Detection and Partial Characterisation of New ...

J Sci Food Agric 1996,70,204-208

Detection and Partial Characterisation of New Anthocyanin-Derived Pigments in Wine Paulo-Jorge Cameira-dos-Santos,* Jean-Marc Brillouet, Veronique Cheyniert and Michel Moutounet INRA-IPV, Laboratoire des Polymtires et des Techniques Physico-Chimiques, 2 place Viala, 34060 Montpellier cedex 1, France (Received 24 April 1995; revised version received 5 July 1995; accepted 18 August 1995)

Abstract: New anthocyanin-derived red-orange pigments were detected in wine in the course of cross-flow microfiltration experiments. Analysis of the sugar and

organic acid released respectively by acid and alkaline hydrolysis showed that the two major products were respectively a glucoside and the corresponding p-coumaroylglucoside. Mass spectrometry indicated molecular masses of 755.5 for the p-coumaroylglucoside and of 447.5 for its aglycone. Therefore, the new pigments are likely to result from condensation reaction between grape

anthocyanins (possibly the 3-glucoside and 3-p-coumaroylglucosideof malvidin) and another wine component, which is not a flavanol. This reaction may participate in the mechanism of colour change during ageing of red wines. Key words : red wines, anthocyanins, pigments.

INTRODUCTION

and flavanols has been suggested (Liao et al 1992; Brouillard and Dangles 1994). Besides, according to Somers (197l), ‘quinone-like’ structures, arising from the oxidation of the anthocyanin-derived pigments, may also contribute to the yellow tints of aged wines. Grape anthocyanins are now well documented. Sixteen anthocyanins, including the 3-glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin, their acetic and p-coumaric esters, and caffeic ester of malvidin-3-glucoside have been reported in Vitis uinifera varieties (Wulf and Nagel 1978; Bakker and Timberlake 1985). The occurrence of condensation reactions between various flavanols and malvidin-3-glucoside, the major pigment in all V uinifera varieties, or malvidin3,5-diglucoside, found in non-uinifera Vitis species, has been studied in model systems (Timberlake and Bridle 1976; Baranowski and Nagel 1983; Liao et al 1992). Acetaldehyde-linked products have been obtained in solutions containing malvidin-3-glucoside and catechin (Timberlake and Bridle 1976; Baranowski and Nagel 1983) and partly characterised by mass spectrometry (Archier 1992). However, given the bathochromic shift resulting from conversion of grape anthocyanins (A,, 540 nm) to these molecules (A, 548 nm), they are more likely to participate in the colour of young red wines than to account for the tile-like orange-red of mature

The evolution of the colour of red wine is a complex phenomenon. Whereas the purple-red colour of young wines is essentially due to anthocyanins extracted from red grapes in the course of vinification, the tawny tint of older wines is usually attributed to specific wine pigments resulting from interactions between anthocyanins and other phenolic compounds, in particular flavanols. However, the mechanisms of these interactions as well as the structure of the new pigments generated are still poorly understood. Two major reaction pathways have been postulated. The first one involves the formation of acetaldehyde bridges between anthocyanins and flavanols (Singleton et al 1964; Timberlake and Bridle 1976; Baranowski and Nagel 1983). The second reported pathway consists in direct condensation of anthocyanins with flavanols, yielding yellow-orange pigments (Somers 1971; Liao et a1 1992). The role of copigmentation, ie hydrophobic stacking interactions between anthocyanins and other phenolic compounds, as the first step in the covalent binding between anthocyanins

* On leave from INIA-Estaqiio Vitivinicola Nacional, Dois Portos, 2575 Runa, Portugal. t To whom correspondence should be addressed. 204

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wines. Direct condensation between anthocyanins and flavanols, probably involving formation of intermediate yellow xanthylium salts and condensation via the anthocyanin 4-position, occurred slower than acetaldehyde-bridged condensation (Timberlake and Bridle 1976; Baranowski and Nagel 1983). Nevertheless, this mechanism may represent a major factor in the evolution of red wine colour during ageing (Liao et a1 1992). The work reported herein is part of a study aiming to determine the constituents participating in the fouling of polymeric membrane during cross-flow microfiltration of wine. The phenolic composition of extracts obtained from membranes fouled by cross-flow microfiltration of wines was analysed by HPLC and compared with that of the original wine. Unknown red-orange pigments preferentially adsorbed on the membrane matrix were detected. The present paper reports the occurrence in wine and partial characterisation of these compounds which may be important intermediates in the formation of the complex anthocyanin pigments of aged red wines.

a U6K manual injector, an automated gradient controller and a 990 diode array detector. UV-visible spectra were recorded from 250 to 600 nm. The column was reversed-phase Lichrospher 100-RP18 (5 pm packing) (250 x 4 mm id) protected with a guard column of the same material (Merck, Darmstadt, Germany). Phenolic compounds were eluted under the following conditions: 1 ml min-' flow rate; oven temperature 30°C; solvent A, 20 ml litre-' formic acid in water; solvent B, acetonitrile/water/formic acid (80 : 18 : 2, v/v); elution with linear gradients from 5 to 30% B in 40 min, from 30 to 50% B in 20 min, from 50 to 80% B in 10 min, followed by washing and reconditioning of the column. Characterisation of the major grape anthocyanins present in the wines and membrane extracts was achieved by HPLC analysis and comparison of the elution order and UV-visible spectra with those of reference compounds and literature data (Wulf and Nagel 1978; Bakker and Timberlake 1985).

EXPERIMENTAL Wine-making procedures and preparation of pigment extracts Wines were prepared at the INRA experimental winery (Station Exptrimentale de Pech Rouge-Narbonne, Gruissan, France) from Vitis uinifera var Carignane grapes harvested in 1993. The wine-making process was as follows : destemming and crushing, fermentation on skins, racking after 8, 15 or 30 days and storage in tanks. The wines were then submitted to batch crossflow microfiltration experiments performed using a pilot plant equipped with a module of 96 capillaries of modified polyethersulfone provided by X-Flow (Almelo, The Netherlands). The operating conditions were as follows : tangential velocity 2 m s- ; transmembrane pressure 1-2 bar; temperature 18°C; volume of filtered wine 180 litres; concentration factor ca 12. After each microfiltration experiment, the membrane was washed with hot (60°C) distilled water to remove the residual wine and colloid surface deposit and then twice with 2.5 litres of methanol-HC1 (99:1, v/v) to extract the adsorbed fouling components prior to membrane regeneration by the usual cleaning procedures. The methanol extracts were concentrated under vacuum by rotary evaporation prior to HPLC analysis. HPLC analyses The wines and membrane extracts were analysed by HPLC using a Waters-Millipore (Millipore Corp., Milford, MA, USA) system including two pumps M510,

Pigment purification The membrane methanol extracts were evaporated to dryness, dissolved in 3 ml ethanol and added with 2 M NaOH (2.4 ml). The first purification step consisted in liquid chromatography on normal-phase silica gel (Si60, Merck, Darmstadt, Germany). Elution was carried out successively with 100 ml ethanol (fraction l), 500 ml methanol/water (90 : 10, v/v) (fraction 2) and 100 ml methanol/trifluoracetic acid (TFA) (99-9 : 0.1, v/v) (fraction 3). Each collected fraction was taken to dryness by rotary evaporation and dissolved in 1.5 ml methanol/TFA (99.9 : 0.1, v/v) prior to HPLC analysis. The unknown orange pigments were isolated from fraction 2 by HPLC at the semi-preparative scale. The latter was performed using the same equipment as described above but under the following conditions: column, pBondapak RP-18 (10 pm packing) (300 x 7.8 mm id) (Millipore Corp, Milford, MA, USA); flow rate, 2 ml min-'; oven temperature, 30°C; solvent A, 2.5% acetic acid in water; solvent B, acetonitrile/ solvent A (80 : 20, v/v); elution with linear gradients from 5 to 20% B in 20 min, from 20 to 50% B in 30 min and from 50 to 80% B in 10 min. Fractions containing the orange pigments were collected, pooled, concentrated under vacuum and lyophilised. Sugar analysis After hydrolysis of pigment B by heating at 120°C for 75 min in 2 M TFA (Albersheim et a1 1967), neutral sugars were determined by gas chromatography (GC) of the alditol acetate derivatives (Harris et a1 1984) at 210°C on a fused-silica DB-225 capillary column (30 m x 0.32 mm id, 25 pm film; J&W Scientific, Folsom, CA, USA) with hydrogen as the carrier gas.

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Fig 1. HPLC chromatogram at 510 nm of (A) a red wine made from V vin$efera var Carignane, and (B) of the methanol-HCl(99 : 1, v/;) extract obtained b; washing a polymer& membrane fouled by cross-flow microfiltration of this wine. Mv-3-G, mavidin-3glucoside; Mv-3-CG, malvidin-3-p-coumaroylglucoside; A, B and 1 to 3, new pigments.

The positions of glycosidic linkages on the sugar moiety(ies)were determined by methylation of the sugar free hydroxyl groups by the Hakomori method (Hakomori 1964) as described by Jansson et al (1976), followed by acid hydrolysis, conversion of the partially methylated sugars to alditol acetates and analysis on DB-1 and DB-225 capillary columns (Saulnier et al 1988). Identifications were based on retention times and confirmed by GC-MS, using the DB-225 column (oncolumn injection at 50°C; injector, temperature gradient from 50 to 250°C at 180°C min-'; oven, temperature gradient from 150 to 180°C at 50°C min-' for 15 min, then 5°C min-' to 210°C; He as carrier gas at 2 ml min-') coupled to a Finnigan Mat ITD 700 mass spectrometer. Desterification reaction Pigment B (9.8 pl of a 0.53 g litre-' methanolic solution) was added with sodium methylate (25 pl of a 2 M solution in methanol) and allowed to react at ambient temperature under argon for 2 h. After acidification with 265 p1 of a methanol/TFA (99.5 : 0.5, v/v) solution, the released compounds were analysed by HPLC as described above.

late on the reversed-phase chromatographic system used), including in particular a series of unknown redorange pigments (peaks noted A, B, 1-3), were preferentially adsorbed onto the membrane and found in larger concentration in the methanol extract (Fig 1B) than in the original wine (Fig 1A). These pigments exhibited characteristic UV-visible absorbance spectra (Fig 2A), differing from those of known grape anthocyanins (Fig 2B) by their lower maximum absorption wavelength in the visible range (2 505-508 nm in the acidic HPLC solvent). Therefore, they might be anthocyanin-derived pigments formed by condensation mechanisms, as reported earlier in model studies (Timberlake and Bridle 1976; Liao et a1 1992). The two major pigments detected in wine, referred to as A and B, eluting, respectively, at 57 min and 62.5 min under our chromatographic conditions, were isolated from the methanol extracts of fouled microfiltration membranes. HPLC analysis of the three fractions collected after chromatography of the extracts on Si60 silica gel showed that pigments A and B were

Mass spectrometry Mass spectrometry analysis of pigment B was performed by electrospray MS in the positive mode using a MS Engine mass spectrometer (Hewlett-Packard). Partially methylated alditol acetates were analysed by GC-MS as described above.

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RESULTS AND DISCUSSION Polymeric membranes fouled by cross-flow microfiltration of red wines were washed successively with water and with acidified methanol in order to estimate the respective role of external deposit and of hydrophobic adsorbed material in the fouling process. The HPLC chromatogram of a red wine and of the methanol extract obtained from a membrane fouled by microfiltration of this wine are presented in Fig 1. Among wine polyphenols, rather unpolar compounds (eluting

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Fig 2. UV-visible spectra of (A) the new red-orange wine pigments A and B, and (B) of malvidin-3-glucoside and malvidin3-p-coumaro ylglucoside.

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eluted mostly in fraction 2, which was therefore used for further purification. Fraction 3, eluted with acidified methanol, also contained small amounts of the orange pigments but they were contaminated with red material appearing as a broad unresolved peak on the chromatographic profile, which may be polymeric pigments. In spite of its lower concentration in wine (Fig 1A) and because it was preferentially adsorbed on the membrane material (Fig lB), pigment B was obtained in larger amounts than pigment A and thus characterised first. Reversible colour change from red-orange to blue was observed when raising the pH, suggesting the presence of a flavylium chromophore in the pigment structure. A first series of experiments was performed in order to detect and determine possible glycosylation. Sugar analysis after hydrolysis under acidic conditions revealed glucose as the sole sugar, as expected from grape anthocyanin composition. GC-MS analysis of the partially methylated alditol acetate derivatives indicated that glucose was originally linked through the hydroxyl in C1 position, like in grape anthocyanins. Relatively large amounts (ca 20%) of 1,2,5-acetyl, 3,4,6-methyl glucitol were also formed, suggesting a substitution in the 2-position. However, sugar analysis after methylation of malvidin-3-glucoside showed the same secondary product in addition to the expected 1,5-acetyl, 2,3,4,6methyl glucitol. Thus, it seems that, in the case of anthocyanins, the glucose hydroxyl in the 2-position was not completely methylated under our experimental conditions. HPLC analysis of the TFA hydrolysis medium showed the presence of a major product (pigment C), along with small amounts of residual pigment B and of several degradation products (Fig 3). The lower polarity and higher absorption maximum (a,,, 523 nm) of pigment C (Fig 3) suggest that it might be the aglycone of pigment B. The UV-visible spectrum of pigment B (Fig 2A), unlike that of pigment A (Fig 2A) and of the probable aglycone C (Fig 3), shows absorbance in the 300320 nm region, characteristic of anthocyanins acylated with p-coumaric acid (Wulf and Nagel 1978) (eg malvidin-3-p-coumaroylglucoside,Fig 2B). This could explain the low polarity of B compared to that of other

wine pigments (eg pigment A). Moreover, it is in agreement with the postulated anthocyanin origin of the newly detected pigments as Vitis uinifera var Carignane was reported to contain large proportions of pcoumaroyl anthocyanins (Roggero et al 1988). Desterification of pigment B under alkaline conditions was carried out to check this hypothesis. HPLC analysis of the desterification medium after acidification showed the presence of three major products. The first two coeluted respectively with the methyl esters of cisand trans-p-coumaric acid and had the same UV-visible spectra as reference compounds, suggesting that they were artefacts formed by methyl esterification of released p-coumaric acid after acidification of the methanoljsodium methylate solvent used for the desesterification reaction. The third one had the same retention time and UV-visible spectrum as pigment A, suggesting that pigment B is a p-coumarate ester of pigment A. The mass spectrum of pigment B, recorded by electrospray-MS in the positive mode, yielded essentially two ions, respectively at mjz = 755.5 and mjz = 447.5. Given the mild ionisation technique used, the former is believed to be the molecular ion of pigment B whereas the latter is a fragment ion resulting from the loss of a p-coumaroylglucose moiety (m = 308), agreeing with the interpretation of degradation experiments. The molecular mass of this fragment, which should correspond to the aglycone of pigments A and B, exceeds that of all known grape anthocyanidins, suggesting that it derives from complexation of an anthocyanidin with another molecule, as postulated. Note that 0-8 mol of p-coumaric methyl esters per mol of pigment B was formed by desesterification, assuming a molecular mass of 755 for pigment B, and using the response factor of p-coumaric acid for the integration of methyl esters peak areas at 310 nm. This suggests the presence of one p-coumaric acid moiety in pigment B structure, as expected both from MS data and UV-visible spectra. Acidic hydrolysis followed by GC analysis of the alditol acetate derivatives yielded only 0.5 mol of glucose per mol of pigment B, again assuming a molecular mass of 755. However, the presence of residual pigment B on the HPLC profile obtained after hydrolysis indicates that neither the gly-

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Fig 3. HPLC Chromatogram at 280 nm of the solution obtained by acid hydrolysis of pigment B and UV-visible spectrum of the major pigment released (C).

P-J Cameira-dos-Santos et a1

208 cosidic linkage nor the ester bond were totally hydrolysed under the reaction conditions, so that the method used for sugar determination appears qualitative rather than quantitative. Since the 3-glucoside and 3-p-coumaroylglucoside of malvidin are the major anthocyanins in the Carignane wine used in this experiment, as reported earlier for Vitis oinfera var Carignane grapes (Roggero et al 1988), pigments A and B are likely to be derivatives formed from these two molecules by the same reaction. As well, minor pigments exhibiting absorbance maxima in the range 505-508 nm (retention times 55, 58.8 and 59.5 min, respectively) detected on the membrane extract HPLC traces (Fig lB, peaks 1-3) might be generated by similar mechanisms from other grape anthocyanins. The partial resistance of the complex to hydrolysis (Fig 3) as well as the lack of fragment corresponding to grape anthocyanidins (or to their p-coumaroylglucosides) in the mass spectrum indicate the existence of strong linkages between the anthocyanidin moiety and its substituent. This is in agreement with the xanthylium salt structure resulting from direct condensation between anthocyanins and flavanols proposed by other researchers (Jurd 1967, 1969; Timberlake and Bridle 1976; Liao et a1 1992). However, the difference calculated between the mass of the new pigment and that of known grape anthocyanidins (from 116 to 160, respectively, with malvidin and cyanidin as reference) is too small to be accounted for by a flavanol moiety. Besides, the absorption maximum of the new pigments in the visible range is higher than that reported for xanthylium salts (440-450 nm) (Jurd 1969; Liao et a1 1992). Thus, it seems that condensation between anthocyanins and nucleophilic molecules other than tannins takes place in the course of wine ageing although the new wine pigments reported may also arise from the degradation of intermediate tannin-anthocyanin complexes. Work is in progress to isolate pigments A and B in larger quantities to achieve structural determination and elucidate the mechanism by which they are generated. To our knowledge, this is the first report of anthocyanin-derived pigments in red wines. In spite of the small amounts detected, their formation is likely to be an important step in the conversion of grape anthocyanins to complex wine pigments. ACKNOWLEDGEMENT

The authors thank Dr Fabre-Bonvin (Service Central d’Analyse, CNRS, Vernaison, France) for assistance

with mass spectrometry analysis and very helpful discussion. The work was supported by CMMC (Chalonnes sur Loire, France) and the cross-flow microfiltration membrane kindly provided by X-flow (Almelo, the Netherlands).

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