Impact of a post-fermentative maceration with overripe seeds on the color stability of red wines

1 IMPACT OF A POST-FERMENTATIVE MACERATION WITH OVERRIPE SEEDS ON THE COLOR STABILITY OF RED WINES. Francisco J. Rivero, M. José Jara-Palacios, Belén Gordillo, Francisco J. Heredia and M. Lourdes González-Miret* Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain Francisco J. Rivero: friver[email protected] M. José Jara-Palacios: [email protected] Belén Gordillo: [email protected] Francisco J. Heredia: [email protected] M. Lourdes González-Miret: [email protected] * Corresponding author: M. Lourdes González-Miret Food Colour & Quality Lab., Área de Nutrición y Bromatología. Facultad de Farmacia. Universidad de Sevilla. 41012-Sevilla, Spain Tel.: +34 955420938 e-mail: [email protected] *Manuscript Click here to view linked References 2 Abstract 1 With the purpose of modulating the copigmentation equilibria of red wines, an 2 environmentally sustainable process was performed based on post-fermentative 3 addition of overripe seeds (OS). Simple (SW) and double (DW) addition were 4 performed to produce different enrichment of phenolics from seeds, hence different 5 copigmentation/polymerization ratios. 6 The determination of the phenolic composition showed different global increases in 7 OS-macerates wines (catechin, epicatechin, gallic acid and procyanidins B1 and B2). 8 The double post-maceration (DW) was more effective than the simple post-maceration 9 addition to improve the phenolic structure of wines. 10 The application of Differential Tristimulus Colorimetry could assess the effects of this 11 practice on the color characteristics and stability of wines. Results highlighted that 12 both simple and double assays underwent colorimetric improvements against the 13 control wines (CW, no seeds addition). DW led to the highest chromatic stability, 14 showing lower lightness, higher chroma values and bluish hues than CW. This color 15 difference was visually detectable. 16 17 18 19 Keywords: Overripe grapes; copigmentation; phenolic composition; red wine color; 20 post-fermentative maceration 21 22 3 1. INTRODUCTION 23 Fermentative maceration is one of the more important steps of winemaking process 24 since the must remains in contact with the raw materials (grapes seeds and skins), and 25 hence phenolics, polysaccharides, nitrogen compounds, minerals and volatile 26 compounds can be extracted. Thus, the wines achieve the aromatic, tasteful and visual 27 structure. Later, the wine develops important sensory characteristics along the 28 stabilization and ageing steps. 29 The chemical compounds pass from grapes to the must/wine differently during the 30 maceration process, depending on their nature and extractability characteristics. Long 31 macerations lead to a depletion of the grapes (Kocabey, Yilmaztekin & Hayaloglu, 32 2016) subsequently, the solids constitute wastes once finished the extraction stage. 33 Phenolic compounds belong to one of the more important chemical groups related to 34 the winemaking process, having crucial influence on the organoleptic structure of the 35 wines (Santos-Buelga & de Freitas, 2009). Differences on extractability exist among the 36 different families within this group. On the one hand, the anthocyanins (located in the 37 skin) and the low-molecular weight flavanols (located largely in the seeds) are 38 extracted in aqueous medium, mainly during the first steps of maceration. 39 On the other hand, high-weight flavanols (also located mainly in the seeds), flavonols 40 (in the skins) and phenolic acids (in both skins and seeds) are more soluble in alcoholic 41 solutions (Jara-Palacios, Gordillo, González-Miret, Hernanz, Escudero-Gilete & Heredia, 42 2014a; González-Neves, Gil, Favre, Baldi, Hernández & Traverso, 2013; Gambuti, 43 Capuano, Lecce, Fragasso & Moio, 2009). 44 Some phenolics, such as gallic acid and protocatechuic acid, need long macerations 45 periods to achieve high extraction levels (Liu, Zhang, He, Duan & Shi, 2016; Zou, 46 4 Kilmartin, Inglis, & Frost, 2002). Therefore, the proper extraction of phenolics depends 47 greatly on both time and type of medium (hydroalcoholic) (Gambuti et al., 2009). 48 Nevertheless, extended macerations could involve sensory problems, mainly for 49 providing taste sensations of bitterness and astringency to the wine (Casassa et al., 50 2013). The bitterness of wine is primarily triggered by flavan-3-ols. This taste could be 51 also caused by some flavonols, hydroxycinnamates and benzoic acid derivatives. On 52 the other hand, polymeric procyanidins (or tannins) are the main responsible for the 53 astringency. Both bitterness and astringency, which have high importance on the 54 gustative evolution of the wines, become mellower along the time (Ma, Guo, Zhang, 55 Wang, Liu & Li, 2014; Chira, Jourdes & Teissedre, 2012). 56 In addition, the phenolics are crucial to stabilize the anthocyanins along the time 57 through copigmentation phenomenon, protecting the sensible coloured forms of 58 monomeric anthocyanins and avoiding their degradation (Gordillo et al., 2014). It is 59 crucial to reach the adequate pigment/copigment ratio in order to achieve good 60 copigmentation capacity to prevent the degradation of the monomeric anthocyanins. 61 Furthermore, it is important to consider the adequate types of copigments because 62 some of them have a more suitable structure for the pigment protection (Jara-Palacios 63 et al., 2014a; Berké & de Freitas, 2007). 64 Previous studies carried out in traditional winemaking (Bimpilas, Panagopoulou, 65 Tsimogiannis & Oreopoulou, 2016; Rivero, Gordillo, Jara-Palacios, González-Miret & 66 Heredia, 2017) showed that the percentage of copigmentation decrease about 20% 67 (from 30% to 10%) during the early 6 months; being approximately 15% the 68 degradation of monomeric anthocyanins during the early 3 months and this rate 69 increases in subsequent months. On the other hand, Rivero et al. (2017) found that 70 5 wines elaborated with the addition of copigments during the fermentative maceration 71 process had lower anthocyanin degradation rate during the stabilization. Therefore, 72 this could indicate that adding copigments at the beginning of stabilization stage could 73 be an alternative practice to favour the copigmentation phenomenon, and hence to 74 attenuate the later degradation of monomeric anthocyanin. 75 Several macerations techniques (cold pre-fermentative maceration, post-fermentative 76 heating, carbonic maceration, délestage or enzymatic maceration) have been 77 developed as alternatives in red winemaking to improve the phenolic extractions from 78 red grapes, skins and seeds (Lukić, Budić-Leto, Bubola, Damijanić & Staver, 2017; 79 Gustavo González-Neves, Favre, Piccardo & Gil, 2016). This increment achieves the 80 subsequent formation of new and more stable pigments, which provides more stable 81 color of wines (Cejudo-Bastante, Gordillo, Hernanz, Escudero-Gilete, González-Miret & 82 Heredia, 2014) 83 Oak chips, enzymatic hydrolysate extracts or white grape pomace have been assayed 84 as exogenous sources for the addition of phenolics during the fermentative stage (Soto 85 Vázquez, Río Segade & Orriols Fernández, 2010; Baca-Bocanegra, Nogales-Bueno, 86 Hernández-Hierro & Heredia, 2018; Cejudo-Bastante, Rodríguez-Morgado, Jara-87 Palacios, Rivas-Gonzalo, Parrado & Heredia, 2016; Gordillo et al., 2014). Recently, the 88 addition of an extra amount of seeds from overripe white grapes during the alcoholic-89 fermentative maceration has obtained encouraging results (Rivero et al., 2017), 90 enhancing the bluish hues of wines as well as the stability of anthocyanins. 91 Nevertheless, this method showed some operational limits coming from the difference 92 on the harvesting time between white and red grapes, and hence overripe seeds from 93 white grapes are not available when red wine fermentation occurs. Therefore, we 94 6 need to have OS from previous vintage, which have had to be stored, usually frozen to 95 avoid spoilages. On the other hand, the length of OS-wine contact is determined by the 96 run-off time (red skin and seeds removal). 97 The overripe seeds are obtained from high-maturate grapes processed in pasera sites, 98 places where grapes are submitted to postharvest direct-sun dehydration, in order to 99 increase the sugar content for the elaboration of typical sweet wines from Andalucía 100 (south of Spain). The long maturation time, the high temperatures and the long sun 101 periods needed to produce the dehydration and the sugar concentration lead to the 102 synthesis and the polymerization of phenolics (Dumitriu, Peinado, Peinado & de Lerma, 103 2015). Thus, these seeds are important source of phenolics and copigments such as 104 epicatechin, gallic acid and procyanidin B2-3-O-gallate. 105 The aim of this work is to assess the overripe seeds by-products as a source of 106 phenolics when adding in a subsequent post-fermentative seed-maceration. In this 107 way, it is possible to strengthen an eventual low copigment/pigment ratio, and thus, to 108 avoid the anthocyanin degradation. Moreover, this post-fermentative seed-maceration 109 could be longer than the fermentative maceration (not conditioned by the run off), 110 and therefore, could produce a higher extraction of phenols from the overripe seeds, 111 which have low bitterness due to their high polymerization grade (Liu, Pan, Yan, He & 112 Duan, 2010). In addition, overripe seeds can be obtained within the same vintage, so it 113 does not need the storage. 114 Thus, post-fermentative seed-maceration involves important operational, economic 115 and environmental benefits to the industry of wine. The sustainability of process is 116 becoming a mandatory standard, and it makes relevant the oenological proposals 117 based on reusing agro-industrial wastes. 118 7 2. MATERIAL AND METHODS 119 2.1. Winemaking protocols and samples 120 The overripe seeds (OS) were obtained from Vitis vinifera L. cv. Pedro Ximénez (PX) 121 high-maturate grapes (D.O. Montilla-Moriles, Southwest Spain, 2016 vintage, 24 °Bé of 122 sugar content). The seeds were manually separated from grape pomace. Around 5400 123 g of these seeds (average total phenolics: 5535 mg/100 g of dry seeds) were used for 124 the elaboration red wines from V. vinifera cv. Syrah grapes (900 kg harvested at 125 optimum technological maturity) grown in D.O. Condado de Huelva (Southwestern 126 Spain). 127 The fermentation mash (destemmed and crushed grapes) was distributed in six 128 stainless steel tanks of 220 L capacity to perform the alcoholic fermentation by adding 129 25 g/hL of selected Saccharomyces cerevisiae yeast (Viniferm BY, Agrovin, Ciudad Real, 130 Spain). Skin maceration was developed manually punching down each tank once a day 131 during 6 days. After this, the mash was drawn off to remove the solid parts, and the 132 free run wines were racked to nine 50 L stainless steel tanks. Based on results from 133 previous studies (Rivero et al., 2017) three types of experimental post-fermentative 134 treatment were performed: 135  SW (3 tanks) single post-fermentative maceration: addition of 600 g of overripe 136 seeds per tank, macerated during 30 days (12 g/L seeds, 30 days) 137  DW (3 tanks) double post-fermentative maceration: addition of 600 g of overripe 138 seeds per tank, macerated during 30 days, and a further second addition of 600 g 139 OS, macerated 30 days more (12 g/L seeds, 30 days, and a second addition of 12 g/L 140 seeds, 30 days). 141 8  CW (3 tanks) wines made by traditional winemaking (without post-fermentative 142 addition of overripe seeds), as control wine. 143 Simultaneously to the OS addition (without OS in control wines), selected Oenococcus 144 oeni lactic acid bacteria (VINIFERM Oe 104, 14 mL/hL, Agrovin, Ciudad Real, Spain) 145 were inoculated to develop the malolactic fermentation. 146 At the end of malolactic fermentation, sulfur dioxide levels were adjusted (total sulfur 147 dioxide about 100 mg/L and free sulfur dioxide about 60 mg/L in all wines). The wines 148 were kept in the stainless steel tanks during 150 days until the end of the stabilization 149 process. 150 The winemaking was carefully followed by analysing wine samples under oenological 151 point of view. For the study, samples (50 mL) were taken at three points: 152 - initial point: 0 days, addition of overripe seeds 153 - seeds removal: after 30 days for SW and 60 days for DW 154 - final point: 150 days after overripe seeds addition (end of stabilization) 155 2.2. Oenological parameters 156 The conventional analysis of oenological parameters (pH, total and volatile acidity, free 157 and total SO2, malic and lactic acids and reducing sugars) (Table 1) were performed 158 according to the Official Methods established by European Union. 159 2.3. Copigmented and Polymerized Anthocyanin Determination 160 The contribution of copigmented anthocyanins to the total wine color at pH 3.6 (% 161 copigmented anthocyanins) and the degree of anthocyanin polymerization (% 162 polymeric pigments) were determined following the method proposed by Boulton 163 (1996). The pH values of wine samples were first adjusted to 3.6 using 1M NaOH or 164 HCl. 165 9 2.4. Colorimetric analysis 166 The visible spectra (380-770 nm) was measured in triplicate at constant intervals (∆=2 167 nm) with an Agilent 8453 UV-Vis spectrophotometer (Palo Alto, USA), using 2 mm path 168 length glass cells and distilled water as white reference. The CIELAB colour parameters 169 (L*, a*, b*, C*ab and hab) were calculated from the transmittance spectra by using the 170 original software CromaLab® (Heredia, Alvarez, González-Miret & Ramírez, 2004), 171 following the recommendations of the Commission Internationale de l’Eclairage (CIE, 172 2004); 10° Standard Observer and D65 Standard Illuminant were used as references. 173 Colour differences (∆E*ab) were calculate as the Euclidean distance between two 174 points in the three-dimensional space defined by L*, a*, and b*: ∆E*ab = [(∆L*)2 + 175 (∆a*)2 + (∆b*)2]1/2. 176 2.5. HPLC-DAD analysis of phenolic compounds 177 The monomeric anthocyanins and flavonols were determined in triplicate according to 178 the method reported by Cejudo-Bastante et al., (2016), which performs identification 179 based on the retention times and HPLC-DAD-ESI-MSn. 180 The HPLC separation and quantification were performed in an Agilent 1200 181 chromatographic system, equipped with quaternary pump, UV-VIS diode-array 182 detector, automatic injector, and ChemStation software (Agilent Technologies, Palo 183 Alto, USA). The samples were filtered through a 0.45 m Nylon filter prior to direct 184 injection; then, a volume of 50 L was injected onto a Zorbax C18 column (250 x 4.6 185 mm, 5 m particle size). Acetonitrile, formic acid and water were used as solvents, 186 being 3:10:87 solvent A and 50:10:40 solvent B (mL:mL:mL). The elution profile was: 0-187 10 min with 6% B; 10-15 min with 11% B; 15-20 min with 20% B; 20-25 min with 23% B; 188 25-30 min with 26% B; 30-35 min with 40% B; 35-38 min with 50% B; 38-46 min with 189 16 decrease and increase, respectively. In such a way, the global process led to slightly 334 darker and more vivid those wines submitted to double OS maceration (DW), being 335 wines with lower L* values and higher C*ab than CW (L* = 72.31 vs 74.55 units; C*ab = 336 25.66 vs 24.23 units, respectively). However, C*ab and L*values were similar in SW and 337 CW (L*=73.69 vs 74.55 units and C*ab=23.46 vs 24.23 units, respectively). Regarding 338 the hue, after an initial increase towards positive values (less bluish-red) during the 339 first step (mainly the OS macerated wines, SW and DW), the wines finally were close to 340 pure-red hues (close to 0°), being the lowest values for SW. Hue angle (hab) showed 341 significant differences (p<0.05) between DW and CW at the end of the stabilization 342 stage. The wines with double addition of PX overripe seeds were more bluish than the 343 CW (hab=3.7° vs 4.9°). This could be due to a higher copigmentation of monomeric 344 anthocyanins in DW caused for the larger copigment/pigment ratio (Sum 345 Copigments/Sum Total Anthocyanin= 10.4 vs 6.4, DW and CW, respectively, measured 346 by HPLC and showed in Table 2), which can result in a higher bathochromic effect or 347 positive shift toward higher wavelength within the visible range (Boulton, 2001). 348 Considering these parameters, the color differences among the wines at the end of the 349 stabilization stage were 1.52 units for CW-SW, 2.70 units for CW-DW and 2.64 units for 350 SW-DW. That is to say, the double assay (DW) lead to color changes that can be 351 perceptible (Gordillo et al, 2014), being the single (SW) similar in color to the control 352 wine. 353 Figure 1 shows the location of the initial (0 days) and final (150 days) wines (CW, SW 354 and DW) on the CIELAB (a*b*)-diagram, where can be seen the chroma (the distance 355 from the origin of coordinates to the color point) and the hue (the angle formed with 356 the semiaxis +a*) for every color point. As observed, the different OS maceration led to 357 17 differences in wine color at the end of the stabilization stage, and the color points of 358 SW, DW and CW appear separated within the a*b*-plane. 359 Considering the global process, the determination of the color differences (ΔE*ab) 360 occurring from the OS addition to the end of the stabilization period allows evaluating 361 the color stability of each wine. Therefore, in order to assess these observations, the 362 CIELAB color difference (ΔE*ab) and the differences of the color parameters (L*, C*ab 363 and hab) were calculated for every wine (CW, SW and DW). The assessment was made 364 considering the end of the maceration process (30/60 days) as well as the end of the 365 winemaking process (150 days), regarding the initial point (0 days) (Table 4). SW and 366 DW showed higher color changes (color difference ΔE*ab and individual color 367 parameters ΔL*, ΔC*ab, Δhab) along the OS-maceration time than their corresponding 368 control wine (CW 30 days for SW and CW 60 days for DW). In the case of hue, the 369 wines submitted to OS-maceration showed higher increases (less bluish-red). This 370 could be due to a combined effect of the higher synthesis of polymeric anthocyanins 371 (yellowish) with the copigments extracted from seeds during the post-fermentative 372 maceration (Burtch, Mansfield & Manns., 2017), together with the light yellowish 373 colors provided directly by copigments such as flavan-3-ols and procyanidins (Ashraf-374 Khorassani & Taylor, 2004) supplied by the overripe seeds. This colorimetric behavior 375 reverted during the stabilization stage (from seeds removal), in both quantitative (C*ab 376 and L*) and qualitative (hab) terms. 377 Along the global process (from initial to end of stabilization) the lowest color 378 difference (ΔE*ab) was found for DW. All wines underwent losses of chroma (ΔC*ab); 379 DW showed the lowest change followed by SW and CW, being significantly (p<0.05) 380 different among them. With respect to lightness (L*), CW and SW showed few 381 18 increases (towards clearer color) and few decrease in DW (towards darker color), but 382 the differences were not significant. In the case of hue (hab), similar increases 383 occurred in CW and DW while SW showed a significant (p<0.05) lower change (5.8°, 384 5.1°, 4.6°, respectively). 385 The comparison of the final wines (at 150 days) allows assessing the effect caused by 386 the OS-maceration. With this purpose, the color differences (ΔE*ab) between the OS-387 macerated wines and their corresponding control wines (SW150 vs CW150 and DW150 vs 388 CW150) were determined. ΔE*ab was higher in the double overripe seed addition than 389 simple addition (3.25 and 1.58 units, respectively). This could be due to copigments 390 from Pedro Ximénez overripe seeds (larger amount in DW) such as benzoic acids or 391 procyanidins, which are involved in the increment of polymeric anthocyanins during 392 the stabilization stage (Liu et al., 2016; Berké & de Freitas, 2007; Rivero et al., 2017). 393 Thus, the polymerization and copigmentation processes led to the highest color 394 difference (ΔE*ab) between CW and DW, which only in DW were visually perceptible by 395 the human eye (Gordillo et al., 2014). 396 Regarding the contribution of the individual colorimetric variables to the color 397 differences (lightness %L, chroma %C and hue %H), it was found that the double 398 addition of overripe seeds induced greater change on lightness followed by chroma 399 and hue (%ΔL*= 77%, %ΔC*ab= 20% and %ΔH= 3%), which is in accordance with 400 previous works (Rivero et al., 2017). 401 4. CONCLUSIONS 402 The addition of overripe seeds to red wine elaboration could represent an alternative 403 technique to strengthen the eventual low copigment/pigment ratio of wines by 404 increasing the content of some compounds, mainly flavanols, benzoic acids and 405 19 procyanidins. Moreover, adding OS during post-fermentative steps allows performing 406 long macerations periods. This addition improves the copigment proportion, avoiding 407 the anthocyanin degradation (by copigmentation and polymerization processes), and 408 leads to more suitable wines for the aging process. In this study, double addition of 409 overripe seeds led to better results. 410 This process exerted positive effects on the wine color, giving wines with lower 411 lightness and higher chroma values (darker and more vivid colors) and more bluish 412 hues than the wines traditionally produced. Results proved the effectiveness of the 413 post-fermentative maceration with overripe seeds, mainly the double addition, to 414 stabilize the color of wines and to provoke lower color modifications along the time, 415 producing wines chromatically more stable for a better ageing. 416 This study obtained interesting and promising results for wines with unbalanced 417 copigment/pigment proportion that is the case of winemaking in warm-climates. In 418 addition, it involves different benefits: environmental, achieved though the reuse of 419 overripe seeds (a waste of the wine industry), operational and economical due to the 420 possibility of using overripe seeds within the same vintage, avoiding the storage. 421 422 ACKNOWLEDGMENTS 423 Authors thank the Ministerio de Economía y Competitividad, Gobierno de España 424 (project AGL2014-58486-C2-2-R) and V Plan Propio de Investigación de la Universidad 425 de Sevilla (Acción II.3) for the financial support; also thank the assistance of the 426 technical staff of Biology Service (SGI, Universidad de Sevilla). 427 Note 428 The authors declare no competing financial interest. 429 20 REFERENCES 430 Ashraf-Khorassani, M. & Taylor, L. T. (2004). Sequential Fractionation of Grape Seeds 431 into Oils, Polyphenols, and Procyanidins via a Single System Employing CO 2 -432 Based Fluids. 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