Complex Carbohydrates of Red Wine: Characterization of the Extreme

Article pubs.acs.org/JAFC

Complex Carbohydrates of Red Wine: Characterization of the Extreme Diversity of Neutral Oligosaccharides by ESI-MS Thierry Doco,*,† Pascale Williams,† Emmanuelle Meudec,‡ Véronique Cheynier,†,‡ and Nicolas Sommerer‡ †

Team BCP2, and ‡Polyphenols Platform, UMR1083 Sciences pour l’Œnologie, INRA, 2 Place Viala, F-34060 Montpellier, France ABSTRACT: The major neutral oligosaccharides of a Carignan red wine have been characterized for the first time. The oligosaccharides were prepared after removal of phenolic compounds by polyamide chromatography and of polysaccharides by alcohol precipitation and then were fractionated by anion exchange and size-exclusion chromatography. In a second step, the glycosyl composition and linkages of wine oligosaccharides were determined. Oligosaccharide fractions were analyzed by mass spectrometry (MS) with an electrospray ionization (ESI) source and an ion trap mass analyzer after separation by hydrophilic interaction liquid chromatography on a Nucleodur HILIC column (zwitterionic sulfoalkyl betaine stationary phase). Glycosyl residue composition analysis showed the predominant presence of arabinose, with galactose, rhamnose, and mannose in lower proportion. Neutral oligosaccharides were present at a concentration of 185 mg/L in this wine. The MS spectra in the negative ion mode of the oligosaccharide fractions showed a series of oligosaccharidic structures corresponding to oligo-arabinans often linked to the basic unit α-L-Rhap-(1 → 4)-α-D-GalpA. The wine oligosaccharides identified correspond to arabinooligosaccharides, rhamno-arabino-oligosaccharides, and different rhamnogalacturonan-arabino-oligosaccharides with DP ranging from 5 to 49, resulting from the degradation of grape cell wall pectins. Oligosaccharides have an extreme diversity, with more than 100 peaks detected in HPLC-ESI-MS spectra corresponding each to at least one oligosaccharidic structure. KEYWORDS: wines, oligosaccharides, HILIC, HPLC-ESI-MS/MS

■

INTRODUCTION Wine polysaccharides have been thoroughly studied because of their technological and sensory properties in wines. They have been shown to interact with tannins, 1,2 to decrease astringency,3 to inhibit hydrogen tartrate crystallization,4 to interact with wine aroma compounds,5 to prevent or limit aggregation and flocculation and the formation of protein haze in white wine,6−10 and to form specific coordination complexes with Pb2+ ions.11,12 For sparkling wines, foam properties have been correlated with the type, the molecular weight, and the composition of polysaccharides.13−16 Although their presence in wine is known,17−22 information on oligosaccharide composition is still limited, because of the difficulty in separating and characterizing them. Sucrose and other diholosides have been identified in wines,22 and a global fraction of oligomers of homo- and rhamno-galacturonan was described and shown to decrease during wine aging.23 The structure and amounts of polysaccharides and oligosaccharides released into the wines also depend on the grape varieties and the wine-making process and can be modified by enzyme treatment.18,20,24−27 Oligosaccharides can be found in various areas, in food, and in nonfood applications.28 Their main applications include elicitation of plant defenses29 and a role in prebiotics,30 but they are also significant for their physicochemical properties such as chelations of cations31 which can be important for wines. It is important to determine the exact composition of oligosaccharides in wines and to analyze their molecular structures in order to better understand the technological and organoleptic properties associated with them. Characterization of oligosaccharide structure has been classically performed by © XXXX American Chemical Society

glycosyl and linkage analyses, by NMR, or by mass spectrometry techniques.32 The direct analysis of oligosaccharide mixtures by MS has been made possible by the development of soft ionization techniques such as ESI (electrospray ionization).33−35 The ion fragmentation patterns obtain by MS/MS or MSn spectra provided information for structural characterization of oligosaccharides.36 Acidic oligosaccharides have been identified previously,17,18 and some neutral oligosaccharides have been characterized by high resolution and high mass accuracy matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) in Grignolino and Chardonnay wines.19 We describe herein the purification of neutral and acidic oligosaccharides from a red wine of Carignan and the characterization of the main neutral oligosaccharides after chromatographic separation using a HILIC column by mass spectrometry coupled to an electrospray ionization source and an ion trap mass analyzer. The MS technique proved to be particularly efficient for the analysis of mixtures of wine oligosaccharides without prior derivatization.

■

EXPERIMENTAL SECTION

Wine Samples. Carignan red wine was made from the grapes of Vitis vinifera cv. Carignan grown at the INRA Experimental Unit Station (Gruissan, Southern France) and harvested in 2004 at

Received: October 8, 2014 Revised: December 9, 2014 Accepted: December 20, 2014

A

DOI: 10.1021/jf504795g J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Scheme of fractionation of wine oligosaccharides by decolorization, alcohol precipitation, and anion-exchange and size-exclusion chromatography. commercial maturity (21.8°Brix). A 100 kg amount of grapes was crushed and destemmed using a destemmer−crusher and put in 100-L stainless steel tanks. Alcoholic fermentation was carried out in tanks equipped with temperature control (28 °C), enabling regulation of fermentation kinetics. At the end of alcoholic fermentation, the must was pressed, the wine was stored in 50-L tanks, and lactic bacteria was subsequently added to induce malolactic fermentation. At the end of malolactic fermentation, the wine was racked in a 30-L inox tank and stored at low temperature (−4 °C) to induce tartaric stability. The wine was then bottled and stored in a cellar at 18 °C until analysis.18,26 Isolation of Oligosaccharide Fractions. As described in Figure 1, wines (500 mL) added to 1 M NaCl were partially decolorized on a column of MN Polyamide CC6 (28 × 2.8 cm) previously equilibrated with 1 M NaCl. Wine polysaccharides and oligosaccharides not retained on the polyamide column were eluted by two bed volumes of 1 M NaCl.37 The wine complex carbohydrates were precipitated by ethanolic dehydration. This was performed by adding ethanol 95% acidified by 0.5% HCl to obtain a final concentration of 80% ethanol.23,24 After one night at 4 °C, the supernatant, which contained the total oligosaccharide fraction, was recovered by centrifugation (30 min, 6288g, 4 °C) and dialyzed extensively against distilled water (MWCO 1 kDa), concentrated, and freeze-dried. Then a step of anion-exchange chromatography was performed. The solution containing oligosaccharides was loaded on a DEAE-MACROPREP column (17 × 5 cm; Bio-Rad, Hercules, CA) equilibrated with 50 mM sodium acetate buffer pH 4.8. An unbound fraction (nonretained fraction) was recovered, and a bound fraction (retained fraction) of oligosaccharides was eluted by 1 M NaCl in the same buffer. The unbound fraction and the bound fraction from DEAE-Macroprep were extensively dialyzed against distilled water (MWCO 1 kDa), concentrated, and freeze-dried. The oligosaccharides of unbound (initial nonretained fraction) and bound (initial retained fraction) fractions were finally purified by size exclusion chromatography. The chromatography was performed by loading 2 mL of initial nonretained or initial retained fractions containing 30 mg of the freeze-dried fraction on a Superdex 30-HR column (60 × 1.6 cm, GE Healthcare, Uppsala, Sweden) with a precolumn (0.6 × 4 cm), equilibrated at 1 mL/min in 30 mM ammonium formate pH 5.6. The elution of oligosaccharides was monitored with an Erma-ERC 7512 (Erma, Tokyo, Japan) refractive index detector combined with Chromeleon software (Dionex,

Sunnyvale, CA). Four and six fractions, according to elution time, were collected for the unbound DEAE fraction (initial nonretained fraction) and bound DEAE fraction (initial retained fraction), respectively. The isolated fractions, called nonretained fractions A, B, C, and D and retained fractions A, B, C, D, E, and F were freeze-dried, redissolved in water, and freeze-dried again four times to remove completely the ammonium salt (Figure 1). The molecular weight distribution of oligosaccharides present in the nonretained fractions A, B, C, D and retained fractions A, B, C, D, E, and F was determined by high-performance size-exclusion chromatography (HPSEC) using a LC-10 AS Shimadzu pump (Kyoto, Japan). HPSEC elution was performed on two serial Shodex Ohpak KB-803 and KB-805 columns (0.8 × 30 cm; Showa Denkko, Japan) connected to a ERC-7512 refractometer (Erma, Japan), at 1 mL/min flow rate in 0.1 M LiNO3. The apparent molecular weights were deduced from the calibration curve established with a pullulan calibration kit (P-400, MW = 380 000 P-200, MW = 186 000; P-100, MW = 100 000; P-50, MW = 48 000; P20, MW = 23 700; P-10, MW = 12 200; P-5, MW = 5800; Showa Denko K.K., Japan). The calibration equation was log MW = 27.746 − 1.03 × tR (tR = column retention-time at peak maximum, and r2 = 0.997). Neutral Sugar Composition as Alditol Acetates. Neutral sugars were determined as alditol acetates by GLC after TFA hydrolysis.38 Separation was carried out on a DB225 column (30 m × 0.25 mm i.d.; 0.25 μm film; J&W Scientific, Agilent Technologies, Santa Clara, CA) with hydrogen as carrier gas (0.6 bar inlet pressure) on a Shimadzu GC-2010plus gas chromatograph. The alditol acetates were identified from their retention times by comparison with those of standard monosaccharides. Neutral sugars amounts were calculated relative to the internal standards. Allose and myo-inositol were used as internal standards.39 Neutral and Acidic Sugar Composition as Trimethylsilyl Derivatives. The neutral and acidic sugar composition was determined after solvolysis with anhydrous MeOH containing 0.5 M HCL (80 °C, 16 h), by GC of their per-O-trimethylsilylated (TMS) methyl glycoside derivatives.40 The TMS derivatives were separated on two DB-1 capillary columns (30 m × 0.25 mm i.d., 0.25 μm film) (temperature programming 120−145 °C at 1.5 °C/min, then 145− 180 °C at 0.9 °C/min, and finally−180−230 °C at 50 °C/min), coupled to a single injector inlet through a two-holed ferrule, with H2 as the carrier gas on a Shimadzu GCMS-QP2010SE gas chromatoB


Article


Table 1. Composition Analysis of Nonretained and Retained Oligosaccharide Fractions on a DEAE-Macroprep Column (mol %) initial nonretained fraction nonretained fraction A nonretained fraction B nonretained fraction C nonretained fraction D initial retained fraction retained fraction A retained fraction B retained fraction C retained fraction D retained fraction E retained fraction F a

Rha

Ara

Xyl

Gal

Glc

Man

GalA

GlcA

ratio Ara/Gal

MWa

concb

3.0 1.0 1.4 1.7 8.9 27.5 17.6 27.2 35.0 39.6 49.9 38.0

75.6 70.9 89.1 82.1 29.3 29.9 48.5 39.2 24.0 15.1 3.4 -

6.0 -

6.3 4.0 5.2 7.0 6.1 8.8 12.1 9.1 7.4 7.1 2.4 -

3.0 4.8 1.2 1.9 10.9 2.5 3.7 2.4 2.0 2.2 2.2 9.0

8.4 19.4 2.3 5.0 30.4 0.8 3.4 Trc Trc Trc Trc 0.7

2.7 0.8 1.1 6.2 29.2 13.6 20.5 29.7 34.1 40.9 52.3

1.1 1.1 2.4 1.2 1.2 1.3 1.3 1.5 0.8 -

12.00 17.72 17.13 11.72 4.80 3.39 4.00 4.30 3.24 2.12 1.41 -

8187 3828 2250 929 7641 2591 1790 1516 1255 966

184.8 4.0 44.4 85.0 36.2 261.6 25.6 110.0 16.8 34.8 30.0 11.6

Molecular weight. bConcentration mg/L. cTr = trace, less than 0.5%.

graph. The outlet of one column was directly connected to a FID at 250 °C, and the second column via a deactived fused-silica column (0.25 m × 0.11 μm i.d.) was connected to a mass detector. Samples were injected in the pulsed split mode with a split ratio of 20:1. The transfer line to the mass was set at 280 °C. EI mass spectra were obtained from m/z 50 to 400 every 0.2 s in the total ion-monitoring mode using an ion source temperature of 200 °C, a filament emission current of 60 μA, and an ionization voltage of 70 eV. Glycosyl-Linkage Determination. The glycosyl-linkage compositions were determined by GC-MS of the partially methylated alditol acetates. Two milligrams of polysaccharides in 0.5 mL of dimethyl sulfoxide were methylated using methyl sulfinyl carbanion and methyl iodide.41 Only for the retained fractions A, B, C, D, E, and F, half of the methylated sample was then carboxyl-reduced with lithium triethylborodeuteride (Superdeuteride, Aldrich, Milwaukee, WI).42 Both methylated and methylated-carboxyl-reduced samples were hydrolyzed with 2 M trifluoroacetic acid (75 min at 120 °C). The released methylated or carboxyl-reduced monosaccharides were converted to their corresponding alditols by treatment with NaBD4 and then acetylated.38 Partially methylated alditol acetates were analyzed by GC GC-EI-MS using a DB-1 capillary column (30 m × 0.32 mm i.d., 0.25 μm film); temperature programming 135 °C for 10 min and then 1.2 °C/min to 180 °C, with H2 as the carrier gas on a Shimadzu GCMS-QP2010SE gas chromatograph.43 Identity of each methyl ether was confirmed by EIMS, and their areas were corrected by response factors. UPLC-ESI-MSn. Separations of oligosaccharide fractions were performed using a Waters Acquity UPLC-DAD system (Milford, MA), on a (100 × 1 mm i.d.) Nucleodur HILIC column, 1.8 μm (zwitterionic sulfoalkyl betaine stationary phase, Macherey-Nagel, Düren, Germany), operated at 35 °C. The mobile phase consisted of acetonitrile/formic acid (99.5/0.5, v/v) (eluent A) and water/formic acid (99.5/0.5,v/v) (eluent B). Flow rate was 0.3 mL/min. The elution program was as follows: 1−40% B (0−20 min), 40−90% B (20−21 min), isocratic with 90% B (21−25 min), 90−1% B (25−26 min), isocratic with 1% B (26−33 min). ESI-MSn analyses were performed with a Bruker Daltonics Amazon (Bremen, Germany) mass spectrometer equipped with an electrospray source and an ion trap mass analyzer. The spectrometer was operated in the negative ion mode (capillary voltage, 4 kV; end plate off set: −300 V; temperature, 200 °C; nebulizer gas: 36 psi and dry gas, 7 L/ min). Collision energy for fragmentation used for MS2 and MS3 experiments was set at 1. The mass spectra were acquired over a mass range of 300−2500 Da with target mass at 1200 Da, and 500−3000 Da with target mass at 2000 Da.

orized on Polyamide CC6 and then were precipitated by acidified alcohol in order to separate polysaccharides (mannoproteins, polysaccharides rich in arabinose and galactose (PRAGs), RGII, and glucans) from oligosaccharides (Figure 1). The passage of the supernatant through a column of anion exchange allowed us to separate wine oligosaccharides into two fractions, a first fraction not retained on the column and a second charged and retained on the column and then eluted by 1 M of NaCl. The glycosyl residue composition determined by GC after methanolysis and trimethysylilation,40 is summarized in Table 1. The nonretained fraction contained low amounts of uronic acids, 2.7 mol % for galacturonic acid, and 1.1 mol % for glucuronic acid and is predominantly composed of arabinose (75.6%). In the retained fraction, galacturonic acid, rhamnose, and arabinose presented the highest molar percentages (29.2, 27.5, and 29.9, respectively). The total amount of oligosaccharides before the anion exchange represented a concentration of 446 mg per liter (184 mg for initial nonretained fraction and 261 mg for the initial retained fraction, Table 1). These results are slightly higher than those previously reported for Merlot and Carignan,17,18 or Grignolino and Chardonnay wines,19 but they were similar to those reported for Monastrell wine oligosaccharide fractions.20 The initial nonretained fraction and the initial retained fraction were finally separated by SEC on superdex HR30. The MW determined for the oligosaccharide fractions obtained by SEC are given in Table 1. The nonretained fraction and retained fractions yielded four and six subfractions, respectively, with molecular weight (MW) ranging from 8000 Da (fraction A) to 1000 Da (fractions D and F, respectively). Table 1 also shows the glycosyl residue composition of the oligosaccharide fractions obtain after solvolysis by GC of their per-Otrimethylsilylated (TMS) methyl glycoside derivatives. They contained most of the sugars known to take part in the composition of wine carbohydrates. Oligosaccharides of nonretained fractions A, B, C, and D and retained fractions A, B, C, D, E, and F included sugars such as rhamnose, arabinose, galactose, and galacturonic acid derived from the pecto-cellulosic cell walls of grape berries but also mannose and glucose released from yeast and/or bacteria polysaccharides.17−21 The nonretained fractions A, B, and C were rich in arabinose (70.9%, 89.1%, and 82.1%, respectively), whereas nonretained fraction D contained similar proportions of arabinose and mannose (30%), and larger proportions of

■

RESULTS AND DISCUSSION Purification and Characterization of Oligosaccharide Fractions. Wine carbohydrates from Carignan were decolC


Article


Table 2. Glycosyl Linkage Composition (mole percentage) of Oligosaccharides Fractions Isolated from Carignan Red Wine nonretained fractions methyl ether 2,3,4-Rhaa 3,4-Rha 3-Rha total rhamnoseb 2,3,5-Ara 2,5-Ara 2,3-Ara 2-Ara Ara total arabinose 3-Xyl total xylose 2,3,4,6-Gal 2,3,4-Gal 2,4,6-Gal 2,3,6-Gal 2,4-Gal 2,6-Gal 2 -Gal total galactose 2,3,4,6-Glc 2,3,4-Glc 3,4,6-Glc 2,4-Glc 2,3-Glc 3,4-Glc 2-Glc total glucose 2,3,4,6-Man 3,4,6-Man 2,4,6-Man 2,3,6-Man 2,3,4-Man 2,4-Man mannose total mannose 2,3,4-GlcAd total glucuronic acid 2,3,4-GalAd 2,3-GalAd 2 -GalAd total galacturonic acid ratio terminal/branched ratio linear/terminal

retained fractions

linkage

A

B

C

D

A

B

C

D

E

F

terminal 2-linked 2,4-linked

0.8 0.8 4.3 5.8 29.3 22.5 61.9 -

0.9 0.9 7.9 4.7 46.6 25.1 2.2 86.5 -

1.7 1.7 8.2 2.6 44.9 19.6 1.3 76.6 -

0.9 5.4 13.8 20.1 16.7 1.7 18.7 7.8 44.9 -

1.4 14.1 12.3 27.9 14.6 1.0 15.4 5.6 36.6 -

2.4 29.1 6.0 37.4 11.4 Tr 10.2 1.2 23.1 -

3.4 36.5 3.6 43.2 9.0 4.6 Tr 13.9 -

11.3 42.9 1.3 55.4 1.9 1.9 -

13.8 34.7 Trc 48.8 0.5 0.5 -

1.0 1.0 Tr 2.7 5.2 8.3 Tr Tr 0.9 1.8 -

Tr 1.1 1.0 1.4 1.6 5.3 Tr 3.4 5.2 9.1 1.2 1.7 1.1 1.0 5.0 -

1.8 1.1 1.6 3.3 2.0 6.6 1.1 17.5 0.8 1.3 2.1 0.8 0.6 1.4

1.1 0.6 1.8 3.6 0.6 7.3 Tr 15.5 Tr 0.8 1.1 -

0.7 0.4 2.3 3.3 6.3 Tr 13.2 Tr 0.7 1.0 -

Tr 1.6 4.0 6.1 12.2 Tr 0.9 1.2 -

Tr Tr 1.9 1.6 4.5 Tr 1.5 1.8 -

1.4 Tr 1.8 1.4 1.0 2.4 -

terminal

Tr 1.6 2.6 4.6 0.7 3.3 3.5 4.5 1.7 13.7 5.1 5.1 8.0 18.2 -

1.5 5.9 7.4 6.9 1.0 15.8 4.9 28.6 5.1 5.1 0.9 2.0 2.4 5.0 2.5 12.8 2.0 4.0 2.4 3.2 11.6 8.4 9.4 5.1 1.8 2.4 1.3 28.4 -

-

-

-

-

-

12.6 1.2

0.5 16.5

1.6 1.6 1.4 25.9 1.0

1.5 1.5 2.6 33.5 0.7

-

terminal 4-linked 3,4-linked

0.9 0.9 1.0 21.5 1.1

11.6 34.9 Tr

0.21 4.70

0.20 6.06

13.8 0.63 2.20

18.1 0.67 2.86

23.5 1.17 3.90

28.0 1.50 4.57

36.6 4.88 4.84

46.5 39.0 2.64

terminal 3-linked 5-linked 3,5-linked 2,3,5-linked 2,4-linked terminal 6-linked 3-linked 4-linked 3,6-linked 3,4-linked 3,4,6-linked terminal 6-linked 2-linked 3,6-linked 4,6-linked 2,6-linked 3,4,6 linked terminal 2-linked 3-linked 4-linked 6-linked 3,6-linked 2,3,4,6-linked

0.05 29.21

0.84 2.93

a 2,3,4-Rha: 1,5-di-O-acetyl-2,3,4-tri-O-methyl rhamnitol, etc. bRelative mole percent of each parent sugar family (sum of ethers from one sugar type) within total sugars. cTr = trace, less than 0.5%. d6,6′-dideuterated ether only for the retained fractions.

rhamnose and glucose than the other three fraction, as well as xylose and galacturonic acid (6% each). In the retained fractions, the proportions of rhamnose and galacturonic acid increased from 15% to 50% in fractions A through F while that of arabinose decreased (48% in retained fraction A to 0% in the retained fraction F, Table 1). This composition indicated that the retained fractions with lower MW correspond to structures of the rhamnogalacturonan of type-I (RGI). The glycosyllinkage compositions for nonretained and retained fractions are given in Table 2. Nonretained fractions A, B, C, and D are mainly constituted by arabinose linked at position 5 or

arabinose linked at 3,5 (Table 2). Presence, after the methylation analysis, of these methyl ethers indicates the presence of a branched (1 → 5)-oligo-arabinan composed of the following repeating unit [ → 5)-Araf-(1 → 5)[Araf-(1 → 3)]-Araf-(1 → 5)-Araf-(1→ ], with more arabinosyl residues involved in the linear (1 → 5)-arabinan chain, and terminal arabinose linked in 3 on the (1 → 5)-arabinan chain. Analysis of glycosidic linkages (Table 2) indicates the presence of other oligosaccharides, in particular originating from mannoproteins, but at low levels in the nonretained fractions. Oligosaccharides from retained fractions are mainly constituted by rhamnose D


Article


Figure 2. UPLC-MS analysis of nonretained fraction B: (a) total ion chromatogram; (b) full-scan mass spectrum of nonretained fraction B; (c) 2D projection of the full-scan mass spectra as a function of oligosaccharide retention times on the HILIC column.

of the oligosaccharides are too large (up to 8 kDa) to be analyzed in our ESI-MS spectrometer. Analysis of the full-scan mass spectra of nonretained fractions B, C, and D (Figures 2b, 4b, and 5b) show a multitude of mass signals between m/z 800 and m/z 2500 Da. The 2D projection of the full-scan mass spectra as a function of oligosaccharide retention times on the HILIC column (Figures 2c, 4c, and 5c) shows the separation as a function of the masses. Due to the low background noise in negative-ion mode, this 2D representation makes it possible to highlight oligosaccharides with different degrees of polymerization (DP). The negative ionization consists of a selective deprotonation of the anomeric hydroxyl at the reducing end of the oligosaccharide chain. Once the negative charge is localized at the reducing end, the ion fragmentation of the ring occurs rapidly and the mass losses observed are found to be diagnostic of the glycoside type. The signals obtained correspond to negatively charged oligosaccharides as deprotonated [M − H]−, [M − 2H]2−, and [M − 3H]3− ions determined by the isotopic distribution (1 for single deprotonated, 0.5 for doubly deprotonated, and 0.33 for triply deprotonated ions). In Figure 2c, a series of ions was observed at m/z 1557.0, 1622.2, 1688.6, 1753.7, 1819.7, and 1886.8, which correspond to a difference of 66 Da between the different ions (M = 3116 Da to M = 3776 Da). A second series of ions was observed at m/z 1196.3, 1263.0, 1329.0, 1395.1, 1461.1, 1527.1, 1592.6, 1659.1, 1725.7, 1791.7, 1857.2, 1923.2, 1989.3, 2055.8, and 2121.8 which also correspond to a difference of 66 Da. The ions of the two series correspond to oligosaccharide molecules detected as doubly deprotonated [M − 2H]2− ions. The difference of 66 Da (m/z 132 with z=2) between the first two series of ions corresponds to the presence of a pentose, likely an arabinose residue (Table

linked at position 2 and 2,4, and by galacturonic acid linked at 4 (Table 2, per-O-methylated-6,6′-dideuterated ethers for uronic acids), indicating that fractions were oligo-rhamnogalacturonans organized with a repeat unit of [(→2)-α-L-Rhap-(1 → 4)-α-D-GalpA-(1 → ]n.44,45 The presence of 2,4-linked rhamnose along with methyl ethers from arabinose and galactose, obtained after methylation analysis (Table 2), indicated that side chains were attached to the oligorhamnogalacturonan backbone through the rhamnose moieties.45 These oligosaccharides arise from the rhamnogalacturonan regions of pectins as a result of degradation of the grape berry cell wall by pectinases during grape maturation and/or wine making46 and have been isolated for oligosaccharides of Carignan or Merlot wines.17 Analysis of the glycosidic linkages enabled determination of the structure of oligosaccharides that were released into the wines but not their structural organizations, for example the degree of polymerization (DP) of the oligosaccharides. UPLC-ESI-MSn of the Nonretained Oligosaccharide Fractions. Figures 2a, 4a, and 5a show the total ion chromatogram of nonretained fractions B, C, and D on the HILIC column. HPLC coupled with mass spectrometry is a very powerful technique for oligosaccharide analysis and characterization, and ESI has been a good technique for the introduction of sugar components into an MS.35,36 Recently, hydrophilic interaction liquid chromatography (HILIC), an alternative method for separation of highly polar compounds such as carbohydrates, and negative-ion ESI-MSn were applied for structural analysis of oligosaccharides.47−49 The nonretained fraction A was also injected on the column but did not give a total ion chromatogram by MS. The masses E


Article


Table 3. Degrees of Polymerization (DP) of the Different Series of Oligosaccharides from Nonretained Fractions B, C, and Da fraction nonretained B DP

(Ara)n-RhaGalA

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

1557.0 1622.2 1688.6 1753.7 1819.7 1886.8 -

(2−) (2−) (2−) (2−) (2−) (2−)

(Ara)n 1196.5 1263.0 1329.0 1395.1 1461.1 1527.1 1595.6 1659.1 1725.7 1791.7 1857.2 1923.2 1989.3 2055.8 2121.8 1546.1 1590.2 1634.0 1678.2 1722.4 1766.1 1810.4 1854.2 1898.7 1942.6 1986.7 2030.8 2074.5 2118.5 2162.8

(2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−) (2−)

(3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−) (3−)

fraction nonretained C

(Ara)n-Rha-GalARha 1790.7 1834.6 1878.6 1922.7 1966.5 2010.0 2054.4 2098.8 -

(3−) (3−) (3−) (3−)

(Ara)n-RhaGalA 1027.4 1093.9 1159.4 1225.9 1291.9 1357.5 1424.0 -

(2−) (2−) (2−) (2−) (2−) (2−) (2−)

(Ara)n

(Ara)n-Rha

998.9 (2−) 1064.9 (2−) 1131.0 (2−) 1196.9 (2−) 1263.0 (2−) 1328.5 (2−) 1395.1 (2−) 1461.1 (2−) 1527.6 (2−) 1595.0 (2−) -

1005.3 1071.9 1137.9 1203.4 1269.5 1336.0 1401.9 -

(2−) (2−) (2−) (2−) (2−) (2−) (2−)

fraction nonretained D (Ara)n-Rha-GalARha 1299.4 (2−) 1365.0 (2−) 1431.0 (2−) 1497.0 (2−) 1563.1 (2−) 1629.1 (2−)(2−) 1695.6 (2−) 1173.7 (3−) 1217.5 (3−) 1261.8 (3−) 1305.7 (3−) 1349.9 (3−) 1393.8 (3−) 1437.8 (3−) 1482.1 (3−) -

(Ara)n-Rha

(Ara)n

(Ara)n

823.3 955.5 1087.5 1219.5 1352.2 1484.2 741.2(2−) 807.3 (2−) 873.3 (2−) 939.3 (2−) 1005.4 (2−) 1071.9 (2−) 1137.4 (2−)

943.4 1076.0 1208.1 1340.3 1472.2 867.4 (2−) 933.4 (2−) 999.8 (2−) 1065.9 (2−) 1131.4 (2−) 1197.4 (2−)

818.7 (2−) 899.3 (2−) 980.7 (2−) 1061.4 (2−) 1142.9 (2−) 1223.9 (2−) -

-

-

(3−) (3−) (3−) (3−)

The ions of the series correspond to negatively charged oligosaccharides detected as deprotonated [M − H]−, [M − 2H]2−, and [M − 3H]3− ions. The charge is indicated in parentheses.

a

1). The first series starting at m/z 1557.0 corresponds to the deprotonated oligosaccharides with the following structures: [Ara]n-(1 → 4)-α-L-Rhap-(1 → 4)-α-D-GalpA, where the number n of arabinose residue is between 21 to 26, and the first arabinose residue is linked at (1 → 4) on the rhamnose residue (Table 2). The second series starting at m/z 1196.3, 1263.0,.... corresponds to doubly deprotonated [M − 2H]2− oligo-arabinans with DP ranging from 18 (m/z 1196.3, M = 2396) to 32 (m/z 2121.8, M = 4246). A third series of ions at m/z 1546.1, 1590.2, 1634.0, 1722.4, 1766.1, 1810.4, 1854.2,

1898.7, 1942.7, 1986.8, 2074.5, 2118.5, and 2162.8 presented a difference of 44 Da between all the ions of the series. The difference of 44 corresponds of a triply charged arabinose residue (m/z 44 where m = 132 and z = 3), and this series of ions correspond to [M − 3H]3− ions of oligo-arabinans with a DP between 35 (m/z 1546.1, M = 4641) and 49 (m/z 2162.8, M = 6492). The separation on the HILIC column depends on the number of hydroxyl groups that each oligosaccharide has and the number of hydrogen bonds established with the stationary phase. Within a homologous series, e.g. oligoF


Article


Figure 3. Spectra of MS3 fragmentation by ESI-TI in negative mode of ion parent at m/z 1262.5 (a). MS spectrum of ion at m/z at 1262 [M − 2H)2‑. (b). MS2 spectrum of the ion at m/z 1262.5 [M − 2H)2−. (c). MS3 spectrum of the ion at m/z at1217.94 [M − 2H)2− from the m/z 1262.5 ion [M − 2H)2−. (red →) Loss of a fragment of m/z 66.

(45 amu from the doubly charged parent ion), i.e. ion at m/z 1217.9 for parent ion at m/z 1262.5 (Figure 3b, MS2 spectrum). The loss of this fragment corresponds to a fragmentation 0,2Xn of a residue of arabinose in a nonreducing terminal position of the oligosaccharide studied.50 Then from this ion, we observe a series of fragment ions produced by loss of arabinose residues: the fragment ion at m/z gives an ion at m/z 1151.9 and m/z 1085.9 and so on until a fragment ion at m/z 887.3 corresponding to the loss of five residues of arabinose from the ion at m/z 1217.9. All fragment ions obtained are doubly charged ions. In the same manner, the MS3 fragmentation (Figure 3c) of the fragment ion at m/z 1217.9 showed the presence of fragment ions arising from successive losses of arabinose residues. The last fragment ion at m/z 623.1 corresponds to the loss of nine residues of arabinose from the fragment ion at m/z 1217.9. The fragment ion at m/z 623.1 corresponds to a doubly charged fragment, but it is also present as a singly charged fragment ion [M − H]− at m/z 1247.6. This fragment ion at m/z 1247.6 then loses an arabinose residue to give an ion [M − H]− at 1115.4 amu. All the ions present in the spectrum of the nonretained fraction B (Figure 2) were fragmented in MS2 and MS3 (data not shown). All ions identified on Figure 2C provide a MS/MS fragmentation spectrum similar to that of the ion at m/z 1262.5. Figures 4c and 5c show mass spectra of wine oligosaccharides from the nonretained fractions C and D. We observe several series of oligosaccharides with different structures and degrees of polymerization. The results are summarized in Table 3. A

arabinans, this allows the prediction of elution positions as a function of DP. The oligo-arabinan of DP 18 (m/z 1196.3, M = 2396) has a retention time of 15.2 min, while the oligo-arabinan with a DP 49 (m/z 2162.8, M = 6492) has a retention time of 20.1 min. In this series, other oligo-arabinans with DP between 18 and 49 are eluted between 15 and 20 min on the column. The results of nonretained fractions B are summarized in Table 3. The MSn fragmentations of each oligosaccharide from nonretained fractions B, C and D, were performed by ESIIT-MSn in the negative ion mode. Figure 3 shows, as an example, the MSn spectra of the [M − 2H]2− ion at m/z 1262.5 obtained from the nonretained fraction B. Under negativeionization ESI-MSn conditions, the fragmentation process of oligosaccharides involves the glycosidic cleavage between two sugar residues and cross-ring opening of sugar (cleavage of two bonds within the sugar ring). MSn spectra of deprotonated oligosaccharides can be read ”from right to left”. The fragment ions observed in the MSn spectra are usually named according to the nomenclature of Domon and Costello (1988).50 The MS2 fragmentation (Figure 3b) of the parent ion at m/z 1262.5 showed the presence of a fragment ion at m/z 1196.5, due to the loss of an arabinose residue (m/z 66 where m = 132 and z = 2). This fragment ion at m/z 1196.5 loses a first arabinose residue to give the fragment ion at m/z 1130.5 and after successive losses of arabinose residues yields fragment ions at m/z 1064.4, 998.3,... Another point of the MS2 spectra was the presence of a fragment ion due to the loss of 90 mass units G


Article


Figure 4. UPLC-MS analysis of nonretained fraction C: (a) total ion chromatogram; (b) full-scan mass spectrum; (c) 2D projection of the full-scan mass spectra as a function of oligosaccharide retention times on the HILIC column.

series starting from the [M − 2H]2− ion at m/z 1027.4 corresponds to the deprotonated oligosaccharides with the following structures: disaccharide α-L-Rhap-(1→4)-α-D-GalpA substituted on the Rha by a lateral chain of arabinans. The first and last oligosaccharides of the series correspond to a DP of 15 and a DP of 21, respectively. The following two series, identified as [M − 2H]2− ions, correspond to the structure of oligo-arabinans with a DP ranging from 15 to 24, and rhamnooligo-arabinans consisting of one residue of Rha substituted at the 4 position by arabinans of DP 15 to DP 21 (Table 3). The last series identified in the nonretained fraction C (Table 3) correspond to oligosaccharide molecules with the following structures: α- L -Rhap-(1→4)-α- D -GalpA-(1→2)-α- L -Rhap, where one residue of rhamnose was substituted at (1→4) by n residues of arabinose with n between 16 to 30 (see Table 3). Our results indicated that these oligosaccharides are constituted by a core structure of degraded RGI, in which the oligorhamnogalacturonan backbone was substituted through the rhamnose moieties by (1→5)-arabinan branched at the 3position by terminal nonreducing arabinose. These types of arabinan chains have been found in red wine as the side chains of RGI51 while arabinans were rarely found.22,52 RGI originates from grape cell walls. It is present in low amounts in wine,22 presumably because it is hydrolyzed by glycoside hydrolases and/or “polysaccharidases” from grapes or from yeast used during the fermentation step. The detection of these oligosaccharides suggests that they have been released from grape cell wall polysaccharides under the action of rhamnogalacturonase, arabinanase, and α-arabinofuranosidase activities at least. The oligosaccharide series identified in the nonretained Fraction D (Table 3) correspond to oligosaccharide with the following structures: One residue of rhamnose substituted by

arabinan with a DP between 7 and 18, and oligo-hexose, where hexoses are galactose, glucose, or mannose (see Table 1) with a DP between 10 to 15. UPLC-ESI-MSn of the Retained Oligosaccharide Fractions. The retained fractions A, B, C, D, E, and F, corresponding to acidic oligosaccharides (Table 1), were also analyzed by LC-ESI-MS with the HILIC column. No mass signal was detected in retained fraction A, while retained fractions B and C provided mass signals of low intensity (Figure 6a−d) and the retained fractions E and F gave mass spectra corresponding to the oligosaccharides already identified.17,18,20 The majority of masses could be assigned to an oligosaccharide structure. All oligosaccharide molecules were detected as the deprotonated [M − H]− and doubly deprotonated [M − 2H]2− ions. The predominant ions (the most intense) observed in the mass spectra were ions at m/z 485, 545, 661, 807, 837, 983, 1130, 1160, 1306, and 1452 for oligosaccharides from retained fractions E and F. The ion observed at m/z 661 corresponds to the deprotonated tetrasaccharide: [(→2)-α-L-Rhap-(1→4)-α-DGalpA(1→]2, the ion at m/z 837 corresponds to the deprotonated pentasaccharide: α-D-GalpA(1→[(→2)-α-LRhap-(1→4)-α-D-GalpA(1→]2, the ion at m/z 983 to the hexasaccharide: [(→2)-α-L-Rhap-(1→4)-α-D-GalpA(1→]3, the ion at m/z 1160 to the heptasaccharide and the ion at m/z 1306 to the octasaccharide: [(→2)-α-L-Rhap-(1→4)-α-DGalpA(1→]4. The ions observed at m/z 485, 661, 807, 983, 1130, 1306, and 1452 correspond to the deprotonated rhamnogalacturonan oligosaccharides where the first oligosaccharide (m/z 485) has the following structure [α-L-Rhap-(1→ 4)-α-D-GalpA(1→2)-α-L-Rhap]. The last ion of the series at m/ z 1452 is an oligosaccharide of DP 9 with the following structure: [α-L-Rhap-(1→4)-α-D-GalpA(1→2)-]4‑α-L-Rhap. The ion at m/z 545 present in the MS spectrum of the H


Article


Figure 5. UPLC-MS analysis of nonretained fraction D: (a) total ion chromatogram; (b) full-scan mass spectrum; (c) 2D projection of the full-scan mass spectra as a function of oligosaccharide retention times on the HILIC column.

mass spectra of fractions of neutral sugars eluted from a HILIC column, used for the first time for separating wine oligosaccharides, showed a high variety of oligosaccharides (detected as [M − H]−, [M − 2H]2−, and [M − 3H]3− ions) corresponding to arabino-oligosaccharides composed of 8 to 49 arabinose residues, rhamnose substituted with arabinooligosaccharides with a DP between 5 to 21, and different rhamnogalacturonan arabino-oligosaccharides composed of one or two residues of rhamnose, one residue of galacturonic acid, and side chains of 13 to 30 residues of arabinose. The oligosaccharides rich in arabinose are present in significant amounts (185 mg/L) in red wine, and are degradation products originating from the pectic polysaccharides of grape cell walls. Arabinans are known to participate in haze formation by selfaggregation when they have a low degree of branching. However, oligosaccharides rich in arabinan side chains isolated and identified in this study are highly branched with low molecular masses. It would be interesting to determine the impact of these structures on wines, especially their implications in haze development. Arabinans are known to be involved in clarification and stabilization problems of apple juices and concentrates.53 No information is available on the properties of oligosaccharides rich in arabinose. However, our preliminary results show that in a wine model solution, oligosaccharides rich in arabinose can interact with purified tannins and form haze, but these results need to be confirmed. Knowledge of these oligosaccharides should allow us to study their physicochemical properties, their interactions with other

retained fraction F corresponds to the trigalacturonic acid coming from the homogalacturonan backbones of the pectins. These acidic oligosaccharides arise from the rhamnogalacturonan or homogalacturonan regions of pectins as a result of degradation of grape cell wall berries by pectinases during grape maturation and/or wine making. In conclusion, we have isolated and characterized the neutral and acidic oligosaccharide fractions of Carignan red wines by complementary methods: glycosyl composition analysis, glycosidic-linkage analysis, and mass spectrometry. Mass spectrometry is a powerful tool used to identify and determine the structure of the oligosaccharides and in particular those coming from pectins.36 Its use has enabled us to identify the various structures of oligosaccharides present in the fractions isolated from wines. We particularly focused on neutral wine oligosaccharide fractions (nonretained fractions A, B, C, and D), and for the first time, we have characterized by ESI-TOF MS a large number of neutral oligosaccharides rich in arabinose. We also found acid oligosaccharide structures previously characterized in our laboratory.17,18,20 These molecules, neutral and acidic oligosaccharides identified in our previous works, and other oligosaccharides identified in Grignolino and Chardonnay wines by Bordiga et al.19 (hexose-oligosaccharides, xyloglucans, and arabinogalactans), represent the degraded structures of polysaccharides originating from the grape berry cell wall, as a result of endogenous (present in grapes) or exogenous (added by the winemakers) enzyme activities present during the various stages of the wine making. The I


Article


Figure 6. Negative ESI-TOF spectrum of oligosaccharide from retained fractions A, B, C, D, E, and F.

Notes

components present in wine (polyphenols, proteins, and aroma compounds), and possibly their beneficial health effects as prebiotics.

■

The authors declare no competing financial interest.

■

REFERENCES

(1) Riou, V.; Vernhet, A.; Doco, T.; Moutounet, M. Aggregation of grape seed tannins in model wine - effect of wine polysaccharides. Food Hydrocolloids 2002, 16, 17−23. (2) Poncet-Legrand, C.; Doco, T.; Williams, P.; Vernhet, A. Inhibition of grape seed tannin aggregation by wine mannoproteins:

AUTHOR INFORMATION

Corresponding Author

*Tel: +33 499612585; fax: +33 499612857; e-mail: thierry. [email protected]. J


Article

Journal of Agricultural and Food Chemistry Effect of polysaccharide molecular weight. Am. J. Enol. Vitic. 2007, 58, 87−91. (3) Vidal, S.; Courcoux, P.; Francis, L.; Kwiatkowski, M.; Gawel, R.; Williams, P.; Waters, E.; Cheynier, V. Use of an experimental design approach for evaluation of key wine components on mouth-feel perception. Food Quality Preference 2004, 15, 209−217. (4) Gerbaud, V.; Gabas, N.; Laguerie, C.; Blouin, J.; Vidal, S.; Moutounet, M.; Pellerin, P. Effect of wine polysaccharides on the nucleation of potassium hydrogen tartrate in model solutions. Trans. Inst. Chem. Eng. 1996, 74, 782−790. (5) Chalier, P.; Angot, B.; Delteil, D.; Doco, T.; Gunata, Z. Interactions between aroma compounds and whole mannoprotein extract or fractions of mannoproteins isolated from saccharomyces cerevisiae strains. Food Chem. 2007, 100, 22−30. (6) Waters, E. J.; Pellerin, P.; Brillouet, J.-M. Saccharomyces mannoprotein that protects wine from protein haze. Carbohydr. Polym. 1994, 23, 185−191. (7) Moine-Ledoux, V.; Dubourdieu, D. An invertase fragment responsible for improving the protein stability of dry white wines. J. Sci. Food Agric. 1999, 79, 537−543. (8) Dupin, I.; Mc Kinnon, B. M.; Boulay, M.; Markides, A. J.; Jones, G. P.; Williams, J. P.; Waters, E. J. Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: Their release during fermentation and lees contact and a proposal for their mechanism of action. J. Agric. Food Chem. 2000, 48, 3098−3105. (9) Lomolino, G.; Curioni, A. Protein haze formation in white wines: Effect of Saccharomyces cerevisiae cell wall components prepared with different procedures. J. Agric. Food Chem. 2007, 55, 8737−8744. (10) Schmidt, S. A.; Tan, E. L.; Brown, S.; Nasution, U. L.; Pettonilo, F.; MacInttyre, O. J.; Lopes, M. D.; Waters, E. J.; Anderson, P. A. Hpf2 glycan structure is critical for protection against protein haze formation in white wine. J. Agric. Food Chem. 2009, 57, 3308−3315. (11) O’Neill, M. A.; Warrenfeltz, D.; Kates, K.; Pellerin, P.; Doco, T.; Darvill, A. G.; Albersheim, P. Rhamnogalacturonan II, a pectic polysaccharide in the walls of growing plant cells, forms a dimer that is covalently cross-linked by a borate ester. J. Biol. Chem. 1996, 271, 22923−22930. (12) Pellerin, P.; O’Neill, M. A.; Pierre, C.; Cabanis, M. T.; Darvill, A.; Albersheim, P.; Moutounet, M. Le plomb est complexé dans les vins par les dimères de rhamnogalacturonane II, un polysaccharide pectique du raisin. J. Int. Sci. Vigne Vin 1997, 33−41. (13) Andrés-Lacueva, C.; Lamuela-Raventós, R. M.; Buxaderas, S.; de la Torre-Boronat, M. C. Influence of variety and aging on foaming properties of cava (sparkling wine). J. Agric. Food Chem. 1997, 45, 2520−2525. (14) López-Barajas, M.; López-Tamames, E.; Buxaderas, S.; TorreBoronat, M. C. Effect of vinification and variety on foam capacity of wine. Am. J. Enol. Vitic. 1998, 49, 397−402. (15) Abdallah, Z.; Aguié-Béghin, V.; Abou-Saleh, K.; Douillard, R.; Bliard, C. Isolation and analysis of macromolecular fractions responsible for the surface properties in native Champagne wines. Food Res. Int. 2010, 43, 982−987. (16) Martínez-Lapuente, L.; Guadalupe, Z.; Ayestarán, B.; OrtegaHeras, M.; Pérez-Magariño, S. Changes in polysaccharide composition during sparkling wine making and aging. J. Agric. Food Chem. 2013, 61, 12362−12373. (17) Ducasse, M.-A.; Williams, P.; Meudec, E.; Cheynier, V.; Doco, T. Isolation of Carignan and Merlot red wine oligosaccharides and their characterization by ESI-MS. Carbohydr. Polym. 2010, 79, 747− 754. (18) Ducasse, M.-A.; Williams, P.; Canal-Llauberes, R.-M.; Mazerolles, G.; Cheynier, V.; Doco, T. Effect of macerating enzymes on the oligosaccharide profiles of merlot red wines. J. Agric. Food Chem. 2011, 59, 6558−6567. (19) Bordiga, M.; Travaglia, F.; Meyrand, M.; German, J. B.; Lebrilla, C. B.; Coisson, J. D.; Arlorio, M.; Barile, D. Identification and characterization of complex bioactive oligosaccharides in white and red wine by a combination of mass spectrometry and gas chromatography. J. Agric. Food Chem. 2012, 60 (14), 3700−3707.

(20) Apolinar-Valiente, R.; Williams, P.; Mazerolles, G.; RomeroCascales, I.; Gómez-Plaza, E.; López-Roca, J. M.; Ros-García, J. M.; Doco, T. Effect of enzyme additions on the oligosaccharide composition of Monastrell red wines from four different wine-growing origins in Spain. Food Chem. 2014, 156, 151−159. (21) Quijada-Morín, N.; Williams, P.; Rivas-Gonzalo, J. C.; Doco, T.; Escribano-Bailón, M. T. Polyphenolic, polysaccharide and oligosaccharide composition of Tempranillo red wines and their relationship with the perceived astringency. Food Chem. 2014, 154, 44−51. (22) Pellerin, P.; Cabanis, J. C. Les Glucides. In Elements d’œnologie. Fondements scientifiques et techniques; Technique et Documentation; Flanzy, C., Ed.; Lavoisier: Paris, 1998; pp 40−93. (23) Doco, T.; Quellec, N.; Moutounet, M.; Pellerin, P. Polysaccharide patterns during the aging of Carignan noir red wines. Am. J. Enol. Vitic. 1999, 50 (1), 25−32. (24) Ayestaran, B.; Guadalupe, Z.; Leon, D. Quantification of major grape polysaccharides (Tempranillo v.) released by maceration enzymes during the fermentation process. Anal. Chim. Acta 2004, 513 (1), 29−39. (25) Guadalupe, Z.; Palacios, A.; Ayestaran, B. Maceration enzymes and mannoproteins: A possible strategy to increase colloidal stability and color extraction in red wines. J. Agric. Food Chem. 2007, 55 (12), 4854−4862. (26) Doco, T.; Williams, P.; Cheynier, V. Effect of flash release and pectinolytic enzyme treatments on wine polysaccharide composition. J. Agric. Food Chem. 2007, 55, 6643−6649. (27) Ducasse, M.-A.; Canal-Llauberes, R.-M.; Lumley, M. D.; Williams, P.; Souquet, J.-M.; Fulcrand, H.; Doco, T.; Cheynier, V. Effect of macerating enzyme treatment on the polyphenol and polysaccharide composition of red wines. Food Chem. 2010, 118, 369− 376. (28) Qiang, X.; YongLie, C.; QianBing, W. Health benefit application of functional oligosaccharides. Carbohydr. Polym. 2009, 77, 435−441. (29) Darvill, A. G.; Albersheim, P. Phytoalexins and their elicitors-A defense against microbial infection in plants. Annu. Rev. Plant Physiol. 1984, 155, 507−516. (30) Broekaert, W. F.; Courtin, C. M.; Verbeke, K.; Van de Wiele, T.; Verstraete, W.; Delcour, J. A. Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and xylooligosaccharides. Crit. Rev. Food Sci. Nutr. 2011, 51, 178−194. (31) Cescutti, P.; Rizzo, R. Divalent cation interactions with oligogalacturonides. J. Agric. Food Chem. 2001, 49, 3262−3267. (32) Duarte, F. I.; Godejohann, M.; Braumann, U.; Spraul, M.; Gil, A. M. Application of NMR spectroscopy and LC-NMR/MS to the identification of carbohydrates in beer. J. Agric. Food Chem. 2003, 51 (17), 4847−4852. (33) Reis, A.; Coimbra, M. A.; Domingues, P.; Ferrer-Correia, A. J.; Domingues, M. R. M. Fragmentation pattern of underivatised xylooligosaccharides and their alditol derivatives by electrospray tandem mass spectrometry. Carbohydr. Polym. 2004, 55, 401−409. (34) Quemener, B.; Ordaz-Ortiz, J. J.; Saulnier, L. Structural characterization of underivatized arabino-xylo-oligosaccharides by negative-ion electrospray mass spectrometry. Carbohyd. Res. 2006, 341, 1834−1847. (35) Fernández, L. E. M. Introduction to ion trap mass spectrometry: Application to the structural characterization of plant oligosaccharides. Carbohyd. Polymers 2007, 68, 797−807. (36) Ralet, M.-C.; Lerouge, P.; Quéméner, B. Mass spectrometry for pectin structure analysis. Carbohyd. Res. 2009, 334, 1798−1807. (37) Brillouet, J.-M.; Moutounet, M.; Escudier, J.-L. Fate of yeast and grape pectic polysaccharides of a young red wine in the cross-flow microfiltration process. Vitis 1989, 28, 49−63. (38) Harris, P. J.; Henri, R. J.; Blakeney, A. B.; Stone, B. A. An improved method for the methylation analysis of oligosaccharides and polysaccharides. Carbohyd. Res. 1984, 127 (1), 59−73. (39) Hilz, H.; Williams, P.; Doco, T.; Schols, H. A.; Voragen, A. G. J. The pectic polysaccharide rhamnogalacturonan II is present as a dimer in pectic populations of bilberries and black currants in muro and in juice. Carbohyd. Polym. 2006, 65 (4), 521−528. K


Article

Journal of Agricultural and Food Chemistry (40) Doco, T.; O’Neill, M. A.; Pellerin, P. Determination of the neutral and acidic glycosyl residue compositions of plant cell polysaccharides by GC-EI-MS analysis of the trimethylsilyl methyl glucoside derivatives. Carbohydr. Polym. 2001, 46, 249−259. (41) Hakomori, S. I. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem. (Tokyo) 1964, 55, 205−208. (42) Pellerin, P.; Vidal, S.; Williams, P.; Brillouet, J.-M. Characterization of five type II arabinogalactan-protein complexes from red wine with increasing uronic acid content. Carbohyd. Res. 1995, 190, 183− 197. (43) Vidal, S.; Williams, P.; Doco, T.; Moutounet, M.; Pellerin, P. The polysaccharides of red wine: Total fractionation and characterisation. Carbohydr. Polym. 2003, 54, 439−447. (44) Schols, H. A.; Voragen, A. G. J.; Colquhoun, I. J. Hairy (ramified) regions of pectins. Isolation and characterization of rhamnogalacturonan oligomers, liberated during degradation of pectic hairy regions by rhamnogalacturonase. Carbohydr. Res. 1994, 256, 97− 111. (45) Mohnen, D. Pectin structure and biosynthesis. Curr. Opin. Plant Biol. 2008, 11 (3), 266−277. (46) Arnous, A.; Meyer, A. S. Quantitative prediction of cell wall polysaccharide composition in grape (Vitis vinifera L.) and apple (Malus domestica) skins from acid hydrolysis monosaccharide profiles. J. Agric. Food Chem. 2009, 57 (9), 3611−3619. (47) Liu, Y.; Urgaonkar, S.; Verkade, J. G.; Armstrong, D. W. Separation and characterization of underivatized oligosaccharides using liquid chromatography and liquid chromatography−electrospray ionization mass spectrometry. J. Chromatogr. A 2005, 1079, 146−152. (48) Leijdekkers, A. G. M.; Sanders, M. G.; Schols, H. A.; Gruppen, H. Characterizing plant cell wall derived oligosaccharides using hydrophilic interaction chromatography with mass spectrometry detection. J. Chromatogr. 2011, 1218 (51), 9227−9235. (49) Fu, Q.; Liang, T.; Li, Z.; Xu, X.; Ke, Y.; Jin, Y.; Liang, X. Separation of carbohydrates using hydrophilic interaction liquid chromatography. Carbohyd. Res. 2013, 379, 13−17. (50) Domon, B.; Costello, C. E. Asystematic nomenclature for carbohydrate fragmentation in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate J. 1988, 5, 397−409. (51) Vidal, S.; Williams, P.; O’Neill, M. A.; Pellerin, P. Polysaccharides from grape berry cell walls. Part I: Tissue distribution and structural characterization of the pectic polysaccharides. Carbohydr. Polym. 2001, 45, 315−323. (52) Belleville, M. P.; Williams, P.; Brillouet, J.-M. A linear arabinan from a red wine. Phytochemistry 1993, 33, 227−229. (53) Mehrlander, K.; Dietrich, H.; Sembries, S.; Dongowski, G.; Will, F. Structural characterization of oligosaccharides and polysaccharides from apple juices produced by enzymatic pomace liquefaction. J. Agric. Food Chem. 2002, 50, 1230−1236.

L


Complex Carbohydrates of Red Wine: Characterization of the Extreme

Recommend Documents