Analytica Chimica Acta 513 (2004) 5765

Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine
Stphane Vidal a,b,1 , Leigh Francis a,b , Ann Noble c , Mariola Kwiatkowski a,b , Vronique Cheynier d , Elizabeth Waters a,b,
a The Australian Wine Research Institute, P.O. Box 197, Glen Osmond SA 5064, Australia Cooperative Research Centre for Viticulture, P.O. Box 154, Glen Osmond SA 5064, Australia c University of Davis, Davis CA, USA Unit Mixte de Recherche Sciences pour lOenologie, INRA, 2, place Viala, 34060 Montpellier cedex, France b

Received 28 July 2003; accepted 8 October 2003 Available online 3 December 2003

Abstract The taste and mouth-feel properties of three different types of tannin-like polyphenolic compounds, representative of some of the tannin-like polyphenolic compounds found in red wines, were determined using descriptive sensory analysis. Ethyl-bridged avanols were produced by reaction of (+)-catechin with acetaldehyde under acidic conditions. Red coloured tannin-like polyphenolic compounds from wine and from wine pomace were isolated by multi layer coil counter-current chromatography (MLCCC). Mouth-feel attributes and bitterness of the fractions dissolved in a model wine (MW) medium were rated while the fractions were held in the mouth and after expectoration. The sensory properties of the fractions described above were compared to those of apple procyanidins (tannins) with degree of polymerisation (dp) 3 and 9. Both wine and pomace-derived coloured tannin-like polyphenolic compounds were rated as signicantly less astringent when compared to both the apple tannin fractions but were similar to the MW. The ethyl-bridged avanols (with mean dp of 5) were rated signicantly more bitter than both the apple procyanidin fractions. Astringency of the ethyl-bridged avanols was lesser than the apple procyanidin fraction with dp9 and more than that with dp3. Highly puried grape anthocyanidin monoglucosides and monoglucoside coumarates, prepared either by solid-phase extraction or by multi layer coil counter-current chromatography were rated similarly to the MW indicating that free anthocyanins, like the coloured tannin-like polyphenolic compounds from wine and pomace, do not contribute astringency nor bitterness to wine. 2003 Elsevier B.V. All rights reserved.
Keywords: Wine; Tannins; Procyanidins; Proanthocyanidins; Anthocyanins; Sensory descriptive analysis; Astringency; Bitterness; Ethyl-bridged avanols

1. Introduction Bitterness and astringency are two sensory terms of crucial importance for describing the sensory properties of wine. Whereas bitterness is a taste mediated by sensory receptors, astringency is considered to be a tactile sensation resulting from the precipitation of salivary proteins and leading to a loss of mouth lubrication. Wine astringency is mainly attributed to the presence of the phenolic compounds called tannins. Wine tannins include a variety of phenolic compounds as described elsewhere [1]. Proanthocyanidins or condensed tannins originate from grapes and can be extracted from

Corresponding author. Tel.: +61-883036600; fax: +61-883036601. E-mail address: elizabeth.waters@awri.com.au (E. Waters). 1 Present address: Inter Rh ne, 2260 Route du Gr` s, 84100 Orange. o e

seeds, skins and stems during wine-making. Proanthocyanidin structures vary in the nature of constitutive sub-units, degree of polymerisation (dp) or chain-length and linkageposition. Grape seed proanthocyanidins (procyanidins) are composed of (+)-catechin, ()-epicatechin and ()epicatechin-3-O-gallate ( 20%) resulting from acylation of epicatechin by gallic acid [2]. Skin proanthocyanidins are composed of the same units with only 5% ()-epicatechin3-O-gallate and additionally include ()-epigallocatechin ( 20%) [3] and are consequently referred to as prodelphinidins. Stem proanthocyanidins have a composition between that of skin and seed proanthocyanidins [4] but only account for 5 to 10% of total grape proanthocyanidins. Mouth-feel properties of grape seed and skin proanthocyanidins have been examined through many studies [5,6]. A recent study showed that astringency increased with the dp and that an increasing degree of galloylation was responsible

0003-2670/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.10.017

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S. Vidal et al. / Analytica Chimica Acta 513 (2004) 5765

for an increasing coarse perception of the proanthocyanidins [7]. Increasing the degree of B-ring trihydroxylation (given by epigallocatechin content) seemed to decrease astringency. Once extracted from grapes, phenolic compounds, including the seed and skin proanthocyanidins undergo chemical reactions during vinication to form tannin-like polyphenolic compounds in wine, following two main reaction pathways. The rst involves acetaldehyde-mediated condensation between avanols [8] leading to molecules referred to as ethyl-bridged avanols. This type of condensation can also occur between anthocyanins [9] or between avanols and anthocyanins. In the latter case, both ethyl-bridged avanolanthocyanin [10] and pyranoanthocyaninavanol adducts have been reported [11]. The second type of wine tannin-like polyphenolic compound is derived from direct reactions between avanols and anthocyanins. Because these compounds can act as electrophiles or nucleophiles at wine pH, both anthocyaninavanol and avanolanthocyanin adducts can be formed. The decrease of astringency which occurs during wine-ageing may be the result of the formation of these tannin-like polyphenolic compounds, which have been suggested to be less bitter and astringent than the parent compounds [12,13]. Because of the difculty in isolating and characterising these types of molecules, only limited studies of their mouth-feel properties have been made to date. The aim of the present study was to improve our understanding of the mouth-feel properties of wine tannin-like polyphenolic compounds. Descriptive analysis of the taste and mouth-feel properties was conducted on isolated and characterised wine tannin-like polyphenolic compound fractions, native proanthocyanidins and native anthocyanins in a wine-like model solution.

Skin extract was obtained from Vitis vinifera cv. Shiraz grapes from Pech-Rouge (France) that had been harvested at commercial maturity. Skins (600 g) were removed from frozen grapes, refrozen in liquid nitrogen and ground using a Dangoumau blender (Prolabo, France). The resulting powder was extracted with 7 l of ethanol/water (75/25) containing 2% of acetic acid at 4 C overnight. The extract was sieved on nylon mesh (Scrynel, 500 m, Poly Labo, Strasbourg, France) and ltered through GF/C glass microbre lter (Whatman, Maidstone, UK). The extract was concentrated under vacuum to 2 l, which contained 6 g of malvidin 3-glucoside equivalents (as determined by high performance liquid chromatography with a diode array detector (HPLC-DAD)). This extract was loaded onto 11 l of Toyopearl TSK HW-50(F) gel (Tosoh, Tokyo, Japan), packed in a semi-preparative scale column (50 25 cm) which had been equilibrated in water. The ow rate of 100 ml/min was maintained by a Dynamax SD-1 pump (RAININ, Oakland, CA, USA). The chromatographic conditions were adapted from those described elsewhere [14]. The column was rst washed with water (three bed volumes) and eluted with ethanol/water/TFA (20/80/0.05, v/v) (two bed volumes) to recover most of the anthocyanidin glucosides. The material was freeze-dried to give the anthocyanidin glucosides, coded GLC. Finally the column was eluted with ethanol/water/TFA (80/20/0.05, v/v) to give a fraction enriched in acylated anthocyanins that was further puried using multi layer coil counter-current chromatography (MLCCC). 2.2. MLCCC fractionation The MLCCC system was a Quattro (AECS, UK) equipped with four coils on two holders (bobbins of 190 mm diameter). Each bobbin is composed of two coils (each with a 1.6 mm internal diameter teon tubing) with volume capacity of 100 and 250 ml. The MLCCC system, maintained at 25 C, was operated under conditions described elsewhere [15]. Revolution speed was set at 800 rpm and the ow rate was delivered at 2 ml/min by a Waters 600E pump. Elution was monitored by an UPC-900 detector (Amersham Biosciences, Sydney, Australia) set at 546 nm. The aqueous phase was used as the stationary phase (retention was in the range of 75 to 85%). The two 100 ml coils were mounted in series and lled with the lower phase of the system of tert-butyl methyl ether/n-butanol/acetonitrile/water acidied with triuoroacetic 0.02% (2/2/0.1/5). The elution was performed following a binary stepwise gradient of solvent A (upper phase of the system tert-butyl methyl ether/n-butanol/acetonitrile/water acidied with triuoroacetic 0.02% (2/2/0.1/5)) and solvent B (upper phase of the system tert-butyl methyl ether/n-butanol/acetonitrile/water acidied with triuoroacetic 0.02% (2/2/2.5/5)): isocratic for 10 min with 100% A, then with 46% B for 10 min then with 70% B for 90 min. The samples (0.25 g; a crude coumaroylated anthocyanin fraction eluted from Toyopearl, a wine

2. Experimental 2.1. Materials (+)-Catechin, acetaldehyde and acetic acid were purchased from Sigma (Sydney, Australia). The two fractions of native apple proanthocyanidins with mean dps of 3 and 9 (Adp3 and Adp9) used in this study were the same as those described elsewhere [7]. The powdered apple tannin samples were stored under argon between studies. Commercial marc extract from a ros wine marc was provided by Socit Franaise de Distillerie (Vallon-Pont-dArc, France). Wine extract was prepared from a 1999 Shiraz wine (1 l) from McLaren Vale (South Australia) that was vinied without wood contact. The extract was prepared by dealcoholisation under vacuum before adsorption on a XAD2 column containing 400 ml of resin equilibrated in water. Polar compounds were eluted with two bed volumes of water and the polyphenolic extract was recovered by methanol elution (two bed volumes). Methanol was evaporated under vacuum and the polyphenolic extract was re-dissolved in water and freeze-dried to yield the wine extract.

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59

and a marc extracts) were dissolved in a 50/50 mixture (5 ml) of the upper and lower phases of system A prior to injection through a Rheodyne injection loop. A coumaroylated anthocyanin fraction, coded COUM, was recovered from the MLCCC of the fraction eluted from Toyopearl TSK HW-50(F) with ethanol/water/TFA (80/20/0.05, v/v). The polar-coloured phenolic material remaining in the stationary phase after MLCCC of wine and marc extracts were freeze-dried to give SP-W and SP-M fractions, respectively. 2.3. Production of ethyl-bridged avanols by hemisynthesis Ethyl-bridged avanols were prepared using the conditions described by Fulcrand et al [8] but starting with 2.5 g of (+)-catechin. After a 75 min incubation, the reaction was stopped by pouring the solution into 400 ml of DVB-PS resin (400 ml, pre-conditioned with water), on a paper lter (GF/C prolabo) in a Bchner funnel and by washing the resin extensively with water to eliminate protonated acetaldehyde from the resin (this species forms as the rst step of the reaction [8]). Un-reacted catechin was removed by elution with diethyl ether (800 ml). The resin was eluted successively with ethyl acetate (800 ml) and methanol (500 ml). The ethyl acetate fraction was evaporated under vacuum, re-dissolved in water and freeze-dried (ethyl-B). 2.4. HPLC-DAD and ESIMS/MS HPLC analyses were performed using a Waters Millenium HPLC-DAD system (Milford, MA, USA) as described previously by Souquet et al [4]. ESIMS/MS analyses were performed with a ThermoFinnigan LCQ Advantage (San Jose, CA, USA) mass spectrometer equipped with an electrospray source and an ion trap mass analyser controlled by the LCQ navigator software. The spectrometer was operated in the positive ion mode (source voltage, 4.5 kV; capillary voltage, 23.5 V; capillary temperature, 250 C). 2.5. Thiolysis Analysis of avanol fractions was performed by thiolysis followed by HPLC under the conditions previously described [3]. 2.6. UVVis Spectrophotometry Solutions of samples (1 mg/ml) were prepared either in methanol or in 12% ethanol solution in water containing 3 g/l of tartaric acid and buffered at pH 3.5 by addition of concentrated NaOH. Absorbance of the wine-like solution was measured at 520 nm directly and after addition of 0.015 volume of a 20 g/l sodium metabisulte to 1 volume of sample and incubation for 10 min. For the methanolic solution, absorbance was measured at 280 nm.

2.7. Mouthfeel Descriptive Sensory Analysis 2.7.1. Experimental samples All fractions were evaluated in a model wine (MW) consisting of ethanol/water (13/87, v/v) saturated with potassium hydrogen tartrate of pH 3.6. The fractions were dissolved in MW immediately before the session, each at 0.5 g/l (w/v) and two drops ( 70 l) of a food colourant, an aqueous 1% carmoisine solution (Cake and Confectionary Colour Rose Pink, Queen Fine Foods, Alderley, Qld., Australia) were added to assist in masking colour differences between the samples. Two drops of the dye was also added to the MW before it was rated with the other samples. A panel of 15 volunteer judges (11 males and 4 females, average age 35 years) from The Australian Wine Research Institute and from Adelaide University was convened. All panelists except two had participated on previous sensory evaluations of mouth-feel properties of proanthocyanidins [16]. 2.7.2. Training Training was carried out for two weeks using puried or commercial tannins in MW, red wines and standards (quinine sulfate (15 mg/l), aluminum sulphate (2.5 g/l) and carboxy methyl cellulose (2 g/l)). Judges decided to evaluate the fractions in the MW solution used in a previous study. The judges agreed by consensus that the terms used in the earlier study were appropriate to describe the samples although two new terms were introduced: texture length and fullness. The terms and their denitions are listed in Table 1. For the terms describing the surfacesmoothness sensations, touch standards (emery papers and silk cloth) were used as recommended by Gawel et al. [17]. After a series of discussion sessions, training sessions were carried out in isolated booths to familiarise the panelists with the intensity scale used, a modied labelled magnitude scale [18], with scores from 0 to a maximum of 10 and the computerised protocol. Data were collected using Fizz software (Biosystemes, Couternon, Version 1.3). 2.7.3. Formal rating sessions The experimental samples and the MW base were evaluated in coded black plastic disposable cups, with assessments carried out in isolated booths under sodium colour masking lights at 2224 C. Two or three coded 20 ml samples were presented per session with a randomised presentation order across panelists. Panelists scored the intensity of the attributes while holding wine in their mouth and after expectoration. Judges rinsed with water and rested 30 sec between samples. The solutions were presented in triplicate over eight sessions. 2.7.4. Data analysis Data were analysed with a mixed model analysis of variance (ANOVA) with judges treated as a random effect. Separate principal component analyses (PCA) were carried

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Table 1 Sensory attributes rated by the panel and their denitions Attribute In mouth and after expectoration Surface smoothness Fine grain Medium grain Coarse grain Dry Chalky After expectoration Pucker Adhesive Texture length Fullness Overall astringency Bitter Denition Textures felt on mouth surfaces when the different surfaces come in contact with each other Fine texture, related to the feel of silk cloth Medium-sized texture, related to the feel of ne grade emery paper Rough texture, related to the feel of coarser grade emery paper Feeling of desiccation or lack of lubrication in the mouth Feeling of ne particulate matter that mouth movements can displace A reexive action of cheek surfaces being brought together and released in an attempt to lubricate mouth surfaces (most obvious immediately after spitting) A sensation that mouth surfaces are sticking or adhering to one another (notably between front lips and gums) Extent of duration of the astringent sensation Feeling of full, rounded sensation in the mouth, related to a 2 g/l carboxymethyl cellulose aqueous solution Overall level of astringent sensation, encompassing the above terms Bitterness, related to a 15 mg/l aqueous quinine sulphate solution

out on the mean ratings for the in-mouth attributes and for those rated after expectoration. The PCA was conducted using the correlation matrix with no rotation. Statistical analyses were performed with JMPTM (Version 3.1, SAS Institute, Cary, NC, USA) software. 2.8. Paired comparison sensory analysis GLC and COUM fractions were evaluated in ethanol/water (5/95, v/v). The anthocyanin fractions were dissolved immediately before the session, each at 0.5 g/l (w/v). A panel of 15 volunteer judges (10 males and 5 females, average age 35 years) from INRA-UMR SPO was convened. All panelists had previously participated in sensory evaluations of astringency and bitterness of proanthocyanidins using the time-intensity technique [19]. The panelists were trained using the same liquid standards as those used for descriptive sensory analysis by the Adelaide panel.

3. Results and discussion 3.1. Characterisation of polyphenolic fractions Six wine polyphenolic fractions were prepared either by chromatography (MLCCC and gel chromatography) or by hemi synthesis. Two apple native proanthocyanidin (Adp3 and Adp9) fractions that had been prepared for a previous sensory analysis were also used. All the fractions were analysed by HPLC-DAD and ESIMS/MS and the main compounds identied on the basis of their UVvisible, retention time and m/z values. The GLC fraction was composed of ve anthocyanidinglucosides (malvidin, peonidin, petunidin, delphinidin and cyanidin 3-glucosides), malvidin and peonidin being the most abundant. The ve anthocyanins represented 98% of the total absorbance at 280 nm of the chromatogram. The COUM fraction was mainly composed of the ve anthocyanin coumarates, with malvidin 3

coumaroyl-glucoside accounting for 60% of the coumaroylated forms. Malvidin and peonidin 3-caffeoyl-glucoside were also present and accounted, when taken together, for 8% of the 520 nm trace. Other phenolic compounds present in this fraction that accounted for 13% of the 280 nm trace were also detected (data not shown). They were essentially avonols (quercetin 3-glucoside (m/z 465), quercetin 3-glucuronide (m/z 479), myricetin 3-glucuronide (m/z 495), isorhamnetin 3-glucoside (m/z 479)). For proanthocyanidin and tannin-like fractions, SP-W, SP-M, Adp9, Adp3 and ethyl-B, information gathered from direct HPLC-DAD was not meaningful. The chromatograms of all these fractions exhibited an unresolved broad hump suggesting a polydisperse distribution of molecules in these fractions. It is however important to note that the ethyl-B fraction did not contain catechin and that the red-coloured fractions SP-W and SP-M were almost completely devoid of free anthocyanins. The absorbances of SP-W and SP-M at 520 nm were 17 and 28%, respectively, of that of GLC when dissolved at the same concentration in wine like solution at pH 3.5. Furthermore, after addition of sodium metabisulphite to SP-W and SP-M, 65 and 53% of the absorbance at 520 nm, respectively, was retained. All fractions were submitted to acid-catalysed de-polymerisation in the presence of a thiolytic reactant (thiolysis), followed by HPLC-DAD analysis of the released products. This method, used for proanthocyanidin characterisation, is based on de-polymerisation occurring under acidic cleavage of interavanic linkages (C4C6 or C4C8) and provides data about the sub-unit composition of the fractions. This method discriminates between terminal units that are released as avan-3-ol monomers and extension units released as thiol derivatives (Fig. 1) resulting from the trapping by the thiolytic agent of the cation generated through the breakdown of the interavanic linkage. As a result, a mean degree of polymerisation (mdp) can be calculated as the ratio of total units to terminal units [3]. Thiolysis of COUM and GLC fractions did not release any avanol unit indicating,

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61

OH HO 8 O 4 OH HO H
+

OH HO 8 O 4 OH OH HO OH H O 4 OH OH OH OH HO O 4 OH OH OH OH OH HO 8 O 4 OH

OH

Extension units
OH

OH OH O 4 OH

OH OH

Terminal unit

OH

Fig. 1. Schematic mechanism of the thiolysis reaction of native proanthocyanidins.

as expected, the absence of proanthocyanidin-like structures in these two fractions. Adp3 and Adp9 showed a composition marked by the sole presence of epicatechin units and by mdp of 3 and 9.6, respectively. Characterisation of SP-M and SP-W was more complicated as thiolysis analysis failed to give meaningful structural information. Thiolysis gave rise to both avanols, present either as terminal units or as extension units, and anthocyanin units, released solely as native anthocyanins. However, a very low yield of conversion into constitutive units (ca. 10%) was observed and, by HPLC, the fraction still exhibited an unresolved broad hump as it did before thiolysis. No ions corresponding to known proanthocyanidin oligomers could be detected in SP-W and SP-M when the fractions were analysed by electrospray direct injection using increasing collision energy to generate small fragments from large molecular weight species. By comparing these results with that obtained from the native apple procyanidins, it could be concluded that SP-W and SP-M did not contain detectable amounts of native grape seed and skin proanthocyanidins. Absorbance at 280 nm of SP-M and SP-W fractions account for 87 and 76%, respectively, of the Adp3 fraction dissolved at the same concentration in methanol, a range of values consistent with the SP-M and SP-W fractions being polyphenolic in nature. Whilst we cannot completely rule out that the material in SP-M and SP-W that was resistant to thiolysis was protein and polysaccharide in nature, this seems highly unlikely because the expected extinction coefcients for red wine proteins and polysaccharides are insufcient to account for this level of absorbance. In addition the sample preparation for these fractions on the divinylbenzenepolystyrene resin should exclude proteins and polysaccharides at several points. It is more likely that the material in SP-M and SP-W that was resistant to thiolysis was polyphenolic in nature as has been very recently reported for anthocyaninavanol adducts [20] linked by both carboncarbon and ether interavanoid bonds as encountered in A-type proanthocyanidins or for direct avanolanthocyanin adducts [21]. Flavanol units in such structures are not sensitive to acidic de-polymerisation. The coloured nature of SP-M and SP-W and their resistance to bleaching by SO2 also suggests that anthocyaninavanol or avanolanthocyanin adducts could be present in these

fractions. In summary, the data presented here supports the hypothesis that both SP-W and SP-M consisted primarily of derived pigmented wine tannin-like polyphenolic compounds. Further structural characterisation of these fractions is currently being undertaken and will be reported separately. Thiolysis of ethyl-B gave rise to a more complex chromatogram than the other fractions but was, nevertheless, in accordance with what had been previously described when ethyl-bridged avanols were submitted to thiolysis in the presence of mercaptoethanol [22,23]. In contrast to native proanthocyanidins, ethyl-bridged avanols are tannin-like structures in which units are linked by ethyl-bridges through C6 or C8, yielding different products originating from the combinations of linkages between C6C6, C8C8 and C6C8 (in the latter case both R and S forms can exist because of the presence of an asymmetric carbon) [10]. After thiolysis, catechin was released along with ethyl-thioether derivatives that were identied by MS. Epicatechin was also detected because of an epimerisation phenomenon that occurred when the solution was heated. Three peaks corresponding to three forms of monothioether-ethyl catechin were identied by their m/z. The theoretical fourth form of monothioether-ethyl catechin was not detected, as observed previously [23]. Two peaks eluting after the thiolytic reactant corresponded to dithioether-ethyl-catechin. The presence of these dithioether derivatives revealed that random cleavage of the linkages that occurs can generate false terminal units as counterparts (Fig. 2). This situation is different from what happened with native proanthocyanidins, in which the interavanic linkages are between C4 and C8 and/or C4 and C6. Acidic cleavage of these interavanic linkages in the presence of a thiolytic reactant results in the thiol ether always located on C4 (Fig. 1), thus giving only one free avanol unit (terminal unit) per proanthocyanidin. To calculate mdp for ethyl-bridged avanols, these dithioether derivatives have to be taken in account and the number of moles of dithiols has to be subtracted from the moles of apparent terminal units. As we do not have standards for thioether and dithioether ethyl-derivatives, we assumed that their response coefcients were similar to those of corresponding thio ether catechin.

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OH OH OH OH HO O OH H3C HO H3C OH HO O HO H3C CH O H3C OH HO HO OH HO OH OH OH HO O OH OH HO O OH OH CH OH HO OH CH O OH CH OH OH OH OH H3C HO O OH CH OH H3C

Flavanol monomer
HO O OH

S-CH-
CH

OH

OH OH

dithioether-ethyl-bridge

S-CH- Thiolysis, 2 min, 90C S-CH- monothioether-ethyl-bridge

Flavanol monomer

Fig. 2. Schematic mechanism of the thiolysis reaction applied to ethyl-bridged avanols and the potential release of false terminal units.

As a result, the calculation of mdp of ethyl-bridged avanols was done according to the formula: mdp = [terminal + monothiol + dithiols] [terminal dithiols]

Without correction for the additional apparent terminal units due to the dithiol derivatives, ethyl-B had a mdp of 3, and, with correction, a mdp of 5.4. The oligomeric ethyl-bridged avanol fraction was further analysed by direct infusion ESIMS. A series of intense ions were detected in the negative mode that corresponded to dimers (m/z = 605), trimers (m/z = 921), tetramers (m/z = 1237), pentamers (m/z = 1553) and hexamers (m/z = 1869) of ethyl-bridged avanols. The dimers and trimers were the prominent ions and the presence of vinyl monomers (m/z = 315), revealed the possible cleavage of ethyl-bridge bonds in the source. No multi-charged species could be detected. The distribution observed by ESIMS was in good accordance with the calculated mdp of 5.4. 3.2. Sensory analysis The scores for the sensory terms differed signicantly across the experimental samples with the exception of in mouth fullness and medium grain after expectoration which

were not signicantly different. The mean values and least signicant differences are shown in Table 2. To give an overview of the results, the PCAs of terms rated whilst the samples were in the mouth and after expectoration are presented in Figs. 3 and 4, respectively. For both PCAs, PC1 separated Adp3, Adp9 and ethyl-B from the other samples. The PCA of in mouth terms (Fig. 3) contrasts Adp3, Adp9 and ethyl-B with the rest of the samples on PC1 on the basis of their high intensity of coarse grain, dry and chalky terms and low intensity of ne grain. With the exception of the wine derived pigmented tannin-like fraction (SP-W), the pigmented samples are clustered close to the model system reecting their low astringency. As shown in Fig. 4, Adp3, Adp9 and ethyl-B were very high in intensity of all post expectoration astringency sub-quality terms, except for medium and ne grain. In contrast, MW, SP-W, SP-M, COUM and GLC were low in intensity in the astringency sub-qualities after expectoration. As shown in Table 2, MW did not differ signicantly from GLC or COUM with one exception: COUM was signicantly higher than MW in intensity of in mouth dryness. Taken as a whole, these results indicate that, at the concentration found in wine (typically 0.3 to 1.2 g/l), anthocyanins do not contribute signicantly to bitterness nor mouth-feel properties, in contrast with what had been reported twice previously [6,24]. In the two previous studies,

S. Vidal et al. / Analytica Chimica Acta 513 (2004) 5765 Table 2 Mean of the scores and the least signicant difference (LSD, P = 0.05) values for each attribute Fine In mouth COUM 1.25ab GLC 1.55a MW 1.45a SP-M 1.35a SP-W 1.20ab Adp3 0.63c Ethyl-B 0.89bc Adp9 0.72bc LSD 0.44 After expectoration COUM 0.70b GLC 1.19a MW 0.94ab SP-M 1.21a SP-W 0.83ab Adp3 0.66b Ethyl-B 0.57b Adp9 0.53b LSD 0.41 Medium 0.91ab 0.71b 0.78b 0.82b 1.09ab 1.29a 1.20ab 1.05ab 0.44 1.45a 1.10a 1.19a 1.02a 1.44a 1.17a 1.22a 0.94a Coarse 0.62c 0.26c 0.33c 0.38c 0.34c 1.10b 1.28b 1.99a 0.41 0.75c 0.40c 0.34c 0.69c 0.48c 1.55b 1.83b 2.54a 0.44 Chalk 1.20bc 1.17bc 1.02c 1.27bc 1.25bc 1.59b 1.80a 2.03a 0.44 1.00c 1.04c 0.93c 1.20bc 1.16bc 1.60ab 1.58ab 1.82a 0.42 Dry 1.80c 1.52cd 1.34d 1.67cd 1.58cd 2.32b 2.36b 2.87a 0.44 2.06c 1.80c 1.88c 2.00c 2.08c 2.67b 2.97a 3.29a 0.42 Full 2.25a 2.12a 2.11a 2.19a 1.96a 2.05a 1.86a 1.97a Adhesive Pucker Astringent Bitter

63

Length

1.43c 1.18c 1.06c 1.29c 1.30c 1.97b 2.27ab 2.56a 0.49

1.31bc 0.94c 0.81c 0.97c 1.04c 1.61b 1.88ab 2.20a 0.49

2.39c 1.98c 1.95c 2.19c 2.19c 2.94b 3.39ab 3.78a 0.47

0.94b 0.86b 0.72b 0.93b 0.74b 0.88b 1.35a 0.76b 0.38

3.16b 2.54b 2.61b 2.72b 2.80b 3.98a 4.29a 4.38a 0.78

Within a column, scores with the same letter are not signicantly different at the 5% level.

it could not be ruled out that the level of astringency associated with anthocyanins could be attributed to polyphenolic contaminants present in the anthocyanin fractions assessed. It is for this reason that the procedure used in this study to isolate anthocyanin material was optimised in order to reach a very high level of purity. However, in the present study,

astringent materials such as Adp3, Adp9 or ethyl-B were assessed along with the anthocyanin fractions. The lack of astringency found for anthocyanins could therefore be attributable to contrast effects versus the astringent fractions. To conrm that this was not the case, a separate sensory experiment with a different panel of judges using paired

In mouth

PC2, 11 %

fullness

COUM SP-M GLC MW fine grain

coarse grain dry chalk Adp3

Adp9

PC1, 80% ETHYL-B SP-W medium grain

Fig. 3. Biplot of principal components 1 and 2 for mean scores (n = 15 judges 3 replicates) of the in mouth sensory descriptors.

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PC2, 13% After spitting

medium grain COUM bitter SP-W texture Adp3 adhesive pucker overall astringency dry coarse grain ETHYL-B

MW GLC SP-M fine grain chalk

PC1, 78% Adp9

Fig. 4. Biplot of principal components 1 and 2 for mean scores (n = 15 judges 3 replicates) of the sensory descriptors rated after expectoration.

comparison was undertaken. For this experiment, the anthocyanin fractions were assessed in un-buffered 5% ethanol, in order to reduce any background astringency due to acid and background bitterness due to high ethanol that may have masked the bitterness and astringency of the samples when they were assessed in MW. When dissolved in 5% ethanol, neither GLC nor COUM were perceived signicantly more astringent or more bitter than the 5% ethanol solution, a result supporting our hypothesis that anthocyanins do not intrinsically contribute astringency or bitterness. The marc and wine extracts (SP-M and SP-W) did not differ from the MW in intensity of any term. Scores of SP-W and SP-M were very close even if SP-W tended to be rated more medium grain (in mouth and after spitting) but also less in coarse grain, ne grain in mouth than SP-M. This result suggests that, provided sufcient levels are formed and thus sufcient levels of the astringent precursors are depleted, the derived pigmented tannin-like polyphenolic compounds that form during wine-making from grape seed and skin tannins and avanols may lead to a decrease of astringency of the wine. Adp9 was rated signicantly higher than Adp3 for coarse grain and dry both in mouth and after spitting and for adhesive, pucker and overall astringency after spitting. These fractions had been previously distinguished by Vidal et al using the same descriptors (except for adhesive that did not signicantly discriminate between the two fractions). This result further validates the use of astringency sub-qualifying attributes in this kind of sensory descriptive analysis and demonstrates the consistency by the panel in using these terms. Ethyl-B (with mdp = 5) was rated intermediately between Adp3 and Adp9 for all the descriptors except for ne

grain in mouth and bitterness. It was rated as signicantly lesser in coarse and dry in mouth and coarse after spitting than Adp9. These results are in good agreement with our previous study that conrmed that, with the exceptions of ne grain and medium grain, the perceived intensity of astringency sub-qualifying terms increased with the dp. Consequently, it can be implied that the insertion of ethyl bridges between avanol units may not inuence astringency. The results obtained for SP-M, SP-W and ethyl-B are in fact in good accordance with the conclusions drawn by Brossaud et al. who highlighted the fact that astringency was highly correlated with the amount of avanol units accessible to thiolysis [6]. The ethyl-bridged avanol fraction was also rated signicantly higher in bitterness than both Adp3 and Adp9 suggesting that the insertion of an ethyl-bond between each of the catechin units contributed bitterness. In this model system with relatively high ethanol levels, as are encountered in red wine, relative differences in bitterness could possibly have been masked by the intrinsic bitterness of ethanol itself [25].

4. Conclusion Our results established that anthocyanins in their glucoside or coumaroylated forms did not inuence either astringency or bitterness of MW solutions. The pigmented tannin-like polyphenolic compounds isolated both from wine and from marc were rated very similarly to the anthocyanins. Ethyl-bridged avanols, provided they were present in sufcient quantity in wine, could contribute astringency to wine through their avanic composition as do the proan-

S. Vidal et al. / Analytica Chimica Acta 513 (2004) 5765

65

thocyanidins and additionally could increase bitterness. This study, taken as a whole, suggests that the pigmented tannin-like polyphenolic compounds that are formed during wine-making and upon storage from avanols may lead to a decrease of astringency in red wine, provided they are present in sufcient levels. This could occur rstly through the transformation of these astringent materials into the pigmented tannin-like polyphenolic compounds and secondly, by these rst reactions competing with reactions leading to the formation of astringent tannin-like polyphenolic compounds such as the ethyl-bridged avanols. Research efforts are needed for the structural characterisation of those compounds that are formed during vinication and ageing.

Acknowledgements The judges involved in the sensory panel are gratefully thanked for their great contribution to this work. The authors wish to thank Peter Hj for his encouragement and critical reading of the manuscript. We also thank George Skouroumounis and Holger Gockowiack for their contribution to the development of the MLCCC method and comments on the manuscript. Markus Herderich, Yoji Hayasaka, and Patrik Jones are also thanked for their comments on the manuscript. This work was supported by Australias grape-growers and wine-makers through their investment body the Grape and Wine Research and Development Corporation, with matching funds from the Federal government, and by the Commonwealth Co-operative Research Centre Program. The work was conducted by the Australian Wine Research Institute and Unit Mixte de Recherche Sciences pour lOenologie, INRA, and forms part of the research program of the Co-operative Research Centre for Viticulture in Australia. The European Union (Maxfun QLK1-CT-2002-02364) is also thanked for funding part of this work. References
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