Or Seeds During Winemaking

Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Addition of Mannoproteins and/or Seeds during Winemaking and Their Effects on Pigment Composition and Color Stability Cristina Alcalde-Eon,† Claudia Peŕ ez-Mestre,† Rebeca Ferreras-Charro,† Francisco J. Rivero,‡ Francisco J. Heredia,‡ and María Teresa Escribano-Bailoń *,† †

Grupo de Investigación en Polifenoles, Facultad de Farmacia, University of Salamanca, Salamanca 37007, Spain Food Color and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, Sevilla 41004, Spain

‡

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S Supporting Information *

ABSTRACT: The increase of temperature can cause a decoupling of sugar and anthocyanin accumulation in grapes, leading to wines poor in color. Different enological techniques are nowadays under study to overcome this problem. Among them, the present study has evaluated the color and pigment composition modifications caused by the addition during Syrah winemaking of either Pedro Ximénez seeds (intended to increase chemical stability) or/and two different mannoproteins (color-protective and astringency-modulator) to increase colloidal stability. Color and pigment composition modifications were assessed from CIELAB color parameters and from HPLC-DAD-MSn results before and after cold-treatment (used to force colloidal instability). The addition of both seeds and mannoproteins increased color stability against cold and, additionally, against SO2bleaching in the case of mannoproteins. However, the initial pigment composition and color of the samples were differently affected by these additions, being clearly affected (ΔE*ab > 3) in the cases of seeds and with the astringency-modulator mannoprotein and hardly modified with the other one. KEYWORDS: Vitis vinifera L. Syrah, Pedro Ximénez, seeds, mannoproteins, CIELAB, HPLC-DAD-MSn, anthocyanins, SO2-bleaching, colloidal stability

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aroma, taste, or mouthfeel perception.5,6 For this reason, the development of vineyard practices and enological techniques aiming at compensating for the decoupling of sugars and anthocyanin accumulation associated with climate change is nowadays a concern of wine producers. The addition of Pedro Ximénez grape pomace (byproduct of the fermentation of Pedro Ximénez white grapes) during the fermentative step of Syrah winemaking has been reported to improve the color characteristics of young Syrah wines from a warm climate7 probably by supplying copigments and favoring polymerization, thus increasing color stability. However, if the percentage of grape pomace added is high, adsorption of the pigments onto it can also occur.7 For this reason, the use of seeds without skins from overripe Pedro Ximénez grapes that have been subjected to postharvest dehydration has recently been evaluated.8 It was concluded that the addition of this byproduct during the fermentation step of the Syrah winemaking allowed higher pigment extraction and better diffusion of copigments to the wine as well as a higher chemical stabilization during the last steps of the winemaking process. These results make this technique a good candidate for compensating color deficiency in Syrah wines from a warm climate. The main drawback is that fermentative addition of seeds from overripe grapes cannot be done from grapes of the current vintage. A postfermentative addition of this byproduct

INTRODUCTION Nowadays, there is no doubt of the influence of global climate on vineyards and on wine quality.1 The impact is mainly caused by the alteration of the rainfall patterns and the increase in the frequency of days with extremely high temperatures. It has been suggested that the effects of climate change are not likely to be uniform for all of the grape varieties and winemaking regions.2 However, one of the main effects that has been observed in different winemaking regions is the advancement of harvest date, which has been more perceptible in the last 10−30 years.1 Furthermore, this advancement of harvest date has also been projected for some of the major wine grape varieties (Cabernet Sauvignon, Syrah, and Chardonnay).3 Nevertheless, this earlier technological maturity does not imply earlier phenolic maturity, and, consequently, at harvest date, grapes may have reached an adequate content of total soluble solids but not an adequate phenolic composition, which can directly affect wine quality. In the case of Vitis vinifera L. cv Syrah grapes, Sadras and Moran4 have demonstrated that increased temperatures can cause decoupling of sugars and anthocyanins accumulation, probably due to a delayed onset of anthocyanin accumulation. Consequently, if the content of soluble solids (°Brix) optimum for the intended Syrah wine is reached earlier, grapes will be harvested earlier, and the wine made from them will show lower color. Because color is the first wine organoleptic attribute that is perceived by consumers, this lower coloration can condition the acceptance of a given wine. Color can inform about the age of the wine and can also point out inadequate storage conditions. It can even condition other attributes such as © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

December 12, 2018 March 14, 2019 March 20, 2019 March 20, 2019 DOI: 10.1021/acs.jafc.8b06922 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Scheme of the winemaking process of the wines employed in the present study (a) and design of the two different experiments performed (b).

directly cause their precipitation. The addition of mannoproteins (MPs) could be useful for improving colloidal stability because MPs can limit self-aggregation of tannins13 and can contribute to the delay of tannin polymerization in red wines14 with direct consequences on the evolution of wine astringency and coloring matter.15 More recently, in a study carried out in our laboratory,11 MPs seemed to stabilize some anthocyaninderived pigments from a colloidal point of view. Furthermore, color changes induced by cold treatment were lower in the wines treated with MPs.11 However, despite all MPs being glycoproteins of the Saccharomyces cerevisiae cell wall, the commercial MP preparations do not seem to exert the same effects. These different effects can be attributed to differences in their compositions and in their purities,14,16 which, in turn, depends on the strain of the fermentative yeast from where they come from.15 In addition, MPs can either be excreted to the wine during alcoholic fermentation or be released during yeast autolysis,17 and it seems that higher stabilizing effects are obtained with the latter ones.18 Consequently, the method of

would allow the use of seeds from overripe grapes and Syrah grapes from the same vintage, reducing costs of storage. Other techniques such as the addition of oak chips9 or enological tannins10,11 during alcoholic fermentation have also been demonstrated to improve wine color features. By the use of these treatments, wine color can be stabilized from a chemical point of view, because they can favor the formation of anthocyanin-derived pigments, whose colors are often more resistant to pH changes or to bleaching by SO2 than those of the grape native anthocyanins. However, in red wines, it is also important to achieve a colloidal stabilization of the coloring matter. Cold has been traditionally used to stabilize wines from a colloidal point of view, because it causes the precipitation of tartrates and unstable colloidal substances avoiding further undesirable precipitation and wine turbidity.12 Nevertheless, cold can also precipitate coloring matter. On the one hand, pigments can adsorb on the tartrate crystals or on tannin aggregates and precipitate with them. On the other hand, cold itself can reduce the colloidal stability of the pigments and B

DOI: 10.1021/acs.jafc.8b06922 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


The first wine was neither added with seeds nor with MPs and served as control wine (C) for the first experiment. The second wine (S) was treated after alcoholic fermentation with seeds from Pedro Ximénez overripe grapes (grown in D.O. Montilla-Moriles, southwestern Spain, harvested in September 2016, and subjected to off-vine postharvest dehydration in the pasera site; 24° Bé; see Table S1 for more details on flavanol composition of the seeds). Two consecutive additions of 12 g of seeds per liter of wine were carried out, replacing the seeds added the first time by a new batch of seeds after 1 month of maceration. This second batch of seeds was also macerated 1 month in the wine (end of the winemaking process). The third and fourth wines (wines SF1 and SF2) were the same as wine S but treated with two different doses of MP-F at the end of alcoholic fermentation (50 and 125 mL/hL) and were the problem samples in the second experiment. The triplicates of a given wine sampled at the end of the winemaking process were first gathered to homogenize the samples at the beginning of experiments 1 and 2. Experiment 1: Effects of Seeds, Mannoprotein M, and Cold Treatment on Wine Color and Pigment Composition. From each of the homogenized wines C and S (Figure 1b, left), six aliquots of 125 mL were separated. For each type of wine, three of them served as control samples (triplicates of the samples C and S), and the other three were treated with MP-M (triplicates of the samples CM and SM) at the dose recommended by the manufacturer (300 mL/hL). In the aliquots nonadded with the MP, the same volume of an acidic (HCl) aqueous solution at the same pH as that of the MP (pH 3) was added to avoid the color changes between samples that might be associated with the dilution effect and pH differences after the addition of the MP. All of the aliquots were kept in darkness at 19 °C for 6 days. Before cold treatment was applied, color and pigment composition were analyzed. Cold treatment consisted of cooling samples at 4 °C and in darkness for 7 days. The samples then were centrifuged at 5000 rpm for 10 min at 5 °C, and the color and pigment composition of the supernatants were analyzed again (samples C#, CM#, S#, and SM#). Experiment 2: Effects of Mannoprotein F at Two Different Doses and Cold Treatment on the Color and Pigment Composition of Wines Added with Seeds. At the end of the winemaking process, three aliquots of 125 mL were separated from each homogenized SF1 and SF2 wines (triplicates of the wines SF1 and SF2). To make the results of wines SF1 and SF2 comparable to those obtained for wine S in experiment 1, the aliquots of wines SF1 and SF2 were also added with the same volume of the acidic aqueous solution indicated above. From this moment, both experiments were conducted simultaneously, and aliquots of SF1 and SF2 were also kept in darkness at 19 °C for 6 days, analyzed prior to cold treatment, cold treated for 7 days (samples SF1# and SF2#), centrifuged, and analyzed again (Figure 1b, right). Colorimetric Measurements. Absorption spectra (190−770 nm) were recorded in a Hewlett-Packard UV−vis HP 8453 (Agilent Technologies, Waldbronn, Germany) spectrophotometer in 2 mm path length quartz cells. The analysis of color was made only from the visible spectra (380−770 nm) data, using the CIE standard illuminant D65 and the CIE 1964 standard observer (10° visual field) as references. CIELAB color parameters (L*, a*, b*, C*ab, and hab) were calculated using the software CromaLab.19 Color differences between different wines and treatments were also determined by means of the CIELAB color difference formula:

their obtaining would also condition the composition and the final activity. On the basis of semiempirical observations and the results of scientific studies,14 different MPs are marketed for different goals, with the improvement of the astringency and the color stability being some of the most requested by oenologists. Thus, the objective of the present study was to evaluate the chemical and colloidal stabilization of the coloring matter of a red wine made from Syrah grapes from a warm climate after the combined addition of Pedro Ximénez overripe seeds (postfermentative addition) and one of two different MPs (MP-M and MP-F) marketed for different purposes (to improve wine color stability and wine astringency, respectively). Two different experiments were conducted separately, one for each MP, conditioned by the different instructions for use recommended by the manufacturer of the MPs. In both cases, wine samples were cold treated to provoke colloidal instability. Whereas CIELAB parameters were used to assess color modifications due to the different treatments, changes in pigment composition were evaluated by HPLC-DAD-MSn. Resistance against SO2-bleaching was also evaluated and compared in all of the types of wines.

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MATERIALS AND METHODS

Yeast MPs. Mannoproteins MP-M and MP-F were supplied in liquid forms by Laffort España S.A. (Renterı ́a, Spain). According to the manufacturer, MP-M could exert a stabilizing effect over the coloring matter because it contributes to the tartaric and colloidal stabilization of the wine. On the contrary, the main effect attributed by manufacturer to MP-F would be the improvement of tactile sensation in the oral cavity. As recommended by the manufacturer, MP-M was added in finished wine (before bottling) at the dose of 300 mL/hL and MP-F at the end of alcoholic fermentation. In the case of MP-F, two doses were assayed (50 and 125 mL/hL), which are both within the recommended range. Samples and Experimental Design. Two different experiments were carried out simultaneously to evaluate the effects on the coloring matter of the postfermentative addition of Vitis vinifera L. cv. Pedro Ximénez overripe seeds and/or two types of MPs as well as to evaluate the ability of these techniques to stabilize the coloring matter against precipitation by cold treatment. It was expected that seeds would promote chemical stability, whereas MPs would promote colloidal stability (in addition to the effects claimed by the manufacturer for them and indicated above). It was necessary to conduct two different experiments because the moment of addition of each tested MP was different. Figure 1a shows a scheme of the winemaking process of all of the wines, whereas Figure 1b shows the design of both experiments. The same batch of Vitis vinifera L. cv. Syrah grapes grown in D.O. Condado de Huelva (Southwestern Spain) and collected in September 2016 at technological maturity (14°Bé) was used to make all of the wines of both experiments. Alcoholic fermentation (inoculation of selected Saccharomyces cerevisiae yeast-VINIFERM BY, 25 g/hL, Agrovin, Ciudad Real, Spain) lasted 7 days, and then the wine obtained from that single batch of grapes was devatted and separated into 12 30 L stainless steel tanks (3 tanks per type of wine), where the additions of seeds and/or MPs were carried out. At this moment, the features of the wine were the following (mean ± SD, n = 3): pH 3.74 ± 0.05; total acidity (g/L as tartaric acid), 5.75 ± 0.17; volatile acidity (g/L as acetic acid), 0.47 ± 0.01; free SO2 (mg/L), 65 ± 2; total SO2 (mg/L), 95 ± 0.00; reducing sugars (g/L), 1.7 ± 0.20; malic acid (g/L), 1.47 ± 0.10; lactic acid (g/L), 0.29 ± 0.03; alcohol (ethanol) content (% vol), 13. Lactic bacteria (VINIFERM Oe 104, 14 mL/hL, Agrovin, Ciudad Real, Spain) were also inoculated at this stage in all of the tanks. Malolactic fermentation (MLF) lasted 18 days. The different wines were kept in these tanks for seven additional weeks, and then they were sampled to begin both experiments.

ΔE*ab = ((ΔL*)2 + (Δa*)2 + (Δb*)2 )1/2 The effect of the bleaching of the wines by SO2 and the possible protective effect of MPs were also evaluated in the present study. UV−vis absorption spectra were measured after the incorporation of a concentrated aqueous solution of sodium bisulphite (40%, 40 μL) to an aliquot of each sample (2 mL). The same volume of an aqueous solution of acetaldehyde (5%) was added to another aliquot (2 mL) of each sample, to release pigments from SO2 that could remain linked to them after the different winemaking steps that involve the use of SO2. The UV−vis spectra of these aliquots were also recorded and used as control ones. The CIELAB parameters then were C


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Table 1. Chromatic Features (CIELAB Parameters) and Quantitative Composition (Expressed as mg/L of Malvidin 3-OGlucoside and as Percentage) of the Samples of Experiment 1 (Mean Values, n = 3) samples VARa CIELAB Parameters L* 1 a* 2 b* 3 C*ab 4 hab 5 Monoglc acetyl p-coum caffeoyl F-A+ F-et-A+ Vit A Vit B Vit VF DRP total Monoglc acetyl p-coum caffeoyl F-A+ F-et-A+ Vit A Vit B Vit VF DRP

6 7 8 9 10 11 12 13 14 15

C

CM

64.70 ab 32.97 c 7.37 b 33.79 b 12.60 b

64.13 a 33.20 c 7.53 b 34.04 bc 12.78 b

45.35 bc 27.69 bc 15.60 abc 0.18 ab 0.45 b 1.15 a 1.90 bc 0.42 c 0.24 a 56.80 ab 152.12 bc

45.23 b 27.67 bc 15.28 ab 0.18 ab 0.45 b 1.40 a 1.74 ab 0.43 c 0.24 a 55.26 ab 150.30 abc

30.28 a 18.56 a 10.52 ab 0.12 a 0.29 c 0.77 a 1.27 b 0.28 c 0.15 b 37.47 c

30.54 a 18.77 a 10.48 ab 0.12 a 0.30 cd 0.95 b 1.20 ab 0.29 c 0.15 b 36.89 bc

C#

CM#

65.14 b 64.34 ab 33.48 cd 34.03 d 6.85 a 6.78 a 34.17 bc 34.70 c 11.56 a 11.26 a Concentration (mg/L) 40.23 a 41.24 ab 24.23 a 24.86 ab 12.16 a 12.29 a 0.16 a 0.17 a 0.45 b 0.46 b 1.25 a 1.24 a 1.65 ab 1.72 ab 0.38 bc 0.39 bc 0.17 a 0.18 a 48.64 a 50.17 ab 131.51 a 135.00 ab Percentage 31.03 a 30.98 a 18.79 a 18.77 a 9.61 a 9.46 a 0.12 a 0.12 a 0.34 d 0.34 d 0.96 b 0.94 b 1.30 b 1.31 bc 0.29 c 0.29 c 0.12 ab 0.13 ab 37.14 c 37.32 c

S

SM

S#

SM#

73.57 c 23.29 ab 10.18 c 25.42 a 23.62 d

73.90 c 23.02 a 10.04 c 25.11 a 23.57 d

73.18 c 23.44 ab 10.05 c 25.50 a 23.20 d

73.44 c 23.73 b 9.85 c 25.70 a 22.54 c

49.49 d 29.98 c 20.33 c 0.21 ab 0.33 a 1.17 a 2.34 d 0.38 bc 0.2 a 58.23 b 165.19 c

49.44 cd 30.11 c 20.21 bc 0.23 b 0.33 a 1.36 a 2.09 c 0.40 bc 0.19 a 56.71 ab 163.66 c

44.30 ab 27.23 bc 16.91 abc 0.17 a 0.34 a 1.11 a 1.56 a 0.31 a 0.14 a 52.17 ab 146.54 abc

44.34 ab 27.27 bc 16.89 abc 0.16 a 0.38 a 1.24 a 1.58 a 0.35 ab 0.15 a 52.47 ab 147.15 abc

30.35 a 18.47 a 12.66 c 0.13 a 0.20 a 0.72 a 1.46 c 0.23 ab 0.12 ab 35.38 a

30.62 a 18.73 a 12.71 c 0.14 a 0.20 a 0.84 ab 1.32 bc 0.25 b 0.11 ab 34.76 a

30.64 a 18.93 a 11.89 bc 0.11 a 0.23 ab 0.77 a 1.10 a 0.21 a 0.09 a 35.75 ab

30.54 a 18.87 a 11.90 bc 0.11 a 0.26 b 0.86 ab 1.10 a 0.24 ab 0.09 a 35.80 ab

a

VAR, number of the variable in PCA; Monoglc, monoglucosides; acetyl, acetyl derivatives; p-coum, p-coumaroyl derivatives; caffeoyl, caffeoyl derivatives; F-A+, flavanol-anthocyanin direct condensation products; F-et-A+, flavanol-anthocyanin acetaldehyde-mediated condensation products; Vit A, A-type vitisins; Vit B, B-type vitisins; Vit VF, vinylphenol-type pyranoanthocyanins; DRP, difficult resolution pigments. Different letters in the same row indicate significant differences (p < 0.05). 5000 V, and the temperature of the probe (TEM) at 600 °C. Both quadrupoles were set at unit resolution. Three types of mass experiments were performed: a full mass analysis (EMS mode, collision energy (CE) 10 V), where all of the ions were detected, an MS2 analysis (EPI mode, CE 30 V), where the major ion of the full mass analysis was fragmented, and a MS3 analysis (CE 30 V, excitation energy (AF2) 80 V), where the major fragment ion of the MS2 analysis was, in turn, fragmented. Spectra were recorded between m/z 150 and 1400. Compounds were identified from the data supplied by the HPLCDAD-MSn analyses of the samples (retention time, UV−vis spectra, m/z ratio, and fragmentation patterns) and by comparison to data previously obtained in our laboratory for wine samples analyzed in the same conditions.20 Thirty-nine anthocyanins and derivative pigments were determined (see Figure S1 and Table S2 for more details). Individual and total pigment contents of each sample were calculated by means of a malvidin 3-O-glucoside calibration curve. For comparative purposes, pigments were then grouped according to their structures in monoglucosides (5 compounds), acetyl derivatives (5 anthocyanin 3-O-acetylglucosides), p-coumaroyl derivatives (7 anthocyanin 3-O-p-coumaroylglucosides; 5 trans isomers and 2 cis isomers), caffeoyl derivatives (2 compounds), flavanol-anthocyanin direct condensation products (3 F-A+ dimers) and flavanolanthocyanin acetaldehyde-mediated condensation products (7 F-etA+ dimers), A-type vitisins (3 compounds) and B-type vitisins (4 compounds), and vinylphenol-type pyranoanthocyanins (3 compounds). The presence of difficult resolution pigments (DRP), such

calculated for each sample, and the differences in each parameter between bleached and control samples were calculated. HPLC-DAD-ESI/MSn Analyses. The anthocyanins and their derivative pigments were analyzed by HPLC-DAD-MSn in all of the wine samples (cold-treated and noncold-treated). Wine samples were diluted 1:1 with acidified water (pH 1.4, HCl) and filtered through a 0.45 μm Millex syringe-driven filter unit (Millipore Corp., Bedford, MA) before HPLC-DAD-MSn analysis. Analyses were performed in a Hewlett-Packard 1100 series liquid chromatograph (Agilent Technologies, Waldbronn, Germany). An Aqua C18 reversed-phase, 5 μm, 150 mm × 4.6 mm column (Phenomenex, Torrance, CA) thermostated at 35 °C was used. The HPLC-DAD conditions have been previously employed with satisfactory results in our laboratory in the analysis of other wine samples.20 Detection was carried out at 520 nm as the preferred wavelength. Spectra were recorded from 220 to 600 nm. The mass spectrometer was connected to the HPLC system via the DAD cell outlet. MS detection was performed in an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany) equipped with an ESI source and a hybrid triple-quadrupole ion trap mass analyzer that was controlled by Analyst 5.1 software. Detection was carried out in positive mode (ESI+). Zero grade air served as nebulizer (GS1) and turbo gas (GS2) for solvent drying. Nitrogen served as curtain (CUR) and collision gas (CAD). Settings were optimized by direct infusion of a solution of malvidin 3-O-glucoside (Extrasynthese, Genay, France). GS1 and GS2 were set at 40 and 50 psi, respectively. CUR was set at 10 psi, and CAD was set as “high”. Declustering potential (DP) was set at 20 V, entrance potential (EP) at 10 V, ion spray voltage (IS) at D


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Journal of Agricultural and Food Chemistry as, among others, dimeric anthocyanins,21 was also taken into account, because an elevation of the baseline was clearly observable in the chromatograms recorded at 520 nm. Their contribution to the total pigment content was calculated from the difference between the total area and the sum of the areas of the resolved peaks. Finally, the percentage of each compound over the total content was also calculated. Statistical Analysis. For each experiment, the significance of the differences observed among samples was assessed by Tukey’s honestly significant difference test (p < 0.05). In addition, Principal component analysis (PCA), an unsupervised pattern recognition technique, was used to observe relationships between samples and/or between variables. PCA was applied to the correlation matrix of the original variables. The original variables (15 variables, see Tables 1 and 3 for the exact identity of each variable) included the CIELAB parameters and the concentration of each family of compounds. The mean values (n = 3) of all of the samples of both experiments were used together. The IBM-SPSS Statistics 23 for Windows software package was used to perform the statistical analysis.

relevance of wine composition on the effects caused by the addition of MPs. To evaluate the ability of MP-M to protect the wine coloring matter against precipitation by cold or against bleaching by SO2, the CIELAB color parameters were measured in all of the types of wines (with MP-M, samples CM and SM, or without it, samples C and S) before and after applying the destabilizing agent (cold or NaHSO3, depending on the experiment). Regarding cold treatment, some differences were observed in the behavior of control and seed-added wines (Table 1). Whereas cold caused a decrease in b* coordinate (yellow component when positive) and in hab in control wines (comparison of C and C# in Table 1) meaning that coldtreated wines showed less orange hues, no significant changes could be observed in any of the CIELAB parameters after coldtreatment in seed-added ones (comparison of S and S# in Table 1). Thus, it seems that the addition of the seeds can be useful to stabilize color against cold. This type of stabilization was also observed11 in wines treated with an enological tannin containing condensed and hydrolyzable tannins, which points to a protective role of tannins against precipitation of the coloring matter caused by cold. However, the initial color change caused by these additions (either seeds or enological tannins) cannot be neglected when considering this approach to increase color stability against cold. The presence of MP-M in control wines did not prevent the color changes induced by cold-treatment, and, as in the case of C and C# samples, b* coordinate and hue decreased after cold treatment (comparison of CM and CM#). Additionally, in MP-M treated samples, an increase in a* coordinate (red component when positive) could be observed after cold treatment. This effect was also observed in seed-added wines treated with MP-M. This means that the red component of color is stabilized against cold in the presence of MP-M. Despite this positive effect in red coordinate, color differences (ΔE*ab) due to cold treatment were slightly higher for the wines added with mannoprotein (1.14 and 0.88 for CM−CM# and SM−SM#, respectively) than for the wines not added with mannoprotein (0.85 and 0.44 for C−C# and S−S#, respectively), because of the higher changes in hue in the former ones. Table 2 shows the values of the increments (Δ) for all of the CIELAB parameters caused by SO2 treatment in C, CM, S, and SM samples, calculated as the difference between the value after SO2 treatment and the value before SO2 treatment. Furthermore, color differences (ΔE*ab) are also shown in the table. In all of the samples, color differences were much higher than 3, meaning that the changes caused by SO2-bleaching were visible for human eye. Considering each CIELAB parameter independently, it can be observed that SO2 treatment caused an increase in lightness, in b* coordinate, and in hue and a decrease in a* coordinate and in color purity in all of the samples. This is in accordance with the mechanism of the bleaching caused by SO2. SO2 usually binds to C4 in the structure of the anthocyanin.24 However, this binding is not possible in some anthocyanin-derived pigments because they have this position occupied or have a steric hindrance that prevents their bleaching. Because the color of anthocyanins is redder than that of many of the bleaching-resistant derivatives, and anthocyanins are usually the most abundant pigments in wine, a bleaching of red pigments but not of other orangebrown ones reduces the total color expression and increases the expression of the color of the resistant pigments. This fact explains the increase in lightness and in hue (positive Δ values)

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RESULTS AND DISCUSSION CIELAB Color Parameters. Experiment 1: Effects of Seeds and Mannoprotein M on Wine Color and on Its Stabilization against Cold Treatment or SO2-Bleaching. The effects of the addition of the Pedro Ximénez seeds on the color of Syrah wines was assessed by the comparison of the CIELAB parameters of the wines made without seeds (column C in Table 1) to those of the wines fermented with seeds (column S in Table 1). The addition of the seeds caused statistically significant increases in lightness (L*) and in hue (hab), together with a decrease in chroma (C*ab). This means that the wines fermented in the presence of seeds were less dark than control wines and that their colors were more orange and less pure than those shown by control wines. To verify if these changes in color parameters were visually perceptible, color difference (ΔE*ab) between C and S samples was calculated (ΔE*ab = 13.43). Human eye is able to detect color differences higher than 3.22 Consequently, the statistically significant color changes caused by the addition of this type of seeds were also distinguishable by human eye. The increase in lightness and the decrease in chroma were also reported in a previous study on Syrah wines fermented in the presence of Pedro Ximénez grape pomace.7 However, in that study, the treatments with grape pomace led to wines with more bluish hues. The differences in the behavior of the hue in these two studies can be due to the presence of the skins in the grape pomace in addition to the seeds. These skins can supply other phenolic compounds to the wine, such as flavonols or hydroxycinnamic acids, which are better copigments than the flavanols supplied by seeds23 and whose interactions cause a shift of the color toward more bluish hues than the interaction with flavanols.23 Unlike the addition of seeds, the addition of MP-M did not produce significant changes in the CIELAB parameters either in C or S wines (comparison of C vs CM and S vs SM in Table 1; ΔE*ab: CM-C 0.63 and SM-S 0.45). This contrast with the results of a previous study carried out in our laboratory with this same MP but in wines made from Vitis vinifera L. cv Tempranillo grapes, where the addition of the MP during alcoholic fermentation caused color changes that were visible to human eye.11 Differences between the results of both studies might be due, among other factors, to the different grape variety and different winemaking practices employed in them, which directly condition the initial pigment composition and, consequently, the effect of the MP. This fact highlights the E


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and the decrease in color purity (negative Δ values) observed in all of the treated samples. Concerning lightness, no significant differences were observed among the different types of wines: the increase was very similar independently of the addition of MP-M or/and seeds. However, the increase in hue was significantly higher in the wines added with seeds (comparison of CSO2−C vs SSO2−S in Table 2). This might be due to the presence in the wine of other orange/brown colored compounds related to the addition of seeds whose influence in wine color is partly buffered by anthocyanins, but expressed after anthocyanin bleaching. Thus, the addition of seeds would slightly increase the effect of SO2 on hue. On the contrary, the addition of MP-M in control wines (comparison of CSO2−C vs CMSO2−CM) seems to partly prevent the decrease in a* coordinate and in color purity (ΔC*ab) observed after SO2 addition, because the decreases are significantly lower in the wines treated with MP-M (−23.35 for Δa* and −18.46 for ΔC*ab in CM wines vs −24.89 for Δa* and −20.09 for ΔC*ab in C wines). This is a relevant finding, because in addition to the colloidal stabilization ability demonstrated for some MPs,11 their use in wines might partly avoid the loss of red component and color purity due to the use of SO2 at different stages of the winemaking process.

Table 2. Modifications of the CIELAB Parameters Caused by the Treatment with SO2 of the Samples of Experiments 1 and 2 (Mean Values, n = 3)a Experiment 1 Δ due to SO2

CSO2−C

ΔL Δa* Δb* ΔC*ab Δhab ΔE*ab

24.99 a −24.89 a 9.53 ab −20.09 a 32.99 a 36.54 a

CMSO2−CM 23.31 a −23.35 b 9.33 a −18.46 b 32.48 a 34.29 a Experiment 2

SSO2−S

SMSO2−SM

24.53 a −24.38 ab 9.45 ab −19.74 ab 34.22 bc 35.86 a

24.59 a −24.15 ab 9.39 ab −19.62 ab 34.12 bc 35.73 a

Δ due to SO2

SSO2−S

SF1SO2−SF1

SF2SO2−SF2

ΔL Δa* Δb* ΔC*ab Δhab ΔE*ab

24.53 bc −24.38 ab 9.45 bc −19.74 a 34.22 c 35.86 a

24.96 c −24.62 a 9.29 b −19.95 a 33.62 b 36.28 a

25.09 c −24.25 ab 9.04 a −19.57 a 32.19 a 36.04 a

a

Different letters in the same row indicate significant differences (p < 0.05).

Table 3. Chromatic Features (CIELAB Parameters) and Quantitative Composition (Expressed as mg/L of Malvidin 3-OGlucoside and as Percentage) of the Samples of Experiment 2 (Mean Values, n = 3) samples VARa L* a* b* C*ab hab Monoglc acetyl p-coum caffeoyl F-A+ F-et-A+ Vit A Vit B Vit VF DRP total Monoglc acetyl p-coum caffeoyl F-A+ F-et-A+ Vit A Vit B Vit VF DRP

S

1 2 3 4 5

73.57 b 23.29 b 10.18 a 25.42 b 23.62 a

6 7 8 9 10 11 12 13 14 15

49.49 d 29.98 cd 20.33 cd 0.21 b 0.33 a 1.17 a 2.34 d 0.38 c 0.2 b 58.23 bc 165.19 bc 30.35 b 18.47 b 12.66 cd 0.13 c 0.20 a 0.72 a 1.46 b 0.23 ab 0.12 a 35.38 a

S#

SF1

CIELAB Parameters 73.18 b 75.19 c 23.44 b 21.62 a 10.05 a 11.76 d 25.50 b 24.61 a 23.20 a 28.54 b Concentration (mg/L) 44.30 b 47.91 cd 27.23 b 30.90 d 16.91 ab 21.61 d 0.17 a 0.22 b 0.34 a 0.29 a 1.11 a 0.70 ab 1.56 a 2.29 c 0.31 a 0.40 cd 0.14 a 0.19 ab 52.17 a 59.19 bc 146.54 a 170.01 c Percentage 30.64 b 30.52 b 18.93 c 18.49 bc 11.89 ab 13.08 d 0.11 a 0.13 c 0.23 a 0.17 a 0.77 ab 0.69 a 1.10 a 1.39 b 0.21 a 0.24 b 0.09 a 0.10 a 35.75 a 34.94 a

SF1#

SF2

SF2#

75.74 c 21.41 a 11.65 d 24.37 a 28.55 b

71.31 a 24.83 c 11.00 c 27.16 c 23.88 a

71.51 a 24.64 c 10.62 b 26.84 c 23.31 a

42.84 b 27.94 bc 18.75 bc 0.17 a 0.33 a 0.60 a 1.65 ab 0.34 b 0.15 ab 54.41 a 153.3 ab

44.78 bc 29.37 bcd 19.16 bc 0.20 b 0.34 a 0.73 b 1.99 bc 0.37 c 0.19 ab 60.31 c 163.54 bc

39.17 a 24.60 a 15.87 a 0.17 a 0.47 b 0.64 ab 1.86 ab 0.40 d 0.17 ab 55.67 ab 145.04 a

29.70 a 18.28 b 12.07 bc 0.12 bc 0.21 a 0.74 a 1.26 ab 0.23 ab 0.11 a 37.01 b

29.50 a 17.29 a 11.29 a 0.12 ab 0.33 b 0.85 b 1.32 b 0.28 c 0.11 a 38.55 c

30.42 b 18.59 bc 12.60 bcd 0.11 a 0.21 a 0.76 ab 1.11 a 0.22 ab 0.09 a 35.64 a

a

VAR, number of the variable in PCA; Monoglc, monoglucosides; acetyl, acetyl derivatives; p-coum, p-coumaroyl derivatives; caffeoyl, caffeoyl derivatives; F-A+, flavanol-anthocyanin direct condensation products; F-et-A+, flavanol-anthocyanin acetaldehyde-mediated condensation products; Vit A, A-type vitisins; Vit B, B-type vitisins; Vit VF, vinylphenol-type pyranoanthocyanins; DRP, difficult resolution pigments. Different letters in the same row indicate significant differences (p < 0.05). F


Article

Journal of Agricultural and Food Chemistry Experiment 2: Effects of Mannoprotein F at Two Different Doses on the Color of Wines Added with Seeds and on Its Stabilization against Cold Treatment or SO2-Bleaching. This second experiment aimed at evaluating the modifications of wine color caused by the addition of another type of MP (MPF), which is marketed to solve, above all, astringency problems. This interesting aspect should be considered in the studies concerning MPs, because, taking into account the similarities between the structures of some of the compounds responsible for astringency and those of the compounds responsible for wine color, an effect on more than one type of phenolic compounds might be expected. Thus, the present study intended to know whether the color is affected or not by interaction of the MP with anthocyanins and anthocyaninderived pigments. Table 3 shows the CIELAB parameters of the wines considered in this second experiment (wines S, SF1, and SF2). Wines SF1 and SF2 were, as well as wine S (control wine for experiment 2), wines made from Syrah grapes fermented with Pedro Ximénez seeds, but contrarily to wine S, wines SF1 and SF2 were additionally treated with two doses of MP-F (SF1, low dose; SF2, high dose). It was striking to discover that MP-F affected color parameters differently at the different doses and that this effect was not dose dependent; that is, the effect did not increase with an increase in the dose. On the contrary, for most of the CIELAB parameters, the different doses caused opposite effects, which highlights the relevance of selecting an appropriate dose of MP. Concerning lightness, the high dose (SF2) caused a reduction of L* value, whereas the low dose (SF1) produced wines that were lighter than the control itself (wine S). Color purity (C*ab) increased in the wines treated with the high dose of the MP (SF2) and decreased in the wines treated with the low dose (SF1) in relation to wine S. Hue (hab) increased in the wines treated with the low dose of MP-F (SF1), but remained stable in those treated with the high dose (SF2). Thus, in what concerns color, the high dose seems to be more beneficial because it significantly increased color purity and darkness without affecting the hue. The low dose of MP-F significantly decreased chroma and increased hue in relation to control wine (wine S), which, along with the increase in lightness, are effects similar to those caused by aging25 and, therefore, usually not desirable for young wines. Furthermore, these statistically significant changes in relation to control wine led to color differences (ΔE*ab) between MP-treated wines and wine S next to 3 (2.81 for SF1 and 2.86 for SF2). These changes in color due to the addition of MP-F were, in turn, greater than those indicated above for the addition of MP-M. Cold treatment hardly caused changes in the CIELAB parameters of any of the samples of this experiment (Table 3, comparison of S vs S#, SF1 vs SF1#, and SF2 vs SF2#). Therefore, color differences between the samples before and after cold treatment were in all cases much lower than 3 (0.44 for S, 0.60 for SF1, and 0.47 for SF2), and consequently not detectable by human eye. It has to be borne in mind that all of the wines of this second experiment were fermented with Pedro Ximénez seeds, and, as it was observed in the first experiment, the presence of seeds increased the stability of color parameters against cold treatment. From the similar values of color differences (ΔE*ab) among these samples before and after cold treatment, it can be deduced that the additional presence of MP-F did not positively or negatively affect the stability supplied by seeds against cold. However, the color changes (ΔE*ab) induced by the additional presence of

this MP were higher than those caused by cold and, as indicated above, next to 3. These changes in color in relation to wines only treated with seeds have to be taken into account when considering the addition of the MP, because they are next to the limit of being detectable by human eye. Regarding the effect of MP-F against color bleaching by SO2, Table 2 shows the differences in the CIELAB parameters (ΔL*, Δa*, Δb*, ΔC*ab, and Δhab) for all of the samples of this second experiment before and after the addition of SO2 (SSO2−S, SF1SO2−SF1, and SF2SO2−SF2). As observed for MP-M in the first experiment, MP-F was not able to prevent, at any of the tested doses, the increase of lightness due to the bleaching of the coloring matter by SO2 (comparison of ΔL* between SSO2−S, SF1SO2−SF1, and SF2SO2−SF2). MP-F was neither able to reduce the decrease of color purity caused by SO2 (comparison of ΔC*ab among samples). However, a significant reduction of the increase in hue (Δhab) could be observed in the wines treated with MP-F. This protective effect was, in turn, dose dependent. Thus, the lowest changes due to the addition of SO2 were observed in the wines treated with the highest dose of MP-F. Thus, according to the results of experiments 1 and 2, MPs can exert a protective effect for the coloring matter against SO2-bleaching. Previous studies on MPs11,13 propose that their role in preventing the precipitation of some types of flavonoids is due to their adsorption on the particles formed by the phenolic compounds, which generate a steric hindrance that prevents their aggregation and subsequent precipitation. Similarly, the observed protection against SO2 might also be due the adsorption of the MPs on the pigment particles, which could make more difficult the attack of the susceptible positions of the anthocyanidin nucleus by SO2. However, it is important to remark that each MP was able to protect different color attributes (color purity in the case of MP-M and hue in the case of MP-F), which could be related to differences in the compositions or structures of both MPs. Pigment Composition. Thirty-nine anthocyanins and anthocyanin-derived pigments were identified in the samples of this study (see Figure S1 and Table S2 for more details). To evaluate the effect of the addition of the MPs and of the cold treatment, pigments were grouped according to their structures, and then the concentrations and percentages of each group were compared before and after the treatments. Furthermore, the contribution of the difficult resolution pigments (DRP) to the total pigment content was also evaluated as well as its modification after the treatments. These DRP usually correspond to oligomeric pigments that coelute with other compounds causing an elevation of the baseline, which, in the case of the samples studied in the present work (see Figure S1), is important from a quantitative point of view. Experiment 1: Effects of Seeds, Mannoprotein M, and Cold Treatment on Wine Pigment Composition. Neither the addition of seeds (comparison of C vs S in Table 1) nor the addition of MP-M (comparison of C vs CM and S vs SM in Table 1) caused significant changes in the total pigment content. However, some significant differences were observed among samples when considering the main families of compounds. Respecting grape native anthocyanins (nonacylated and acylated monoglucosides), the addition of seeds caused a significant increase in the content of monoglucosides, which was in agreement with previous studies on Syrah wines treated either with Pedro Ximénez grape pomace7 or with overripe seeds of this white variety.8 It has been proposed that tannins supplied by seeds could increase the extraction of G


Article


small changes were also observed when the proportions were considered, and only the proportion of flavanol-anthocyanin acetaldehyde-mediated condensation products (F-et-A+) increased after the addition of MP-M in control wine. This little modification of the anthocyanin profile after the addition of MP-M agrees with the small changes observed in the CIELAB parameters after the addition of MP-M. Respecting cold treatment and the behavior of the different compounds (comparison of C vs C#, CM vs CM#, S vs S#, and SM vs SM# in Table 1), a high stability against cold was observed for F-A+ and vinylphenol-type pyranoanthocyanidins (Vit VF). Their contents remained quite stable after cold treatment independently of the presence of the MP. Consequently, the expected protection of this MP against the effects of cold treatment is barely noticeable in these two groups of anthocyanin-derived pigments due to their own stability against cold. This behavior was also reported in a previous study carried out in our laboratory,11 where it was observed that most of the F-A+ compounds that were present in the samples before cold treatment were absent in the precipitates obtained after cold treatment. Conversely, small amounts Vit VF could be detected in the precipitates after cold treatment,11 which was pointing to a lower stability against cold than F-A+ compounds, but always greater than the rest of pigments. In the present study, the concentrations of all of the grape native anthocyanins and especially those of the monoglucosides were drastically reduced after cold treatment in all of the samples (C vs C# and S vs S# in Table 1). However, in control wines and in the presence of MP-M (CM vs CM# in Table 1), the decreases were smaller, pointing out to a protective effect of this MP against precipitation by cold. Similarly, the levels of A-type and B-type vitisins seemed to be affected by cold treatment, and MP-M was able to reduce these decreases. However, differences in the results were observed in seed-treated wines. The presence of higher levels of tannins in the wines treated with seeds might be one the causes of these different effects, because it affects the ratio between MP, flavanols, anthocyanins, and other compounds involved in the synthesis of anthocyanin derivatives. As previously reported,11 cold also seemed to produce a decrease in the levels of DRP. However, the reported protective effect of MP-M against cold for this family of compounds was not significant in the present study. Considering the percentages of the different types of compounds, some changes were detected after cold treatment and with the use of MP-M (Table 1). The main significant changes were observed for F-A+, whose percentages increased in all of the samples after cold treatment as a consequence of their higher stability against cold. In addition, in the samples added with seeds, the increase was higher in the presence of MP-M. The percentages of F-et-A+ also increased after cold treatment, which was indicative of a greater stability against cold in relation to other types of pigments. However, in the samples treated with MP-M, greater percentages of F-et-A+ were observable even before cold treatment and maintained after it. Thus, it seems that in the case of F-A+, the addition of MP-M is useful to increase the colloidal stability in extreme conditions (cold treatment), whereas in the case of F-et-A+, the addition of MP-M increases their stability in relation to other types of pigments in normal conditions. In any case, the changes caused by cold in the percentages of the main pigment families were always smaller than those caused by the addition of seeds. This is in accordance with the results of color

anthocyanins through weak interactions between them (such as copigmentation), favoring the extraction from grape skins to the must.7,8 This effect was also observed when the tannins were supplied to the wines through an enological tannin instead of seeds.10,11,26 Furthermore, the additional presence of these copigments during winemaking and aging can be responsible for lower losses of anthocyanins during these stages due to the increased chemical stability that can be conferred by copigmentation.27,28 Taking into account that in the present study the seeds were added after fermentation, the increase observed in monoglucosides can only be attributed to the chemical stabilization of the anthocyanins by the additional presence of copigments and not to the higher extraction from grape skins. Concerning acylated anthocyanins and contrarily to the results reported for Syrah wines supplied during fermentation with grape pomace7 or with overripe seeds,8 the postfermentative addition of seeds did not produce significant changes in any of the groups of acylated anthocyanins in the present study. This different effect on acylated compounds might be related to the moment of addition of the byproducts. In previous studies,7,8 they were added when grapes were still in contact with wine, whereas in the present one, seeds were added after devatting. In addition, the differences in the shifts in hue observed in previous studies after the addition of these byproducts (shift toward bluish hues) and the shift observed in the present study (shift toward more orange hues) could be partly related to the different behavior against acylated compounds. Respecting anthocyanin-derived pigments, the addition of seeds also caused an increase in the levels of A-type vitisins, which are chemically more resistant than original anthocyanins. Furthermore, their color is less red and more orange than that of the anthocyanin from which they are synthesized, and consequently they can also contribute to the shift observed in the hue after the addition of seeds. On the contrary, the addition of seeds decreased the levels of F-A+ dimers, despite the additional supply of oligomeric flavanols that might take part in this type of reactions. These lower levels might be related to the reactivity of this type of anthocyanin derivatives, which could be broken, and then react again with other anthocyanins or flavanols, giving rise to new oligomeric structures that would integrate DRP or other fractions not easily detectable by HPLC-DAD as resolved peaks. Bearing in mind that the color expressed by wines depends not only on the total content of pigments but also on the proportions of the different families of compounds (pigment profile), the percentages of the different types of pigments were also calculated and compared in all of the types of wines (Table 1). The addition of seeds caused significant changes in different pigment families. Whereas the percentages of monoglucosides and acetyl derivatives remained quite stable, those of p-coumaroyl derivatives increased, as well as those of A-type vitisins. On the contrary, the percentages of B-type vitisins, F-A+ dimers, and DRP were lower in the wines treated with seeds. These differences in the pigment profiles between wines C and S could explain the differences observed in the CIELAB parameters of these two wines better than the total pigment content, in which differences were not significant. Similarly, total pigment content was not significantly affected by the addition of MP-M (comparison of C vs CM and S vs SM in Table 1). Among the different pigment families, only Atype vitisins showed a significant decrease in their concentration after the addition of the MP, but only in seed-treated wines (comparison of S vs SM in Table 1). Accordingly, only H


Article


Figure 2. Scores (a) and loadings (b) plots obtained by principal component analysis (PCA) from the data matrix containing the mean (n = 3) values of the CIELAB parameters and concentration of the main pigment families of all of the samples of both experiments (C, control samples; S, seed added samples; M, mannoprotein M added samples; F, mannoprotein F added samples (1, low dose; 2, high dose); #, samples after cold treatment). See Tables 1 and 3 for the identification of the variables in the loadings plot.

linked through a C−C bond involving C4 in the flavanol and C8 or C6 in the anthocyanin. This different type of bond probably causes greater changes in the structures of the residues in relation to the free compounds in the case of the FA+, where the direct linkage forces the condensation product to adopt a more rigid conformation, less similar to that of the free compounds and consequently less affected by MP-F than them. Respecting pyranoanthocyanins, the main effects of the addition of this MP were observed for A-type vitisins, whose levels significantly decreased at both doses of this MP but slightly more at the high dose. On the contrary, the high dose seemed to slightly increase the content of DRP, but differences among samples were small. These changes in the concentrations of the different groups also caused modifications in the proportions. With the high dose, the percentage of monoglucosides significantly decreased and that of DRP significantly increased. With the low dose, only a significant increase in the proportion of coumaroylderivatives could be detected in relation to nontreated samples. This study has revealed an important difference between MPs M and F: whereas the addition of MP-M hardly changed the pigment composition of the wines (Table 1, comparison of S and SM), the addition of MP-F caused important changes in several groups of pigments, mainly in monoglucosides (Table 3, comparison of S, SF1, and SF2). This fact points to different target compounds for each MP and different mechanisms, despite both being MPs. In the case of MP-F, the modifications in the coloring matter could be due to adsorption phenomena, which seemed to be greater at higher doses. It has been reported that the different anthocyanins can be differently adsorbed onto yeast cell walls29−31 and that this adsorption can, in turn, be conditioned by the strain of the yeast.30,31

analyses, which showed that the addition of seeds caused greater changes in CIELAB parameters (ΔE*ab = 13.43) than the addition of MP-M (ΔE*ab = 0.63 or 0.45 on C or S wines, respectively) or cold treatment (ΔE*ab = 0.83, mean value of those of all of the samples). Experiment 2: Effects of Mannoprotein F at Two Different Doses and Cold Treatment on the Pigment Composition of Wines Added with Seeds. Table 3 shows the concentration of the main groups of pigments in the samples of this experiment before (S, SF1, and SF2) and after (S#, SF1#, and SF2#) cold treatment. The addition of mannoprotein F did not cause significant changes in the total pigment content. However, it caused a reduction in the content of the anthocyanin monoglucosides, which was significantly greater when the high dose of MP-F was used. On the contrary, MP-F increased the levels of coumaroyl derivatives, but only at the low dose. Acetyl- and caffeoyl-derivatives were less affected by MP-F addition with no significant differences between samples. Concerning anthocyanin derivatives, the addition of MP-F decreased the content of F-et-A+, clearly detectable at the high dose of MP-F. Such an interaction was not unexpected. On the one hand, these derivatives contain anthocyanin monoglucosides, which, as previously indicated, were strongly affected by the addition of the MP. On the other, they contain flavanols, which are expected to be one of the main target compounds of this MP because this MP is marketed to modulate wine astringency. In contrast to this, MP-F did not affect the concentration of F-A+. Differences have to be due to differences in the structure, which are conditioned, in turn, by the type of linkage between the flavanol and the anthocyanin residues. In the F-et-A+ they are linked through an ethyl bridge, whereas in the F-A+ both residues are directly I


Article


of the Pedro Ximénez overripe seeds (separation between C and S). Furthermore, after cold treatment, the samples treated with MP-M were always more similar to the samples before cold treatment than the samples not treated with MP-M (differences between C and C# were higher than those between CM and CM#; the same behavior can be observed for the wines added with seeds: differences between S and S# were higher than those between SM and SM#). Respecting the addition of MP-F, it was observed that the changes associated with its addition were, at any of the assayed doses, more noticeable than those provoked by MP-M (compare the differences between S and SM and the differences between S, SF1, and SF2 in Figure 2a). MP-F also seemed to partly prevent the loss of pigments by precipitation after cold treatment, slightly better at the highest dose (differences between S and S# as compared to differences between SF1 and SF1# and those between SF2 and SF2#). However, as previously indicated, the high dose seemed to affect the coloring matter of the wine more than the low dose. In summary, the present study has first shown that the addition of seeds from overripe Pedro Ximénez grapes caused changes in the pigment composition of Syrah wines that caused, in turn, color modifications perceptible for the human eye (ΔE*ab = 13.43). Among the different families of compounds, the addition of seeds increased the levels of native monoglucoside anthocyanins probably by protecting them from degradation through copigmentation phenomenon. This technique also increased the levels of some anthocyanin derivatives, which are more stable from a chemical point of view. In addition, when the samples were cold treated, the CIELAB color parameters changed less in the presence of seeds. The main drawback of the addition of Pedro Ximénez seeds that has been observed in the present study is that the color of the wines added with seeds was lighter, with lower chroma and higher hues than that of the nontreated wines, which might be associated with the color corresponding to older wines. Respecting the addition of MP-M, which intended to improve color stability, the main advantage was that its addition was almost imperceptible in terms of color and pigment composition and that it was able to reduce the decreases caused by cold-treatment in most of the types of anthocyanin and anthocyanin-derived pigments. When MP-M was added to seed treated wines, some of these beneficial effects were reduced with a predominance of the effects due to the seeds addition. It has been very interesting to discover in the second experiment of this study that MP-F, which is intended to improve astringency, can have important effects on the coloring matter. Unlike MP-M, the addition of MP-F at both doses assayed caused the decrease of grape native anthocyanins and of some anthocyanin derivatives. These changes in the composition are in line with the results observed in color analyses, where color differences (ΔE*ab) between MP-F treated and nontreated wines were next to the limit of being perceptible to the human eye (ΔE*ab next to 3). Color parameters hardly changed in this second experiment after cold treatment, which might be related to the presence of seeds in all of the samples. However, looking at the pigment composition, the presence of MP-F seemed to protect certain types of pigment against cold treatment. The results of this second experiment highlight the relevance of studying the effects of the addition of MPs in families of compounds different from the targeted ones, to evaluate the benefit/risk

Consequently, a differential adsorption of the pigments onto the MPs could be expected depending on the yeast strain from which the MP has been obtained or even on the method employed for their obtaining.32 This could explain the differences observed in the coloring matter of the wines after the addition of the two types of MPs assayed in the present study. Concerning the effect of cold on the coloring matter (S vs S#, SF1 vs SF1#, and SF2 vs SF2#), in the wines not added with MP-F (S), a significant decrease in the levels of most of the anthocyanin and anthocyanin-derived pigments could be detected. As observed in the first experiment, flavanolanthocyanin condensation products (both acetaldehydemediated and direct condensation products) remained quite stable after cold treatment. In the wines added with MP-F, independently of the dose employed, the decreases due to cold were slightly lower in the case of A- and B-type vitisins and vinylphenol-type pyranoanthocyanins, which could be indicative of a protective role of this MP against precipitation by cold for these anthocyanin-derived pigments. On the contrary, in the case of the grape native anthocyanins, the lowest levels after cold-treatment were observed in the wines added with the high dose of MP. Thus, in general, the high dose of the MP-F did not achieve better protection against cold than the low dose and caused more changes in the coloring matter. Consequently, after cold treatment, the samples treated with the high dose of the MP-F (SF2#) were those whose percentage compositions most differed from the control sample (S) (Table 3), with lower percentages of grape native anthocyanins and greater percentages of F-A+, F-et-A+, and Btype vitisins, and, above all, with the highest percentages of DRP among all of the samples. Principal Component Analysis (PCA). Figure 2 shows the results of the PCA, which was applied to the correlation matrix of the original variables (15 variables, including both colorimetric and pigment composition variables; for the exact identity of these variables, see Tables 1 and 3). The mean values (n = 3) of the CIELAB parameters and of the concentration of each group of pigments in each sample of both experiments were considered. Colorimetric data and composition data were, therefore, evaluated together. The results of that PCA were quite satisfactory, because they clearly summarize the main findings of the present study. PC1 and PC2 explained 85.76% of the variance. Along PC1, samples were mainly separated according to the addition or not of seeds. Positive values correlated with grape native pigments, and negative ones with some anthocyanin-derived pigments. Samples added with seeds showed positive values and control samples showed negative values, which is in accordance with the greater grape native pigment contents observed for the former ones. Along PC2, samples were separated according with the application of cold treatment: the positive values corresponded to the samples not treated, and the negative ones with cold treated wines. This fits well with the decrease caused by cold observed in the contents of all of the pigments, because all of the variables related to pigment content showed positive values. In relation to the addition of MP-M, it was observed that the changes caused in the coloring matter by its addition were lower than the changes provoked by the addition of seeds. In fact, the separations between samples due to the addition of MP-M (see the separation between C and CM and between S and SM in Figure 2a) are the lowest of all in the study and much lower than the separation due to the addition J


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grape by-products during red wine fermentation on wine color and phenolic composition. LWT - Food Science and Technology 2017, 84, 544−550. (9) Gordillo, B.; Baca-Bocanegra, B.; Rodríguez-Pulido, F. J.; González-Miret, M. L.; García-Estévez, I.; Quijada-Morín, N.; Heredia, F. J.; Escribano-Bailón, M. T. Optimisation of an oak chips-grape mix maceration process. Influence of chip dose and maceration time. Food Chem. 2016, 206, 249−259. (10) Alcalde-Eon, C.; García-Estévez, I.; Ferreras-Charro, R.; RivasGonzalo, J. C.; Ferrer-Gallego, R.; Escribano-Bailón, M. T. Adding oenological tannin vs. overripe grapes: effect on the phenolic composition of red wines. J. Food Compos. Anal. 2014, 34, 99−113. (11) Alcalde-Eon, C.; García-Estévez, I.; Puente, V.; Rivas-Gonzalo, J. C.; Escribano-Bailón, M. T. Color stabilization of red wines. A chemical and colloidal approach. J. Agric. Food Chem. 2014, 62, 6984−6994. (12) Ribéreau-Gayon, P.; Glories, Y.; Maujean, A.; Dubourdieu, D. Handbook of Enology, Vol. 2: The Chemistry of Wine Stabilization and Treatments, 1st ed.; John Wiley & Sons Ltd.: Chichester, England, 2000. (13) Riou, V.; Vernhet, A.; Doco, T.; Moutounet, M. Aggregation of grape seed tannins in model wine-effect of wine polysaccharides. Food Hydrocolloids 2002, 16, 17−23. (14) Rodrigues, A.; Ricardo-Da-Silva, J. M.; Lucas, C.; Laureano, O. Effect of commercial mannoproteins on wine color and tannins stability. Food Chem. 2012, 131, 907−914. (15) Escot, S.; Feuillat, M.; Dulau, L.; Charpentier, C. Release of polysaccharides by yeast and the influence of released polysaccharides on color stability and wine astringency. Aust. J. Grape Wine Res. 2001, 7, 153−159. (16) Del Barrio-Galán, R.; Pérez-Magariño, S.; Ortega-Heras, M.; Guadalupe, Z.; Ayestarán, B. Polysaccharide characterization of commercial dry yeast preparations and their effect on white and red wine composition. LWT-Food Science and Technology 2012, 48, 215− 223. (17) Ribéreau-Gayon, P.; Dubourdieu, D.; Donèche, B.; Lonvaud, A. Handbook of Enology, Vol. 1: The Microbiology of Wine and Vinifications, 1st ed.; John Wiley & Sons Ltd.: Chichester, England, 2000. (18) Pellerin, P.; Cabanis, J. C. Los glúcidos. In Enologı ́a: Fundamentos Cientı ́ficos y Tecnológicos; Flanzy, C., Ed.; AMV Ediciones, Ediciones Mundi-Prensa: Madrid, Spain, 2003; pp 66−96. (19) Heredia, F. J.; Á lvarez, C.; González-Miret, M. L.; Ramírez, A. CromaLab Análisis de Color; Registro General de la Propiedad Intelectual: Sevilla, Spain, 2004. (20) Alcalde-Eon, C.; Escribano-Bailón, M. T.; Santos-Buelga, C.; Rivas-Gonzalo, J. C. Changes in detailed pigment composition of red wine during maturity and ageing. A comprehensive study. Anal. Chim. Acta 2006, 563, 238−254. (21) Alcalde-Eon, C.; Escribano-Bailón, M. T.; Santos-Buelga, C.; Rivas-Gonzalo, J. C. Identification of dimeric anthocyanins and new oligomeric pigments in red wine by means of HPLC-DAD-ESI/MSn. J. Mass Spectrom. 2007, 42, 735−748. (22) Martínez, J. A.; Melgosa, M.; Pérez, M. M.; Hita, E.; Negueruela, A. I. Note. Visual and instrumental color evaluation in red wines. Food Sci. Technol. Int. 2001, 7, 439−444. (23) Gómez-Míguez, M.; González-Manzano, S.; Escribano-Bailón, M. T.; Heredia, F. J.; Santos-Buelga, C. Influence of different phenolic copigments on the color of malvidin 3-glucoside. J. Agric. Food Chem. 2006, 54, 5422−5429. (24) Berké, B.; Chèze, C.; Vercauteren, J.; Deffieux, G. Bisulfite addition to anthocyanins: revised structures of colorless adducts. Tetrahedron Lett. 1998, 39, 5771−5774. (25) García-Puente Rivas, E.; Alcalde-Eon, C.; Santos-Buelga, C.; Rivas-Gonzalo, J. C.; Escribano-Bailón, M. T. Behaviour and characterisation of the color during red wine making and maturation. Anal. Chim. Acta 2006, 563, 215−222. (26) Chen, K.; Escott, C.; Loira, I.; Del Fresno, J. M.; Morata, A.; Tesfaye, W.; Calderon, F.; Benito, S.; Suárez-Lepe, J. A. The effects of

equilibrium of the addition of the MP. Furthermore, the present study has revealed a relative protective effect of both types of MPs against SO2-bleaching of the pigments.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b06922. Figure S1, chromatogram recorded at 520 nm of a Syrah control sample (C); Table S1, flavanol composition of the seeds added in the present study coming from overripe Vitis vinifera L. cv Pedro Ximénez grapes; values are the mean of three replicates (n = 3); Table S2, chromatographic and spectral features (m/z of the molecular ion, fragment ions in the MS2 and MS3 analyses, and absorption maxima in the UV−vis spectra) of the main pigments detected in Syrah samples (DOCX)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 923 29 45 37. E-mail: [email protected]. ORCID

Cristina Alcalde-Eon: 0000-0002-8526-2442 Francisco J. Heredia: 0000-0002-3849-8284 María Teresa Escribano-Bailón: 0000-0001-6875-2565 Funding

We thank the Spanish MINECO (project ref AGL2017-84793C2-1-R cofunded by FEDER). R.F.-C. thanks FEDERInterreg España-Portugal Programme (project ref 0377_IBERPHENOL_6_E) for a research contract. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are due to Laffort España S.A. for providing the MPs. REFERENCES

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