Effect of winemaking techniques on polysaccharide composition of Cabernet Sauvignon, Syrah and Monastrell red wines

Effect of winemaking techniques on polysaccharidecomposition of Cabernet Sauvignon, Syrah

and Monastrell red wines

R. APOLINAR-VALIENTE1, I. ROMERO-CASCALES1, P. WILLIAMS2, E. GÓMEZ-PLAZA1,J.M. LÓPEZ-ROCA1, J.M. ROS-GARCÍA1 and T. DOCO2

1 Departamento de Tecnología de Alimentos, Nutrición y Bromatología, Facultad de Veterinaria,Universidad de Murcia, 30100 Murcia, Spain

2 INRA, Joint Research Unit 1083 Sciences for Enology, 2 Place Viala, F-34060 Montpellier, FranceCorresponding author: Dr Thierry Doco, email [email protected]

AbstractBackground and Aims: Several authors have demonstrated the interesting properties of wine polysaccharides.These compounds act as protective colloids and are able to interact with tannins and anthocyanins in wines, reducingtheir reactivity and increasing colour stability. Little, however, is known about the release of polysaccharidesby winemaking technologies. We examine the effect of several winemaking techniques – cold prefermentativemaceration, dry ice addition and grape skin freezing, and addition of two maceration enzymes – on the quantityand composition of polysaccharides extracted from the three red wine cultivars – Cabernet Sauvignon, Syrah andMonastrell.Methods and Results: The molecular mass distribution and composition of polysaccharides were determined,respectively, by high-performance size-exclusion chromatography and by gas chromatography. The amount ofsome polysaccharide fractions extracted depended on the grape cultivar. The addition of commercial pectic enzymepreparations released a greater quantity of polysaccharides in all three cultivars studied and altered thepolysaccharide composition of the Cabernet Sauvignon wine. The effect of the other treatments on the amount ofpolysaccharides depended on the cultivar.Conclusion: This study confirms that grape cultivar and winemaking technique have a significant impact on thequantity and composition of polysaccharides extracted from grapes into wine. The concentration of polysaccharidesrich in arabinose and galactose, and in rhamnogalacturonan II was greater in the Syrah wine than that in theCabernet Sauvignon and Monastrell wines. Both enzymatic treatments and also dry ice addition had a significantinfluence on the polysaccharide concentration and composition of the wines made from a given cultivar, whereascold prefermentative maceration or grape skin freezing had no effect.Significance of the Study: This is the first report that shows that the polysaccharide composition of CabernetSauvignon, Syrah and Monastrell wines is affected by winemaking technique.

Keywords: cultivar, pectic enzyme, polysaccharide, refrigeration technique, wine

IntroductionPolysaccharides that are derived from microorganisms and cellwalls of grapes are included in the macromolecular fraction ofwines. The polysaccharide fraction in wine has been identifiedin previous studies (Pellerin et al. 1996, Vidal et al. 2000, 2003,Ayestarán et al. 2004) and can be grouped into three majorfamilies. Two of these families [polysaccharides rich in arabinoseand galactose (PRAGs) and rhamnogalacturonan II (RG-II)]originate in the pectocellulosic cell walls of grape berries (Docoet al. 2003, Vidal et al. 2003, Ayestarán et al. 2004), whilethe other family comprises yeast-derived mannoproteins(MPs) (Pellerin and Cabanis 1998) released during fermentation(Pellerin et al. 1996, Vidal et al. 2003, Ayestarán et al. 2004) andduring the aging of wines on lees (Doco et al. 2003). The impactof these macromolecules on the chemical and sensory propertiesof wine has been largely confirmed; they are known to haveseveral properties, including acting as protective agents in the

prevention of protein haze in white wine (Waters et al. 1994)and in tartrate stability (Gerbaud et al. 1997). Polysaccharidesare able to interact and aggregate with phenolic substances(Saucier et al. 1997, Riou et al. 2002), and it has been shownthat the addition of arabinogalactan proteins (AGPs), MPs orRG-II to a wine increases the ‘fullness’ sensation (Vidal et al.2004). Escot et al. (2001) found that some polysaccharidesincreased the colour stability of wines.

The origin of the grape-derived polysaccharides is mainlythe grape cell walls. In intact grapes, the skin cell wall forms abarrier against the diffusion of anthocyanins, the compoundslargely responsible for wine colour (Vidal et al. 2001), which iswhy some winemaking techniques are specifically designed tofavour cell wall degradation, because this may affect the winepolysaccharide content.

The influence of exogenous pectic enzymes on the amount ofphenolic substances especially anthocyanins, in wines has been

62 Polysaccharides of wines from different varieties Australian Journal of Grape and Wine Research 20, 62–71, 2014

doi: 10.1111/ajgw.12048© 2013 Australian Society of Viticulture and Oenology Inc.

mailto:[email protected]

reported by several authors (Clare et al. 2002, Arnous and Meyer2010, Ducasse et al. 2010). There are fewer studies, however,on the efficiency of polysaccharide extraction using pecticenzymes, a winemaking treatment that can modify the amountof polysaccharide released in red wines (Doco et al. 2007,Guadalupe et al. 2007). It has been observed that the addition ofcommercial enzymes to musts increased the quantity of total acidand neutral polysaccharides (Ducruet et al. 2000), while Docoet al. (2007) and Ducasse et al. (2010) observed an increase inRG-II and a decrease in PRAGs when enzymes were added.

Barnavon et al. (2000) observed during grape ripening anincrease in β-galactosidase activity, which could be involved inthe hydrolysis of galactose residues of polysaccharide chainsfrom AGPs (Kotake et al. 2005). We have found, however, nostudies relating the polysaccharide composition of wines withβ-galactosidase addition during winemaking.

Low temperature techniques are another tool for degradingthe cell wall and for achieving greater extraction of pheno-lic substances and polysaccharides. The ‘cold’ needed can beobtained in several ways: cold prefermentative macerationfavours the selective diffusion of the water-soluble compoundsin aqueous media (Sacchi et al. 2005); addition of dry ice atthe beginning of the fermentation favours the release ofpolysaccharides and phenolic substances (Zamora 2004);and grape skin freezing provides an easy exit for pigments(Couasnon 1999). The effect of these low temperature tech-niques on the polysaccharide composition of wines has not beenspecifically studied, although the effect on phenolic substancesand aromas in wines has been demonstrated.

Few studies have been made on the influence of thecombination of cultivar and winemaking technique on thepolysaccharide composition of wines. In the present study,Cabernet Sauvignon, Syrah and Monastrell grapes were vinifiedby five winemaking techniques: cold prefermentative macera-tion, dry ice addition, grape skin freezing, and the addition ofβ-galactosidase and of a commercial maceration enzyme; theresulting wines were analysed for the impact of these tech-niques on the polysaccharide composition of the wines of thethree cultivars.

Materials and methods

GrapesVitis vinifera cvs Cabernet Sauvignon, Syrah and Monastrellwere grown in three plots located near Murcia in southeasternSpain, and the grapes were harvested at commercial ripenessduring the 2007 vintage. The physicochemical characteristics ofthe grape berry samples are shown in Table 1.

Preparation of the control winesThree 90 kg lots of grapes from each of the three cultivars weredestemmed and crushed. The crush grapes were macerated in

100 L stainless steel tanks to yield triplicate control lots codedCACO (Cabernet Sauvignon), SYCO (Syrah) and MOCO(Monastrell). At the same time, bisulfite (8 g/100 kg grapes) wasadded. This was the basic winemaking procedure followed forall wines produced with the treatments described later.

Cold prefermentative macerationTanks containing the crushed grapes were held in a chamber at10°C for 10 days before being transferred to the winery at 25°C.These wines were coded CACM, SYCM and MOCM.

Dry ice additionDry ice (−78°C) was added at a rate of 100 kg per tank directlyinto the tanks and mixed with the crushed grapes. The dry icekept the must frozen for 3 days at a temperature lower than−3°C. The resultant wines were coded CAIA, SYIA and MOIA.

Grape skin freezingThree 90-kg lots of grapes were frozen in three steps: first,grapes were cooled overnight at 5°C and then cooled at −4°C for12 h and finally at −10°C (Syrah grapes for 3 h, and CabernetSauvignon and Monastrell grapes for 5 h). The resulting wineswere coded CAFR, SYFR and MOFR.

Commercial enzyme additionCommercial enzyme preparation was added to the tanks(3 g/100 kg), and the resulting wines were coded CACE, SYCEand MOCE. The enzyme contained the following activitiesas described by the supplier (Agrovin Company, Alcázar de SanJuan, Spain): polygalacturonase activity, 546.6 IU/g; pectineste-rase activity, 7.3 IU/g; pectin lyase activity, 2.8 IU/g; andβ-glucanase activity (179.6 IU/g).

Galactosidase enzyme additionInstead of the commercial enzyme, a mixed enzyme consistingof α- and β-galactosidase (Agrovin Company, Alcázar de SanJuan, Spain) was added to the tanks (1 g/100 kg). The wineswere coded CAGE, SYGE and MOGE.

FermentationAll samples of crushed grapes were fermented out in 100-Lstainless steel tanks, equipped with temperature control (25°C);fermentations were initiated with Levuline Gala (commercialdry Saccharomyces cerevisiae yeast, OenoFrance, Bordeaux,France) inoculated at 10 g/hL.). Each lot was fermented tocompletion [monitored by reducing sugar analysis using theofficial method of the Organisation Internationale de la Vigne etdu Vin (1990)], at which time each lot was pressed at 150 kPa ina 75-L tank membrane press (Hidro 80L, Ausavil, Spain). Free-run and press wines of each trial were combined and storedin 50-L tanks. One month later, the wines were racked. Afterspontaneous malolactic fermentation, the wines were rackedagain, and 25 mg/L of sulfur dioxide was added. The wines werenot clarified or filtered but cold-stabilised (−3°C) for 1 month,and bottled and stored in the experimental wine cellar at 18°Cuntil analysis.

Wine analysisThe dry extract, alcohol content, and total and volatile acidity ofthe wines were determined according to the official methods ofthe European Commission (1990).

Isolation of total soluble polysaccharides from winesWine polysaccharides were isolated as previously described(Vidal et al. 2003). An aliquot (2.5 mL) of each wine wasevaporated in a centrifugal evaporator (EZ-2, Genevac, Ipswich,

Table 1. Physicochemical characteristics of the CabernetSauvignon, Syrah and Monastrell grapes.

Cultivar Total solublesolids (°Bé)

Titratableacidity (g/L)†

pH

Cabernet Sauvignon 13.9 ± 0.4b 2.9 ± 0.2a 3.9 ± 0.1a

Syrah 14.3 ± 0.7c 3.2 ± 0.3b 3.8 ± 0.1a

Monastrell 13.3 ± 0.6a 3.3 ± 0.2b 3.9 ± 0.1a

†g/L tartaric acid. Different letters within the same column represent significantdifferences according to a least significant difference test (P < 0.05).

Apolinar-Valiente et al. Polysaccharides of wines from different varieties 63

© 2013 Australian Society of Viticulture and Oenology Inc.

England), and the residue was dissolved in 0.5-mL water toobtain wine concentrated five times. A 2.66-mL aliquot ofethanol (95%) acidified by 0.5% hydrochloric acid, was added toobtain a final concentration of 80% ethanol. After one night at4°C, the wine polysaccharides were precipitated, and the super-natant was eliminated after centrifugation (16 500 × g, 10 min).The pellet, which corresponds to total wine colloids, was dis-solved in 1 mL of H2O (Millipore, Billerica, MA, USA). Theoligosaccharides and salts contained in the total colloids wereeliminated by retention on an ion exchange column (4 mL mixedresin: Mix Bed Resin AG 501-X8, Bio-Rad Laboratories, Inc.,Hercules, CA, USA). Wine polysaccharides that were not retainedwere eluted by 2.5 bed volumes of H2O. Total solublepolysaccharides were obtained after freeze-drying of the water-eluted materials.

Analysis of polysaccharidesThe molecular mass (MM) distribution of wine polysaccharideswas established by high-performance size-exclusion chromatog-raphy (HPSEC), the system being composed of a 234-Gilsonsampling injector (Gilson S.A.S., Villiers le Bel, France) andan LC-10 AS Shimadzu pump (Shimadzu Corporation, Kyoto,Japan). Polysaccharides were eluted from HPSEC on two serialShodex OHPAK KB-803 and KB-805 columns (0.8 × 30 cm;Showa Denko K.K., Tokyo, Japan) connected to an ERC-7512refractometer (Erma, Inc., Tokyo, Japan), at a 1 mL/min flowrate in 0.1 M LiNO3. The apparent MM of the polysaccharideswas calculated from the calibration curve established witha Pullulan calibration kit (P-400, MM = 380 000; P-200,MM = 186 000; P-100, MM = 100 000; P-50, MM =48 000; P-20, MM = 23 700; P-10, MM = 12 200; P-5, MM =5800; Showa Denko). The calibration equation was logMM = 28.321 − 1.04 × tR (tR = column retention time at peakmaximum, and r2 = 0.997).

Neutral monosaccharides were released after hydrolysisof the wine polysaccharides by treatment with 2 mol/Ltrifluoroacetic acid for 75 min at 120°C (Albersheim et al.1967). They were then converted to the correspondingalditol acetate derivatives by reduction and acetylation, andquantified by gas chromatography (GC) analysis using a fusedsilica DB-225 (210°C) capillary column (30 m × 0.32 mm i.d.,0.25 μm film), with hydrogen as the carrier gas, on a Hewlett-Packard Model 5890 gas chromatograph (Hewlett-Packard, PaloAlto, CA, USA). The different alditol acetates were identifiedfrom their retention time by comparison with that of standardmonosaccharides. Neutral sugar amounts were calculated rela-tive to the internal standard (myoinositol).

Polysaccharide concentrationThe polysaccharide composition of each wine was estimatedfrom the concentration of individual glycosyl residues, as deter-mined by GC after hydrolysis, reduction and acetylation. Cal-culation of wine polysaccharide concentration took into accountthe composition of characteristic monosaccharides as well as thehydrolysis yield (Vidal et al. 2001).

Statistical data treatmentAverage values, standard deviation and statistical significanceaccording to a least significant difference (LSD) test (P < 0.05)were calculated and undertaken with the package StatgraphicsPlus 5.1 (Statpoint Technologies, Inc., Warrenton, VA, USA).

Results and discussion

Composition of Cabernet Sauvignon, Syrah and Monastrell winesThe composition of the Cabernet Sauvignon, Syrah andMonastrell wines was within the normal value range of dry

young wines (Table 2), although some differences betweencultivars and treatments were observed. For example, thedry extract increased in wines when the commercial enzyme(Cabernet Sauvignon) and the galactosidase enzyme (Syrah)were used. This behaviour may reflect greater cell wall degrada-tion with these treatments (see later).

Cabernet Sauvignon wines showed a higher alcohol contentwhen the five treatments were applied. Gardner et al. (2011)detected a 1.5% increase in the ethanol content when coldprefermentative maceration was applied. In contrast, Doco et al.(2007) and Guadalupe et al. (2007) observed no differences inthe ethanol produced when enzymatic treatments were appliedcompared with that of the control.

Effect of the cultivar on the polysaccharide content of CabernetSauvignon, Syrah and Monastrell winesThe MM distribution of wine polysaccharides was studied byHPSEC (Figure 1). The population eluting between 14 and16.5 min corresponded mainly to the MPs released from yeastduring fermentation (Waters et al. 1994, Doco et al. 1996, 2007,Ducasse et al. 2010). A second population that eluted between16.5 and 18 min corresponded to a complex mixture of AGPs,arabinans and a few MPs (Pellerin et al. 1996, Doco et al. 2003,Vidal et al. 2003, Ducasse et al. 2010). The third peak, whicheluted between 18 and 19.2 min corresponded mainly to RG-IIwith PRAGs and MPs of lower MM (Pellerin et al. 1996, Docoet al. 2003, Vidal et al. 2003, Ducasse et al. 2010). Thepolysaccharides isolated from the wines in this work show adistribution similar to that described in the literature, forexample in Carignan (Doco et al. 1999), Tempranillo (Ayestaránet al. 2004) and Merlot (Ducasse et al. 2010) wines.

In this study, all winemaking processes that could influencethe amount of MPs, PRAGs and RG-II were similar. The profilesof Cabernet Sauvignon (CACO), Syrah (SYCO) and Monastrell(MOCO) control samples differed;, Monastrell clearly showinga higher peak corresponding to the first population (MP), andSyrah two high peaks corresponding to the second (PRAGs) andthird (RG-II) populations. This may reflect higher polysaccharidecontent or the easier degradation of cell walls in Syrah grapescompared with that in Cabernet Sauvignon and Monastrellgrapes.

Table 3 presents the glycosyl residue composition of thepolysaccharides. The presence of all the known neutral sugars(mannose, rhamnose, arabinose, galactose and fucose) comingfrom the pectocellulosic cell walls of grapes and from yeastpolysaccharides confirms the presence of mannan-, arabino-galactan-, homogalacturonan- and rhamnogalacturonan-likestructures in the polysaccharides in the red wines studied (Docoet al. 1996, 2003, Pellerin et al. 1996, Vidal et al. 2000, 2003,Ayestarán et al. 2004). The presence of xylose residues indicatesthat traces of hemicelluloses might be solubilised from grape berrycell walls (Carpita and Gibeaut 1993). The identification of severalrare sugars, such as apiose, 2-O-methyl-fucose and 2-O-methyl-xylose, indicates the presence of RG-II (Pellerin et al. 1996, Docoet al. 2003). Glucose is not known as a component of pecticpolysaccharides but may arise from microbial polysaccharides oranthocyanins. These results agree with those obtained by Ducasseet al. (2010), who also found mannose, galactose and arabinose tobe the main sugars of the wine polysaccharides.

Our data pointed to a significantly higher quantity of severalsugars (2-O-methyl-fucose, galactose and glucose) in the Syrahwine than that in the wines from the other two cultivars. TheCabernet Sauvignon wines had an amount of mannose lowerthan that of the Syrah and Monastrell wines. Doco et al. (2007)



also found a difference between the polysaccharide sugars fromdifferent cultivars (Carignan and Grenache).

The concentration of MPs, PRAGs and RG-II in control winesis shown in Figure 2, and it was estimated from the concentra-tion of individual glycosyl residues, as determined by GC afterhydrolysis, reduction and acetylation. All the mannose can beattributed to yeast MPs (Waters et al. 1994). The sum of galactoseand arabinose residues was used to estimate PRAGs, representingmainly AGPs, arabinogalactans and arabinans in wines. Theconcentration of RG-II is calculated from that of 2-O-methyl-fucose and 2-O-methyl-xylose. Our results are coherent with

those obtained by other authors (Vidal et al. 2001, Ayestaránet al. 2004, Doco et al. 2007, Ducasse et al. 2010).

The MP concentration was lower in Cabernet Sauvignonwines (CACO: 154 mg/L) than in Syrah and Monastrell wines(SYCO: 277 mg/L and MOCO: 331 mg/L). Considering that theyeast strain used in all wines was the same and that all themannose can be attributed to yeast MPs (Waters et al. 1994),the higher MP amounts observed in Cabernet Sauvignon winescould be caused by changes in yeast conditions, such as thedifferent alcohol content of the wines. Several factors, such asthe winemaking conditions (Ribéreau-Gayon et al. 2000) or theinitial colloid content in must (Rosi and Giovani 2003, O’Neillet al. 2004), are related with the MPs released by yeasts. Docoet al. (2007) found a higher concentration of MPs in Carignanwines than in Grenache wines probably because of differentripening degrees at harvest implying a better yeast fermentationwith the release of micronutrients.

The concentration of PRAG was significantly higher inSyrah (SYCO: 242 mg/L) than in Cabernet Sauvignon (CACO:188 mg/L) and Monastrell (MOCO: 151 mg/L) wines. Syrahwines also showed a statistically higher amount of RG-II (SYCO:282 mg/L) compared with that of Cabernet Sauvignon (CACO:224 mg/L) and Monastrell (MOCO: 186 mg/L) wines. Thedata obtained confirm the interpretation of MM distributionprofiles (Figure 1). Ortega-Regules et al. (2008a) detectedβ-galactosidase activity in Monastrell grape skins lower thanthat in Cabernet Sauvignon and Syrah skins, potentiallyexplaining the lower PRAG amount released into wines. Docoet al. (2007) found differences in PRAG and RG-II concentrationbetween different wines (Carignan and Grenache) and attrib-uted these differences to different ripening states at harvest.

Table 2. Composition of Cabernet Sauvignon, Syrah and Monastrell wines.

Cultivar/treatment Experimentalnomenclature

Dry extract(g/L)

Alcohol(% v/v)

pH Titratableacidity (g/L)†

Volatile acidity(g/L)‡

Cabernet Sauvignon

Control CACO 28.9 ± 0.3a 12.9 ± 0.1a 3.7 ± 0.0a 4.7 ± 0.3a 0.35 ± 0.04a

Prefermentative cold maceration CACM 28.9 ± 0.3a 14.1 ± 0.2b 3.5 ± 0.1a 5.2 ± 0.4a 0.25 ± 0.01a

Dry ice addition CAIA 28.9 ± 0.2a 14.3 ± 0.1b 3.5 ± 0.1a 5.2 ± 0.4a 0.19 ± 0.02a

Frozen grape skin CAFR 28.4 ± 0.3a 13.8 ± 0.5b 3.6 ± 0.1a 4.7 ± 0.3a 0.28 ± 0.05a

Commercial enzyme addition CACE 31.1 ± 0.4b 14.2 ± 0.2b 3.5 ± 0.1a 5.6 ± 0.4a 0.27 ± 0.02a

Galactosidase enzyme addition CAGE 28.4 ± 0.1a 14.0 ± 0.2b 3.5 ± 0.1a 5.6 ± 0.4a 0.27 ± 0.07a

Syrah

Control SYCO 31.6 ± 1.3ab 15.4 ± 0.6ab 3.5 ± 0.1a 5.7 ± 0.2a 0.38 ± 0.05b

Prefermentative cold maceration SYCM 27.7 ± 0.4a 14.4 ± 0.1a 3.4 ± 0.1a 5.5 ± 0.1a 0.38 ± 0.05b

Dry ice addition SYIA 29.2 ± 1.2a 14.7 ± 0.4a 3.3 ± 0.1a 6.6 ± 0.6a 0.18 ± 0.02a

Frozen grape skin SYFR 34.2 ± 4.5ab 15.8 ± 1.1b 3.5 ± 0.1a 6.0 ± 0.4a 0.20 ± 0.03a

Commercial enzyme addition SYCE 38.2 ± 3.3b 16.2 ± 0.3b 3.5 ± 0.1a 5.8 ± 0.3a 0.38 ± 0.05b

Galactosidase enzyme addition SYGE 46.1 ± 8.6c 16.2 ± 0.2b 3.5 ± 0.0a 6.1 ± 0.4a 0.40 ± 0.06b

Monastrell

Control MOCO 28.4 ± 0.4a 14.1 ± 0.5a 3.5 ± 0.1a 5.6 ± 0.3a 0.38 ± 0.03a

Prefermentative cold maceration MOCM 25.5 ± 0.7a 13.6 ± 0.4a 3.4 ± 0.1a 5.7 ± 0.4a 0.44 ± 0.03a

Dry ice addition MOIA 29.1 ± 2.6a 14.1 ± 0.5a 3.5 ± 0.1a 5.8 ± 0.4a 0.43 ± 0.02a

Frozen grape skin MOFR 26.8 ± 0.5a 13.4 ± 0.4a 3.4 ± 0.1a 5.7 ± 0.2a 0.40 ± 0.03a

Commercial enzyme addition MOCE 27.5 ± 0.5a 13.4 ± 0.2a 3.4 ± 0.1a 5.5 ± 0.4a 0.41 ± 0.04a

Galactosidase enzyme addition MOGE 25.9 ± 2.5a 13.0 ± 0.5a 3.4 ± 0.1a 5.5 ± 0.3a 0.46 ± 0.02a

†g/L tartaric acid. ‡g/L acetic acid. Different letters within the same cultivar column represent significant differences according to a least significant difference test(P < 0.05).

Figure 1. Molecular mass distribution of polysaccharides inCabernet Sauvignon ( ), Syrah ( ) and Monastrell ( ) controlwines.



Tabl

e3.

Glyc

osyl

resi

due

com

posi

tion

ofpo

lysa

ccha

rides

from

Cabe

rnet

Sauv

igno

n,Sy

rah

and

Mon

astre

llw

ines

.

Cu

ltiv

ar/t

reat

men

tG

lyco

syl

resi

du

eco

mp

osit

ion

(mg/

L)

2-O

MeF

uc

Rh

aFu

c2-

OM

eXyl

Ara

Ap

iX

ylM

anG

alG

lc

Cab

erne

tSa

uvig

non

CA

CO

1.6

±0.

0†a

14.9

±1.

7a1.

3±

0.5a

1.9

±0.

5b79

.2±

10.5

a1.

9±

0.5a

1.6

±0.

0a12

3.5

±35

.7a

90.4

±15

.2a

32.3

±9.

4a

CA

CM

1.6

±0.

0a12

.5±

4.4a

1.3

±0.

5a1.

1±

0.5a

b68

.8±

7.9a

1.3

±0.

5a1.

9±

0.9a

133.

3±

25.1

a92

.0±

20.1

a29

.1±

14.4

a

CA

IA2.

1±

0.9a

13.2

±2.

6a1.

3±

0.5a

0.7

±0.

2a72

.2±

4.2a

1.9

±0.

5a1.

7±

0.6a

132.

1±

20.7

a94

.1±

16.4

a30

.1±

11.4

a

CA

FR1.

1±

0.5a

10.8

±3.

0a1.

6±

0.0a

1.1

±0.

5ab

61.3

±15

.0a

1.6

±0.

8a1.

3±

0.5a

99.6

±18

.0a

81.7

±25

.8a

26.9

±10

.4a

CA

CE

5.2

±1.

4b39

.7±

12.4

b3.

1±

1.0b

3.7

±1.

2c15

9.1

±29

.4a

2.4

±2.

1a6.

8±

0.7b

121.

5±

13.0

a14

0.3

±27

,0a

30.5

±18

.1a

CA

GE

1.4

±0.

2a12

.7±

2.2a

1.1

±0.

5a1.

1±

0.5a

b64

.4±

8.3a

1.6

±0.

0a1.

9±

0.5a

123.

6±

17.5

a96

.0±

14.4

a33

.3±

13.0

a

Syra

h

SYC

O2.

5±

0.2a

b20

.3±

0.5b

2.1

±0.

5a1.

9±

0.2a

b78

.9±

1.8a

2.1

±0.

5a4.

5±

0.5c

221.

9±

12.0

a13

3.3

±4.

6bc

56.3

±5.

1a

SYC

M3.

2±

0.8b

c21

.1±

5.1b

2.3

±0.

8a2.

0±

0.4b

111.

7±

42.3

a1.

9±

0.9a

4.1

±1.

3bc

176.

3±

36.2

a13

3.3

±22

.2bc

34.0

±16

.4a

SYIA

2.0

±0.

4a10

.8±

2.3a

1.7

±0.

2a1.

0±

0.5a

45.7

±6.

9a1.

5±

0.2a

2.7

±0.

5a19

9.3

±59

.9a

83.9

±9.

4a42

.5±

18.4

a

SYFR

2.1

±0.

5a14

.0±

3.2a

1.7

±0.

6a1.

5±

0.8a

b94

.3±

43.0

a2.

0±

1.1a

3.1

±1.

0ab

175.

1±

42.0

a11

2.8

±28

.5b

27.7

±13

.5a

SYC

E4.

5±

0.5d

26.4

±1.

4c2.

4±

0.0a

b2.

9±

0.5c

115.

2±

25.8

a2.

7±

0.5a

5.3

±0.

5c18

9.3

±47

.2a

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Table 4 presents the arabinose/galactose ratio, a characteris-tic ratio of wine PRAGs (Ducasse et al. 2010), which has beenreported to be affected by the degradation of wine type II AGPsor arabinans during the aging of wines on lees (Doco et al. 2003)

or by the release of polysaccharides rich in arabinose-likearabinans (Belleville et al. 1993). The ratio was higher in theCabernet Sauvignon wines than in the other two cultivars,suggesting greater release of arabinose or polysaccharides richin arabinose. This could be linked with the observations ofOrtega-Regules et al. (2008b), who found a higher concentra-tion of arabinose in Cabernet Sauvignon grape skin than inSyrah and Monastrell grape skins.

Effect of the winemaking treatments on the polysaccharideconcentration of Cabernet Sauvignon, Syrah andMonastrell winesFigure 3 presents the MM distribution of the winepolysaccharides measured in the wines obtained by the coldprefermentative maceration, dry ice addition, grape skin freez-ing, β-galactosidase and commercial enzyme addition treat-ments. In the same way, Figure 4 shows the concentration ofMPs, PRAGs and RG-II in the control and treated wines elabo-rated with Cabernet Sauvignon, Syrah and Monastrell grapes.

The second (PRAGs) and the third (RG-II) populationsincreased in the Monastrell wine when ice was added(Figure 3e), which would appear to suggest a higher concentra-tion of PRAG and RG-II in Monastrell wines treated by dry iceaddition. The data of Figure 4c, however, did not confirm thisinterpretation, suggesting that the greater release of PRAGs andRG-II with dry ice treatment for Monastrell wine was not signifi-cant. Álvarez et al. (2006) showed that dry ice addition toMonastrell grapes produced wines with a content of phenolicsubstances and anthocyanin greater than that in control grapes,which may indicate greater cell wall degradation and subsequentpolysaccharide release.

In contrast, compared with the control, the second (PRAGs)and the third (RG-II) peaks decreased, and the first peak (MPs)increased in the Syrah wine when ice was added, while there wasa slight decrease in the third peak in the Syrah wine after grapeskin freezing (Figure 3c). In contrast, our data, shown inFigure 4b, confirm that this slight decrease was not significant.Ducasse (2009) found that cold prefermentative maceration didnot modify the MM distribution profiles of wine polysaccharides.The same author, however, observed that a longer prefer-mentative maceration step could benefit metabolite diffusionfrom fermentation yeasts, leading to greater MP release.

Added commercial pectic enzyme preparation increased thethird population in the polysaccharide profiles of the wines of allthree cultivars and increased the second population in CabernetSauvignon and Syrah wines (Figure 3b, d). This could beexplained by their increased release from grape skin cell wallsbecause of the action of pectic enzymes. The second population(PRAGs) also increased in the Syrah wines when galactosidaseenzyme was added. The same treatment led to an increase inthe third population (RG-II) in Syrah and Monastrell wine pro-files, suggesting an effect linked to the galactosidase enzymatictreatment. In general, our profiles agree with those obtainedby other authors (Doco et al. 1999, Ayestarán et al. 2004,Guadalupe and Ayestarán 2007). Ducasse et al. (2010),however, detected that PRAG peaks were lower in enzyme-treated wines than that in control wines, which could beexplained by the degradation of polysaccharides of lower MM.

Of the sugars present in the polysaccharides of the wines ofCabernet Sauvignon, Monastrell and Syrah where the must wastreated with refrigeration techniques (Table 3), the amount of2-O-methyl-xylose was lowest when dry ice had been added tothe Cabernet Sauvignon grapes. Similarly, the lowest quantityof rhamnose and xylose was detected in Syrah wines subjected

Figure 2. Concentration of mannoproteins (MPs), polysaccharidesrich in arabinose and galactose (PRAGs), and rhamnogalacturonan II(RG-II) in control wines of cultivars Cabernet Sauvignon ( ), Syrah( ) and Monastrell ( ). Different letters within the same poly-saccharide major family represent significant differences according toa least significant difference test (P < 0.05).

Table 4. Arabinose/galactose ratio of polysaccharides fromCabernet Sauvignon, Syrah and Monastrell wines.

Cultivar/treatment Arabinose/galactose

Cabernet Sauvignon

CACO 0.88 ± 0.08†b

CACM 0.75 ± 0.10ab

CAIA 0.77 ± 0.12ab

CAFR 0.75 ± 0.18ab

CACE 1.13 ± 0.04c

CAGE 0.67 ± 0.10a

Syrah

SYCO 0.59 ± 0.01a

SYCM 0.84 ± 0.44a

SYIA 0.54 ± 0.03a

SYFR 0.84 ± 0.44a

SYCE 0.78 ± 0.36a

SYGE 0.67 ± 0.21a

Monastrell

MOCO 0.56 ± 0.03a

MOCM 0.61 ± 0.03a

MOIA 0.58 ± 0.06a

MOFR 0.54 ± 0.03a

MOCE 0.55 ± 0.01a

MOGE 0.54 ± 0.08a

†Each value is the average of three measurements and standard deviation.Different letters within the same cultivar column represent significant differ-ences according to a least significant difference test (P < 0.05). CA, CabernetSauvignon; CE, commercial enzyme; CM, prefermentative cold maceration; CO,control; FR, frozen grape skin; GE, galactosidase enzyme; IA, dry ice; MO,Monastrell; SY, Syrah.



to grape skin freezing. The concentration of rhamnose, xyloseand galactose in Syrah wines was lower than that of the controlwine after cold prefermentative maceration.

Following the addition of the pectic enzyme preparation, therewas statistically a higher amount of 2-O-methyl-fucose and 2-O-methyl xylose in all wines, of rhamnose in Cabernet Sauvignonand Syrah wines, and of fucose, arabinose and xylose in theCabernet Sauvignon wine. Following galactosidase enzyme treat-ment, there was a statistically higher concentration of 2-O-methyl-fucose and fucose only in the treated Syrah wines (Table 3).Arnous and Meyer (2010) detected a greater monosacchariderelease (mainly galacturonic acid) when pectic enzymes wereadded, compared with that of the control wines. The same authorsalso found that phenolic acids were accessible only when a certainpectin quantity had been released from the cell wall matrix.

The Syrah wine elaborated with dry ice addition showed aconcentration of RG-II (192 mg/L) and PRAGs (147 mg/L) sig-nificantly lower than that of the control wine (RG-II: 282 mg/L,and PRAGs: 242 mg/L) (Figure 4b). This is in contrast withother findings, which showed that dry ice addition favours therelease of phenolic substances (Couasnon 1999, Álvarez et al.2006, Gil-Muñoz et al. 2009). The release of phenolic sub-stances shows a strong linear correlation with cell wall degra-dation in different fruits (Bagger-Jørgensen and Meyer 2004).The discrepancy may be attributable to the effect of dry ice onthe natural pectic enzymes from grape skin, weakening theiraction on cell wall degradation and decreasing subsequentpolysaccharide release.

Variation in the composition of the cultivars could explainwhy only Syrah wine showed this behaviour when dry ice wasadded. Moreover, Álvarez et al. (2006) observed that dry iceaddition to wines produced from grapes of 13°Baumé was lesseffective at extracting phenolic substances than those producedfrom grapes of 12°Baumé.

The addition of a commercial pectic enzyme significantlyincreased RG-II in all three wines (CACE: 570 mg/L, SYCE:474 mg/L and MOCE: 461 mg/L) over that of the correspondingcontrol wines (Figure 4a–c). The same treatment led to a signifi-cant difference in PRAGs in Cabernet Sauvignon (CACE: 316 mg/L). These results agree in general with those obtained by otherauthors. Ayestarán et al. (2004) detected that enzyme-treatedwines had a greater concentration of soluble monosaccharide,arabinogalactans and RG-II than that of control wines. The releaseof homogalacturonans during enzymatic hydrolysis has beenobserved by Bonnin et al. (2002) and Hellín et al. (2005). Thecommercial pectic enzyme used in this study had, among other,polygalacturonase and pectin methylesterase activities. RG-IIhas also been isolated alongside endo-polygalacturonase frompurified vegetal cell walls (Darvill et al. 1978). Cell wall structuresmay be weakened by polygalacturonase probably throughattack of homogalacturonans. It appears that the primary cell wallis more affected, although the middle lamella is also attacked bythe joint action of polygalacturonase and pectin methylesterase(Amrani-Joutei et al. 2003, Lohani et al. 2004). Doco et al. (2007)observed the release of low MM polysaccharides and an increase inRG-II release, when pectic enzymes were applied to Carignan

Rel

ativ

e re

frac

tive

inde

x

Retention time (min)

Figure 3. Molecular massdistribution of polysaccharidesin Cabernet Sauvignon (a,b),Syrah (c,d) and Monastrell (e,f)wines elaborated with thefollowing winemakingtechniques: (a,c,e) control,CACO, SYCO and MOCO ( );cold prefermentativemaceration, CACM, SYCM andMOCM ( ); dry ice addition,CAIA, SYIA and MOIA ( );and grape skin freezing, CAFR,SYFR and MOFR ( ) and(b,d,f) control, CACO, SYCOand MOCO ( ); commercialpectic enzyme, CACE, SYCEand MOCE ( ); andgalactosidase enzyme, CAGE,SYGE and MOGE ( ).



grapes, directly affecting the sensory and physicochemi-cal attributes of the wines. Ducasse et al. (2010) also found thatRG-II release increased when pectic enzymes were applied toMerlot grapes, which agrees with our results. The same authors,however, detected that the concentration of PRAGs was stronglyinfluenced by the harvest year. In our study, the higher concen-tration of PRAGs observed in treated Syrah and CabernetSauvignon wines could be due to an increase in arabinose andgalactose extraction from grape skin accompanied by the lower

degradation of the PRAGs. The original composition in grapes, thecomponents of enzymatic products and even the harvest yearcould affect the way in which the enzymatic treatment acts. Docoet al. (2007) and Ducasse et al. (2010) found no difference in MPswhen pectic enzymes were applied, which agrees with our results.

The addition of the galactosidase enzyme preparation signifi-cantly increased RG-II in Syrah wines (SYGE: 384 mg/L) com-pared with that of the control (Figure 4b). The galactosidase actiononly on the Syrah wines could be related to the differences incomposition of grapes from the three cultivars studied. In this way,greater complexity and stiffness of the grape skin cell wall structurein Monastrell grapes could hinder the access of galactosidaseenzyme to the substrate (Ortega-Regules et al. 2008a).

The observed difference in polysaccharide compositioninduced by the treatments applied in this study may affectthe colloidal structure and the mouth-feel properties inwines. Carvalho et al. (2006) showed that most acid fractionsfrom AGP in wines inhibited protein-tannin aggregation, whichcould decrease astringency. It was also shown that an acidicarabinogalactan could reduce the self-aggregation of tannins inmodel wine solutions (Riou et al. 2002). In the same way, RG-IIenhances tannin particle size, suggesting the co-aggregationof RG-II and tannins. When the intrinsic sensory properties oftwo wine polysaccharide fractions were investigated (Vidal et al.2004), the ‘fullness’ sensation was significantly increased whena mixture of AGPs and MPs was added. Therefore, our PRAGsand RG-II data could suggest an impact of some winemakingtechniques on the chemical and sensory properties of wines.

The arabinose/galactose ratio in treated and controlwines (Table 4) increased to a statistically significant extentin Cabernet Sauvignon wine when commercial pectic enzymewas added, however decreased statistically in the same winewhen galactosidase enzyme was added. This could suggest thatthere is statistically significant effect on the composition of thereleased polysaccharides only when the enzymatic treatmentswere applied to Cabernet Sauvignon grapes. The increase in thearabinose/galactose ratio with commercial pectic enzyme addi-tion indicates that this treatment modifies the total PRAGs com-position. This could be explained by greater release of arabinoseand suggests the release of arabinose or polysaccharides rich inarabinose from the hairy region of the pectic framework. Thisseems to contrast with the results of Doco et al. (2007), whoobserved a decrease in arabinose in Carignan wine PRAGs whenenzymes were added.

ConclusionsOur work suggests that there may be a potential cultivar effecton the polysaccharide concentration of wines, demonstrated inparticular with the concentration of PRAGs and RG-II in Syrahwines higher than that in Cabernet Sauvignon and Monastrellwines. Similarly, the polysaccharide composition is different inCabernet Sauvignon, as the arabinose/galactose ratio demon-strated. Addition of commercial pectic enzyme (rich in pectinmethylesterase, polygalacturonase, among other enzymaticactivities) increases the amount of released polysaccharides:PRAGs in Cabernet Sauvignon and RG-II in all three winecultivars studied. This treatment changed the polysaccharidecomposition in Cabernet Sauvignon wines. Galactosidaseenzyme increased the polysaccharides released only in Syrahwine and changed the polysaccharide composition of CabernetSauvignon wines compared with that of the control wine.Cold prefermentative maceration or grape skin freezing had noeffect on the polysaccharide composition or concentration ofany of the wines. No effect on polysaccharides was detected

Figure 4. Concentration of mannoproteins (MPs), polysaccharidesrich in arabinose and galactose (PRAGs) and rhamnogalacturonan II(RG-II) in (a) Cabernet Sauvignon, (b) Syrah and (c) Monastrellwines elaborated with the following winemaking techniques: control( ); cold prefermentative maceration ( ); dry ice addition ( ); grapeskin freezing ( ); commercial pectic enzyme preparation ( ); andgalactosidase enzyme ( ). Different letters within the same poly-saccharide major family represent significant differences accordingto a least significant difference test (P < 0.05).



with dry ice addition in Cabernet Sauvignon or Monastrellwines, while this treatment led to a decrease in the quantityof polysaccharides released in Syrah wine. The grape cultivarappears to influence the polysaccharide composition andquantity in wines, and it could seem advisable to adapt thewinemaking treatment according to the grape cultivar beingused. But, further studies should be conducted with othercultivars to provide a better understanding of the impact ofwinemaking technique on polysaccharide composition of wine.

AcknowledgementsThis work was made possible by financial assistance of theMinisterio de Ciencia e Innovación of Spain, Project AGL2006-11019-C02-01/ALI. Dr Rafael Apolinar-Valiente is the holder ofa FPI fellowship from the Spain Government.

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Manuscript received: 21 February 2013

Revised manuscript received: 11 June 2013

Accepted: 13 June 2013



Documents

Effect of winemaking techniques on polysaccharide composition of Cabernet Sauvignon, Syrah and Monastrell red wines