Food Chemistry 179 (2015) 311–317

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Oligosaccharides of Cabernet Sauvignon, Syrah and Monastrell red wines Rafael Apolinar-Valiente a,⇑, Inmaculada Romero-Cascales a, Pascale Williams b, Encarna Gómez-Plaza a, José María López-Roca a, José María Ros-García a, Thierry Doco b a b

Departamento de Tecnología de Alimentos, Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain INRA, Joint Research Unit 1083, Sciences for Enology, 2 Place Pierre Viala, F-34060 Montpellier, France

a r t i c l e

i n f o

Article history: Received 14 October 2014 Received in revised form 20 January 2015 Accepted 31 January 2015 Available online 7 February 2015 Keywords: Wine Oligosaccharides Cabernet Sauvignon Syrah Monastrell SEC-MALLS

a b s t r a c t Wine oligosaccharides were recently characterized and their concentrations, their composition and their roles on different wines remain to be determined. The concentration and composition of oligosaccharides in Cabernet Sauvignon, Syrah and Monastrell wines was studied. Oligosaccharide fractions were isolated by high resolution size-exclusion chromatography. The neutral and acidic sugar composition was determined by gas chromatography. The MS spectra of the oligosaccharides were performed on an AccuTOF mass spectrometer. Molar-mass distributions were determined by coupling size exclusion chromatography with a multi-angle light scattering device (MALLS) and a differential refractive index detector. Results showed significant differences in the oligosaccharidic fraction from Cabernet Sauvignon, Syrah and Monastrell wines. This study shows the influence that the grape variety seems have on the quantity, composition and structure of oligosaccharides in the finished wine. To our knowledge, this is the first report to research the oligosaccharides composition of Cabernet Sauvignon, Syrah and Monastrell wines. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Due to their importance for the properties of wines, polyphenols, proteins, and polysaccharides have been widely studied. However, the identification, quantification and composition of oligosaccharides in wine have only recently been the object of study (Bordiga et al., 2012; Ducasse, Williams, Meudec, Cheynier, & Doco, 2010; Ducasse et al., 2011). Oligosaccharides contain between three and fifteen monosaccharide residues covalently linked through glycosidic bonds. Important medicinal, food and agricultural applications are associated with these molecules (Gibson & Roberfroid, 1995; Qiang, YongLie, & QianBing, 2009). Oligosaccharides are related to plant defense responses (Darvill & Albersheim, 1984). In addition, these molecules are well established as dietary antioxidants (Otaka, 2006) and are thought to provide various health benefits (Lama-Muñoz, Rodríguez-Gutiérrez, Rubio-Senent, & Fernández-Bolaños, 2012). In wine, oligosaccharides were identified for a long time as sucrose and various diholosides (Doco, Williams, Vidal, & Pellerin, 1997), and they are found too as short chain galacturonic acid (2–6 DP). These molecules are originated in the degradation of the smooth ⇑ Corresponding author. Tel.: +34 868 887662; fax: +34 868 884147. E-mail addresses: [email protected] (R. Apolinar-Valiente), [email protected] (I. Romero-Cascales), [email protected] (P. Williams), [email protected] (E. Gómez-Plaza), [email protected] (J.M. López-Roca), [email protected] (J.M. Ros-García), [email protected] (T. Doco). http://dx.doi.org/10.1016/j.foodchem.2015.01.139 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

region of pectin (Pellerin & Cabanis, 1998). As regards wine quality, some oligosaccharides have physicochemical properties such as an ability to chelate cations (Cescutti & Rizzo, 2001). Besides, QuijadaMorín, Williams, Rivas-Gonzalo, Doco, and Escribano-Bailón (2014) reported than astringency perception is positively related to certain monosaccharides in the oligosaccharide fraction from Tempranillo wines. The first isolation and characterization of the oligosaccharide fractions in red wines was made by Ducasse et al. (2010). Recently, Bordiga et al. (2012) have isolated and characterized forty-five complex free oligosaccharides in red and white wines. Monastrell, also known internationally as the French name of ‘‘Mourvèdre’’, is the main wine grape cultivar in southeastern Spain. Cabernet Sauvignon and Syrah, two of the most widely used varieties in the world, are also used in this region to complement Monastrell wines. However, to our knowledge there is no study comparing the oligosaccharide content of the wines obtained with these three grape cultivars. The aim of the present work was to study the oligosaccharide fractions of Cabernet Sauvignon, Syrah and Monastrell wines. 2. Materials and methods 2.1. Grape materials The raw materials used for this study were berries from Vitis vinifera cvs. Cabernet Sauvignon, Syrah and Monastrell grown in

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Murcia (southern Spain). The grapes were carefully harvested at commercial maturity with a total of soluble solids content between 24 and 26°Brix during the 2007 vintage. 2.2. Preparation of Trials Three 90 kg lots of grapes from each variety (Cabernet Sauvignon, Syrah and Monastrell) were destemmed and crushed, and distributed into 100 L stainless steel tanks to yield triplicate control lots. At the same time, Potassium bisulfite (8 g/100 kg grape) was added. 2.3. Fermentation All fermentations were started by adding commercial dry yeast (Levuline Gala, OenoFrance, Bordeaux, France) at 10 g/hL and were carried out in 100-L stainless steel tanks equipped with temperature control (25 °C) which enabled the fermentation kinetics to be regulated. Each lot was fermented to completion, and when alcoholic fermentation was finished (as attested by sugar analysis), the musts were pressed at 150 kPa in a 75-L tank membrane press (Hidro 80L, Ausavil, Spain). Free-run juice and press wines of each trial were combined and stored in 50-L tanks. One month later the wines were racked. After spontaneous malolactic fermentation, the wines were racked again and supplied with 25 mg/L sulfur dioxide. The wines were cold stabilized ( 3 °C), bottled and stored in the experimental wine cellar at 18 °C until analysis. 2.4. Isolation of oligosaccharide fractions The oligosaccharide fractions were isolated as previously described (Ducasse et al., 2010, 2011). Cabernet Sauvignon, Syrah and Monastrell wines (5 mL) were partially depigmented by decolourization on a column of MN Polyamide SC6 (5  1 cm) previously equilibrated with 1 M NaCl. The wine oligosaccharides not retained on the polyamide column were eluted by 2 bed volumes of 1 M NaCl (Brillouet, Moutounet, & Escudier, 1989). High-resolution size exclusion chromatography was performed by loading 2 mL of the previously concentrated fraction on a Superdex 30-HR column (60  1.6 cm, Pharmacia, Sweden) with a precolumn (0.6  4 cm), equilibrated at 1 mL/min in 30 mM ammonium formiate pH 5.6. The elution of polysaccharides was followed with an Erma-ERC 7512 (Erma, Japan) refractive index detector combined with a Waters Baseline 810-software. One fraction was collected between 60 and 93 min. The isolated fraction was freeze-dried, re-dissolved in water, and freeze-dried again four times to completely remove the ammonium salt. The resulting fraction corresponded to the Cabernet Sauvignon, Syrah and Monastrell oligosaccharide fraction. 2.5. Sugar composition as trimethylsilyl derivatives The monosaccharide composition was determined after solvolysis with anhydrous MeOH containing 0.5 M HCl (80 °C, 16 h), by gas chromatography (GC) of their per-O-trimethylsilylated methyl glycoside derivatives (Doco, O’Neill, & Pellerin, 2001). The separation of the TMS derivatives was previously described (Apolinar-Valiente et al., 2014). 2.6. Glycosyl-linkage determination The glycosyl-linkages composition was determined by GC–MS of the partially methylated alditol acetates. One milligram of polysaccharides in 0.5 mL dimethylsulfoxide was methylated using methyl sulfinyl carbanion and methyl iodide (Hakomori, 1964). The methylated materials were then treated with 2 M TFA

(1.15 h at 120 °C). The released methylated monosaccharides were converted to their corresponding alditols by treatment with NaDH4 and then acetylated (Harris, Henri, Blakeney, & Stone, 1984). Partially methylated alditol acetates were analyzed by GC–EI–MS using a DB-1 capillary column (30 m  0.25 mm i.d., 0.25 lm film); temperature programming 135 °C for 10 min, then 1.2 °C/min to 180 °C, coupled to a HP5973 MSD (Vidal, Williams, O’Neill, & Pellerin, 2001). 2.7. ESI mass spectrometry Wine oligosaccharide samples (50 lg) in 1:1 MeOH–water (5 lL) were injected directly into an AccuTOF (AccuTOF™ JMS-T100 LC, Jeol, Japan) mass spectrometer equipped with an ESI source and a time-of-flight (TOF) mass analyzer in negative ion modes. The source voltage was set at 2000 V (negative ESI), the orifice voltage at 45 V (negative ESI), the desolvating chamber temperature at 250 °C and the orifice temperature at 80 °C, the mass ranging from 200 to 4000 Da. ESI-TOF spectra were obtained and extracted as ASCII files. 2.8. Determination of molar mass Molar-mass distributions were determined at 25 °C by coupling size exclusion chromatography with a multi-angle light scattering device (MALLS) and a differential refractive index detector. SEC elution was performed on OHPAK guard column followed by four serial Shodex Ohpak KB-803, KB-804, KB-805 and KB-806 columns (0.8  30 cm; Shodex Showa Denkko, Japan) at 1 mL/min flow rate in 0.1 M LiNO3 after filtration through 0.1 lm filter unit. The MALLS photometer, a DAWN-HELEOS from Wyatt Technology Inc. (Wyatt Technology Corporation, Santa Barbara, CA, USA), was equipped with a GA–AS laser (k = 658 nm). The concentration of each eluted oligosaccharide was determined using the differential refractive index detector (Optilab TrEX, Wyatt Technology Inc. USA). All collected data were analyzed using Astra V 6.0.6 software with the zimm plot (order 1) technique for molar-mass estimation and a dn/dc of 0.145 mL/g (Redgwell, Schmitt, Beaulieu, & Curti, 2005). 2.9. Statistical data treatment Average values, standard deviation and statistical significance were calculated and performed with the Statgraphics Plus 5.1. Package. 3. Results and discussion 3.1. Cabernet Sauvignon, Syrah and Monastrell wine oligosaccharide fractions: quantification and characterization The fraction eluted on Superdex 30-HR column contained a complex mixture of small sugars, was taken to represent the oligosaccharide fraction of the studied wines and were distributed similarly as described previously in Carignan, Merlot and Monastrell wines (Apolinar-Valiente et al., 2014; Ducasse et al., 2010). When the glycosyl residue composition of Cabernet Sauvignon, Syrah and Monastrell oligosaccharides was analyzed (Table 1), all the wine oligosaccharides included rhamnose, arabinose, galactose, xylose and galacturonic and glucuronic acids coming from the pecto-cellulosic cell walls of grape berries. Other sugars such as mannose and glucose were released from the yeast polysaccharides. The glycosyl residue composition (Table 1) showed most of the sugars known to form part of the composition of complex carbohydrates in wine (Ayestarán, Guadalupe, & León, 2004; Doco,

2.87 ± 0.06a 5.10 ± 0.09b 0.73 ± 0.06b 0.67 ± 0.07b Different letters within the same column represent significant differences according to a least significant difference test (P < 0.05). a Average of three measurements and standard deviation. b Rha, Rhamnose; Fuc, Fucose; Ara, Arabinose; Xyl, Xylose; Man, Mannose; Gal, Galactose; Glc, Glucose; Glc A, Glucuronic acid; 4-OMeGlc A, 4-O methyl Glucuronic acid.

1.96 ± 0.20c 1.53 ± 0.18b 7.5 ± 3.7a 3.7 ± 0.5aa 3.5 ± 3.0c 0.8 ± 0.0a 11.1 ± 4.8a 4.4 ± 0.7a 79.9 ± 16.9b 24.7 ± 1.6a 72.4 ± 16.9b 58.5 ± 5.3b 56.7 ± 12.0b 32.8 ± 1.4a 55.9 ± 11.5b 39.7 ± 1.9a 18.1 ± 4.3b 9.6 ± 0.0a 6.6 ± 2.2b 2.5 ± 0.2a 58.0 ± 8.7b 16.4 ± 0.7a

109.9 ± 13.2c 50.8 ± 3.5b

4.77 ± 0.90b 0.47 ± 0.07a 1.18 ± 0.19a 5.9 ± 1.7a 1.6 ± 0.8b 5.6 ± 1.4a 21.6 ± 5.5a 34.0 ± 6.0a 21.2 ± 2.4a 34.4 ± 5.0a 13.3 ± 2.4ab 24.9 ± 4.3a 2.0 ± 0.4a 9.9 ± 2.0a

Cabernet Sauvignon Syrah Monastrell

Gal Aa,b Glca,b Gala,b Mana,b Xyla,b Araa,b Fuca,b Rhaa,b Cultivar

Table 1 Glycosyl composition (mg/L) and characteristic ratios of Oligosaccharides from Cabernet Sauvignon, Syrah and Monastrell wines.

Glc Aa,b

Xylitola,b

4-OMeGlc Aa,b

Ara/Gala,b

Rha/Gal Aa,b

(Ara + Gal)/ Rhaa,b

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Williams, Pauly, O’Neill, & Pellerin, 2003; Ducasse et al., 2010; Vidal, Williams, Doco, Moutounet, & Pellerin, 2003; Waters, Pellerin, & Brillouet, 1994). The identification of these sugars confirms the presence of mannan-, arabinogalactan-, homogalacturonan- and rhamnogalacturonan-like structures in the wine oligosaccharides, as previously reported (Apolinar-Valiente et al., 2014; Ducasse et al., 2010). The presence of xylose, glucuronic and 4-O-Me glucuronic acid residues indicated that traces of hemicelluloses might have been solubilized from grape berry cell walls (Doco, Vuchot, Cheynier, & Moutounet, 2003; Ducasse et al., 2010) and recovered as oligosaccharide structures in wines. The Syrah wine showed significantly higher values for most of the detected sugars except glucuronic acid and 4-O-Me glucuronic acid. This data could result in differences as regards sensory properties: Quijada-Morín et al. (2014) observed that mannose and galactose amounts in the oligosaccharide fraction are positively related to astringency perception. The lowest arabinose and glucose amounts were detected in Cabernet Sauvignon wine and the lowest xylose and xylitol amount in Monastrell wine. The total oligosaccharide amount was calculated as the sum of individual monosaccharide amounts measured by GC. Oligosaccharide concentrations from the isolated fractions were higher in Syrah wine (480 mg/L) than in Cabernet Sauvignon (174 mg/L) and Monastrell (244 mg/L) wines. Therefore, obtained profiles of the studied wines (Fig. 1) are corroborated by chemical quantitative analysis. We previously presented that oligosaccharide concentration of Monastrell wines from different origin varied between 195 and 288 mg/L (Apolinar-Valiente et al., 2014), which is in agreement with our data concerning this grape variety. The highest oligosaccharide content in wines that we have found in literature was reported by Ducasse et al. (2010), who measured 332 mg/L of oligosaccharides in Carignan wine. However, our Cabernet Sauvignon and Monastrell values were more similar than those reported for Merlot (252 mg/L) (Ducasse et al., 2010), although they were higher than those reported for Grignolino and Chardonnay wines (127 and 102 mg/L, respectively) (Bordiga et al., 2012). Quijada-Morín et al. (2014) observed total oligosaccharides contents ranged from 75 to 325 mg/L in thirteen commercial wines elaborated with Tempranillo. To increase our knowledge of the oligosaccharide sugar structures, several characteristic ratios were calculated: Arabinose to Galactose (Ara/Gal), Rhamnose to Galacturonic acid (Rha/GalA), and Arabinose + Galactose to Rhamnose (Ara + Gal)/Rha (Table 1). The Arabinose/Galactose ratio is characteristic of the PRAGs-like structures (Doco, Williams, et al., 2003; Vidal et al., 2003). This ratio obtained for the oligosaccharide fractions show significant differences between Cabernet Sauvignon (1.18 ± 0.19), Monastrell (1.53 ± 0.18) and Syrah wines (1.96 ± 0.20). This behavior could indicate a higher release of arabinose or oligosaccharides rich in arabinose arising from the pectic framework in Syrah wines (Vidal et al., 2003). The observed data is in agreement with a previous work, which is where we reported values of Ara/Gal ratio for Monastrell wines between 0.77 and 1.72 (Apolinar-Valiente et al., 2014). On the other hand, Ducasse et al. (2010) found higher values of Ara/Gal ratio for Carignan wine (2.2) and for Merlot wine (2.8). The Rha/GalA ratio allows the relative richness of wine oligosaccharides in homogalacturonans versus rhamnogalacturonans like structures to be deduced (Arnous & Meyer, 2009). The detected value for this ratio in Cabernet Sauvignon was 0.47 ± 0.07, while in Syrah and Monastrell was close to 1 (0.73 ± 0.06 and 0.67 ± 0.07, respectively). The low value detected for the Rha/GalA ratio in Cabernet Sauvignon oligosaccharide fraction (0.47 ± 0.07) reflected a majority of homogalacturonan structures. The obtained values for this ratio are in agreement with these obtained by Ducasse et al. (2010) for Carignan (0.97) and Merlot (0.40) wines, whereas we previously found values for the Rha/GalA ratio

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Fig. 1. Evolution of major oligosaccharide families (relative mole percentage) isolated from Cabernet Sauvignon (CA), Syrah (SY) and Monastrell (MO) wines.

between 0.10 and 1.07 for Monastrell wines (Apolinar-Valiente et al., 2014). The ratio of (Ara + Gal) to Rhamnose was calculated to estimate the relative importance of the neutral side-chains to the rhamnogalacturonan backbone, since it is assumed that most of the arabinose and galactose are associated with pectin hairy regions. This ratio was considerably lower in Syrah (2.87 ± 0.06) oligosaccharides than in Cabernet Sauvignon (4.77 ± 0.90) and Monastrell (5.10 ± 0.09), which could indicate that Syrah oligosaccharides contain more structures from the hairy regions of pectins (rhamnogalacturonan-like structures carrying neutral lateral chains). Ducasse et al. (2010) found that the ratios of (Ara + Gal) to Rhamnose were at 2.8 and 3.1 for Carignan and Merlot oligosaccharides, respectively. In a previous work we obtained values for this ratio in Monastrell oligosaccharides between 3.4 and 7.5 (Apolinar-Valiente et al., 2014). Taken together, the results obtained could suggest the influence of grape variety on the concentration, composition and structure of the wine oligosaccharide. It is therefore essential to mention that differences in maturity stages of grapes according to the cultivar properties could modulate the release of polysaccharides and oligosaccharides during vinification process (Ducasse et al., 2010; Nunan, Sims, Bacic, Robinson, & Fincher, 1998; Vicens et al., 2009). 3.2. Glycosyl-linkage determination: structure of oligosaccharide fractions from Cabernet Sauvignon, Syrah and Monastrell wines Table 2 shows the results of analysis of the glycosidic linkages, which permit us to determinate the structure of oligosaccharide fractions from studied wines. Mannose linked in ?2, in ?3, in ?3,6 and in non-reducing terminal position correspond to those typically found in yeast mannoproteins (Saulnier, Mercereau, & Vezinhet, 1991; Waters et al., 1994). It has been also detected mannose linked in ?2,3, in ?6, in ?4,6 and in ?2,6. The oligosaccharides contained arabinose linked in ?5 and in ?3,5 characteristic of branched arabinans on the rhamnogalacturonan chain of the pectins (presence of 2- and 2,4-Rha) (Vidal et al., 2003), and terminal arabinose which may arise from both AGPs and arabinans. Values for arabinose linked in ?5 are higher in Monastrell wine oligosaccharides (6.6%) compared with Cabernet Sauvignon (3.7%) and Syrah (3.5%) wines. It has been also detected arabinose linked in ?2, in ?3 and in ?2,5, but in these cases only arabinose linked in ?2 showed differences between

Syrah wine (0.1%) and Cabernet Sauvignon and Monastrell wines (1.1%). It also contained all methyl ethers corresponding to the galactose linked in ?3; ?4; ?6; ?3,4; ?3,6; ?4,6 and ?3,4,6, linkages that are found in AGPs (Pellerin, Vidal, Williams, & Brillouet, 1995; Vidal, Williams, O’Neill, & Pellerin, 2001). The presence of 3,4-di-Omethyl-D-xylopyranose in the methylation analysis showed that the wine oligosaccharides contain xylan structures, arising from the (1 ? 2)-linked xylose chain units (Ebringerova & Heinze, 2000). The main oligosaccharide residue of Cabernet Sauvignon, Syrah and Monastrell wines was mannose (28.3%, 21.9% and 25.2% respectively, Table 2), although rhamnose residue also showed similar values in the case of Syrah wines (21.2%). In contrast, rhamnose residues were lower in Cabernet Sauvignon and Monastrell wines (14.1% and 9.9%, respectively). On the other hand, glucose residue of Monastrell wines showed higher values (21.7%) compared with Cabernet Sauvignon (16.9%) and Syrah (16.8%) wines. Galactose residue also showed different values, the highest values corresponding to Monastrell wines (17.1%) and the lowest values to Syrah wines (14.4%). Proportions of several families of oligosaccharides from yeasts (mannans and glucans) and grapes (xylans, rhamnogalacturonans, arabinans and arabinogalactans) have been calculated (Fig. 1). The sum of mannose linked in ?2,3 and terminal mannose is considered as OligoMannans (Ducasse et al., 2011), and the sum of glucose linked in ?6 and terminal glucose is considered as OligoGlucans (Ballou, 1982) (Fig. 1). The sum of rhamnose linked in ?2 and linked in ?2,4 was attributed to OligoRhamnogalacturonans (Ducasse et al., 2011). OligoArabinogalactan type I was calculated as the sum of galactose linked in ?4 and in ?3,4 Gal plus terminal galactose and OligoArabinogalactan type II as the sum of galactose linked in ?3 and galactose linked in ?3,6 and equivalent in terminal arabinose to the value for galactose linked in ?3,6, whereas the sum of arabinose linked in ?5 and linked in ?3,5 is considered as OligoArabinans (Ducasse et al., 2011). OligoXyloglucans were estimated as the sum of terminal xylose, terminal fucose, glucose linked in ?4,6 and on third of its proportion in glucose linked in ?4 (Fry et al., 1993). Bordiga et al. (2012) also classified the oligosaccharides of Grignolino and Chardonnay wines in three different main families: hexose oligosaccharides (potentially galacto-oligosaccharides), arabinogalactans and xyloglucans. The cell wall oligosaccharides from yeasts (the sum of OligoGlucans and OligoMannans) varied among the three different variety wines. Wines from Cabernet Sauvignon and Monastrell presented

R. Apolinar-Valiente et al. / Food Chemistry 179 (2015) 311–317 Table 2 Glycosyl-linkage composition (mole percentage) of oligosaccharides fractions isolated from Cabernet Sauvignon, Syrah and Monastrell wines. Glycosyl residue

Linkage

Cabernet Sauvignon

Syrah

234 Rhamnose 34 Rhamnose 4 Rhamnose 3 Rhamnose Total

Terminal 2-Linked 2,3-Linked 2,4-Linked

2.0 10.1 1.1 0.9 14.1

2.8 14.9 2.0 1.4 21.2

1.5 6.8 0.6 1.0 9.9

234 Fucose Total

Terminal

0.7

0.6

0.7

235 Arabinose

Terminal furanose Terminal pyranose 3-Linked 2-Linked 5-Linked 3,5-Linked 2,5-Linked

4.9

2.2

7.5

6.8

8.8

3.6

1.1 1.1 3.7 0.8 1.1 19.5

1.2 1.1 3.5 1.0 1.3 19.1

0.9 0.1 6.6 1.0 0.8 20.7

234 Arabinose 25 Arabinose 35 Arabinose 23 Arabinose 2 Arabinose 3 Arabinose Total

4-Linked 3,4-Linked

3.9 0.8 4.8

4.0 1.6 5.6

3.8 0.9 4.6

2346 Galactose 234 Galactose 246 Galactose 236 Galactose 26 Galactose 24 Galactose 23 Galactose 2 Galactose Total

Terminal

1.0

0.6

0.6

6-Linked 3-Linked 4-Linked 3,4-Linked 3,6-Linked 4,6-Linked 3,4,6-Linked

4.0 2.3 3.2 0.0 2.6 1.9 0.7 15.6

3.7 2.4 3.9 0.0 1.7 2.0 0.0 14.4

4.7 1.5 2.8 1.2 2.9 2.2 1.3 17.1

2346 Glucose 234 Glucose 346 Glucose 236 Glucose 36 Glucose 24 Glucose 23 Glucose Total

Terminal 6-Linked 2-Linked 4-Linked 2,4-Linked 3,6-Linked 4,6-Linked

3.3 6.1 0.8 2.6 0.6 1.7 1.0 16.9

4.4 4.8 1.3 2.5 0.0 1.8 1.2 16.8

3.9 8.4 0.8 2.9 0.8 3.1 1.2 21.7

2346 Mannose 346 Mannose 246 Mannose 234 Mannose 46 Mannose 23 Mannose 34 Mannose 24 Mannose Total

Terminal 2-Linked 3-Linked 6-Linked 2,3-Linked 4,6-Linked 2,6-Linked 3,6-Linked

7.6 8.2 2.6 2.5 0.7 0.6 1.9 4.1 28.3

4.7 6.0 2.5 2.2 1.3 0.6 2.0 2.6 21.9

6.4 8.2 2.9 2.9 0.5 0.4 2.2 2.4 25.2

1.15

1.06

than linear arabinan-oligomers in sugar beet (Kühnel et al., 2010). Additional in-depth characterization would be required to confirm the branching configuration, e.g. by using NMR.

Monastrell

23 Xylose 2 Xylose Total

Ratio terminal/branched residues

315

0.97

similar values (17.7% and 19.2%, respectively), whereas wine from Syrah showed the lowest value (15.2%) (Fig. 1). On the other hand, an important release of OligoRhamnogalacturonans can be observed in Syrah wine (16.3%). In contrast, the highest release of OligoArabinans was detected in Monastrell wine (7.6%), Syrah wine presenting the lowest values for OligoAG type II (9.5%). Besides, Monastrell wine showed the highest values for OligoXyloglucans (2.9%). Other oligosaccharide structures as OligoAG type I did not show differences between the Cabernet Sauvignon, Syrah and Monastrell samples. The ratio of the terminal to the branched residues for Cabernet Sauvignon, Syrah and Monastrell oligosaccharides is 1.15, 1.06 and 0.97, respectively. Physic-chemical properties of wine could be affected by the branching degree. It has been reported that branched arabino-oligosaccharides are more difficult to degrade

3.3. ESI mass spectrometry: analysis of oligosaccharide fractions from Cabernet Sauvignon, Syrah and Monastrell wines The ESI-TOF spectra of the Cabernet Sauvignon, Syrah and Monastrell wine oligosaccharides are given in Fig. 2. The MS spectra show all the oligosaccharide molecules as being deprotonated [M H] ions. The structures of these ions were determined by fragmentation using a mass spectrometer equipped with an ESI source and an ion trap mass analyser and agreed with those described previously for oligosaccharides for Carignan and Merlot wines (Ducasse et al., 2010). Because oligosaccharide species may exhibit different desorption capacities according to their structure, mass spectrometry cannot be considered a good method for their quantification. The MS profiles obtained were highly reproducible for each sample and very different between samples, allowing comparison between the samples. The predominant ions observed in mass spectra were at m/z 499, 605 and 737 for Cabernet Sauvignon oligosaccharides (Fig. 2A), at m/z 605, 661, 807, 837 and 983 for Syrah oligosaccharides (Fig. 2B), and at m/z 235, 605, 661, 737 and 983 for Monastrell oligosaccharides (Fig. 2C). The ion identified at m/z 499 in Cabernet Sauvignon wine corresponds at Rha-[GalA-CH3]-Rha and represents the structures of galacturonans (smooth regions). As regards the ion at m/z 605, it corresponds to 4-OMe-glucuronic acid (4-OMe-GlcA), two xylose residues (Xyl) linked in 1 ? 4, and a xylitol residue in non-reducing position. Reis, Coimbra, Domingues, Ferrer-Correia, and Domingues (2004) described this type of structure in olive fruit glucuronoxylan, a characteristic of xylan families. The presence of glucuronoxylan-like structure in Cabernet Sauvignon oligosaccharides is re-enforced by the presence of the ion at m/z 737, which corresponds to [4-OMe-GlcA-[Xyl]3-Xylitol], whose structure corresponds to glucuronoxylans (Ducasse et al., 2010, 2011). The ions observed in the mass spectra at m/z 661, 837 and 983 corresponded to a double the repetition of the basic unit [(1 ? 4)a-D-GalAp-(1 ? 2)-a-L-Rhap], to the pentasaccharide GalA-[RhaGalA]2 and to [Rha-GalA]3, respectively (Ducasse et al., 2010). These oligosaccharides detected in Syrah wines represent rhamnogalacturonic zones (hairy regions). The ion at m/z 807 corresponds to a-L-Rhap-(1 ? 4)-a-D-GalAp-(1 ? 2)-a-L-Rhap-(1 ? 4)-a-DGalAp-(1 ? 2)-a-L-Rhap, and its presence indicates that oligosaccharides of Syrah wines also contain structures of rhamnogalacturonans but having a residue of rhamnose in terminal position. On the other hand, the ion at m/z 605 has also been detected in Syrah wines, corresponding to the presence of glucuronoxylan-like structure (Ducasse et al., 2010, 2011). The ion at m/z 235 was only identified in Monastrell oligosaccharides, and corresponds at GalA-COCH3. The rest of the ions observed in Monastrell wines (at m/z 605, 661, 737 and 983) have been above described. The ESI mass spectrometry results show differences among the composition of released oligosaccharides in wines from Cabernet Sauvignon, Syrah and Monastrell. 3.4. Determination of molar mass: the structural features by SECMALLS of oligosaccharide fractions from Cabernet Sauvignon, Syrah and Monastrell wines The elution profiles of the oligosaccharide fractions from Cabernet Sauvignon, Syrah and Monastrell wines using HPSEC coupled to on-line differential refractometer, viscosimeter, and multi-angle light scattering (MALLS) were given in Fig. 3 (concentration signal

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Fig. 3. SEC-MALLS chromatograms and weight-average molar mass distributions of the oligosaccharide fraction of Cabernet Sauvignon (CA), Syrah (SY) and Monastrell (MO) wines in 0.1 LiNO3 at 25 °C. Refractive Index (DRI; thin line) and light scattering (Mw; thick line).

to our knowledge, no HPSEC-MALLS data were reported in the literature on the oligosaccharide fraction from red wine. The oligosaccharides refractive index elution profiles displayed two principal populations whose concentration signal peaks are in the ranges 2150–2350 s and 2350–2600 s (Fig. 3, DRI signal). The molar mass of the eluting molecules decreased with increasing elution volume in agreement the normal size exclusion separation mechanism (Fig. 3, Mw signal). Oligosaccharides of Cabernet Sauvignon wine showed the higher molar mass profile, Monastrell showing the lower profile. Table 3 shows the distribution analysis of oligosaccharide fraction from Cabernet Sauvignon, Syrah and Monastrell wines as determined by Size Exclusion Chromatography coupled on-line to Multi Angle Laser Light Scattering (SEC-MALLS) and differential refractometer. Regarding these data, largely differences along five delimited ranges (Molar mass range: range 1 = 500–2500 g/mol; range 2 = 2500–5000 g/mol; range 3 = 5000–7500 g/mol; range 4 = 7500–10,000 g/mol; and range 5 = up to 10,000 g/mol) can be observed among different varietal wines. In the case of Syrah and Monastrell oligosaccharide fractions, 22% and 64% (respectively) of mass can be found in (500–2500 g/mol), whereas Cabernet

Table 3 Distribution analysis determined by light scattering (dn/dc = 0.145 mL/g) obtained of oligosaccharides fractions isolated from Cabernet Sauvignon, Syrah and Monastrell wines. Cultivar

Fig. 2. ESI-TOF spectra of the oligosaccharide fraction of Cabernet Sauvignon (A), Syrah (B) and Monastrell (C) wines.

derived from the differential refractometer and molecular mass derived from light scattering were given). It has been confirmed that SEC-MALLS is effective in measuring molecular weights in the oligomeric range (Xie, Penelle, & Verraver, 2002). However,

Cumulative (%)

Cabernet Sauvignon Molar mass (g/mol) Molar mass (g/mol) Molar mass (g/mol) Molar mass (g/mol) Molar mass (g/mol)

500–2500 2500–5000 5000–7500 7500–10,000 10,000–20,000

0.0 51.9 11.7 20.4 15.4

Syrah Molar Molar Molar Molar Molar

mass mass mass mass mass

(g/mol) (g/mol) (g/mol) (g/mol) (g/mol)

500–2500 2500–5000 5000–7500 7500–10,000 10,000–20,000

21.7 53.3 14 5.6 5.5

Monastrell Molar mass Molar mass Molar mass Molar mass Molar mass

(g/mol) (g/mol) (g/mol) (g/mol) (g/mol)

500–2500 2500–5000 5000–7500 7500–10,000 10,000–20,000

63.6 20.3 9.8 3.2 3.2

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Sauvignon showed 0% of its mass in this range. In contrast, this variety had 20% and 15% of mass in ranges 4 and 5, respectively), whereas Syrah and Monastrell presents lower percentages of masse in these two ranges (SY: 6% and MO: 3%). Obvious differences can be also detected regarding range 2: Monastrell showed 20% whereas in Cabernet Sauvignon and Syrah oligosaccharide fraction the mass percentage were distributed more similarly in range 2 (52% and 53%, respectively). Finally, range 3 showed no clear differences between studied wines (CS: 12%; SY: 14%; MO: 10%). In summary, the results of this study highlight grape variety could impact on wine oligosaccharide concentration, composition and structure. However, more studies should be carried out to further investigate the possible influence that different oligosaccharides from several grape varieties could have on the physic-chemical and sensory properties of wine. Acknowledgements This work was made possible by financial assistance from the Ministerio de Ciencia e Innovación of Spain, Project AGL200611019-C02-01/ALI. Author R. Apolinar-Valiente is the holder of an FPI fellowship from the Government of Spain. References Apolinar-Valiente, R., Williams, P., Romero-Cascales, I., Gomez-Plaza, E., Lopez-Roca, J. M., Ros-Garcia, J. M., et al. (2014). Effect of enzyme additions on the oligosaccharide composition of Monastrell red wines from four different winegrowing origins in Spain. Food Chemistry, 156, 151–159. Arnous, A., & Meyer, A. S. (2009). Quantitative prediction of cell wall polysaccharide composition in grape (Vitis vinifera L.) and apple (Malus domestica) skins from acid hydrolysis monosaccharide profiles. Journal of Agricultural and Food Chemistry, 57(9), 3611–3619. Ayestarán, B., Guadalupe, Z., & León, D. (2004). Quantification of major grape polysaccharides (Tempranillo v.) released by maceration enzymes during the fermentation process. Analytica Chimica Acta, 513, 29–39. Ballou, C. E. (1982). The molecular biology of the yeast Saccharomyces. In J. N. Strathern, E. W. Jones, & J. R. Broach (Eds.), Metabolism and gene expression (pp. 335–360). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Bordiga, M., Travaglia, F., Meyrand, M., German, J. B., Lebrilla, C. B., Coïsson, J. D., et al. (2012). Identification and characterization of complex bioactive oligosaccharides in white and red wine by a combination of mass spectrometry and gas chromatography. Journal of Agricultural and Food Chemistry, 60, 3700–3707. Brillouet, J. M., Moutounet, M., & Escudier, J. L. (1989). Fate of yeast and grape pectic polysaccharides of a young red wine in the cross-flow microfiltration process. Vitis, 28, 49–63. Cescutti, P., & Rizzo, R. (2001). Divalent cation interactions with oligogalacturonides. Journal of Agricultural and Food Chemistry, 49, 3262–3267. Darvill, A. G., & Albersheim, P. (1984). Phytoalexins and their elicitors – A defense against microbial infection in plants. Annual Review of Plant Biology, 155, 507–516. Doco, T., O’Neill, M. A., & Pellerin, P. (2001). Determination of the neutral and acidic glycosyl residue compositions of plant cell polysaccharides by GC–EI–MS analysis of the trimethylsilyl methyl glucoside derivatives. Carbohydrate Polymers, 46, 249–259. Doco, T., Vuchot, P., Cheynier, V., & Moutounet, M. (2003b). Structural modification of arabinogalactan-proteins during aging of red wines on lees. American Journal of Enology and Viticulture, 54, 150–157. Doco, T., Williams, P., Pauly, M., O’Neill, M. A., & Pellerin, P. (2003a). Polysaccharides from grape berry cell walls. Part II: Structural characterization of the xyloglucan polysaccharides. Carbohydrate Polymers, 53, 253–261. Doco, T., Williams, P., Vidal, S., & Pellerin, P. (1997). Rhamnogalacturonan II, a dominant polysaccharide in juices produced by enzymatic liquefaction of fruits and vegetables. Carbohydrate Research, 297, 181–186.

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