Occurrence and Formation Kinetics of Pyranomalvidin-Procyanidin

Article pubs.acs.org/JAFC

Occurrence and Formation Kinetics of Pyranomalvidin-Procyanidin Dimer Pigment in Merlot Red Wine: Impact of Acidity and Oxygen Concentrations Laurent Pechamat,†,‡ Liming Zeng,†,‡ Michael Jourdes,†,‡ Rémy Ghidossi,†,‡ and Pierre-Louis Teissedre*,†,‡ †

University Bordeaux, ISVV, EA 4577 Oenologie, 210, chemin de Leysotte CS 50008, 33882 Villenave d’Ornon cedex, France INRA, ISVV, USC 1366 Oenologie, 210, chemin de Leysotte CS 50008, 33882 Villenave d’Ornon cedex, France

‡

ABSTRACT: Once released from red grape skins, anthocyanins undergo various chemical reactions leading to the formation of more stable pigments such as pyranoanthocyanin, as well as other derivatives. Among these pigments, pyranoanthocyanins linked directly to flavanol dimers have been detected and identified in aged Port wine but not in dry red wine. These pigments are very important with regard to the wine color evolution since they are involved in wine color evolution and stabilization. During this investigation, the occurrence in dry red wine of two pyranomalvidin-procyanidin dimer has been established by low and high resolution HPLC-UV-MS analysis. Moreover, the impact of acidity and oxygen levels on their formation in red wine has been estimated. After four months of evolution, the results showed that, for the same pH, the quantity of this pigment was correlated with oxygen concentrations. Moreover, for the same quantity of oxygen, the concentration of this pigment was related to the acidity level. KEYWORDS: pyranoanthocyanin-procyanidin dimer, red wine, oxygen, pH, acidity, pigment

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INTRODUCTION In red wines, phenolic compounds are mainly represented by anthocyanins and condensed tannins, extracted from grapes during the winemaking process. In red wines, tannin content is estimated using Bate-Smith reaction ranges from 1 to 4 g/L.1 These compounds are responsible for the mouth-feel properties and structure of red wine. From monomers to trimers, the mouth-feel properties range from bitterness to astringency, while for higher polymerized tannins, the astringency increases with galloylation degree.2 The other main compounds found in red wine are anthocyanins, which are the pigments responsible for black grapes and red wine color. In Vitis vinifera varieties, anthocyanins are present under monoglucosylated forms with their glucose moiety that can be acylated by acetyl, caffeoyl, or p-coumaroyl groups.3,4 These anthocyanins undergo equilibrium among several forms according to the pH. The flavylium form which is the red colored form is favored in acidic medium.5 Thus, in the pH range of red wine, between pH 3 and pH 4, the flavylium cation is not the prominent form since it represents around 40% and only 8%, respectively. Once released from red grape skins during the maceration stage, the native anthocyanins progressively disappear during wine maturation due to various chemical reactions leading to the formation of more stable pigments such as anthocyanin− flavanol or anthocyanin−tannins adducts linked together6,7 or through a methylmethine bridge,8,9 pyranoanthocyanins,3,10−13 anthocyanin oligomers,14 xanthylium salts,8,15,16 and anthocyano-ellagitannins.17 These newly formed pigments contribute to the progressive evolution of red wine color from the bright redpurple color of the young red wine to the more red-orange color of the aged red wine. Even if the overall color of red wine evolved to be more red-orange, some of these newly formed © 2014 American Chemical Society

pigments can exhibit a more bluer color than native anthocyanins, as well as some turquoise color.18 Among these pigments, the pyranoanthocyanins are currently acknowledged as one of the most important classes of anthocyanin derivatives. Moreover, these new pigments (especially pyranoanthocyanins) participate to the color stabilization of wine, since they are more resistant to sulfur dioxide bleaching than the malvidin3-O-glucoside.19 The increasing interest in pyranoanthocyanins over the last two decades has led to the detection, identification, and structural elucidation of a large variety of structures in wine model solution as well as in young, and aged red wine. The general mechanism leading to these pyranoanthocyanins pigments involves, as a first step, a cycloaddition reaction between a vinyl group and the 5-OH group and C-4 of the anthocyanin, followed by an oxidation step leading to the aromatization of pyranic ring D. The vinyl group engaged in the cycloaddition derived either from the enolic form of aldehydic or ketonic compounds (i.e., ethanal, pyruvic acid, ...), or from 4vinyl-phenols, vinyl-flavanols, and vinyl-procyanidin.20 However, these vinyl-flavanols and vinyl-procyanidins do not occur naturally in grapes: they in fact derive from the addition of ethanal on catechin or procyanidin under its enolic form, followed by the dehydration of the newly obtained benzylic alcohol or by the decomposition of the methylmethine-linked flavanol adducts.21 Both ways arise from the reaction between flavanols or procyanidin and acetaldehyde, which can be found Received: Revised: Accepted: Published: 1701

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mm, 5 μm Lichrospher 100 RP 18 column. The solvents used for the gradient were as follows: solvent A, water with 0.5% of formic acid, and solvent B, acetonitrile with 0.5% formic acid. The gradient used was as follows: from 100 to 85% of A in 5 min, from 85 to 70% of A in 30 min, from 70 to 0% of A in 1 min, then 0% of A during 4 min. The flow rate was set at 1 mL/min, and the tray temperature was fixed at 12 °C. The wavelengths used were 280 and 520 nm. This HPLC-UV system was also coupled to a Thermo-Finnigan LCQ Advantage spectrometer equipped with an electrospray ionization source and an ion trap mass analyzer. The electrospray ionization mass spectrometry detection was performed in positive mode with the following optimized parameters: capillary temperature 300 °C, capillary voltage 5 V, nebulizer gas flow 1.75 L/min, desolvation gas flow 1 L/min, and spray voltage 5 kV. The collision energy was set at 30% for the MS2 analyses on the ion [M] = 1093 and MS3 on the ion [M] = 931. The pyranoanthocyanin-procyanidin dimers were identified according to their mass spectra and fragmentation. The molecular formula of the molecular ion [M] = 1093 as well as the fragment detected during MS/MS analyses with the ion trap were confirmed by injection on a high resolution Q-TOF mass spectrometer. The UHPLC system used was an Agilent 1290 Infinity Series which consisted of a quaternary pump, a solvent degasser, an autosampler, and a diode-array detector. These analyses were carried out on 2.1 × 100 mm, 1.8 μm Eclipse Plus C18. The solvents used for the gradient were: solvent A, water with 0.1% formic acid, and solvent B methanol with 0.1% formic acid at a flow rate of 400 μL/min. The gradient was as follows: 94% of A during 30 s; 94 to 5% of A during 13.5 min; 5% of A during 3 min. This UHPLC system was coupled to an ESI-Q-TOF-MS Agilent 6530 Series Accurate with an electrospray ion source with Agilent Jet Stream Technology. The mass spectrometer was operated in extended dynamic range of 2 GHz with the mass range up to 1700 Th, with a drying gas flow and temperature respectively set at 9 L/min and 300 °C. The nebulizer pressure was set at 25 psi, and the sheath gas flow and temperature were respectively set at 11 L/min and 350 °C. The capillary voltage was of 4 kV. The collision energy was set at 5, 15, and 30% for MS2 analysis. The fragmentation voltage was set at 150 V. The reference masses used were [M+H] + = 121.050873, 322.048121, and 1221.990637. The software used for the determination of the exact molecular formula of the pyranoanthocyanin-procyanidin dimers molecular ions, as well as their fragments was MassHunter Qualitative Analysis. Quantification of the Pyranoanthocyanin−Procyanidin Dimers by HPLC-UV-MS. The quantification of the pyranoanthocyanin−procyanidin dimers was performed by HPLC-UV-MS using the same HPLC Thermo Finnigan Surveyor system coupled to the ion-trap mass apparatus as described above. The pigment has been quantified using pure malvidin-3-O-glucoside as standard and chlorogenic acid as internal standard. The calibration curve was accomplished by injecting solutions with increasing concentrations of malvidin-3-O-glucoside ranging from 1 to 256 mg/L and containing 50 mg/L of chlorogenic acid. The response factor was established by plotting the concentrations ratios versus the peak-area ratios of malvidin-3-O-glucoside ion (i.e., m/z 493) to the internal standard ion (i.e., m/z 355). The R2 obtained was 0.9998. The quantification of the pyranoanthocyanin-procyanidin dimers was realized using their molecular ion [M]+ = 1093.

in aged red wine as a result of ethanol oxidation. In summary, from a mechanistic point of view, the two key parameters leading to the formation of pyranoanthocyanins in red wine are the acidity, which controls the addition of ethanal on flavanols or procyanidins, as well as the cycloaddition of a vinyl group on the anthocyanin skeleton, and the oxygen level for the oxidative aromatization of pyranic ring D. Pyranoanthocyanins linked directly to flavanol monomers and dimers have been detected and identified in different aged Port wine (i.e., from 2 to 6 year-old). In those wines pyranoanthocyanin−procyanidin dimers appear to be more abundant and more stable than pyranoanthocyanin−flavanol monomers.22 Thus the aim of this work was to estimate the impact of acidity and oxygen level in dry red wine on the formation of pyranomalvidin-3-O-glucoside-procyanidin dimers. For this purpose, the pH of a dry red wine has been adjusted at 3, 3.5, and 4, and each pH has been submitted to four different levels of oxygen (i.e., 0, 2, 8, and 20 mg/L). The occurrence in dry red wine of these two pyranomalvidin−procyanidin dimers, as well as their kinetics of formation, has been followed by HPLC-UV-MS.

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MATERIALS AND METHODS

General. Distilled water was obtained from an ELGA system, and Milli-Q (Millipore) water used for HPLC-UV-MS separation and analysis was prepared using a Sartorius-arium 611 system. Acetonitrile (HPLC grade quality, > 99.8%) was purchased from VWR (Strasbourg, France). Wine Sample. The wine used was a Merlot vintage 2011 from the “Entre deux mers” region of Bordeaux. The chemical characteristics of the red wine were as follows: a total polyphenolic index (TPI) of 61, a pH of 3.78, a total condensed tannins (i.e., estimated by RibereauGayon and Stonestreet procedure) of 3.22 ± 0.25 g/L, total anthocyanins (i.e., determined using the SO2 bleaching method) of 603 ± 1.2 mg/L, molecular anthocyanins content (i.e., anthocyanins quantified by HPLC-UV at 520 nm) of 347 ± 11.9 mg/L, and a condensed tannins mean degree of polymerization (mDP; i.e., determined by phloroglucinolysis method) of 4.38 ± 0.04. All of these parameters were determined using similar procedure as described in Chira et al.23 Experimental Conditions. The wine was placed in 5 L glass containers, which were closed as tightly as possible, using proper stoppers (i.e., silicone stopper with small septum in order to collect the sample and add oxygen without opening the containers), with addition of vacuum grease around the caps. Each 5 L glass container was flushed with N2 prior to red wine filling in order to exclude incorporation of oxygen during filling. The wine was split in three pH groups: pH 3, pH 3.5, and pH 4. The pH was adjusted with NaOH (5 M) and/or sulphuric acid (99%). For each pH group, four quantities of oxygen were added: 0, 2, 8, and 20 mg/L. The oxygen was added into the bottles with a syringe filled with pure oxygen. As red wine consumes oxygen quite fast, oxygen was readjusted to 0, 2, 8, and 20 mg/L each week in order to keep constant levels during the four months. Each modality was performed in triplicate. After four months, 1 mL of each separate sample was filtrated on a 0.45 μm membrane and then injected in the HPLC-UV-MS for identification and quantification. For the quantitative analyses, 900 μL of sample was mixed with 100 μL of chlorogenic acid (500 mg/L) used as internal standard. Identification of the Pyranoanthocyanin−Procyanidin Dimers by HPLC-UV-MS/MS. These analyses were performed on two different systems. The first one was a Thermo-Finnigan Surveyor HPLC-UV system composed of a UV−vis detector (Surveyor PDA Plus), an autosampler (Surveyor autosampler Plus), and a quaternary pump system (Surveyor LC pump Plus) and controlled by a Xcalibur data treatment system. These analyses were carried out on a 250 × 4.6

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RESULTS AND DISCUSSION The first part of our study was to identify pyranoanthocyanin− procyanidin dimers in Merlot red wine since these pigments have only been detected in aged Port red wine which is a sweet red wine.22 This identification has been realized by HPLC-UVMS using both low and high resolution HPLC-MS analysis. During the investigation on Merlot red wine, two peaks with a molecular ion of [M]+ = 1093 have been detected which could be the two pyranomalvidin-3-O-glucoside-procyanidin dimers as previously described in aged Port wine.11,22,24,25 MSn analysis 1702

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ppm) between the measured mass and the calculated one (Table 1). Similarly, the identification and chemical formulas of the fragments have also been confirmed by high resolution mass analysis since the mass differences between the measured and the calculated masses were around 5 ppm. These investigations in HPLC-UV-MS confirmed the identification in dry red wine of two pyranomalvidin−3-O-glucosideprocyanidin dimers. Following this first identification in dry red wine, a quantification method by HPLC-UV-MS using chlorogenic acid as internal standard has been developed and used to estimate the impact of acidity and oxygen levels in wine on the formation of these two pyranomalvidin−3-O-glucoside-procyanidin dimers. As shown in Figure 4, the quantification of both pyranomalvidin−procyanidin dimers showed similar trends among the different studied modalities (i.e., three pH 3, 3.5, and 4 as well as four different level oxygen 0, 2, 8, and 20 mg/ L). Among the same pH, it clearly appeared that the amounts of the quantified compounds constantly increased from 0 to 20 mg/L of oxygen. In the modality without oxygen at pH 3, these compounds were present at the lowest concentration (i.e., around 2 mg/L), while in the modalities with 2, 8, and 20 mg/L of oxygen, the concentrations were respectively of 5.5, 14.5, and 19.7 mg/L. Following a similar trend, for a same quantity of oxygen, the amounts of the pyranomalvidin−procyanidin dimers increased along with the acidity of the red wine. Indeed, for the modality with the highest oxygen concentration, the amounts were 19.7, 12.5, and 4.6 mg/L from pH 3 to pH 4. However, for all studied pH, the lowest level was observed for the modality without oxygen, with concentrations of 2.0, 1.7, and 1.9 mg/L for the pH 3, 3.5, and 4, respectively. A similar trend has been previously observed from pyranomanthocyanins deriving from a vinyl cycloaddition mechanism in red wine with a single oxygen level and with three different pH.26 The observed formation trends according to pH and oxygen levels were of interest and confirmed these key parameters in the mechanism leading to the formation of pyranomalvidin− procyanidin dimers in red wine. Indeed, without oxygen the lowest level of pyranomalvidin−procyanidin dimers were similar for each pH which confirmed that oxygen is absolutely needed for the oxidative aromatization of the newly formed pyranic ring D of the pyranoanthocyanins. Similarly, the acidity of the red wine also appeared to be an important feature since the highest concentrations of pyranomalvidin−procyanidin dimers were observed for the lowest pH in the presence of oxygen.

with an ion trap mass spectrometer has been conducted to confirm this assignation. Fragmentation pattern in MS2 of the molecular ion m/z 1093 leads to the formation of a single ion at m/z 931 which can result of the loss of a glucose moiety (i.e., loss of 162 amu). Then, a MS3 analysis performed on the m/z 931 ion led to the formation of three main fragments at m/z 779, 641, and 623 (Figure 1). The fragment at m/z 641

Figure 1. MS3 fragmentation of the [M]+ = 1093 compound.

resulted from the direct cleavage of the interflavanoid linkage from the pyranomalvidin-procyanidin-dimer aglycone (i.e., m/z 931). This fragment m/z 641 can then undergo a water elimination to form a new fragment at m/z 623. Another important fragmentation pattern of the ion at m/z 931 was attributable to the RDA (Retro-Diels−Alder) ring fission of the C-ring of the upper flavanol unit leading to an ion at m/z 779. This RDA ring fission was a key fragmentation for the structure elucidation, proving the presence of the procyanidin moieties in the molecule. Such fragmentation patterns (Figure 2) are in accordance with the fragmentation pattern of the pyranomalvidin−procyanidin dimer detected in Port wine.22 To confirm this assignation of both the molecular ion as well as the observed fragments, HPLC-UV-MS analysis using a high resolution Q-TOF mass spectrometer has been performed (Figure 3). This analysis clearly showed that the molecular ion of both peaks had a chemical formula of C55H49O24 with a very high identity score and an extremely low difference (i.e., 0.34

Figure 2. Fragmentation pattern of the pyranomalvidin−3-O-glucoside-procyanidin dimer. 1703

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Figure 3. Extract ion chromatogram (EIC) of [M]+ = 1093 compound (right corner) and ion spectrum after MS/MS fragmentation with Q-TOF.

Funding

Table 1. Accurate Mass Analyses of the Fragments fragment [M]+

chemical formula

mass difference (ppm)

1093.2607

C55H49O24

0.34

99.11

931.2026 779.1569 641.1256

C49H39O19 C41H31O16 C34H25O13

5.82 4.88 5.28

X X X

scoreb

L.P. is supported by two French financial grants of Agricultural french Ministry FAM (France Agrimer) Nos. 2011-1162 and 2012-1025 and by a French financial grant of CIVB (Conseil Interprofessionnel des Vins de Bordeaux) No. 30566 (20112014). The authors gratefully thank CIVB (Conseil Interprofessionnel des Vins de Bordeaux) and the Region Aquitaine for providing funds for the acquisition of the HPLC-MS(n)UV/Fluo material.

corresponding fragment pyranomalvidin− procyanidin dimer glucose RDA ring fissiona glucose, catechin

a RDA: (Retro-Diels−Alder) ring fission of the C-ring of the upper flavanol. bScore represent a percentage of identity between the calculate molecular formula and the analyzed mass taking in too account the obtained mass as well as the isotopic abondances.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully thank Christian POUPOT for his help with the wine oxygen delivery experiments.

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(1) Glories, Y.; Maujean, A.; Dubourdieu, D.; Ribéreau-Gayon, P. Traité d’oenologie: Tome 2, Chimie du vin, stabilisation et traitement; 5th ed.; Dunod, 2004. (2) Arnold, R. A.; Noble, A. C.; Singleton, V. L. Bitterness and astringency of phenolic fractions in wine. J. Agric. Food Chem. 1980, 28, 675−678. (3) Bakker, J.; Timberlake, C. F. The distribution of anthocyanins in grape skin extracts of port wine cultivars as determined by High Performance Liquid Chromatography. J. Sci. Food Agric. 1985, 36, 1315−1324. (4) Bakker, J.; Timberlake, C. F. The distribution and content of anthocyanins in young port wines as determined by High Performance Liquid Chromatography. J. Sci. Food Agric. 1985, 36, 1325−1333. (5) Brouillard, R.; Dubois, J.-E. Mechanism of the structural transformations of anthocyanins in acidic media. J. Am. Chem. Soc. 1977, 99, 1359−1364. (6) Remy, S.; Fulcrand, H.; Labarbe, B.; Cheynier, V.; Moutounet, M. First confirmation in red wine of products resulting from direct anthocyanin−tannin reactions. J. Sci. Food Agric. 2000, 80, 745−751. (7) Salas, E.; Atanasova, V.; Poncet-Legrand, C.; Meudec, E.; Mazauric, J. .; Cheynier, V. Demonstration of the occurrence of flavanol−anthocyanin adducts in wine and in model solutions. Anal. Chim. Acta 2004, 513, 325−332. (8) Timberlake, C. F.; Bridle, P. Interactions between anthocyanins, phenolic compounds, and acetaldehyde and their significance in red wines. Am. J. Enol. Vitic. 1976, 27, 97−105. (9) Rivas-Gonzalo, J. C.; Bravo-Haro, S.; Santos-Buelga, C. Detection of compounds formed through the reaction of malvidin 3-monoglucoside and catechin in the presence of acetaldehyde. J. Agric. Food Chem. 1995, 43, 1444−1449. (10) Fulcrand, H.; Santos, P.-J. C.; dos Sarni-Manchado, P.; Cheynier, V.; Favre-Bonvin, J. Structure of new anthocyanin-derived wine pigments. J. Chem. Soc. Perkin Trans. 1 1996, 735−739. (11) Mateus, N.; Silva, A. M. S.; Santos-Buelga, C.; Rivas-Gonzalo, J. C.; de Freitas, V. Identification of anthocyanin-flavanol pigments in

Figure 4. Evolution of the pyranomalvidin−-3-O-glucoside-procyanidin dimer content in red wine according to pH and oxygen level.

From an oenological point of view, the key involvement of red wine acidity in the formation of pyranoanthocyanins is an important parameter for red wine color stabilization since over the last two decades, red wine acidity tends to decrease due to the global warming. Such decrease in wine acidity might result in a diminution of pyranoanthocyanins contents, which are an important contributor to red wine color stability. Further studies on the impact of pH and oxygen levels on the formation of the other pyranoanthocyanins are actually under investigation.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: (+33)557575850. Fax: (+33)557575813. E-mail: p. [email protected]. 1704

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