months 9 months 12 months a b b b d c c c a a b c b b b b d a a a a b c b c a b a b Sum_GL Sum_AC Sum_COUM Sum_PY Sum_ACD+DC Figure 3 Click here to download Figure: Gordillo_SyrahAgeing_Figure3.doc Figure 3. 100% 75% 50% 25% 0% 3 6 9 12 Ageing time (months) 1.1 1.9 3.7 1.2 12.4 59.0 58.9 55.4 51.6 5.7 14.3 4.6 20.0 21.4 11.7 12.0 22.0 29.1 7.5 6.5 Figure 4 Click here to download Figure: Gordillo_SyrahAgeing_Figure4.doc 1 Figure 4. 90 80 70 60 50 40 30 20 10 0 3 6 9 12 Ageing time (months) Different letters for the %Copigmentation and %Polymerization among successive ageing stages mean significant differences (p < 0.05). b b a a a b c d Figure 5 Click here to download Figure: Gordillo_SyrahAgeing_Figure5.doc Figure 5. (a) 3-6 months 30 20 10 0 0 10 20 30 a* (b) 9-12 months 30 20 10 0 0 10 20 30 a* 0° 80° 70° no copigmented wines copigmented wines 60° 50° 40° 30° 20° 10° 0 0° 80° 70° no copigmented wines copigmented wines 60° 50° 40° 30° 20° 10° 0 Figure 6 Click here to download Figure: Gordillo_SyrahAgeing_Figure6.doc Figure 6. (a) 82 81 80 79 78 77 76 75 3 6 9 12 Ageing time (months) (b) 25 24 23 22 21 3 6 9 12 Ageing time (months) (c) (c) 12 10 8 6 4 2 0 -2 3 6 9 12 Ageing time (months) 2 3 months 6 months 9 months 12 months Figure 7 Click here to download Figure: Gordillo_SyrahAgeing_Figure7.doc Figure 7. 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 -2.0 -1.6 -1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 Factor 1