Analytica Chimica Acta 563 (2006) 2632

A rst approach towards the relationship between grape skin cell-wall composition and anthocyanin extractability
A. Ortega-Regules, I. Romero-Cascales, J.M. Ros-Garc a, J.M. L opez-Roca, E. G omez-Plaza
Departamento de Tecnolog a de Alimentos, Nutrici on y Bromatolog a, Facultad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain Received 11 August 2005; received in revised form 8 November 2005; accepted 9 December 2005 Available online 17 January 2006

Abstract The composition of skin cell-walls from four different grape varieties (Vitis vinifera L., cv. Cabernet Sauvignon, Merlot, Syrah and Monastrell, the later harvested in three different locations) has been studied, trying to correlate those differences found in cell-wall composition among varieties with their degree of ripeness and with the easiness of anthocyanin extractability. The results of an anthocyanin extractability assay showed that the anthocyanins from Monastrell grape skins might be more difcult to extract than those from the other varieties. The study of the cell-wall composition showed some differences in uronic acids, cellulosic glucose, proteins and polyphenol content of cell-walls among varieties, and also differences between the Monastrell grapes cultivated in different locations were detected. Little differences could be found among the different samples regarding the non-cellulosic neutral sugars. In trying to look if differences in the extractability index were related to cell-wall composition and to help to clarify some of the mechanism involved in the anthocyanin extractability, a multiple regression analysis was conducted. The results showed that a model could be built up explaining a high percentage of the variability in the extractability index, using the content of the different components of the skin cell-wall as independent variables. Following this approach, the differences on the easiness of anthocyanin extractability in grape samples could be based in differences in pectin and cellulose content, but also, differences occurring on arabinoxylan, arabinogalactan and xyloglucan could be of importance. 2005 Elsevier B.V. All rights reserved.
Keywords: Wine; Anthocyanin extractability; Cell-wall composition

1. Introduction Anthocyanins are the compounds responsible for wine color. They are located in the skin. Inside the cells, they are located in the vacuoles, in a free, non-complex form [14] and they diffuse into the must and wine during the maceration step in winemaking. Evidently, the barrier for these compounds is the skin cell-wall. These cell-walls are highly complex and dynamic, composed of polysaccharides, phenolic compounds and proteins, and stabilized by ionic and covalent linkages. Hemicellulose, pectins and structural proteins are inter-knotted with the network of cellulose microbrils, the skeleton of cell-walls [5]. In grape cell-walls, cellulose and pectins accounts 3040% of the polysaccharide components of the cell-wall [6]. Extraction of anthocyanins during the maceration process requires that the pectin rich middle lamella to be degraded to

Corresponding author. Tel.: +34 96836 7323; fax: +34 96836 4147. E-mail address: encarnag@um.es (E. G omez-Plaza).

release the cells, and the cell-walls to be broken to allow their vacuole contents to be extracted, or to diffuse into the wine [4]. The extraction of anthocyanins from the grape into the wine is, therefore, essentially a diffusion process, and the rate and extent of extraction is inuenced by the grape anthocyanin concentration, the composition of berry cell-walls and processing methods. However, experience has shown that highly colored grapes do not necessarily produce high colored wines, being any difference probably related with the easiness of anthocyanins to be extracted from grape skins into musts [4]. The structural properties of the cell-walls of the different varieties may determine the mechanical resistance, the texture and the ease of processing berries [7]. The loosening of cellwalls is realized by the breakdown of chemical bounds among the structural components [5], therefore, changes in the cell-wall polysaccharide structure could affect the solubility and the tissue disassembling mechanisms [8]. In this study, the composition of skin cell-walls from four different grape varieties (Vitis vinifera L., cv. Cabernet Sauvignon, Merlot, Syrah and Monastrell, the later harvested in three

0003-2670/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.12.024

A. Ortega-Regules et al. / Analytica Chimica Acta 563 (2006) 2632

27

different locations) has been studied, trying to correlate those differences found in cell-wall composition among varieties with their degree of ripeness and with the easiness of anthocyanin extractability, calculated with an extractability assay developed by Saint-Cricq de Gaulejac et al. [9]. 2. Experimental 2.1. Grape samples Grapes from Vitis vinifera L., cv. Cabernet Sauvignon, Merlot and Syrah were harvested in September 2003 from a commercial vineyard located in Jumilla (S.E. Spain). Vitis vinifera L., cv. Monastrell grapes were harvested in three different locations: Monastrell-A and Monastrell-C were harvested in two different commercial vineyards in Jumilla and Monastrell-B in a commercial vineyard in Bullas (Murcia, Spain). Berries samples (ca. 300 g) were immediately take to the laboratory for the analyses. The number of berries and the total weight of each sample were recorded prior to splitting into two sub-samples. One sub-sample was used for the physico-chemical determination and the grapes from the other sub-sample were peeled with the help of a scalpel, and the skins were stored at 20 C until analysis. All analyses were done in triplicate. 2.2. Physico-chemical determinations in grapes Total soluble solids ( Brix) were measured using an Abb etype refractometer. The anthocyanin extractability of grapes was calculated according to the method described by Saint-Cricq de Gaulejac et al. [9]. Grapes were slightly grinded (Gt 550, Robot Coupe, France) and two fractions of 25 g were macerated during 4 h in 25 ml of two solutions at two different pH values (3.6 and 1). The original pH 3.2 solution was changed for one of pH 3.6, which is more suited to the musts from our region. After 4 h, the macerated samples were centrifuged. The anthocyanin contents of the two supernatant (ApH1 and ApH3.6) were then chemically assayed by measuring the absorbance at 520 nm of the supernatants after a 1/20 dilution with 0.1N HCl. The extractability index was calculated as follows: Extractability index = ApH1 ApH3.6 100 ApH1

HEPES, pH 7. The HEPES-insoluble residues were treated for 60 min at 20 C with buffered phenol 5 M in TrisHCl, pH 7.5 (25 ml). The suspensions were centrifuged (16,500 g, 20 min) and the pellet extracted again with 25 ml of buffered phenol. The insoluble residues were washed twice with 80% ethanol and then with acetone to remove phenol, and stirred for 30 min in methanol/chloroform (1:1, v/v). The methanol/chloroforminsoluble material is the cell-wall material and was washed with absolute ethanol, acetone and di-ethyl ether and nally dried at 37 C overnight. 2.4. Analytical methods The neutral sugars composition of the cell-wall material was determined by gasliquid chromatography (GLC) after pretreatment (30 C, 1 h) with aq. 72% sulfuric acid followed by hydrolysis with 1 M sulfuric acid (100 C, 3 h) and conversion of the products into alditol acetates [11]. GLC was carried-out using a SP2380 column from Supelco (Bellefonte, PA, USA). Helium was used as the carrier at a constant ow of 2.0 ml/min. The injector temperature was 260 C. The oven temperature program was as follow: 200 C for 22 min and up-to 235 C at a rate of 5 C/min, then 11 min at 235 C. Detection was carried-out by FID. Pure sugars from Sigma (St. Louis, MO, USA) were used as standards. A different hydrolysis using only 1 M sulfuric acid (100 C, 3 h) was used to determine non-cellulosic glucose. Uronic acids were determined in the sulfuric acid hydrolysate by the colorimetric 3,5-dimethylphenol assay [12]. Pure galacturonic acid from Sigma was used as standard. The methanol and acetic acid content of the cell-wall was determined by HPLC [13] using an ORH-801 column with an ORH-801 guardcolumn, both from Interaction (San Jose, CA, USA). A 5 mM sulfuric acid was used as the eluent at a constant ow of 0.8 ml/min and 30 C. Detection was carried-out by RID. Pure methanol and glacial acetic acid from Panreac (Barcelona, Spain) were used as standards. The proteins and total polyphenols content of the cell-wall material were determined after extraction (100 C, 10 min) with 1 M NaOH by the colorimetric Coomassie Brilliant Blue assay [14] and by the colorimetric Folin reagent assay [15], respectively. Bovine albumin fraction V from J.T. Baker (Deventer, Holland) and pure gallic acid from Sigma were used as standards, respectively. 2.5. Statistical data treatment

2.3. Isolation of cell-walls The cell-wall material was isolated by the procedure described by Vidal et al. [10] from the skin of the grapes. As stated by Barnavon et al. [7], cell-wall preparations were not destarched because, although starch is present in a very low levels in green grape berries, tends to disappear during development. All steps were performed at 4 C to minimize fragmentation of wall polysaccharides by endogenous enzymes. Skin tissues (10 g) were suspended in 20 ml of 40 mM HEPES, pH 7 and homogenized. The suspensions were centrifuged (10,000 g, 15 min) and the insoluble residue washed twice with 40 mM

Analysis of variance, multiple regression analysis and discriminate analysis have been performed with the statistical package Statgraphics 3.1. 3. Results and discussion 3.1. Characteristics of the grapes Monastrell grapes always reached the highest berry weight (Table 1). Monastrell B was the variety that reached the highest

28 Table 1 Physico-chemical characteristics of the grapes Berry weighta Monastrell A Monastrell B Monastrell C Cabernet Sauvignon Syrah Merlot 127.2 c 167.6 d 122.9 c 67.9 a 84.2 b 67.9 a

A. Ortega-Regules et al. / Analytica Chimica Acta 563 (2006) 2632

Brix

mg skin/berry 140.3 b 228.1 d 167.5 c 106.5 a 104.7 a 99.2 a

mg pulp/berry 978.7 d 1277.9 e 870.6 cd 238.1 a 600.8 b 376.8 a

%Skin 10.8 a 13.2 bc 13.6 c 16.5 d 11.9 ab 14.0 c

%Pulp 72.3 cd 73.8 d 70.8 cd 36.7 a 68.1 c 53.3 b

EIb 62 c 52 b 53 b 32 a 32 a 31 a

23.3 a 24.9 c 24.0 b 25.6 d 26.2 e 26.2 e

Different letters (ae) within the same column represents signicant differences according to an LSD test (p < 0.05). a Berry weight expressed as the weight (g) of a hundred of berries. b Extractability index according to Saint-Cricq de Gaulejac et al. [9].

value, two and a half times larger than Cabernet Sauvignon and Merlot grapes. Syrah grapes reached intermediate values. Some differences were found in Brix at the moment of harvest. The lowest value was found in Monastrell A grapes, being Syrah and Merlot grapes the more mature grapes. The percentage of pulp and skin was very similar for Monastrell grapes in the different locations (7177% of pulp and 1113% of skin). Cabernet Sauvignon presented the lowest proportion of pulp and higher proportion of skin. Phenolic maturity is reached when the concentration of grape anthocyanins is maximal. But in addition to phenolic maturity, phenolic extractability is also an important issue. Grapes that are high in phenols do not necessarily produce wines that are also rich in phenolic compounds. A method for evaluating this extractability, the phenolic extractability assay, was proposed by Saint-Cricq de Gaulejac et al. [9]. It consists in macerating the grapes for 4 h at two different pH values (3.6 and 1) and is based on the assumption that at pH 1 there is a complete disorganization of the vacuolar membrane, which facilitates the liberation of phenolic compounds. When the pH of the macerating solution is 3.6, the natural degradation of the cells is respected, a situation that is similar to that occurring during maceration in winemaking [16]. Extractability is considered good when the difference between these two results is small and, therefore, the extractability index low. Values of extractability index as shown in Table 1 would indicate, according to Saint-Cricq de Gaulejac et al. [9], that the anthocyanins from Monastrell skins may be more difcult to extract than those from the other varieties. Two facts could be the responsible for the higher extractability index of Monastrell grapes: a less mature grapes (at the moment of harvest Monastrell grapes presented lower Brix than Merlot, Cabernet Sauvignon and Syrah, as shown in Table 1) and/or the fact that Monastrell grapes are genetically characterized by a more rigid cell-wall structure which difcult anthocyanin extraction. Although some authors maintain that the values of the extractability index decrease as ripening progresses [17,18], we found in previous studies [19] that Monastrell grapes always presented a high extractability index and, during the course of one month, the sugar content changed from 24.4 to 26.2 Brix, with no signicant changes in the extractability index. It is the purpose of this research to try to correlate the differences in EI with the chemical composition of skin cell-walls.

3.2. Isolation of skin cell-wall material Table 2 shows the amount of cell-wall material isolated from the different grape varieties. Merlot and Cabernet Sauvignon presented lower amount of cell-wall material in the skins than Syrah and Monastrell grapes. Monastrell C presented the largest quantities. The quantities of cell-wall material isolated from our skin samples are similar to those reported by Femenia et al. [20]. We cannot be sure if differences in the amount of cellwall material extracted from skins could inuence the properties of the cell-walls regarding the extractability, since Rosli and Civello [21] found that no clear correlation has been established between fruit rmness and cell-wall material content. 3.3. Composition of the isolated cell-wall material The content of polyphenolic compounds in skin cell-wall material is high (Table 3). Nunan et al. [23] also revealed a high content of phenolics in skin cell-walls. The highest quantities
Table 2 Skin cell-wall material (mg g1 of skin) Variety Monastrell A Monastrell B Monastrell C Cabernet Sauvignon Syrah Merlot Skin cell-wall material 55.3 bc 53.1 b 60.4 c 40.2 a 50.8 b 42.5 a

Different letters (ac) within the same column represents signicant differences according to an LSD test (p < 0.05). Table 3 Protein and total polyphenols content of the cell-walls (expressed in mg g1 of cell-wall) Variety Monastrell A Monastrell B Monastrell C Cabernet Sauvignon Syrah Merlot Proteins 102.2 c 81.9 a 85.0 ab 99.2 c 100.6 c 88.7 abc Polyphenols 87.2 ab 75.1 a 82.6 b 73.3 a 106.6 c 94.2 c

Different letters (ac) within the same column represents signicant differences according to an LSD test (p < 0.05).

A. Ortega-Regules et al. / Analytica Chimica Acta 563 (2006) 2632 Table 4 Sugar composition of cell-walls (mg g1 of cell-wall) Variety Monastrell A Monastrell B Monastrell C Cabernet Sauvignon Syrah Merlot Rha 19.2 a 18.0 a 18.5 a 18.5 a 15.3 a 19.8 a Fuc 4.6 a 4.4 a 4.3 a 3.9 a 3.6 a 4.7 a Ara 59.5 a 61.0 a 59.4 a 57.1 a 50.1 a 66.2 a Xyl 24.7 b 26.8 b 26.0 b 23.8 ab 17.5 a 25.4 b Man 9.7 bc 9.9 c 11.3 d 8.5 b 6.7 a 8.5 b Gal 27.9 bc 29.0 c 26.7 b 21.1 a 20.3 a 21.7 a Glu 30.6 bc 39.4 c 26.5 ab 21.0 a 40.3 c 40.5 c Cell-Glua 128.5 ab 171.7 d 150.8 c 112.0 a 143.9 bc 143.0 bc UAb 160.6 b 194.9 c 172.3 bc 177.3 bc 133.8 a 162.7 b Acetic acid 7.1 ab 7.9 b 7.1 ab 8.1 b 6.5 a 7.6 b Methanol 19.0 b 19.3 b 20.1 b 16.1 a 15.5 a 14.3 a DMc 65.9 b 54.5 ab 64.1 b 49.7 a 63.5 b 49.1 a DAd

29

13.0 ab 11.9 a 12.0 a 13.4 ab 14.3 b 14.0 b

Different letters (ad) within the same column represent signicant differences according to an LSD test (p < 0.05). a Cellulosic glucose. b Uronic acids. c Degree of methylation. d Degree of acetylation.

were found in Merlot and Syrah. The lowest values were found in Monastrell B and Cabernet Sauvignon grapes. A large part of the detected polyphenolic compounds could be tannins since tannins integrated in the skin cell-walls have been previously detected [22]. As regard to protein content, the highest values were found for Monastrell A, Syrah and Cabernet Sauvignon (Table 3). These proteins are likely to be structural components that reinforce the wall during berry expansion (extensin), since they are believed to form a brilar network [23]. The sugar composition of the skin cell-walls of the different grape varieties indicates no great differences between varieties, as shown in Table 4. However, it is known that gross changes in wall composition may not always be detected; molecular mass, degree of substitution of an individual wall polysaccharide and therefore, the cell-wall properties may be altered without any large change in the total amount of that polysaccharide [23]. Galacturonic acid and glucose (cellulosic glucose plus hemicellulosic glucose) were the main components, being the amount of cellulosic glucose of the same order to that of galacturonic acid. Nunan et al. [6] stated that uronic acids and cellulosic glucose are major grape cell-wall components. Arabinose, noncellulosic glucose, galactose, xylose and rhamnose were present in reasonable amounts, while mannose and fucose were present as minor components. The type and amount of the carbohydrates found as main components of the skin cell-wall indicate the presence of pectic polysaccharides, hemicellulloses and cellulose. The presence of pectic polysaccharides could be inferred from the large amounts of uronic acids, arabinose, galactose and rhamnose (those exists primarily as side chains on the rhamnogalacturan backbone). The cellulose was inferred from the fact that the bulk of glucose could be release only after a Seaman hydrolysis. The presence of xylose, fucose and non-cellulosic glucose was indicative of the occurrence of hemicellulosic polysaccharides. These polysaccharides has been described in grapes by Nunan et al. [6], who deduced from the carbohydrate composition and the linkage composition the presence of galacturonan, arabinan, galactan (AG I), arabinogalactan II, xyloglucan, mannan, glucuronoarabinoxylan and cellulose. The general prole found in our results on sugar composition of the skin cell-walls of Monastrell, Cabernet Sauvignon, Syrah and Merlot grapes is

in agreement with the results on sugar composition of skin cellwall material isolated from Chardonnay and Carignan grapes [24] and Grenache blanc grapes [10]. Differences between Monastrell and the other varieties could only clearly be seen in manose and galactose, the latter being higher in Monastrell grapes. The amount of methanol and acetic acid found in skin cellwalls (Table 4) ranged from 14 to 20 mg of methanol and 6 to 8 mg of acetic acid per gram of cell-wall. Lecas and Brillouet [24] reported a slightly higher amount of methanol (2224 mg g1 of skin cell-wall) and acetic acid (1011 mg g1 of skin cell-wall) in Chardonnay and Carignan grapes. No differences in the quantities of acetic acid among Monastrell and the other varieties but higher quantities of methanol. 3.4. Multiple regression analysis Due to the large number of parameters that can characterize the skin cell-wall, each analytical variable may individually give little information on its importance on the skin cell-wall characteristics so the use of multivariate statistical analysis is necessary. The interest in such models is that they may help to interpret the roles played by different variables in the cellwall characteristics of grape skins. Also, it can be determined whether they provide information needed to differentiate among them. Trying to correlate both the ripeness of grapes and the extractability index with the composition of skin cell-walls, two different multiple regression analyses were carried out. The multiple regression analysis for ripeness ( Brix) showed that the obtained equation was not statistically signicant (data not shown), whereas the multiple regression analysis for EI (Fig. 1) gave a statistically signicant equation with an R-squared of 78.35% (p < 0.05): EI = 34.5917 0.592554 [Arabinose] + 1.02988 [Fucose] + 4.11352 [Galactose] + 0.0663419 [Cellulosic glucose] 0.493977 [Hemicellulosic glucose] 2.41539 [Manose] + 0.19204 [total phenols] 0.0126344 [protein]

30

A. Ortega-Regules et al. / Analytica Chimica Acta 563 (2006) 2632 Table 5 Discriminant function standardized coefcients for varieties Function 1 Arabinose Galactose Hemicellulosic glucose Mannose Xylose 3.29 1.17 1.52 1.01 3.62 Function 2 4.39 0.62 0.90 1.41 5.23 Function 3 0.85 0.50 0.62 0.52 1.44

Table 6 Discriminant function standardized coefcients for Brix Function 1 Fig. 1. Multiple regression analysis for extractability index (EI). Arabinose 29.60 Galactose 6.87 Cellulosic glucose 3.85 Hemicellulosic glucose 4.29 Mannose 7.67 Rhamnose 13.09 Uronic acids 4.17 Xylose 18.28 Function 2 4.51 0.32 1.69 1.52 1.60 0.75 0.80 5.64 Function 3 1.53 0.47 0.33 0.10 2.35 0.59 0.50 3.51 Function 4 3.56 0.58 1.14 0.35 0.24 1.06 0.20 2.28

+ 0.821038 [Rhamnose] 0.112376 [Uronic acids] + 0.64127 [Xylose] + 0.109665 [degree of acetylation] + 0.147877 [degree of methylation]

Table 7 Discriminant function standardized coefcients for extractability index

From the relative magnitude and sign of the coefcients in the above equation, it can be determined how each independent variable inuences the magnitude of EI. According to the equation, a low EI (high extractability) would be found with low concentrations of galactose, total phenols, rhamnose and xylose accompanied with low degree of acetylation and methylation of the pectins. The loss of galactose during ripening is an event reported for several fruits, however, the occurrence and the degree of loss of other pectic sugars such as arabinose and rhamnose seem to be intrinsic aspect of the species considered [25]. A low content of rhamnose is usually associated to the acidic pectins containing small hairy domains of branched rhamnogalacturonan, interspaced by long stretches of polygalacturans [7]. A low content of rhamnose is therefore consistent with the solubilization of rhamnose-type fragments and/or with the turnover of rhamnose from pectic side chains. A low content of xylose could be related with partial breakdown of cellulose-xyloglucan network decreasing the integrity of cell-wall and this is usually associated with signicant changes in fruit texture [26]. Also, for a small extractability index, low total phenol content should be required. The wall bound peroxidase is responsible for the irreversible formation of phenolics-cross-linkage between structural polysaccharides and proteins, so high phenolic might lead to the loss of wall extensibility and the increase of wall strength. A low value for the degree of methylation and acetylation is usually associated with mature grapes, which is consistent with the increasing pectin de-esterication carried out by natural esterases when ripening is in progress [27,28]. Step backward discriminant analysis was also used to check if some measured skin cell-wall components, selected by the statistical analysis treatment, could distinguish among pre-established groups (varieties, degree of ripening or anthocyanin extractability). Tables 57 show the coefcients of the functions used to dis-

Function 1 Arabinose Galactose Cellulosic glucose Hemicellulosic glucose Degree of methylation Mannose 1.37 0.80 2.32 0.40 2.37 0.94

Function 2 0.86 1.19 1.71 1.56 0.70 1.43

criminate amongst the different varieties, Brix and extractability index. From the relative magnitude of the coefcients in these equations, it can be determined how the independent variables are being used to discriminate amongst the groups. Regarding to varieties, three discriminate functions were established (Table 5) and these function allows a 100% of correct classication of the samples (Fig. 2). The classication could be obtained with ve variables, with galactose, xylose and arabinose being the variables with the highest discriminate power when separating Monastrell grapes from the other varieties. Regarding the degree of ripening, samples were previously grouped, those samples with statistically different Brix (as obtained in the multiple range test of the ANOVA analysis shown

Fig. 2. Distribution of the grape samples in the coordinate system dened by the discriminant function used to differentiate among varieties.

A. Ortega-Regules et al. / Analytica Chimica Acta 563 (2006) 2632

31

Table 8 Protein, total phenols and sugar composition of cell-walls of the test sample (mg g1 of cell-wall) Variety Protein PFT Rha Fuc Ara Xyl Man Gal Glu Cell-Glua UAb DMc DAd
a b c d

Fig. 3. Distribution of the grape samples in the coordinate system dened by the discriminant function used to differentiate among groups with statistically different Brix (Mon: Monastrell, Sy: Syrah, M: Merlot, CS: Cabernet Sauvignon).

in Table 1) being assigned to different groups (ve groups) and 100% of correct classication was obtained (Fig. 3). The variables used to discriminate were galactose, cellulosic glucose, hemicellulosic glucose, arabinose, rhamnose, manose, xylose and uronic acids, being arabinose, galactose and rhamnose the variable with the highest discriminate power (Table 6). Those samples with the highest content of xylose, rhamnose and galactose will be classied in the group of grapes with the lower degree of ripening. When we wanted to separate samples regarding the extractability index, samples were grouped as previously described for Brix and three different groups were obtained. Two discriminant functions were obtained (Table 7; Fig. 4). Six parameters were chosen as variables in the discriminant functions: arabinose, galactose, cellulosic glucose, hemicellulosic glucose, manose and degree of methylation. The classication of the samples reached 100%. Those samples with the highest extractability index are located in the positive part of function 1 and therefore characterized by high values of cellulosic glucose, galactose and a high degree of methylation of the pectins. To test the strength of the discriminant model, we used them to classify a Monastrell sample from a very low quality vineyard. This sample presented low Brix (22 Brix), very low anthocyanin content but also low EI (38), closer to that found in the Cabernet Sauvignon, Syrah and Merlot samples than in the Monastrell grapes. The protein, polyphenols and sugar composition of the cell-walls of this test sample are shown in Table 8. This Monastrell sample was not correctly classied as regard variety (it was classied as being a sample of Syrah grapes). It

Monastrell (test sample) 92.9 80.3 20.7 4.7 64.5 25.8 10.1 27.1 38.5 141.8 153.6 61.6 13.7

Cellulosic glucose. Uronic acids. Degree of methylation. Degree of acetylation.

was also incorrectly classied regarding the degree of ripeness, since it was classied as having a high Brix (it was classied in the same group of Cabernet Sauvignon grapes) but it was correctly classied with regards the extractability index (it was classied in the same group of Syrah, Cabernet Sauvignon and Merlot). From these results, it seems that skin cell-wall composition might be highly related to the extractability index and therefore the easiness of anthocyanin extraction from skin to must. 4. Conclusions The results showed that a model could be built up explaining 78% of the variability in the extractability index, using the content of the different components of the skin cell-wall as independent variables. Also, using these variables it is possible to classify the grape samples regarding their extractability index. Following this approach, the differences on polysaccharides based on galactose and arabinose, together with the cellulose content and the degree of methylation of the pectins could be responsible for the different anthocyanin extractability, keeping in mind that differences on the thickness or density of the skin cell-wall could also inuence, since Monastrell grapes, with the highest extractability index, also showed the highest weight of cell-wall material. More research is needed to deeply characterize the grape skin cell-wall since this knowledge will help to understand the mechanisms occurring during red wine maceration, especially those that could facilitate the anthocyanin extraction from grapes to wine. Acknowledgements

Fig. 4. Distribution of the grape samples in the coordinate system dened by the discriminant function used to differentiate among groups with statistically different extractability index (Mon: Monastrell, Sy: Syrah, M: Merlot, CS: Cabernet Sauvignon).

This work was made possible by nancial assistance of the Ministerio de Ciencia y Tecnolog a, Project AGL2003-01957. Author A. Ortega-Regules is the holder of a fellowship from the Government of Mexico (CONACYT).

32

A. Ortega-Regules et al. / Analytica Chimica Acta 563 (2006) 2632 [14] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [15] V.L. Singleton, J.A. Rossi, Am. J. Enol. Viticult. 16 (1965) 144. [16] Y. Glories, C. Saucier, in: J. Rautz (Ed.), The ASEV 50th Anniversary Annual Meeting, ASEV, Davis, CA, 2000, p. 353. [17] Y. Glories, Vignevini 3 (1999) 46. [18] G. Gonzalez-Neves, D. Charamelo, J. Balado, L. Barreiro, R. Bochicchio, G. Gato, G. Gil, A. Tessore, A. Carboneau, M. Moutounet, Anal. Chim. Acta 513 (2004) 191. [19] A.B. Bautista-Ort n, J.M. L opez-Roca, J.I. Fernandez-Fernandez, E. G omez-Plaza, in: L. Dufosse (Ed.), Pigments in Food. More Than Colors, Universit e de Bretagne Occidentale, Quimper, France, 2004, p. 300. [20] A. Femenia, E.S. Sanchez, S. Simal, C. Rosello, J. Agric. Food Chem. 46 (1998) 271. [21] H.M. Rosli, P.M. Civello, Plant Physiol. Biochem. 42 (2004) 823. [22] K. Amrani Joutei, Y. Glories, M. Mercier, Vitis 33 (1994) 133. [23] K.J. Nunan, I.M. Sims, A. Bacic, S.P. Robinson, Plant Physiol. 118 (1998) 783. [24] M. Lecas, J.M. Brillouet, Phytochemistry 35 (1994) 1241. [25] G.D. Manrique, F.M. Lajolo, Postharvest Biol. Tec. 33 (2004) 11. [26] K. Wakabayashi, J. Plant Res. 113 (2000) 231. [27] K.J. Nunan, C. Davies, S.P. Robinson, G.B. Fincher, Planta 214 (2001) 257. [28] L. Barnavon, T. Doco, N. Terrier, A. Ageorges, C. Romieu, P. Pellerin, Phytochemistry 58 (2001) 693.

References
[1] G. Hrazdina, A. Moskowitz, in: D.A. Webb (Ed.), Grape and Wine Centennial Symposium, Universidad de California, Davis, CA, 1980, p. 245. [2] A. Ros Barcelo, A.A. Calderon, J.M. Zapata, R. Munoz, Sci. Hort. 57 (1994) 265. [3] M. Moutounet, J. Rigaud, J.M. Souquet, V. Cheynier, Bull. O. I. V. (1996) 433. [4] K. Amrani Joutei, Y. Glories, Rev. Franc. Oenol. 153 (1995) 28. [5] X.M. Huang, H.B. Huang, Aust. J. Grape Wine Res. 7 (2001) 132. [6] K.J. Nunan, C.A. Sims, A. Bacic, S.P. Robinson, G.B. Fincher, Planta 203 (1997) 93. [7] L. Barnavon, T. Doco, N. Terrier, A. Ageorges, C. Romieu, P. Pellerin, Plant Physiol. Biochem. 38 (2000) 289. [8] T.M. Shiga, F.M. Lajolo, T.M. Filisetti, Food Chem. 84 (2004) 53. [9] N. Saint-Cricq de Gaulejac, N. Vivas, Y. Glories, Rev. Franc. Oenol. 173 (1998) 22. [10] S. Vidal, P. Williams, M.A. ONeill, P. Pellerin, Carbohydr. Polym. 45 (2001) 315. [11] A.B. Blakeney, P.J. Harris, R.J. Henry, B.A. Stone, Carbohydr. Res. 113 (1983) 291. [12] R.W. Scott, Anal. Chem. 51 (1979) 936. [13] A.G.J. Voragen, Food Hydrocolloid 1 (1986) 65.