Application of the differential colorimetry and polyphenolic profile to the evaluation of the chromatic quality of Tempranillo red wines elaborated in warm climate. Influence of the presence of oak wood chips during fermentation

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Food Chemistry on 1 December 2013, available at: https://doi.org/10.1016/j.foodchem.2013.05.014 .” 1 1 Application of the differential colorimetry and polyphenolic profile to the 2 evaluation of the chromatic quality of Tempranillo red wines elaborated in warm 3 climate. Influence of the presence of oak wood chips during fermentation 4 Belén Gordillo, María Jesús Cejudo-Bastante, Francisco J. Rodríguez-Pulido, M. 5 Lourdes González-Miret, Francisco J. Heredia* 6 Food Colour and Quality Lab., Dept. Nutrition and Food Science, Facultad de Farmacia, 7 Universidad de Sevilla, 41012 Sevilla, Spain 8 Belén Gordillo: [email protected] 9 María Jesús Cejudo-Bastante: [email protected] 10 Francisco J. Rodríguez-Pulido: [email protected] 11 M. Lourdes González-Miret: [email protected] 12 Francisco J. Heredia: [email protected] 13 14 15 16 17 18 19 * Corresponding author: 20 Francisco J. Heredia 21 Food Colour & Quality Lab., Dept. Nutrition & Food Science. Facultad de Farmacia. 22 Universidad de Sevilla. 41012-Sevilla, Spain 23 Tel.: +34 954556495 Fax: +34 954556110 24 e-mail: [email protected] 2 25 ABSTRACT 26 The effect of adding American oak wood chips during fermentation on 27 Tempranillo red wines elaborates in a warm climate has been studied. Our 28 attention was focused on the tristimulus colorimetry, differential colorimetry and 29 phenolic compounds related to wine colour. This technique was applied as an 30 oenological alternative to the conventional winemaking for avoiding the 31 common fall of colour of red wines elaborated in warm climates. The addition of 32 oak wood chips promoted the colour enhancement and stabilisation, producing 33 wines with a notably darker colour and with more bluish tonality. This fact was 34 also related to the significantly higher content of some phenolic compounds. On 35 the basis of the results, it could be affirmed that the addition of oak wood chips 36 during fermentation induced visually perceptible colour changes (by the analysis 37 of E*ab, % 2L, % 2C and % 2H), mainly in a quantitative way, and also a 38 lower percentage of diminution of colour. 39 KEYWORDS: Differential colorimetry; polyphenolic compounds; American oak wood 40 chips; Tempranillo red wine; warm climate. 41 3 42 1. INTRODUCTION 43 Phenolic compounds are the main chemical substances responsible for the quality of the 44 red wines, with regard to the colour, astringency and bitterness. Anthocyanins, 45 flavonols, flavan-3-ols and polymeric compounds play an important role on these 46 sensory characteristics (Robichaud & Noble, 1990; Sarni-Manchado, Cheynier, & 47 Moutounet, 1999). The anthocyanins extracted from the solid parts of the grape (mainly 48 skin) provide the red wine colour, whereas the presence of other compounds (the so- 49 called copigments), normally colourless, allow improving the colour stabilization of 50 aged wines (Pérez-Magariño & González-San José, 2004) by means of copigmentation 51 reactions, which are covalent interactions between anthocyanins and copigments, giving 52 rise polymeric pigments (Ribéreau-Gayón, Dubourdieu, Donéche, & Lonvaud, 2003). 53 Thus, the content of anthocyanins significantly decreases during ageing, contrarily to 54 that observed for polymeric pigments. 55 In warm regions, the stressful climate conditions make difficult to obtain high quality 56 red wines, with high intensity and stable colour. This fact normally occurred since the 57 phenolic maturity does not coincide with the technological (sugars) maturity of the 58 grapes, and so, at the moment of harvesting different levels of both phenolic and sugar 59 maturity exist (López, Sánchez, Díaz, Ramírez, & Morales, 2007; Mori, Sugaya, & 60 Gemma, 2005), so that is the grapes have high sugar content but phenolics unripe. Thus, 61 seeds normally remain unripened while the optimal maturity of skins and pulp is 62 reached. Wines made from these grapes, low in pigments and cofactors, are not able to 63 form much copigmentation in the first steps of the winemaking process (Boulton, 2001) 64 and as a result, the colour stabilisation does not correctly develop and, after several 65 months of storage both in bottle or barrels, fall of colour normally occurred. Thus, in 66 these warm regions the seed are normally scarce in tannins, so an extra contribution of 4 67 tannins could be necessary to reach colour stabilisation, and hence the ageing period 68 could be long lasting. 69 Several authors have studied how colour stabilisation was increased by the addition of 70 tannins, both derived from grape and wood (Bautista-Ortín, Martínez-Cutillas, Ros- 71 García, López-Roca, & Gómez-Plaza, 2005; Vivas & Glories, 1996; Zamora, 2003). In 72 particular, phenolics extracted from wood have been described as compounds having 73 great influence on the colour, astringency and bitterness of the wine. They are also 74 involved in changes that take place during ageing (Del Alamo, Bernal, & Gomez- 75 Cordoves, 2000). The high variety of phenolic compounds present in wood, such as 76 benzoic and cinnamic compounds, and ellagitanins, among others, has been studied in a 77 large extent regardless of the regions and raw materials employed (Fernández De 78 Simón, Cadahía, Conde, & García-Vallejo, 1996, 1999; Mämmelä, Savolainen, 79 Lindroos, Kangas, & Vartiainen, 2000; Sanz, de Simón, Cadahía, Esteruelas, Muñoz, 80 Hernández, et al., 2012). In this sense, several authors have demonstrated that cinnamic 81 acids (such as caffeic and coumaric acids) play an important role in the copigmentation 82 reactions with the anthocyanins - , & Boulton, 2001), being 83 described colour enhancements at 520 nm (Jurd & Asen, 1966). 84 Currently, the use of wood chips have been widely used in winemaking as alternative 85 system to the classic ageing, mainly because barrels take up a lot of space in the winery, 86 their lifetime is not too long and the elaboration of aged wines is quite expensive. The 87 use of chips in winemaking is a legal practice in EU countries since 2006, when EU 88 approved the use of wood chips come exclusively from the Quercus genus 89 (Commission Regulation (EC), 2006). The use of oak wood chips as alternative of 90 barrels, and their effect on phenolic compounds and colour characteristics have been 91 widely studied. However, their addition before alcoholic fermentation and the study on 5 92 the stabilisation of the colour have been scarcely studied, and even less as alternative in 93 warm climate vinifications. Among those research studies, Gómez García-Carpintero, 94 Gómez Gallego, Sánchez-Palomo, and González Viñas (2012), Rodríguez-Bencomo, 95 Ortega-Heras, and Pérez-Magariño (2009), and Soto Vázquez, Río Segade, and Orriols 96 Fernández (2010)) studied the effect of the pre-fermentative addition of oak wood chips 97 on phenolic composition and chromatic characteristics, and volatile fraction, 98 respectively. However, the addition of oak chips did not favour the reactions involved in 99 anthocyanin stabilisation neither nor in colour increase in bottled wines, suggesting that 100 this technique could be useful for the elaboration of young red wines. 101 Therefore, with the objective of improving the colour stabilisation of red wines from 102 grapes grown in a warm climate, the main goal of this work was to study the effect of 103 adding oak wood chips during fermentation. The study was performed on red wine 104 made from Tempranillo grape variety, one of the most important red grape cultivars 105 grown in Spain. To date, the addition of oak wood chips during the 106 fermentation/maceration stage has been scarcely studied, even less regarding the effect 107 on colour in relation to a considerable number of phenolics studied. Thus, our interest 108 was focused on the study of phenolic composition (anthocyanins, flavonols, flavan-3- 109 ols, hydroxycinnamic acid derivatives and benzoic compounds), chromatic 110 characteristics by applying differential colorimetry. 111 2. MATERIAL AND METHODS 112 2.1. Winemaking 113 Around 300 kg of grapes of Vitis vinifera cv. Tempranillo cultivated in 114 -western Spain, were harvested at their optimal 115 ripening stage and in good sanitary conditions. After the grapes were destemmed and 116 crushed, the must was distributed in twelve stainless steel tanks of 220 l. Six tanks were 6 117 submitted to the addition of 3 g/l of American oak (Quercus alba) wood medium- 118 toasted chips of 1 cm2 average size (Tonelería Martín y Vázquez, Logroño, Spain) to the 119 must, together with skins, and the other six tanks contained untreated, control wine. For 120 all wines, alcoholic fermentation was spontaneously developed. Skin maceration was 121 developed, manually punching down each tank once a day during 8 days. Subsequently, 122 the malolactic fermentation was induced by inoculation of Oenococcus Oeni lactic acid 123 bacteria (>1010 CFU O. oeni/ml, VINIFERM Oe 104, Agrovin, Spain) at the rate of 14 124 ml/hl at the end of alcoholic fermentation. This second fermentation ended in three 125 weeks, which was confirmed by HPLC determination of malic acid and lactic acid 126 contents, and the wines were then racked. Experiments were carried out in triplicate. 127 2.2 Oenological Parameters 128 The conventional oenological parameters (pH, total and volatile acidity, free and total 129 SO2, malic and lactic acids and reducing sugars) were performed according to the 130 Official Methods established by European Union (UE, 2003). 131 2.3. Spectrophotometric colour measurement 132 The whole visible spectrum (380- 133 with a Hewlett-Packard UV-vis HP8452 spectrophotometer (Palo Alto, CA), using 2 134 mm path length glass cells and distilled water as a reference. The CIELAB parameters 135 (L*, a*, b*, C*ab, and hab) were determined by using the original software CromaLab© 136 (Heredia, Álvarez, González-Miret, & Ramírez, 2004), following the Commission 137 , CIE, recommendations (CIE, 1986): the CIE 1964 10° 138 Standard Observer and the CIE Standard Illuminant D65. Euclidean distance between 139 two points in the three-dimensional space define by L*, a*, and b* were used for 140 calculating colour differences ab ab = 2 + 2 + 2]1/2. 7 141 The percentage contributions of copigmented anthocyanins to the total wine colour at 142 pH 3.6 were determined following the method proposed by Boulton (1996). Wine 143 samples were first adjusted to pH 3.6. 144 2.4. HPLC-DAD analysis of phenolic compounds 145 HPLC separation, identification and quantification of phenolic compounds were 146 performed in an Agilent 1200 chromatographic system equipped with a quaternary 147 pump, an UV-vis diode-array detector, an automatic injector, and ChemStation software 148 (Palo Alto, CA). 149 Nylon filter (E0034, Análisis Vínicos, Spain). All analyses were made in triplicate. 150 The anthocyanin identification was carried out following the method proposed by 151 Heredia et al. (2010). Anthocyanins were separated using a Zorbax C18 column (250 x 152 ºC. Acetonitrile-formic acid-water 153 (3:10:87) as solvent A and acetonitrile-formic acid-water (50:10:40) as solvent B were 154 used. The elution profile was as follows: 0-10 min 94% A-6% B; 10-15 min 70%A-30% 155 B; 15-25 min 60%A-40%B; 25-35 min 55%A-45%B; 35-40 min50%A-50%B; 40-42 156 min 40%A-60% B; 42-43 min 94% A-6% B. The flow rate was 0.8 mL/min, and the 157 l. UV-Vis spectra were recorded from 200 to 800 nm with a 158 bandwidth of 2.0 nm. The quantification was made at 525 nm by comparing the areas 159 and the retention times with the malvidin 3-glucoside standard, and anthocyanin 160 concentration was expressed as mg/l. 161 The method used for the identification of the phenolic compounds (flavan-3-ols, 162 flavonols, hydroxycinnamic acid derivatives and other low molecular weight phenolic 163 compounds), was a modification of the method described by Castillo-Muñoz, Gómez- 164 Alonso, García-Romero, and Hermosín-Gutiérrez (2007). These individual phenolic 165 compounds were separated using 8 166 size) maintained at 40 ºC. Acetonitrile-formic acid-water (3:10:87) as solvent A and 167 acetonitrile-formic acid-water (50:10:40) as solvent B were used. The elution profile 168 was as follows: 0-5 min 94% A-6% B; 5-10 min 89%A-11% B; 10-15 min 80%A- 169 20%B; 15-20 min 77%A-23%B; 20-25 min 74%A-26%B; 25-30 min 60%A-40% B; 170 30-35 min 50%A-50% B; 35-38 min 40%A-60%B; 38-46 min 94%A-6%B. The flow 171 rate was 0.63 ml l. UV-Vis spectra were 172 recorded from 200 to 800 nm with a bandwidth of 2.0 nm. The quantification was made 173 at 280, 320 and 360 nm by comparing the areas and the retention times with the gallic 174 acid, caffeic acid, and quercetin standards, respectively. Phenolic compounds 175 concentration was expressed as mg/l. Total anthocyanins, flavonols and flavan-3-ols 176 were calculated as sum of individual phenolic compounds identified by HPLC. Folin- 177 Ciocalteau reagent was used for the analysis of total phenolics (Singleton & Rossi, 178 1965). 179 2.5. Statistical Analysis 180 Statistical analysis was carried out by using Statistica version 8.0 software (Statistica, 181 2007). Univariate analysis of variance (Tukey test) was applied to discriminate among 182 the means of chemical data and by pairs of control wines and wines with addition of oak 183 wood chips for each studied point. Moreover, multivariate analysis of data (linear 184 discriminant analysis, LDA) was performed in order to classify wine samples according 185 to phenolic compounds and colour parameters. This method was applied to the set of 186 data consisting of 72 rows (wine samples) and 29 columns (individual phenolic 187 compounds and colorimetric variables). 188 3. RESULTS AND DISCUSSION 189 The effect of the fermentative addition of oak wood chips on the colour (by tristimulus 190 colorimetry), differential colorimetry and polyphenolic composition of Tempranillo red 15 340 discriminant function. Therefore, those variables were able to discriminate between both 341 groups of samples (CW and OW) with high levels of significance (p<0.001). 342 According to the classification functions, control wine samples were clearly 343 differentiated from those belonged to wines with the addition of oak wood chips (Figure 344 5). 95.8% of the samples were correctly assigned. Moreover, the percentage of 345 prediction was 100% for CW samples and 91.7% for OW samples. 346 4. CONCLUSIONS 347 It can be concluded that, a priori, the presence of oak wood chips during fermentation 348 and skin-maceration lead to an increase of the content of phenolics that improves the 349 chemical characteristics of the wines for colour stabilisation. Thus, adding oak chips 350 could represent a useful oenological alternative to prevent the excessive and typical loss 351 of pigments in wines obtained from grapes cultivated in warm climates. 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For each 5 day, asterisks denote significant differences (p<0.05), according to Tukey test, between 6 control wines (CW) and wines with the addition of oak wood chips (OW). 7 Fig. 3. CIELAB colour space (a*b*)-plane for control wines (CW) and wines with the 8 addition of oak wood chips (OW) during stabilisation stage. 9 Fig. 4. Colour variation ( E*ab) between control wines (CW) and wines with the 10 addition of oak wood chips (OW) during vinification. 11 Fig. 5. Scatterplot of the canonical variate obtained by LDA: control wines (CW) and 12 wines with the addition of oak wood chips (OW) during the stabilisation stage. Table 1. Mean values of concentration (mg/L) and standard deviations (n=33) for the polyphenolic compounds identified by HPLC-DAD, and the percentage of copigmentation (%CA), in control wines (CW) and wines with the addition of oak wood chips (OW). CW OW Mean SD Mean SD Total phenolics (Folin Ciocalteau) 1546.6 ± 796.94 * 2364.2 ± 736.82 Total anthocyanins 541.95 ± 170.21 627.73 ± 164.53 Total glucoside derivatives 397.10 ± 117.68 458.61 ± 120.62 Total acetate derivatives 59.91 ± 14.31 69.42 ± 15.41 Total p-coumaric derivatives 84.93 ± 26.19 99.69 ± 27.20 Total phenolic acids 139.92 ± 61.05 * 184.00 ± 55.44 Total flavan-3-ols 13.79 ± 8.35 16.52 ± 7.51 Total flavonols 23.26 ± 15.64 * 34.73 ± 14.28 %CA (Boulton) 21.12 ± 9.41 23.35 ± 10.03 Monomeric anthocyanins Delphinidin-3-glucoside 53.05 ± 15.8 54.74 ± 14.21 Cyanidin-3-glucoside 10.98 ± 2.2 10.04 ± 1.83 Petunidin-3-glucoside 62.57 ± 18.32 70.96 ± 18.53 Peonidin-3-glucoside 24.85 ± 5.56 25.06 ± 4.92 Malvidin-3-glucoside 255.33 ± 77.85 297.96 ± 83.44 Petunidin-3-acetyl-glucoside 15.32 ± 3.02 16.91 ± 3.07 Peonidin-3-acetyl-glucoside 9.63 ± 0.90 10.20 ± 1.12 Malvidin-3-acetyl-glucoside 36.78 ± 10.17 42.31 ± 11.52 Petunidin-3-p-coumaroyl-glucoside 17.62 ± 4.43 19.94 ± 4.61 Peonidin-3-p-coumaroyl -glucoside 11.23 ± 1.86 11.35 ± 1.99 Malvidin-3-p-coumaroyl -glucoside 59.19 ± 19.50 68.39 ± 20.95 Benzoic acids Gallic acid 97.89 ± 41.07 * 127.48 ± 20.15 Hydroxycinnamic acid derivatives GRP 8.10 ± 2.60 8.24 ± 2.36 t-caftaric acid 17.09 ± 5.80 19.29 ± 5.46 t-coutaric acid 29.09 ± 10.39 36.19 ± 10.19 p-coumaric acid 0.50 ± 0.83 1.03 ± 0.80 Flavan-3-ols (+)-catechin 21.98 ± 11.19 23.16 ± 12.46 (-)-epicatechin 6.23 ± 3.7 * 10.00 ± 4.16 ± ± Flavonols Myricetin-3-glucuronide 1.71 ± 1.29 * 2.57 ± 1.37 Myricetin-3-glucoside 10.65 ± 6.55 * 15.05 ± 6.03 Quercetin-3-glucuronide 4.16 ± 2.49 5.60 ± 2.53 Quercetin-3-glucoside 5.38 ± 2.87 6.97 ± 2.73 Laricitrin-3-glucoside 1.86 ± 1.67 2.80 ± 1.77 Kaempferol-3-glucoside 0.27 ± 0.28 0.39 ± 0.29 Isorhamnetin-3-glucoside 0.02 ± 0.09 0.13 ± 0.34 Syringetin-3-glucoside 0.76 ± 0.75 1.21 ± 0.84 * Asterisk indicates significant difference according to Tukey test (p<0.05) between control wines (CW) and wines with the addition of oak wood chips (OW); GRP, Grape Reaction Product (2-S-glutathionyl- caftaric acid). CW OW Fermentative maceration Stabilisation 1000 800 600 400 200 0 0 2 4 6 8 15 20 30 45 50 60 Vinification Stage (days) Fig. 1a 3750 3000 2250 1500 750 0 0 2 4 6 8 15 20 30 45 50 60 Vinification stage (days) Fig. 1b CW OW Fermentative maceration Stabilization