customized toasting profiles such as the gradual toasting based on a temperature increase in four steps. In our study, all barrels underwent a gradual toasting (G), and various initial temperatures were compared (180 °C for Sangiovese (G180), 160 and 170 °C for Cabernet Sauvignon (G160 and G170)).

<table><caption>**Table 1 - Nature and origin of wines**</caption><thead><tr><th>Origin</th><th>Grape variety</th><th>Toasting process</th><th>Tannin Potential (TP)</th><th>TPI</th></tr></thead><tbody><tr><td colspan="5" style="text-align: center; font-weight: bold;">Influence of TP</td></tr><tr><td rowspan="2">Estate A<br>Tuscany</td><td>2013</td><td rowspan="2">Sangiovese</td><td rowspan="2">G180</td><td>LTP</td><td>57 / 61</td></tr><tr><td>2012</td><td>LTP</td><td>53 / 56</td></tr><tr><td>Estate B<br>Napa Valley</td><td>2013</td><td>Cabernet sauvignon</td><td>G180</td><td>LTP</td><td>70 / 78</td></tr><tr><td colspan="5" style="text-align: center; font-weight: bold;">Influence of toasting level</td></tr><tr><td>Estate C<br>Sonoma county</td><td>2013</td><td>Cabernet sauvignon</td><td>G160 / G170</td><td>LTP</td><td>58 / 59</td></tr></tbody></table>

*Wine sensory and chemical analyses*

An international panel of 17 trained tasters from the National School of Agricultural Sciences of Bordeaux (BSA) carried out blind sensory analysis. The significant differences are shown in figures 3 and 5 by arrows. The impact of toasting modality on wine structure was evaluated by measuring an index of salivary protein (SPI) as previously described [9, 10]. The assay of wood aromatic compounds was carried out on wines by SBSE/GC/MS and LC/MS (internal methods, Sarco laboratory, Bordeaux). Grape-based tannin composition and concentration were determined by phloroglucinolysis after a solid phase extraction step [11].

**Results and discussion**

As shown in figure 1 and according to recent research [5], the measurement of TP on a sample of more than 2000 staves of various origins and grain sizes confirmed the variability of wood ellagitannin concentration.

<figure><img src="image_357.png" /><figcaption>**Fig. 1 - Variability of ellagitannin content (EGT) in oak staves.**</figcaption></figure>

The relationship between TP and grain was investigated for two grain sizes: growth ring width between 1.5 and 2 mm (FI) and over 3 mm (GR). Accordingly to data literature [12], ellagitannin content increased with the grain size (figure 2). However, this correlation was weak; therefore, grain size was determined not to be a strong predictor of stave ellagitannin content.

*Impact of aging for barrels with different TP at the same toasting level*

For the 2013 Sangiovese (figures 3a and 3b) tasted after 12 months of aging, the wine aged under HTP barrels exhibited significantly more pronounced tannin intensity and the salivary protein index was significantly higher. For the 2012 Sangiovese tasted after 24 months of aging, sensory and chemical analysis could not discriminate between the two samples on the structure criteria (data not shown). In light of these results and in accordance with recent research [5], it seemed HTP barrels release a higher content of ellagitannins compared to LTP barrels. After this first phase of ellagitannin extraction, a decrease in their content is generally observed as a result of their reactivity, both with oxygen and various other wine compounds.
<figure><img src="image_358.png" /><figcaption>**Fig. 2** - Distribution of ellagitannin content according to grain size.</figcaption></figure>

<figure><img src="image_359.png" /><figcaption>**Fig. 3** - Impact of aging under barrels of different TP (LTP/HTP) with a same toasting level (G180) on wine organoleptic properties and level of wood aromatic compounds (Sangiovese 2013).</figcaption></figure>

After 12 months of aging, the condensed tannin concentration measured by phloroglucinolysis increased accordingly to TP from 184.4 mg/L (LTP) to 215.3 mg/L (HTP) (figure 4). The mean degree of polymerization (mDP) was similar between both TP; however, the extension subunit percentage such as (-)-epigallocatechin and (-)-epicatechin was higher in red wine from HTP than LTP. If it is assumed that ellagitannin in oak wood reacted with oxygen as it diffused into the wine, this could explain the observed results. Based upon this hypothesis, grape-based tannins would “age” more slowly in high phenolic barrels as well as barrels toasted at lower temperatures.

Beyond its impact on wine structure and preservation of grape-based tannins, wood sorting according to its TP seemed to have a significant influence on wine aromas (figure 3).

After 12 months of aging, LTP modality is considered significantly more complex and fruity than HTP (figure 3c). That same LTP modality presented more intense fresh wood notes and conversely less toasted notes compared to the HTP modality (figures 3a and 3c). Chemical analysis provided a molecular interpretation of these sensory differences (figure 3d). According to recent research [5], levels of furanic compounds in wines, associated with toasted notes, increase along with wood TP. Regarding whisky lactones, aging under a lower TP resulted in a richer wine. Differences in fruity perception between the two TP classes could also be explained by their respective chemical composition. Indeed, a greater concentration in furanic aldehydes reduces wine fruitiness [13] while whisky lactones can enhance it [14-16].
<figure><img src="image_360.png" /><figcaption>Fig. 4 - Tannin composition and concentration of red wine from barrels of different TP (LTP vs. HTP) with a same toasting level (180 °C) measured by phloroglucinolysis.</figcaption></figure>

Thus, the tannin level of wood seemed to be a determining factor for the levels of extractible compounds. HTP barrels conferred the greatest concentration in compounds responsible for toasted notes. Given their lower whisky lactone concentration, this suggests that ellagitannin content may reflect other constitutive variations in wood composition such as lactones and hemicelluloses.

### Impact of aging with different toasting level and the same TP

Sensory analysis after 10 months of aging clearly discriminated between the two samples from various criteria (figures 5a and 5b). At equal TP, the 170 gradual toasting is judged significantly more fruity and complex; on the palate, it exhibited a better tannin quality (softness) and greater harmony. It is important to remember there was only 10°C difference between the two modalities. In light of these first results, the effective management of the toasting process and the selection of toasting level to each TP, in order to provide homogenous and reproducible barrels, is highlighted.

CS 2013-LTP

5a Mouth feel perception

<figure><img src="image_361.png" /></figure>

5b Olfactory perception

<figure><img src="image_362.png" /><figcaption>Fig. 5 - Impact of aging under barrels of different toasting level (G160/G170) with a same TP (LTP) on wine organoleptic properties (Cabernet Sauvignon, Sonoma county 2013).</figcaption></figure>
Conclusion

The heterogeneity of oak wood composition combined with the lack of toasting standards can lead to substantial differences in wine perception after aging to such an extent that the management of this variability constitutes a major challenge for the cooperage industry. The originality of our production approach lies in the combination of a reliable sorting method, based on the analysis of wood ellagitannin, and an accurate, reproducible toasting tool, matched to wood phenolic content. It is important to note that, to our knowledge, our toasting process is unique in the cooperage industry.

The enological advantage of the rationalization of cooperage oak wood selection combined with a perfect toasting management is evidenced both by sensory and chemical analysis. Moreover, beyond purely enological considerations, we expect that this rationalization of wood sorting will play a role in the years to come to a better use of natural resources, which are not unlimited.

Acknowledgements

The authors would like to kindly thank Mr. Marco Cerqua (Barone Ricasoli, Tuscany, Italy), Mr. Hugh Chapelle (Quivira, Sonoma county, USA) and Mr. Jeffrey Stambor (Beaulieu, Napa, USA) for having carried out the trials.

References

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2. Doussot F, Pardon F, Dedier J et al '2000) Individual, species and geographic origin influence on cooperage oak extractible content (*Quercus robur* L. and *Quercus petraea* Liebl.). *Analusis* **28**: 960-965.

3. Doussot F, De Jeso B, Quideau S et al (2002) Extractives content in cooperage oak wood during natural seasoning and toasting; influence of tree species, geographic location and single-tree effects. *J Agric Food Chem* **50**: 5955-5961.

4. Prida A, Boulet J-C, Ducoussou A et al (2006) Effect of species and ecological conditions on ellagitannin content in oak wood from an even-aged and mixed stand of *Quercus robur* L. and *Quercus petraea* Liebl. *Ann For Sci* **63**: 415-524.

5. Michel J (2012) Classification et influences des polyphénols du bois de chêne sur les qualités sensorielles des vins (Application du procédé Oakscan®). *Thèse de doctorat*, Bordeaux, Université Bordeaux II.

6. Masson G, Moutounet M, Puech JL (1995) Ellagitannin content of oak wood as a function of species and of sampling position in the tree. *Am J Enol Vitic* **46**: 262-268.

7. Masson G, Puech JL, Moutounet M (1996) Composition chimique du bois de chêne de tonnellerie. *Bull OIV* **69**: 634-657.

8. Mosedale JR, Charrier B, Crouch N et al (1996) Variation in the composition and content of ellagitannins in the heartwood of European oaks (*Quercus robur* and *Q petraea*). A comparison of two French forests and variation with heartwood age. *Ann For Sci* **53**: 1005-1018.

9. Gambutti A, Rinaldi A, Pessina R et al (2006) Evaluation of aglianico grape skin and seed polyphenol astringency by SDS-PAGE electrophoresis of salivary proteins after the binding reaction. *Food Chem* **97**: 614-620.

10. Rinaldi A, Gambuti A, Moine-Ledoux V et al (2010) Evaluation of the astringency of commercial tannins by means of the SDS-PAGE-based method. *Food Chem* **122**: 951-956.

11. Kennedy JA, Jones GP (2001) Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. *J Agric Food Chem* **49**: 1740-1746.

12. Vivas N (1995) The notion of grain in cooperage. *J Sci Tech Tonnellerie* **1**: 17-32.

13. Prida A, Chatonnet P (2010) Impact of oak-derived compounds on the olfactory perception of barrel-aged wines. *Am J Enol Vitic* **61**: 408-413.

14. Atanasova B, Thomas-Danguin T, Langlois D et al (2004) Perceptual interactions between fruity and woody notes of wine. *Flavour Fragr J* **19**: 476-482.

15. Atanasova B, Thomas-Danguin T, Langlois D et al (2005) Perception of wine fruity and woody notes: influence of peri-threshold odorants. *Food Qual Prefer* **16**: 504-510.

16. Chaput MA, El Mountassir F, Atanasova B et al (2012) Interactions of odorants with olfactory receptors and receptor neurons match the perceptual dynamics observed for woody and fruity odorant mixtures. *Eur J Neurosci* **35**: 584-597.
# Effect of grape maturity on carbohydrate composition of red sparkling wines

L. MARTÍNEZ-LAPUENTE<fnref n="1" />*, R. APOLINAR-VALIENTE<fnref n="3" />, Z. GUADALUPE<fnref n="1" />, B. AYESTARÁN<fnref n="1" />, S. PÉREZ-MAGARIÑO<fnref n="2" />, T. DOCO<fnref n="3" />, P. WILLIAMS<fnref n="3" />

Red sparkling wine – grape maturity – polysaccharides – oligosaccharides

## Introduction

Sparkling wines elaborated following the traditional method undergo a second fermentation in closed bottles of base wines, followed by aging of wines with lees for at least 9 months. The production of sparkling wines is lower compared to that of still wines, but the economic impact of this product is very important because of its high added value. For this reason winemakers are constantly looking for product improvements and new products. In fact, a new market strategy in the enological industry based on the diversification of wine production and on the exploitation of the characteristics and particularities of different varieties of grapes is emerging. In this sense, although most of the sparkling wines elaborated are white and rosé, the production of red sparkling wines is highly increasing. One of the initial problems in red sparkling wine processing is to obtain suitable base wines that should have moderate alcohol content. Therefore, grapes must be harvested at low maturity stage. This fact could affect the polysaccharide and oligosaccharide content of wines, which in turn could have implications for sparkling wine sensory properties. A previous study has analyzed the composition of polysaccharide families during the winemaking and aging of white and rosé sparkling wines [1], however, none has analyzed the evolution of oligosaccharides. Therefore, this paper aims to analyze the changes occurring in oligosaccharide and polysaccharide families during the red sparkling wine processing by the traditional method, as well as to study the effect of grape ripening stage on carbohydrate composition.

## Materials and methods

Grapes from Tempranillo variety from the Cigales Denomination of Origin (D.O.) were harvested at two maturity levels: prematurity grapes (PM) and grapes at their optimum degree of maturity (M). Then, two red sparkling wines were manufactured using the traditional method champenoise in the enological station of Castilla y León (Valladolid, Spain). Samples for analyses were taken from the base wines and then after 3 months, 6 months and 9 months of aging on yeast lees. Isolation of polysaccharide and oligosaccharide fractions was made according to a previously described method [2]. The polysaccharide composition was estimated from the concentration of individual glycosyl residues determined by GC-MS after hydrolysis, reduction and acetylation as described elsewhere [3]. Oligosaccharide fraction was determined after solvolysis by GC of their per-O-trimethylsilyl methyl glycoside derivatives [4].

## Results and discussion

Figure 1 gives the molecular weight distributions of polysaccharides and oligosaccharides of premature and mature red sparkling wines during their aging on yeast lees. The fraction eluted on Superdex 30-HR column between 40 and 53 min contained the polysaccharide fraction, while the oligosaccharide fraction was collected between 54 and 93 min. The first peak in the range 40–48 min corresponded to the

<footnote n="1">¹Instituto de Ciencias de la Vid y del Vino (Universidad de la Rioja, Gobierno de La Rioja y CSIC), C/ Madre de Dios 51, 26006, Logroño, Spain</footnote>

<footnote n="2">²Instituto Tecnológico Agrario de Castilla y León, Consejería de Agricultura y Ganadería, Ctra Burgos Km 119, Finca Zamaduenas, 47071, Valladolid, Spain</footnote>

<footnote n="3">³INRA, Joint Research Unit 1083, Sciences for Enology, 2 Place Pierre Viala, F-34060 Montpellier, France</footnote>
polysaccharide fraction of highest mass and rich in PRAGs (Polysaccharides Rich in Arabinose and Galactose) and mannoproteins (MPs). The second peak eluted between 49 and 53 min corresponded to the fraction containing mainly rhamnogalacturonan II (RG-II). Evident differences between the profiles of base wines were found. The polysaccharide and oligosaccharide fractions of red base wines elaborated with mature grapes were higher than the fractions in the other base wine due to a different ripening state at harvest. It should be noted the occurrence of peak tailing at 48 min in premature but no in mature red sparkling wines, probably due to a degradation of polysaccharides in mature grapes but no in premature grapes.

<figure><img src="image_363.png" /><figcaption>**Fig. 1** - Purification by high-resolution size-exclusion chromatography of polysaccharide and oligosaccharide fractions isolated on Superdex 30-HR column from premature and mature red sparkling wines during different stages of sparkling wine production: base wines (T0), sparkling wines after 3 months (T3), 6 months (T6), and 9 months (T9) of aging on yeast lees (Refractive index versus Retention Time (Minutes)).</figcaption></figure>

Grape ripening stage affected the content and evolution of polysaccharides and oligosaccharides. Figure 2 shows the concentration of MPs, glucans (GLs), PRAGs and RG-II in premature and mature red sparkling wines during different stages of sparkling wine production. In both wines and in all vinification stages, PRAGs were the most prevalent polysaccharide family detected. However in all vinification stages mature sparkling wines showed higher content of MPs, PRAGs and RG-II than premature ones. The higher MPs content could be caused by changes in yeast conditions, such as the different alcohol content of base wines, during alcoholic fermentation, which could influence the kinetics of the release of mannose. The higher content of polysaccharides from grapes in mature red base wines could be due to an easier degradation of cell walls in mature grapes that could ease the extraction of wine components during the early steps of their processing to base wine. The evolution of polysaccharide families was different during the stages of the sparkling wine processing. In mature red sparkling wines, MPs increased from 3 to 6 months of aging, GLs increased from base wine to 3 months of aging, while PRAGs and RG-II slightly decreased at 9 months. However, in premature red sparkling wines, MPs increased at 6 months of aging, GLs and PRAGs remained and RG-II decreased. As observed with polysaccharide families, grape maturity also affected the arabinose/galactose ratio of the wine PRAGs composition. Mature red sparkling wines showed higher arabinose/galactose ratio than premature ones, which suggests higher release of arabinose. In both wines the ratio remained constant during the aging, which suggests that aging did not change the PRAGs composition (date not shown).

Table 1 shows the glycosyl composition (mg/L) of oligosaccharides from red sparkling wines during aging on yeast lees. In all wines and together with PRAGs, oligosaccharides were the two most prevalent carbohydrates detected in all vinification stages. Oligosaccharides of all wines included sugars such as rhamnose, arabinose, galactose, xylose and galacturonic and glucuronic acids coming from the pecto-cellulosic cell walls of grape berries but also mannose and glucose released from yeast polysaccharides. Galacturonic acid, arabinose, xylose, mannose and glucose presented the highest contents in both wines during all vinification stages.
<figure><img src="image_364.png" /><figcaption>**Fig. 2** - Concentration of mannoproteins (MPs), glucans (GLs), polysaccharides rich in arabinose and galactose (PRAGs), and rhamnogalacturonan II (RG-II) in premature and mature red sparkling wines during different stages of sparkling wine production: base wines (T0), sparkling wines after 3 months (T3), 6 months (T6), and 9 months (T9) of aging on yeast lees.</figcaption></figure>

<table><caption>**Table 1** - Glycosyl composition (mg/L) of oligosaccharides from red sparkling wines during different stages of sparkling wine production: base wines (BW), sparkling wines after 3 months (T3), 6 months (T6), and 9 months (T9) of aging on yeast lees.</caption><thead><tr><th></th><th>Rha <sup>a</sup></th><th>Fuc <sup>a</sup></th><th>Ara <sup>a</sup></th><th>Xyl <sup>a</sup></th><th>Man <sup>a</sup></th><th>Gal <sup>a</sup></th><th>Glc <sup>a</sup></th><th>Gal A <sup>a</sup></th><th>Glc A <sup>a</sup></th><th>Xylitol</th><th>4-OMeGlc A <sup>a</sup></th><th>Total</th></tr></thead><tbody><tr><td>PM<sup>b</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>T0</td><td>4.3</td><td>3.3</td><td>11.6</td><td>17.9</td><td>26.0</td><td>7.5</td><td>41.1</td><td>176.8</td><td>3.1</td><td>1.8</td><td>5.6</td><td>299.0</td></tr><tr><td>T3</td><td>3.4</td><td>2.2</td><td>10.3</td><td>15.1</td><td>21.6</td><td>7.3</td><td>34.5</td><td>144.0</td><td>2.6</td><td>2.0</td><td>4.9</td><td>247.9</td></tr><tr><td>T6</td><td>4.1</td><td>2.6</td><td>12.5</td><td>17.0</td><td>24.4</td><td>7.9</td><td>50.0</td><td>172.9</td><td>3.1</td><td>2.1</td><td>5.3</td><td>301.9</td></tr><tr><td>T9</td><td>2.6</td><td>2.1</td><td>6.7</td><td>10.8</td><td>18.1</td><td>6.2</td><td>36.2</td><td>91.8</td><td>1.7</td><td>1.3</td><td>3.0</td><td>180.5</td></tr><tr><td>M<sup>b</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>T0</td><td>3.2</td><td>2.7</td><td>18.0</td><td>20.9</td><td>24.2</td><td>11.0</td><td>42.2</td><td>174.2</td><td>3.9</td><td>3.3</td><td>7.6</td><td>311.2</td></tr><tr><td>T3</td><td>3.0</td><td>3.0</td><td>16.8</td><td>21.1</td><td>26.0</td><td>9.3</td><td>54.7</td><td>172.8</td><td>3.6</td><td>3.2</td><td>7.2</td><td>320.7</td></tr><tr><td>T6</td><td>2.5</td><td>2.5</td><td>13.7</td><td>16.4</td><td>20.6</td><td>6.9</td><td>44.4</td><td>160.2</td><td>3.7</td><td>2.0</td><td>6.0</td><td>278.9</td></tr><tr><td>T9</td><td>2.5</td><td>2.5</td><td>15.0</td><td>18.8</td><td>21.6</td><td>7.0</td><td>56.8</td><td>147.0</td><td>3.3</td><td>2.8</td><td>6.0</td><td>283.3</td></tr></tbody></table>

Several characteristic ratios were calculated from oligosaccharide sugar composition: Arabinose/Galactose, Rhamnose/Galacturonic acid, and (Arabinose + Galactose)/Rhamnose (Table 2). Red mature sparkling wines showed higher arabinose/galactose ratio than premature ones, which suggests a greater release of arabinose or oligosaccharides rich in arabinose arising from the pectic framework into this wine. The lower value calculated for Rha/Gal A suggests an homogalacturonan structure predominance. (Arabinose + Galactose) to Rhamnose ratio was lower in premature red sparkling wines than mature wines. It could indicate that the rhamnogalacturonan oligomers present in mature red sparkling wines carry more neutral lateral chain.

<footnote><sup>a</sup>Rha, Rhamnose; Fuc, Fucose; Ara, Arabinose; Xyl, Xylose; Man, Mannose; Gal, Galactose; Glc, Glucose; Gal A, Galacturonic acid; Glc A, Glucuronic acid; 4-OMeGlc A, 4-O methyl Glucuronic acid.<sup>b</sup>PM, premature grapes; M, mature grapes.</footnote>
<table><caption>**Table 2** - Glycosyl composition (mg/L) of oligosaccharides from red sparkling wines during different stages of sparkling wine production: base wines (BW), sparkling wines after 3 months (T3), 6 months (T6), and 9 months (T9) of aging on yeast lees.</caption><thead><tr><th></th><th>Ara/Gal <sup>a</sup></th><th>Rha/Gal A <sup>a</sup></th><th>(Ara+Gal)/Rha <sup>a</sup></th></tr></thead><tbody><tr><td colspan="4">Premature</td></tr><tr><td>T0</td><td>1.87</td><td>0.03</td><td>4.49</td></tr><tr><td>T3</td><td>1.68</td><td>0.03</td><td>5.27</td></tr><tr><td>T6</td><td>1.89</td><td>0.03</td><td>5.03</td></tr><tr><td>T9</td><td>1.30</td><td>0.03</td><td>4.98</td></tr><tr><td colspan="4">Mature</td></tr><tr><td>T0</td><td>1.96</td><td>0.02</td><td>9.24</td></tr><tr><td>T3</td><td>2.17</td><td>0.02</td><td>8.89</td></tr><tr><td>T6</td><td>2.37</td><td>0.02</td><td>8.51</td></tr><tr><td>T9</td><td>2.56</td><td>0.02</td><td>9.27</td></tr></tbody></table>

## Conclusion

The results of this study highlight grape ripening stage affected the concentration, composition and evolution of polysaccharides and oligosaccharide during the aging on lees of sparkling wines. Polysaccharides rich in arabinose and galactose and oligosaccharides were the two most prevalent carbohydrates detected in all vinification stages. More studies should be carried out to further investigate the possible influence that different polysaccharides and oligosaccharides from different grape maturity could have on the physico-chemical and sensory properties of sparkling wines.

## Acknowledgements

The authors thank the INIA for financing this study through the projects RTA2012-00092-C02-01 (with FEDER funds).

## References

1. Martínez-Lapuente L, Guadalupe Z, Ayestarán B et al (2013) Changes in polysaccharide composition during sparkling wine making and aging. *J Agric Food Chem* **61**: 12362-12373.

2. Ducasse MA, Williams P, Meudec E et al (2010) Isolation of Carignan and Merlot red wine oligosaccharides and their characterization by ESI-MS. *Carbohydr Polym* **79**: 747-754.

3. Apolinar-Valiente R, Williams P, Romero-Cascales I et al (2013) Polysaccharide composition of Monastrell red wines from four different Spanish terroirs: effect of wine-making techniques. *J Agric Food Chem* **61**: 2538-2547.

4. Doco T, O'Neill MA, Pellerin P (2001) Determination of the neutral and acidic glycosyl-residue compositions of plant polysaccharides by GC-EI-MS analysis of the trimethylsilyl methyl glycoside derivatives. *Carbohydr Polym* **46**: 249-259.

<footnote><sup>a</sup> Ara, Arabinose; Gal, Galactose; Gal A, Galacturonic acid; Rha, Rhamnose.</footnote>
# L'amertume des vins blancs secs

S. CAILLE¹*, C. BEGUE², C. LERICHE², F. DELLOVA², A. SAMSON²

¹UMR 1083 Sciences pour l'œnologie, INRA, Montpellier, France

²UE 999 Pech Rouge, INRA, Gruissan, France

## Amertume – vins blancs – analyse sensorielle

### Introduction

L'amertume est un critère important dans l'acceptation des produits alimentaires et des boissons car elle serait associée, de manière innée, à des produits toxiques ou dangereux pour la santé de l'homme [1]. Cette perception est d'ailleurs rejetée par les nourrissons ; ce n'est que par l'éducation que l'amertume peut devenir pour certains un facteur positif d'acceptation avec des niveaux de perception et d'acceptation très différents d'un individu à l'autre.

Dans le vin, l'amertume est un des paramètres majoritaires pour définir le goût ; elle participe à l'équilibre général avec l'acidité et la sucrosité. Si l'amertume des vins rouges trouve son origine majeure dans leurs richesses en polyphénols dont on sait que certaines conformations peuvent l'exacerber [2], l'amertume des vins blancs est beaucoup plus controversée. Mieux comprendre son origine et agir éventuellement sur son niveau est un enjeu pour la filière afin de répondre aux attentes des consommateurs, de plus en plus sensibles à de faibles niveaux de défaut dans les vins [3].

Certains cépages tels que le muscat à petits grains, le viognier ou le mauzac sont reconnus pour apporter plus fréquemment de l'amertume aux vins. En outre, ces cépages sont particulièrement sensibles à l'oxydation, qui renforcerait elle-même cette sensation [4]. D'autres facteurs engendreraient l'amertume : la concentration en certaines molécules, telles que l'alcool, les terpènes ou l'acroléine [2, 5, 6], ou l'équilibre des perceptions gustatives. L'astringence peut par exemple perturber la perception de l'amertume [7]. A l'inverse, des paramètres peuvent diminuer cette perception comme l'acidité, le taux de sucre ou de glycérol [8, 9].

Pour diminuer l'amertume des vins blancs, des pratiques œnologiques sont bien connues. C'est le cas des collages comme la PVPP (poly vinyl poly pyrrolidone), qui se lie aux polyphénols [10] ou l'édulcoration des vins au conditionnement. Cependant ces solutions sont plus correctives que préventives et ne permettent pas de comprendre l'origine d'une amertume excessive.

Cette étude se propose d'identifier certains facteurs inhérents au raisin ou à la vinification impactant l'amertume des vins blancs et ainsi de mieux en comprendre l'origine pour mettre en place des actions préventives.

### Matériel et méthodes

#### Modalités

Pour cette étude, plusieurs facteurs potentiellement influant ont été sélectionnés : le cépage, la maturité des baies, le contact des parties solides de la baie avec le jus au stade pré fermentaire (extraction et macération pelliculaire) et la chaptalisation du moût. Deux séries de neuf modalités chacune ont ainsi été analysées par des analyses sensorielles et œnologiques (Figure 1).

#### Analyse sensorielle des vins

L'analyse sensorielle des vins est réalisée par un jury expert (20 juges), sélectionné sur ses aptitudes sensorielles et entraîné (AFNOR ISO 8586, [11]). Les juges sont entraînés à identifier et évaluer l'intensité de l'amertume par l'utilisation de solutions standards (caféine et sulfate de quinine). L'intensité de l'amertume est évaluée sur une échelle linéaire, continue et bornée. Les vins sont présentés en comparatif de 3 vins (équilibrage des comparaisons sur l'ensemble du jury et des répétitions), selon un ordre basé sur
un carré latin équilibré sur les effets arrières [12] et dans des verres noirs standardisés INAO.
Une répétition de l'analyse des vins est réalisée. Les données sont recueillies avec un système d'acquisition
de données informatisé (FIZZ software, Biosystemes, Couternon France).

<figure><img src="image_365.png" /><figcaption>Fig. 1 - Caractéristiques des modalités analysées.</figcaption></figure>

Traitement des données

Le traitement statistique est réalisé avec le logiciel XLstat (Addinsoft, France), par des analyses de variances. Au préalable, les performances du jury sont étudiées. Lorsque des différences significatives sont observées (p<0,05), les intensités moyennes sont comparées en utilisant le test de comparaison multiple Tukey (HSD). Des corrélations de Pearson sont calculées entre les données issues de l'analyse sensorielle et les données œnologiques des vins.

Résultats et discussion

Pour les 2 cépages étudiés, le panel est performant puisqu’il est à la fois discriminant, consensuel et répétable.

Pour le cépage muscat, des différences significatives d'amertume existent entre les 9 modalités étudiées (p<0,0001). Les vins 05 et 06 sont significativement plus amers que le vin 02 (Figure 2).

<figure><img src="image_366.png" /><figcaption>Fig. 2 - Classement des vins en fonction de l'amertume obtenu à partir de l'ANOVA à 2 facteurs (juge-vin) pour le cépage muscat.</figcaption></figure>
Quand on étudie plus en détail les différents effets, on constate pour ce cépage que la différence d'amertume est principalement liée à la maturité et la chaptalisation (Figure 3). En effet, les modalités issues de la première maturité sont significativement moins amères que celles de la seconde maturité ou de la première maturité chaptalisée (p<0,0001). Par contre, aucune différence significative n'est observée entre les modalités d'extraction.

<figure><img src="image_367.png" /><figcaption>Fig. 3 - Effet des facteurs sur la perception de l'amertume pour le cépage muscat.</figcaption></figure>

Comme le cépage précédent, on observe pour le viognier une différence significative d'amertume entre les 9 modalités étudiées (p=0,003). Le vin 02bis est significativement plus amer que les vins 01, 02, 03 et 04 (Figure 4).

<figure><img src="image_368.png" /><figcaption>Fig. 4 - Classement des vins en fonction de l'amertume obtenu à partir de l'ANOVA à 2 facteurs (juge-vin) pour le cépage viognier.</figcaption></figure>

L'étude plus précise des effets montre que la différence d'amertume est liée d'une part à la chaptalisation et d'autre part à l'extraction (Figure 5). En effet, les modalités issues de la première maturité sont significativement moins amer que celles de la première maturité chaptalisée (p=0,005). Par contre, il n'y a pas de différences significatives entre la première et la seconde maturité. Pour l'effet de l'extraction, les modalités extraites par le décanteur centrifuge après foulage et macération sont significativement plus amères que les modalités extraites par le pressoir pneumatique.

Les corrélations entre la perception de l'amertume et les données œnologiques existent. Principalement pour le cépage muscat, des corrélations significatives sont observées pour le degré d'alcool (r=0,929) et la DO 420 (r=0,701), ce qui indiquerait que l'amertume des vins augmente avec le taux d'alcool. Et elle serait liée à une couleur des vins plus jaune, liée probablement à une plus grande richesse en polyphénols ou à une oxydation accrue. Ces corrélations ne sont pas significatives pour le cépage viognier mais des tendances existent (alcool r=0,544 ; DO 420 r=0.608).
<figure><img src="image_369.png" /><figcaption>**Fig. 5** - Effet des facteurs sur la perception de l'amertume pour le cépage viognier.</figcaption></figure>

## Conclusion

Ces premiers résultats indiquent qu'il existe un effet de la maturité pour les 2 cépages étudiés. Une maturation des baies plus tardive confère plus d'amertume aux vins en corrélation avec un pourcentage en volume d'alcool supérieur. L'itinéraire pré fermentaire avec un contact accru entre les parties solides et le jus procure davantage d'amertume seulement pour les vins de viognier.

Il semble que parmi tous les facteurs étudiés dans le protocole, l'incidence du pourcentage en volume d'alcool soit pré dominant. En effet, une deuxième année d'expérimentation n'a pas permis de mesurer des différences d'amertume, dès lors que les différences de pourcentage en volume d'alcool entre les vins étaient moindres.

Ce travail sur l'amertume des vins blancs se poursuivra en ciblant l'étude sur d'autres familles de molécule que l'alcool.

## Références

1. Pandey S, Kumar S, Prajapati SK et al (2010) An overview on taste physiology and masking of bitter drugs. *Int J Pharma Bio Sci* **1**: 3.

2. Noble AC (1994) Bitterness in wine. *Physiol Behav* **56**: 1251-1255.

3. Leigh F (2010) Linking wine flavour components, sensory properties and consumer quality perceptions. *Aust NZ Wine Ind J* **25**: 18-23.

4. Nagel C, Graber W (1988) Effect of must oxidation on quality of white wines. *Am J Enol Vitic* **39**: 1-4.

5. Noble AC, Strauss C, Williams P et al (1988) Contribution of terpene glycosides to bitterness in Muscat wines. *Am J Enol Vitic* **39**: 129-131.

6. Bauer R, Cowan A, Crouch A (2010) Importance of 3-hydroxypropionaldehyde and derivatives in production and detection. *J Agric Food Chem* **58**: 3243-3250.

7. Brannan GD, Setser CS, Kemp KE (2001) Interaction of astringency and taste characteristics. *J Sens Stud* **2**: 179-197.

8. Smith AK, June H, Noble AC (1996) Effects of viscosity on the bitterness and astringency of grape seed tannin. *Food Qual Prefer* **3-4**: 161-166.

9. Fontoin H, Saucier C, Teissedre PL et al (2008) Effect of pH, ethanol and acidity on astringency and bitterness of grape seed tannin oligomers in model wine solution. *Food Qual Prefer* **19**: 286-291.

10. Caillet MA (2006) Historique du collage, le collage des moûts et des vins : principe, résultats et perspectives. *Rev Fr Oenol* **217**.

11. ISO 8586 (1994) *Analyse sensorielle – guide général pour la sélection, l'entraînement et le contrôle des sujets*, AFNOR.

12. Macfie H, Bratchell M, Greenhoff K et al (1989) Designs to balance the effect of order of presentation and first order carry-over effects in hall tests. *J Sens Stud* **4**: 129-148.
# Fractionation and determination method of proanthocyanidol mass from grapes and wine. Validation of proanthocyanidol standards used for the calibration of size exclusion chromatography columns

M.F. NONIER<fnref n="1" />*, N. VIVAS DE GAULEJAC<fnref n="1" />, N. VIVAS<fnref n="1" />, I. PIANET<fnref n="2" />, C. ABSALON<fnref n="2" />

SEC analysis – procyanidins – mass distribution – grape seeds and skin

## Introduction

Phenol compounds are found in grapes and wines in the form of non-flavonoids (phenolic acids, stilbenes) and flavonoids such as condensed tannins (e.g. proanthocyanidins, PAs) and monoglucoside anthocyanidins. They are important in the colouration and organoleptic properties of berries and give specific characteristics to the wine. PAs are mainly present in the seeds and skin of grape, and are polymers of flavan-3-ol units and in the case of grape seeds (procyanidins, PCs): catechin and epicatechin.

Size-exclusion chromatography (SEC) is the method routinely employed to determine polymer molecular mass. The molecules are separated according to their hydrodynamic volume, which depends on their molar mass, the solvent and the chemical composition of the polymer. By calibrating the column with known standards, the molar mass of an analyte can be calculated. If the analytes do not have the same relationship between molar mass and hydrodynamic volume as the standards used, the calculated molar mass is only relative to the standards. The gross structure of proanthocyanidin polymer is characterized by its degree of polymerization (DP), i.e. average number of units in the polymer.

In our case, SEC in the organic phase is usually used. Currently, molecular mass values are obtained in polystyrene (PS) or polyethylene glycol (PEG) equivalents as commercial PC standards are not available. But SEC is not an absolute method; structural and conformational differences between PS or PEG and PA must be taken into account. Mass spectrometry (MS) is an effective method for characterizing the molecular mass. In order to evaluate possible differences, MALDI technique was carried out, MALDI being a well-known technique able to accurately determine the mass of low dispersity samples. In this work, we present the new calibration curve and a comparative study on grape seed samples based on the two different calibrations.

The purpose of the study is also to establish a new calibration curve by SEC realistic chemical structures studied and to validate the selected standards and to compare grape seed samples based on the two different calibrations. Our work will permit to devise a reliable method to characterize polymerized forms of tannins, their structural information and mass distribution. All these parameters will enable us to establish a comparative map of PA of different origins.

## Materials and methods

### Procyanidin isolation and fractionation

Grapes were collected at the beginning (A) and at the end (B) of maturation: seeds and skins were separated manually. Isolated seeds (A and B) were lyophilized, reduced to powder and frozen at -18°C until used. Portions (40 g) of seeds (A) and (B) were extracted in 100 ml of EtOH and 100 ml H2O at 1 g/l sodium metabisulfite under nitrogen with mechanical stirring for 12 h. 200 ml CHCl3 were added to

<footnote n="1">¹Tonnellerie DEMPTOS – CESAMO – ISM – Université Bordeaux 1,
351 Cours de la Libération, 33405 Talence, France</footnote>

<footnote n="2">²CESAMO – ISM – Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence, France</footnote>
eliminate lipids and pigments (x3). The mixture was centrifuged for 10 min. The lower green phase corresponding to the chloroform phase was eliminated and the upper yellow phase corresponding to the hydroalcoholic (HA) part was recovered. All the HA extracts were collected, evaporated to dryness, lyophilized and frozen at -18°C until analysis ----> **Seed extract A and Seed extract B were obtained.**

**2 g of seed extract A + 20 ml H2O were extracted in ethyl acetate (x6), and after decantation and centrifugation, organic phases were collected, evaporated to dryness and frozen at -18°C ----> Oligomeric Fraction.**

**4 g of seed extract B + 40 ml H2O were extracted in ethyl acetate (x6), and after decantation, aqueous phases were collected and centrifuged. The liquid phase was collected, evaporated to dryness and frozen at -18°C ----> Polymeric Fraction; the solid phase was collected, evaporated to dryness and frozen at -18°C ----> Highly Polymeric Fraction.**

*Analysis by Size Exclusion Chromatography (SEC)*

The study of the *M*<sub>p</sub> (peak-average molecular mass) distribution of seed fractions was performed using organic columns. SEC analysis was performed using a PL-GPC 50 Plus instrument equipped with three columns (300x7.8 mm i.d.): Styragel HR1, HR3, HR4, in series, protected with a guard column of the same material (Waters). Analysis conditions were: DMF+0,1% LiBr as the eluent, flow-rate: 0,8 ml/min at 20°C, injection volume: 20 µl and analysis time: 60 min. Detection was made at 280 nm, by an UV detector (SPECTRA SERIES™ UV-150). Cirrus™ software was used for data acquisition.

*Low pressure gel chromatography*

Separation was performed using low pressure TSK HW-40(S) gel chromatography with 100% MeOH as eluent. Flow rate: 0,8 ml/min; column: 27±2 cm length and 2,5 cm diameter.

*MALDI analysis*

MALDI-MS spectra were acquired using a TofSpec™ MALDI-TOF mass spectrometer from Micromass™ (Manchester, UK). The instrument is equipped with a pulsed N2 laser (337 nm, 4 ns pulse width) and a time-delayed extracted ion source. Spectra were recorded in the positive-ion mode using the reflectron and with an accelerating voltage of 20 kV.

Samples were dissolved in MeOH at 10 mg/ml. The DHB matrix solution was prepared by dissolving 10 mg in 1 ml of H2O. The solutions were combined in a 10:1 volume ratio of matrix to polymer to cationization agent. One to two microlitres of the obtained solution were deposited onto the target sample and vacuum-dried.

*NMR analysis*

1D and 2D NMR experiments were performed on a Bruker spectrometer equipped with an inverse 5 mm broad-band probe at 600 MHz for 1H. 1H chemical shifts are given in ppm relative to TMS.

**Results and discussion**

Obtaining information on the mass distribution and the calculation of average masses is performed by analysis of Size Exclusion Chromatography (SEC) in the organic phase (DMF). Currently, these values are obtained as polystyrene equivalent (PS) or polyethylene glycol (PEG), because there are no commercially available standards of PA. But the structural and conformational difference between PS, PEG and PA do not have the same behavior in these SEC conditions, suggesting potential differences between the calculated mass and the real masses.

So we fractionated grape seeds by selective extraction with ethyl acetate, and 3 fractions were obtained: Oligomeric Fraction (F. Oligo), Polymerized Fraction (F. Polym.) and Highly Polymerized Fraction (F.H. Polym.).

A SEC analysis was performed on fractions of grape seeds under the previously described conditions. Figure 1 represents the obtained chromatogram.
<figure><img src="image_370.png" /><figcaption>Fig. 1 - SEC chromatogram of seed fractions</figcaption></figure>

The polydispersity of these fractions is sufficiently low that the average masses are calculated from MALDI spectra, and for all fractions, the average degree of polymerization is obtained by NMR. In order to obtain a new calibration curve with PA standards, we used two techniques: MALDI-TOF and NMR.

This spectrum (Figure 2) clearly demonstrates the kind of information that can be achieved: the basic unit found is the flavan-3-ol (epi)catechin (repeat unit: 288 Da) and the galloylation is also observed (152 Da). This fraction presents a DPmax = 9.

The Highly Polymeric Fraction is very polydisperse and one of the well-known limitations of MALDI analysis is the misestimation of molecular masses for polydisperse polymers (high masses are discriminated against low masses).

To overcome this problem, one solution is to fractionate by SEC into very narrow fractions. The oligomeric fraction was refined by low pressure chromatography equipped with a semi-preparative column (TSK gel Toyopearl) and 9 oligomeric fractions were obtained (F.F. Oligo. 1 to Oligo. 9).

<figure><img src="image_371.png" /><figcaption>Fig. 2 - MALDI-TOF spectrum of polymeric fraction</figcaption></figure>
Diffusion-ordered spectroscopy (DOSY) NMR technique was used to separate resonances according to the diffusion coefficients (D) of the different fractions. These results (Figure 3) demonstrate that a separation occurs with respect to procyanidin molecular weight.

<figure><img src="image_372.png" /><figcaption>Fig. 3 - DOSY NMR spectrum of seed fractions</figcaption></figure>

Conclusion

This paper describes the first steps in order to devise a reliable method to characterize polymerized forms
of tannins, their structural information and mass distribution. All these parameters will enable us to
establish a comparative map of PA of different origins.
# Variations in ageing bouquet typicality of red Bordeaux wines and associated aromatic marker levels during ageing in three vineyards

M. PICARD<fnref n="1" />,<fnref n="2" />*, C. THIBON<fnref n="1" />,<fnref n="2" />, M.C. PERELLO<fnref n="1" />,<fnref n="2" />, P. REDON<fnref n="1" />,<fnref n="2" />, P. DARRIET<fnref n="1" />,<fnref n="2" />, G. DE REVEL<fnref n="1" />,<fnref n="2" />, S. MARCHAND<fnref n="1" />,<fnref n="2" />

Ageing bouquet – red Bordeaux wines – dimethyl sulphide – polyfunctional thiols

## Introduction

The tendency of wine to improve with age is one of its most intriguing properties. The overall quality of fine aged wines is conditioned by the development of their ageing bouquet. Similar to a complex perfume, it denotes a range of aromas where individual perceptions do not clearly dominate.

In a previous work, a holistic approach was performed to attain a sensory definition of the ageing bouquet of the red Bordeaux wines [1]. Its olfactory representation is structured around several main aromatic notes, among which undergrowth, truffle, fresh red- and black-berry fruits, empyreumatic and toasty notes play a significant role.

The aim of this work was to investigate how DMS, 2FMT and 3SH could be related to the typicality of the wine ageing bouquet and to which extent they could contribute to the evolution of its aromatic nuances over time.

## Materials and methods

### Wine samples

Three sets of wines, each coming from the same Château and representing three different Bordeaux appellations: Margaux (MX series: 7 wines, vintages: 1995; 1998; 2000; 2004; 2005; 2008 and 2013), Saint-Emilion (SEM series: 8 wines, vintages: 1995; 1996; 1998; 2001; 2003; 2005; 2008 and 2012) and Pomerol (P series: 7 wines, vintages: 1996; 1998; 2001; 2004; 2007; 2010 and 2012), were studied both from sensory and chemical point of views.

### Sensory analysis

Sensory analyses were performed in similar conditions than described in a previous work with typicality assessment and aromatic profiling tasks [1].

### DMS quantitation

DMS was quantified in all studied red wines by HS-SPME-GC/MS developed methods [2]. 3SH and 2FMT were specifically extracted and analyzed by GC-MS according to the methodology described by Tominaga and Dubourdieu [3].

## Results and discussion

In all the red Bordeaux wines analyzed, DMS, 2FMT and 3SH turned out to be significantly discriminant between good and poor wine examples of the ageing bouquet.

The quantitative analyses performed on the three series of 1-19 aged red Bordeaux wines showed that concentrations ranged from 92 to 513 ng/L for 3SH, 12 to 150 ng/L for 2FMT and 11 to 146 µg/L

<footnote n="1">¹Univ. Bordeaux, ISVV, EA 4577 Œnologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²INRA, ISVV, USC 1366 Œnologie, F-33140 Villenave d'Ornon, France</footnote>
for DMS. Figure 1 represents, for each series, the evolution of the ageing bouquet typicality score and the
corresponding DMS, 2FMT and 3SH concentrations over the addressed bottle ageing period.

Overall, in the sets of wines considered, typicality reached a maximum after 10 (MX) or 16 years (P and SEM) in bottle, with DMS, 3SH, and 2FMT levels quite well correlated with ageing bouquet typicality. Indeed, in almost all cases, high levels of the three compounds were aligned with high typicality scores. It was noticed that in the P series, the typicality curve, as well as the three volatile sulphur compound concentration profiles, peaked for the 7-year-old wine, possibly revealing a specific vintage effect in this vineyard. Interestingly, closer examination of the DMS profile revealed that high concentrations perfectly accounted for high typicality scores. It was also seen that the DMS levels aligned with the best typicality notes within each series (i.e the 16-aged wine for MX series; the 7 and 16-aged wines for P series and the 16-aged wine for SEM series) are closed to 100 µg/L, a concentration previously reported for inducing a positive contribution in wine aroma [4].

Considering the evolution of 3SH and 2FMT levels in wines during ageing, the results reported here
revealed a less obvious correlation than for DMS between their concentrations and the corresponding
typicality notes. Even if in most cases, high levels of the two thiols accounted for high typicality, their
highest concentrations did not always correspond to the highest typicality scores, with the exception of the
16-year-old wine in the P series. Interestingly, a modulated impact between DMS and 2FMT on the
typicality score seems to exist. This is depicted within MX series where DMS and 2FMT levels of the
18 and 19-aged wines were evolving in an opposite way whereas the typicality note was kept.

<figure><img src="image_373.png" /><figcaption>**Fig. 1** - Evolution of the typicality score and the DMS, 2FMT and 3SH levels during bottle ageing for three red Bordeaux vineyards (series MX, P and SEM).</figcaption></figure>

**Conclusion**

This study highlighted that DMS, 2FMT and 3SH concentrations evolved in the same way as ageing
bouquet typicality over wine bottle storage. Furthermore, a subtle balance of these three compounds
seemed important to accurately account for the ageing bouquet typicality representativeness over wine
bottle ageing [5].
**Acknowledgements**

The authors express their thanks to the Châteaux of the Bordeaux area who kindly provided wine samples.
Special thanks are also expressed to the wine professionals who participated to the sensory sessions.

**Abbreviations**

DMS, dimethyl sulphide; 2FMT, 2-furanmethanethiol; 3SH, 3-sulfanylhexanol; HS-SPME-GC/MS, Head Space Solid Phase Micro Extraction - Gas Chromatography/Mass Spectrometry.

**References**

1. Picard M, Tempere S, de Revel G, Marchand S (2015) A sensory study of the ageing bouquet of red Bordeaux wines: a three-step approach for exploring a complex olfactory concept. *Food Qual Prefer* **42**: 110-122.

2. Fedrizzi B, Magno F, Moser S et al (2007) Concurrent quantification of light and heavy sulphur volatiles in wine by headspace solid-phase microextraction coupled with gas chromatography/mass spectrometry. *Rapid Commun Mass Spectrom* **21**: 707-714.

3. Tominaga T, Dubourdieu D (2006) Novel method for quantification of 2-methyl-3-furanthiol and 2-furanmethanethiol in wines made from *Vitis vinifera* grape varieties. *J Agric Food Chem* **54**: 29-33.

4. Segurel MA, Razungles AJ, Riou C et al (2004) Contribution of dimethyl sulfide to the aroma of Syrah and Grenache Noir wines and estimation of its potential in grapes of these varieties. *J Agric Food Chem* **52**: 7084-7093.

5. Picard M, Thibon C, Redon P et al (2015) Involvement of dimethyl sulfide and several polyfunctional thiols in the aromatic expression of the aging bouquet of red Bordeaux wines. *J Agric Food Chem* **63**: 8879-8889.
# Characterization of precipitate formation in Bordeaux red wine with solid-state and solution NMR

S. PRAKASH<fnref n="1" />, N. ITURMENDI<fnref n="2" />, A. GRÉLARD<fnref n="1" />, V. MOINE<fnref n="2" />, E.J. DUFOURC<fnref n="1" />*

Precipitate – solid-state NMR – red wine

## Introduction

In recent years, stability of wine has become one of the important quality parameters required for marketing wine. Instability in red wine leads to flocculation and finally to the appearance of a precipitate or deposit. The origin and the composition of this precipitate formation in red wine are not very well known. In previous studies, it has been suggested that the interaction of proteins with tannins leads to aggregate or precipitate formation in red wine [1, 2]. It has also been suggested that polysaccharides could influence this precipitate formation [3-5]. However, no conclusive evidence has been found until now in support of these suggestions.

There are many chemical and biological mechanisms that could lead to instability, flocculation and finally precipitation of the 'colouring matter'. One of the well known mechanisms responsible for red wine instability is due to the high concentration of potassium bitartrate in red wine [1, 6]. Traditionally, a cold stabilization can be employed at a temperature of -4°C to eliminate bitartrate. A cold assay has also been proposed for predicting instability due to colouring matter by placing the wine at 4°C for 48 h [7].

The chemical composition of wine makes it a complex mixture, and to assess the quality of this complex mixture and therefore the origin of the precipitate is not an easy task. Nuclear Magnetic Resonance (NMR) spectroscopy is a well established tool for determining the molecular structure of organic compounds and macromolecules [8]. In addition, it is a non-destructive technique that allows the study of samples both in solution and solid-state environment. NMR is therefore an ideal technique in identifying the molecular components of a mixture in its native state, as in the case of the red wine precipitate.

In the present study we performed a quantitative estimation of the different components in Bordeaux red wine with solid-state and solution NMR during the barrel ageing process. For this purpose, precipitates formed in Merlot and Cabernet Sauvignon wines from the Bordeaux region for the year 2013 were isolated at different stages of the barrel ageing process. Precipitates were obtained by placing the wine at -4°C for 6 days (Pre-freezing precipitate), and at 4°C for 6 days (Long Cold Storage precipitate) for $t = 1$ and 4 months for the year 2013.

## Materials and methods

### *Precipitate preparation from red wine*

Red wine samples of 4 L each were taken from the upper layers of wine in the oak barrels from Merlot and Cabernet Sauvignon (Chateau Reynon; Bordeaux), for year 2013 at time ($t$) = 1 and 4 months from the start of barrel ageing. Two precipitates were obtained at two temperatures according to the following methods: i) Pre-freezing (Pf) precipitate was obtained after placing 2 L of wine at -4°C for 6 days, and ii) Long Cold Storage (LCS) precipitate: Supernatant (2 L) from precipitate preparation after two days was kept for further 6 days at 4°C. In all the cases, after the cold exposure, the wine was centrifuged at 5,000-6,000 rpm in a JLA 8.100 rotor (Beckman Coulter, USA) to yield a dark red-coloured pellet. The supernatant from the Pf and LCS preparation were discarded. Both precipitates were taken in minimum amount of distilled water and lyophilized overnight yielding a dry red powder.

<footnote n="1">¹Institute of Chemistry & Biology of Membranes & Nano-objects, CBMN UMR5248, CNRS University of Bordeaux, Bordeaux Institute of Technology, Allée Geoffroy St Hilaire, 33600, Pessac, France</footnote>

<footnote n="2">²BioLaffort, 126 Quai De La Souys, 33100 Bordeaux, France</footnote>
## NMR measurements

**Sample preparation:** About 50 mg of the lyophilized powder was used for solid-state NMR measurements by placing the genuine powder in 4-mm Zirconia rotors (Cortecnet, Paris) of 70 µL closed with a Kel-F cap. For the solution NMR measurements, 4-5 mg of the lyophilized Pf and LCS precipitates from Merlot and Cabernet Sauvignon 2013, respectively, were placed in 500 µL of 90% H₂O and 10% D₂O. The solution was centrifuged and the supernatant was taken for measurements using a 5-mm NMR glass tube.

**Solid-state NMR measurements:** ¹³C magic angle spinning (MAS) NMR measurements were performed on a 800 MHz Bruker Avance III NMR spectrometer SB (Wissembourg, France) and on a 500 MHz Bruker Avance II NMR spectrometer WB (Wissembourg, France). Samples were spun at the magic angle at a frequency, v<sub>r</sub>, of 10 kHz. All spectra were recorded using a cross polarization, CP, pulse sequence with a two-pulse phase modulation (TPPM) proton decoupling. ¹³C chemical shifts are reported relative to the external reference of glycine whose carbonyl group was set at 176.03 ppm [9].

**Liquid state NMR measurements:** The solution NMR measurements were performed on a 800 MHz Bruker Avance III NMR spectrometer SB (Wissembourg, France) with a 5-mm TCI cryo probe (¹H-¹³C/¹⁵N/²H) and recorded with an excitation sculpting with gradients to suppress the residual water signal (HDO) [10].

## Results

The two different types of precipitates (Pf and LCS) were obtained as described in the Materials and Method section for Merlot and Cabernet Sauvignon 2013. The precipitates obtained after centrifuging the wine were weighed (data not presented here). It was observed that the amount of precipitate depends on temperature and grape variety, with higher amount obtained at -4°C (M<sub>Pf</sub> > M<sub>LCS</sub>) and for the grape variety Cabernet Sauvignon.

To identify the components in all three types of precipitates, ¹³C CPMAS NMR spectra from the precipitates (Pf and LCS) from Merlot and Cabernet Sauvignon 2013 were recorded. All the spectra from the precipitates had the same qualitative profile, but different relative quantities of the components. In Figure 1, the Pre-freezing (Pf) precipitates from Merlot 2013 at t = 1, 4 months (Figure 1A, B) and Cabernet Sauvignon 2013 at t = 4 months (Figure 1C) are shown. The assigned peaks are labelled in Figure 1. The assignment was performed by comparing with the established ¹³C chemical shift values in literature on standard compounds and by 2D NMR spectroscopy on soluble fraction of the precipitate in water. The two pairs of sharp signals at 75.3, 73.8 ppm and 181.2, 176.2 ppm from potassium bitartrate can be clearly observed in the Pf precipitate from Merlot 2013 (Figure 1A). The region from 110-160 ppm contains peaks from procyanidins and anthocyanins [11, 12] with the centre peak at 145.0 ppm assigned to the quaternary carbon C3', 155.3 ppm to C5, 7 and 9, 131 ppm to C1', 147 ppm to C4' and 116.7, 118.7 ppm to C2', 5', 6' of a catechin and a cyanidin unit. The region from 65-90 ppm is characteristic for the contribution from the sugar moieties in the polysaccharides and the region from 10-50 ppm has contribution from methyl groups in the organic acids, free amino acids and amino acids side chains. The peak at 32.7 ppm can be assigned to succinic acid, 21.8 ppm to acetic acid and lactic acid and the peak at 30.7 ppm is a contribution from the Cβ of amino acids [13]. The region from 170-190 ppm has major contributions from all the carbonyl groups.

Classes of components present in precipitates could be identified with ¹³C CPMAS NMR, however the broad peaks in the spectra made it difficult to identify and assign specific molecules in the precipitate. For this reason, precipitates were solubilized in water to perform 2D NMR spectroscopy (data not shown here). ¹H NMR spectra from the soluble fraction of the Pf and the LCS precipitates from Merlot 2013 and Cabernet Sauvignon 2013 at t = 1 month in water are shown in Figure 2A and B respectively. It could be estimated from the insoluble fraction that ~ 70% of precipitates from Merlot are soluble in water as compared to 20-40% of Cabernet Sauvignon.

The intensities of all the spectra in Figure 2 have been normalized to the soluble fraction of Pf precipitate from Merlot 2013. The major components identified and assigned in the soluble fraction of the precipitates include glycerol, succinic acid and lactic acid. Contribution from succinic acid can be seen at 2.5 ppm, lactic acid at 1.27 and 4.06 ppm and the three -CH₄OH groups of glycerol between 3.4-3.8 ppm. In addition, the peaks from the polyphenolic components are in the region from 6-8 ppm. Comparing the intensities of the spectra in Figure 2 and therefore the amounts of the components in the soluble fraction,
it is evident that the Pf precipitate from Merlot 2013 contains the most polyphenols and LCS precipitate
from Cabernet Sauvignon 2013 contains the most lactic acid.

<figure><img src="image_374.png" /><figcaption>Fig. 1 - ¹³C MAS NMR spectra of Pf precipitates from 2013 in a 4-mm rotor as obtained at a magnetic field strength of 18.8 and 11.7 T, 298 K, spinning frequency v=10 kHz, with a CP pulse sequence.</figcaption></figure>

A) Merlot 2013, 18.8 T. B) Merlot 2013, 11.7 T, t = 4 months. C) Cabernet Sauvignon 2013, 11.7 T, t = 4 months. The side bands are marked with an asterisk and the peaks from potassium bitartrate with a (K).

<figure><img src="image_375.png" /><figcaption>Fig. 2 - ¹H-NMR spectra of the water-soluble part of Pf and LCS precipitate for Merlot 2013 and Cabernet Sauvignon 2013 at t = 1 month as obtained at a magnetic field strength of 18.8 T, 298 K, with an excitation sculpting pulse sequence to suppress the water signal (see text). A) Pf precipitate from Merlot 2013 and B) LCS precipitate from Cabernet Sauvignon 2013. Intensities of the spectra have been normalized to the soluble fraction of Pf precipitate from Merlot 2013 in water.</figcaption></figure>

Discussion

NMR of the solid precipitates in Bordeaux red wine has brought a lot of information regarding the nature
and composition of the precipitates, and these results raise many points for discussion. The main findings
can be summarized as follows:
a) Solid state ¹³C-NMR can be considered as a robust analytical method for determining semi-quantitatively the components in wine precipitates.

b) A quick method for analyzing red wines both at 4°C and -4°C can be proposed.

c) Precipitates are dominated by potassium bitartrate, tannins, and polysaccharides.

All of these points are discussed below.

¹³C CPMAS NMR spectra recorded from the various precipitates of wine colouring matter, obtained either at -4°C or 4°C, give at once a complete view of their carbon composition. They are obtained with only a few mg of genuine residue, in a powder form, which is not altered by the experiment. It is possible to readily identify families of components and estimate the relative amounts of components. For example it is noteworthy that after a few months of ageing in oak barrels the NMR signature of potassium bitartrate has completely disappeared, thus bringing a quick diagnostic for the presence or absence of this compound in coloured matter. Other families of major compounds include polyphenols, polysaccharides, glycerol and acids. The solid state NMR technique can be complemented by liquid state NMR on partly solubilized precipitates. The additional resolution brought by the technique allows further identification of compounds.

We have shown that precipitation of several tens of mg of colouring matter occurs both at -4°C and 4°C, two temperatures that are used in the winemaking process. It is noteworthy that the coldest temperature (-4°C) favors the largest amount of precipitate formation. A quick method for analyzing red wines both at 4°C and -4°C can then be proposed.

Tannins are present in large amounts in red wines (3-5 g/L) and it is clear from the NMR data that tannins are also strongly present at all stages of precipitate formation. The predominant peaks observed from polyphenols in the ¹³C CPMAS NMR spectra are from catechin, epicatechin and cyanidin units. It is however difficult to differentiate individual tannin species from oligomeric tannins and no difference at this stage can be made between polyphenols coming from Merlot and those from Cabernet Sauvignon. It is interesting to relate the variability in precipitates obtained from different grape varieties to their water solubility. In general, ~ 70% of precipitates from Merlot are soluble in water as compared to 20-40% of Cabernet Sauvignon. The difference in solubility indicates a difference in composition of the two precipitates. This solubility difference could also be due to the mean polymerization degree (mDP) of wines since Cabernet Sauvignon tannins are more polymerized than Merlot ones [14, 15].

Arabinose units and tentatively mannose units were identified by 2D NMR spectroscopy, indicating the presence of arabinogalactan proteins and mannoproteins in the precipitate. However, it was difficult to distinguish between glucose and mannose units and no signature of peptidic NH resonances were observed in the 2D NMR data that could indicate the presence of proteins. Amino acids that are predominant in the precipitate include proline, alanine, leucine and arginine, among which proline is the most abundant. The fact that only a few amino acids have been identified prompts us to assign the resonances to free species.

Organic acids such as succinic and lactic acids and diol such as glycerol are present in large quantities in the precipitates. It is however surprising to find them as they are hydrophilic molecules and very soluble in alcohol-water solutions. At this stage one may only speculate that they are trapped in complexes with other less soluble compounds.

## Conclusion

Solid state NMR has been shown to be the technique of choice to semi-quantify components in wine precipitates. Analyzing precipitates as a function of temperature and grape variety highlights the role of potassium bitartrate and polyphenols in precipitation phenomena. This opens a new avenue for further characterization of precipitates, and in particular their exact composition that may help wine makers in producing wines of greater stability.

## Acknowledgements

The Conseil Régional d'Aquitaine and Très Grands Equipements de Résonance Magnétique Nucléaire à Très Haut Champ, Fédération de Recherche (TGE RMN THC FR 3050) are gratefully acknowledged.
The authors would like to thank Philippe Louazil and Estelle Morvan for their help and support. Finally, the authors would especially like to thank Château Reynon and Denis Dubourdieu (Béguey, Gironde, France) for the donation of wine samples.

## References

1. Ribéreau-Gayon P, Glories Y, Maujean A et al (2006) *The Chemistry of Wine. Stabilization and Treatments*. 2nd ed. Handbook of Enology. Vol. 2. John Wiley and Sons.

2. Waters EJ, Peng Z, Pocock KF et al (1994) Solid-state ¹³C NMR investigation into insoluble deposits adhering to the inner glass-surface of bottled red wine. *J Agric Food Chem* **42**: 1761-1766.

3. Riou V, Vernhet A, Doco T et al (2002) Aggregation of grape seed tannins in model wine - effect of wine polysaccharides. *Food Hydrocoll* **16**: 17-23.

4. Poncet-Legrand C, Doco T, Williams P et al (2007) Inhibition of grape seed tannin aggregation by wine mannoproteins: effect of polysaccharide molecular weight. *Am J Enol Vitic* **58**: 87-91.

5. Ducasse MA, Canal-Llauberes RM, de Lumley M et al (2010) Effect of macerating enzyme treatment on the polyphenol and polysaccharide composition of red wines. *Food Chem* **118**: 369-376.

6. Lasanta C, Gomez J (2012) Tartrate stabilization of wines. *Trends Food Sci Technol* **28**: 52-59.

7. Lagune-Ammirati L, Glories Y (2001) Produits de clarification, une alternative rapidement exploitable : les dérivés de l’albumine d’œuf. *Rev Fr Œnol* **191**: 25-33.

8. Wagner G (1997) An account of NMR in structural biology. *Nat Struct Biol* **4**: 841-844.

9. Strohmeier M, Stueber D, Grant DM (2003) Accurate C-13 and N-15 chemical shift and N-14 quadrupolar coupling constant calculations in amino acid crystals: Zwitterionic, hydrogen-bonded systems. *J Phys Chem A* **107**: 7629-7642.

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11. Newman RH, Porter LJ, Foo LY et al (1987) High-resolution ¹³C NMR studies of proanthocyanidin polymers (condensed tannins). *Magn Reson Chem* **25**: 118-124.

12. Watanabe M (1998) Catechins as antioxidants from buckwheat (*Fagopyrum esculentum* Moench) groats. *J Agric Food Chem* **46**: 839-845.

13. Pretsch E, Buehlmann P, Affolter C (2000) *Structure Determination of Organic Compounds - Tables of Spectral Data*. Third ed. Germany: Springer-Verlag.

14. Chira K, Jourdes M, Teissedre PL (2012) Cabernet sauvignon red wine astringency quality control by tannin characterization and polymerization during storage. *Eur Food Res Technol* **234**: 253-261.

15. Chira K, Pacella N, Jourdes M et al (2011) Chemical and sensory evaluation of Bordeaux wines (Cabernet-Sauvignon and Merlot) and correlation with wine age. *Food Chem* **126**: 1971-1977.
# Impact des thiols volatils dans l'arôme typique des vins de Vitis vinifera var. Chardonnay

J. GROS<fnref n="1" />,<fnref n="2" />*, F. THIBAUD<fnref n="1" />,<fnref n="2" />, A. MARCHAL<fnref n="1" />,<fnref n="2" />, V. LAVIGNE<fnref n="3" />, V. MOINE<fnref n="4" />, P. DARRIET<fnref n="1" />,<fnref n="2" />

## Thiols volatils – analyse en composante principale – quantification – Chardonnay

### Introduction

Le Chardonnay est le second cépage blanc le plus planté au monde et les vins qui en sont issus rencontrent un succès croissant auprès des amateurs. Ils possèdent leur propre espace sensoriel puisqu'il a été démontré que les experts reconnaissent distinctement leurs signatures parmi des vins issus d'autres cépages dégustés à l'aveugle [1, 2]. Ce phénomène met en avant le principe de typicité [2]. Les vins blancs de Bourgogne constituent ainsi l'expression la plus aboutie et la plus originale du Chardonnay. Plusieurs études sur les arômes des vins de Chardonnay ont été effectuées, intégrant l'analyse de différents marqueurs volatils [3-6].

Lors d'une étude sensorielle des vins par des experts, des odeurs pouvant être associées à des thiols volatils ont été perçues. Cette famille chimique ayant rarement été étudiée dans les vins de Chardonnay, il a semblé intéressant d'examiner la relation entre leur concentration et les descripteurs olfactifs cités par un panel. Pour cela, une méthode de quantification ainsi qu'une approche sensorielle suivie d'analyses en composante principale ont été mises en place.

### Matériel et méthodes

#### Analyses sensorielles

Six vins de Bourgogne élaborés exclusivement à partir de Chardonnay (Chassagne Montrachet, Puligny Montrachet et Pernand Vergelesses) ont été présentés à un panel de 34 jurés. Il leur a été demandé de laisser libre cours à leur imagination et d'utiliser tout le vocabulaire qu'ils souhaitaient pour décrire l'odeur des vins. Les analyses sensorielles sont effectuées dans une salle répondant aux normes AFNOR. La température y est constante (20°C). Les verres utilisés sont noirs opaques afin que le jury ne soit pas influencé par d'éventuelles différences d'aspect visuel des vins.

#### Extraction des vins

Afin de simplifier la composition complexe des vins, l'extraction en phase solide a été privilégiée pour cette étude. Des cartouches contenant une phase Lichrolut®EN (40.120 µm phase copolymère styrène-divinylbenzène) ont été utilisées. Les cartouches sont lavées avec 10 mL de dichlorométhane, séchées à l'aide du vide, puis conditionnées avec 5 mL de méthanol et 10 mL d'un mélange eau/éthanol (90/10). 50 mL de vin sont alors percolés à un débit de 5 mL/min avant rinçage de la cartouche avec 10 mL d'eau et élution par le solvant (10 mL de dichlorométhane). L'extrait séché puis concentré à 100 µL sous flux de diazote est injecté par CPG-olfactométrie sur un appareil HP5890 sur une colonne de type carbowax 20M (BP20) 30 m, 0.22 mm, 0.5 µm.

#### Quantification des thiols volatils

L'extraction des thiols volatils requiert une méthode spécifique développée par Tominaga et Dubourdieu [7]. Son principe est de créer un complexe thiols-pHMB pour que celui-ci soit retenu par une résine DOWEX. Les composés qui n'ont pas été fixés sur la colonne sont éliminés par un lavage avec un tampon acétate puis la percolation des thiols se fait grâce à la cystéine. Une extraction liquide-liquide par dichlorométhane est effectuée. L'extrait est séché puis concentré pour être injecté en CPG-SM avec un détecteur de masse Agilent 5975.

<footnote n="1">¹Univ. de Bordeaux, ISVV, EA 4577, Unité de recherche OENOLOGIE, 33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²INRA, ISVV, USC 1366 OENOLOGIE, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="3">³Seguin Moreau France, Z.I. Merpins, Cognac F-16103, France</footnote>

<footnote n="4">⁴Biolaffort, 126 quai de la Souys, Bordeaux, France</footnote>
# Résultats et discussion

## Etablissement d'un profil sensoriel

Le panel a pu laisser libre cours à son imagination pour décrire les 6 vins de Chardonnay présentés. 35 descripteurs ressortent de cette étude (fréquence de citation >5) (Figure 1a). Un consensus est observé entre les descripteurs donnés par le panel et les zones odorantes d'intérêt trouvées en CPG-olfactométrie (Figure 1b). Paradoxalement, le caractère «pierre à fusil» présente la fréquence de citation la plus importante alors qu'il ne correspond qu'à une seule zone de l'aromagramme. A l'inverse, les arômes «noisette» et «amande» représentent une grande partie de zones odorantes de l'aromagramme tandis qu'ils sont respectivement 5ème et 12ème dans les descripteurs les plus cités de l'analyse sensorielle.

<figure><img src="image_376.png" /><figcaption>**Fig. 1** - Descripteurs donnés aux odeurs senties a) en analyse sensorielle (fréquence de citation = nombre de répétitions du descripteur/nombre total de descripteurs) et b) en analyse CPG-O (zones d'intérêt senties dans les extraits de vins de Chardonnay et absentes dans les vins d'autres cépages blancs).</figcaption></figure>

## Quantification des thiols dans les vins de Chardonnay

La présence de thiols volatils a été suspectée lors de séances d'analyse sensorielle due à une forte corrélation entre les nuances aromatiques perçues dans les vins (pierre à fusil, agrumes, fruits jaunes) et les odeurs de thiols volatils déjà connus des experts. L'addition de sulfate de cuivre (100 mg/L) a permis de confirmer l'extinction de ces notes aromatiques. Le jury n'a pas été capable de reconnaitre le vin de Chardonnay parmi les autres cépages. Fort de ces résultats, la quantification de plusieurs thiols volatils (furfurylthiol ou 2-methanefuranthiol (FFT), 3-sulfanylhexan-1-ol (3SH), 2-méthyl-3-furanthiol (2M3F), benzenemethanethiol (BM)) a été effectuée dans les 6 vins de Chardonnay de Bourgogne (Tableau 1).

<table><caption>**Tableau 1** - Concentrations de 4 thiols volatils obtenus pas GC-MS : FFT, Furfurylthiol; BM, Benzenemethanethiol; 3SH, 3-Sulfanylhexan-1-ol; 2M3F, 2-Méthyl-3-furanthiol</caption><thead><tr><th rowspan="2">Vins</th><th colspan="4">Concentration en ng/L</th></tr><tr><th>FFT</th><th>BM</th><th>3SH</th><th>2M3F</th></tr></thead><tbody><tr><td>A</td><td>4.1</td><td>12.8</td><td>160.4</td><td>41.5</td></tr><tr><td>B</td><td>18.3</td><td>38.2</td><td>380.5</td><td>17.7</td></tr><tr><td>C</td><td>32.1</td><td>21.4</td><td>57.2</td><td>5.9</td></tr><tr><td>D</td><td>64.3</td><td>42.9</td><td>100.7</td><td>3.2</td></tr><tr><td>E</td><td>17.1</td><td>17.2</td><td>251.6</td><td>31.4</td></tr><tr><td>F</td><td>28.8</td><td>8.1</td><td>32.4</td><td>5.3</td></tr></tbody></table>
Le furfurylthiol (FFT) est présent dans les vins C, D et F à des concentrations supérieures à 28 ng/L c'est-à-dire nettement supérieures à son seuil de détection olfactive (Op 0.4 ng/L en solution modèle de composition proche du vin) [8]. Les vins B et D présentent une forte concentration en benzenemethanethiol (BM) (>38 ng/L) qui a un seuil de détection de 0.3 ng/L [9]. Le 2-méthyl-3-furanthiol [10] (seuil de détection de 0.4 ng/L) a, quant à lui, une concentration jusqu'à dix fois supérieure dans les vins A et E par rapport aux autres vins. Les vins B, E, A, D contiennent des concentrations en 3-sulfanylhexanol (3SH) au moins 10 fois supérieures au seuil de détection olfactive de ce composé dans les vins (Op 16 ng/L). Compte tenu du pouvoir odorant de ces composés en regard des concentrations dosées dans les vins, il est vraisemblable que ces thiols contribuent significativement à l'arôme des vins de Chardonnay.

## Analyse des corrélations entre composés volatils et tonalités aromatiques principales

Les aromagrammes obtenus grâce à la CPG-olfactométrie dévoilent plusieurs zones odorantes spécifiques aux vins de Chardonnay elles-mêmes potentiellement attribuées à des thiols volatils. Une analyse en composante principale (Figure 2) a donc été effectuée pour déterminer s'il y avait une corrélation entre ces descripteurs dans les vins et les concentrations en thiols volatils (dimensions des axes).

<figure><img src="image_377.png" /><figcaption>**Fig. 2** - Analyse en Composante Principale qui corrèle les intensités de chaque descripteur cité par le panel et les concentrations en FFT, BM, 3SH et 2M3F (gauche) pour des vins donnés (droite).</figcaption></figure>

Un consensus entre le descripteur « pierre à fusil » et les molécules BM et FFT est observé en dimension 2 impliquant un impact potentiel de ces composés dans les tonalités empyreumatiques des vins de Chardonnay comme déjà suggéré par Tominaga et al. en 2003 [9], tandis que les vins A et E présentent des concentrations en 2M3F importantes et sont associés à des notes « amande » et « noisette ». Cette observation soulève l'hypothèse d'une implication du 2M3F dans l'arôme de noisette caractéristique des vins de Chardonnay.

## Conclusion

Les résultats de cette première étude démontrent des corrélations entre les concentrations en thiols volatils observées dans des vins de Chardonnay et les descripteurs associés à leur typicité aromatique. En particulier, le FFT et le BM apparaissent comme associés aux notes empyreumatiques, en particulier au descripteur « pierre à fusil », ce qui rejoint les conclusions d'études préalables. De façon plus originale, une corrélation entre la teneur en 2M3F et les nuances de fruit à coque (noisette et amande) a également été observée. Ces données suggèrent la contribution des composés soufrés à l'identité aromatique des vins de Chardonnay.

Il paraît intéressant d'étendre les analyses sensorielles et quantitatives à d'autres vins de Chardonnay et d'autres cépages blancs pour préciser l'impact sensoriel des thiols sur la typicité des vins de Chardonnay.
D'autres types d'analyses sensorielles telles que la supplémentation ou la reconstitution pourraient aider à affirmer ou infirmer cette étude. Une approche inductive impliquant conjointement la CPG-olfactométrie et la spectrométrie de masse pourra par ailleurs permettre de rechercher d'autres molécules contribuant à l'arôme des vins de Chardonnay.

Références

1. Ballester J (2004) Mise en évidence d'un espace sensoriel et caractérisation des marqueurs relatifs à l'arôme des vins issus du cépage Chardonnay. *Thèse de doctorat*, Université de Bourgogne.

2. Charters S, Pettigrew S (2007) The dimensions of wine quality. *Food Qual Prefer* **18**: 997-1007.

3. Moio L, Schlich P, Etievant P (1994) Acquisition and analysis of aromagrams of Chardonnay Burgundy wines. *Sci Aliments* **14**: 601-608.

4. Lee SJ, Noble AC (2003) Characterization of odor-active compounds in Californian Chardonnay wines using GC-olfactometry and GC-mass spectrometry. *J Agric Food Chem* **51**: 8036-8044.

5. Ballester J, Dacremont C, Le Fur Y et al (2005) The role of olfaction in the elaboration and use of the Chardonnay wine concept. *Food Qual Prefer* **16**: 351-359.

6. Jaffré J, Valentin D, Meunier JM et al (2011) The Chardonnay wine olfactory concept revisited: a stable core of volatile compounds, and fuzzy boundaries. *Food Res Int* **44**: 456-464.

7. Tominaga T, Dubourdieu D, Murat ML (1998) Development of a method for analyzing the volatile thiols involved in the characteristic aroma of wines made from *Vitis vinifera* L. cv. Sauvignon Blanc. *J Agric Food Chem* **46**: 1044-1048.

8. Tominaga T, Baltenweck-Guyot R, Peyrot des Gachons C et al (2000) Contribution of volatile thiols to the aromas of white wines made from several *Vitis vinifera* grape varieties. *Am J Enol Vitic* **51**: 178-181.

9. Tominaga T, Guimberteau G, Dubourdieu D (2003) Contribution of benzenemethanethiol to smoky aroma of certain *Vitis vinifera* L. wines. *J Agric Food Chem* **51**: 1373-1376.

10. Bouchilloux P, Darriet P, Dubourdieu D (1998) Identification d'un thiol fortement odorant, le 2-méthyl-3-furanthiol, dans les vins. *Vitis* **37**: 177-180.
# Origine de notes olfactives « mastic » dans les eaux-de-vie nouvelles de cognac

L. URRUTY*, D. LAURICHESSÉ, L. MORNET, B. LOISEAU, J.-C. MATHURIN

E. Rémy Martin & C°, avenue de la Grande Champagne, ZI de Merpins, 16100 Merpins, France

Distillation – défaut – GC-MS/O – OAV

## Introduction

La Maison Rémy Martin élabore des cognacs à partir d'eaux-de-vie nouvelles rigoureusement sélectionnées. Ces eaux-de-vie de vin, fraîchement distillées, seront ensuite vieillies puis assemblées.

Chaque eau-de-vie nouvelle est dégustée, avant achat, par une commission d'agrément. Lors de ces dégustations, les descripteurs « mastic », « huile de lin » sont parfois cités par certains juges. Quand ce caractère est très marqué, l'eau-de-vie est jugée défectueuse et peut être refusée par la commission d'agrément.

L'objectif de ces travaux est d'identifier l'origine de ces notes olfactives « mastic ».

## Matériel et méthodes

### Analyse par GC-MS/O

L'eau-de-vie nouvelle (50 mL) est mélangée à 8 g de NaCl, 80 mL d'eau ultra-pure et 20 mL d'une solution de carbonate de sodium à 42 g/L. Ce mélange est extrait par 8 mL de dichlorométhane pendant 5 minutes. La phase organique est récupérée et concentrée sous flux d'azote jusqu'à 0.5 mL. Cet extrait est injecté sur un système composé d'un chromatographe en phase gazeuse Agilent GC6890 couplé à un spectromètre de masse Agilent MSD5975 et d'un port d'olfaction Gerstel ODP3. La séparation est effectuée sur une colonne DB-waxetr de 60 m, 0.32 mm de diamètre interne et 0.25 µm d'épaisseur de phase.

### Analyse quantitative par GC/MS

10 mL d'eau-de-vie nouvelle sont mélangés à 15 mL d'eau ultra-pure. Après addition de 100 µL d'une solution de *trans*,*trans*-2,4-undécadiénal à 10 mg/L (étalon interne), 2 mL de solvant (pentane:dichlorométhane 80:20) sont ajoutés. L'extraction est réalisée sur rolling pendant 30 minutes. La phase organique est récupérée et concentrée sous flux d'azote jusqu'à 0.2 mL. Cet extrait est injecté sur le même GC/MS que celui utilisé en GC/MS-O mais le port d'olfaction est déconnecté et l'analyse est réalisée en mode SIM (m/z 81, 138, 152 et 166).

## Résultats et discussion

Six eaux-de-vie nouvelles de la récolte 2013 jugées « mastic » par la commission d'agrément ont été analysées en GC-MS/O. Deux zones olfactives « mastic » ont été mises en évidence, aux indices de rétention $IR_{DBwax}$ 1692 et 1795. Les composés responsables de ces zones ont été identifiés à partir de leur indice de rétention et de leur spectre de masse (Tableau 1). Il s'agit du *trans*,*trans*-2,4-nonadiénal et du *trans*,*trans*-2,4-décadiénal, déjà mis en évidence dans des eaux-de-vie de vin [1].

Ces deux zones « mastic » ont également été retrouvées dans deux eaux-de-vie 2013 ne présentant, à la dégustation, aucune note « mastic » mais elles semblent moins intenses dans ces deux derniers échantillons. Afin de vérifier ce point, une méthode de dosage du *trans*,*trans*-2,4-nonadiénal et du *trans*,*trans*-2,4-décadiénal a été mise en place et a été appliquée à des eaux-de-vie élaborées à partir de raisins récoltés en 2013 et 2014. Dans ces échantillons, les teneurs observées varient de quelques ng/L à 15 µg/L pour le *trans*,*trans*-2,4-nonadiénal et 60 µg/L pour le *trans*,*trans*-2,4-décadiénal. Généralement, le *trans*,*trans*-2,4-décadiénal est majoritaire.
<table><caption>**Tableau 1 - Identification des composés responsables des zones olfactives « mastic »**</caption><thead><tr><th>IR<sub>DBwax</sub></th><th>Composé</th><th>lons m/z</th></tr></thead><tbody><tr><td>1692</td><td>trans,trans-2,4-nonadiénal</td><td>81<sub>(100%)</sub> ; 95<sub>(9%)</sub> ; 138<sub>(11%)</sub></td></tr><tr><td>1795</td><td>trans,trans-2,4-décadiénal</td><td>81<sub>(100%)</sub> ; 95<sub>(10%)</sub> ; 152<sub>(7%)</sub></td></tr></tbody></table>

La commission de dégustation a classé ces échantillons en 3 catégories :

- Sans défaut (n=15) : eaux-de-vie ne présentant aucune note « mastic » ;

- Suspect (n=30) : eaux-de-vie pour lesquelles au moins un juge a utilisé le descripteur « mastic » ;

- Défaut (n=11) : eaux-de-vie refusées car présentant un caractère « mastic » marqué.

Les eaux-de-vie défectueuses se révèlent plus riches en *trans,trans-2,4-nonadiénal* et *trans,trans-2,4-décadiénal* (Figure 1).

<figure><img src="image_378.png" /><figcaption>**Fig. 1 - Dosage du *trans,trans-2,4-nonadiénal* et du *trans,trans-2,4-décadiénal* dans des eaux-de-vie présentées pour agrément (récoltes 2013 et 2014).**</figcaption></figure>

Le seuil de perception du *trans,trans-2,4-nonadiénal* est de 2.4 µg/L en solution hydroalcoolique à 40 % vol., celui du *trans,trans-2,4-décadiénal*, 10.1 µg/L [2]. Les OAV déterminés à partir de ces valeurs indiquent que ces deux composés peuvent expliquer le caractère « mastic ».

La sélection d'eaux-de-vie de la récolte 2013 a également été dégustée par 14 juges de la commission d'agrément, afin de noter l'intensité du caractère « mastic ». Cette note « mastic » a été comparée aux OAV calculés : les résultats de la dégustation sont cohérents avec le calcul des OAV (Figure 2). Le caractère « mastic » est cependant plus difficile à déceler dans les eaux-de-vie les plus riches en composés volatils aromatiques comme les esters éthyliques d'acides gras.

Le *trans,trans-2,4-nonadiénal* et le *trans,trans-2,4-décadiénal* proviendraient de l'auto-oxydation de l'acide linoléique présent dans les vins de distillation et leurs lies [3]. Des températures élevées favoriseraient cette transformation.

La Maison Rémy Martin conseille de limiter le préchauffage des vins à 2 heures maximum, à des températures ne dépassant pas 40°C. Le préchauffage des brouillis, quant à lui, est fortement déconseillé. D'autre part, la chaudière, le bassin d'imparfaits et le réchauffe-vin doivent être régulièrement nettoyés, afin d'éviter l'accumulation d'acides gras.
<figure><img src="image_379.png" /><figcaption>**Fig. 2** - Relation entre l'intensité de la note « mastic » et la somme des OAV du *trans,trans*-2,4-nonadiénal et *trans,trans*-2,4-décadiénal dans les eaux-de-vie de la récolte 2013.</figcaption></figure>

## Conclusion

Ce travail a permis d'identifier le *trans,trans*-2,4-nonadiénal et le *trans,trans*-2,4-décadiénal comme responsables des notes « mastic » détectées parfois dans les eaux-de-vie de cognac. Ils proviendraient de l'oxydation de l'acide linoléique mais une bonne maîtrise du préchauffage des vins, une absence de préchauffage des brouillis et le nettoyage régulier des alambics limitent la formation de ces composés.

## Abréviations

GC-MS/O, chromatographie en phase gazeuse couplée à la spectrométrie de masse et l'olfactométrie ;
OAV, odor activity value.

## Références

1. Williams PJ, Strauss CR (1978) Spirit recovered from heap-fermented grape marc: nature, origin and removal of the off-odour. *J Sci Food Agric* **29**: 527-533.

2. Bureau National Interprofessionnel du Cognac (2014) *Rapport d'activité de la Station Viticole - campagne 2013-2014*.

3. Bourgeois G, Vivas N, Glories Y et al (1995) Identification par spectrométrie de masse couplée à la chromatographie en phase gazeuse des produits de dégradation des hydroperoxydes de l'acide linoléique. *Sci Aliments* **15**: 625-630.
# Purification and characterization of three new condensed tannin oligomers in red wine

L. ZENG<sup>1,2</sup>, T. RICHARD<sup>3</sup>, P.-L. TEISSEDRE<sup>1,2</sup>, M. JOURDES<sup>1,2*</sup>

<sup>1</sup>Univ. Bordeaux, ISVV, EA 4577, Œnologie, 210 Chemin de Leysotte, F-33140 Villenave d'Ornon, France

<sup>2</sup>INRA, ISVV, USC 1366 Œnologie, F-33140 Villenave d'Ornon, France

<sup>3</sup>Univ. Bordeaux, ISVV, GESVAB EA 3675, 210 Chemin de Leysotte, F-33140 Villenave d'Ornon, France

Tannin oligomer – red wine – phloroglucinolysis – high resolution mass spectrometry

## Introduction

Procyanidins, also known as condensed tannins, are one of the most important classes of polyphenolic compounds in red wine. They are responsible for important sensory properties such as astringency and bitterness, which are the main factors contributing to the balance of red wine [1]. Besides, the potential health benefits of condensed tannins such as anti-oxidant, anti-inflammatory, cardiovascular system amelioration, and hyper tension diminution effect have been proved in several studies [2]. Procyanidins or condensed tannins are complex polymers of flavan-3-ols. In red wine, there are five monomeric units: (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Monomeric units can be linked together by direct inter-flavanoid linkage or mediated by aldehydes [3].

During our investigation on red wine condensed tannins, three surprisingly polar tannin oligomers (one tetramer and two pentamers) were detected and it was noticed that their concentrations remained stable during wine aging. Thus the objective of this study was to establish a purification method for these three unknown condensed tannin oligomers observed in red wine and to determine their structures by a high resolution mass spectrometry, chemical depolymerization strategy, as well as NMR.

## Materials and methods

### Reagents

Deionized water was purified with a Milli-Q water system (Millipore, Bedford, MA). Methanol (analytical and HPLC grade), ethanol (HPLC grade), L-ascorbic acid and hydrochloric acid were purchased from Prolabo-VWR (Fontenays/Bois, France). Water (Optimal® LC/MS), methanol (Optimal® LC/MS) and formic acid (Optimal® LC/MS) were obtained from Fisher Scientific (Geel, Belgium) for high resolution mass spectrometry analysis. Phloroglucinol was obtained from Extrasynthese (Z.I Lyon Nord, France).

### Purification of three novel condensed tannin oligomers in red wine

A three-step purification method was established with the first step of C-18 solid phase extraction, the second step of Toyopearl HW-40S gel filtration chromatography and the last step of C-18 HPLC semipreparative.

### Phloroglucinolysis: depolymerization of pure tannin oligomers

A fraction of pure tetramer after HPLC-semipreparative was dissolved in 200 µL methanol. The phloroglucinolysis reagent solution (200 µL) containing 0.1 N HCl in methanol, 50 g/L phloroglucinol and 10 g/L ascorbic acid was then added to the 200 µL of the methanol fraction above. Then, the reaction mixture was placed at 50°C for 20 min and 1 mL of 40 mM acetate sodium aqueous solution was added to stop the reaction. The reaction mixture was then analyzed by UPLC-UV-Q-TOF.
## UPLC-Q-TOF

The UPLC-MS system used was an Agilent 1290 Infinity equipped with an ESI-Q-TOF-MS (Agilent 6530 Accurate Mass). Chromatographic separation was carried out on an Eclipse Plus C18 column (2.1 x 100 mm, 1.8 µm). The solvents used were: water with 0.1% formic acid for solvent A and methanol with 0.1% formic acid for solvent B at a flow rate of 0.4 mL/min. The gradient of solvent B for oligomer analysis was as follows: 4% of B during 10 min; from 4 to 95% of B in 4 min; 95% during 4 min and UPLC column was equilibrated for 3 min using the starting condition. ESI conditions were as follows: gas temperature and flow were set at 300°C and 9 L/min respectively; sheath gas temperature and flow were set at 350°C and 11 L/min respectively; capillary voltage was set at 4000V. The fragmentor was always set at 200 V. The data obtained were treated by Mass Hunter Qualitative Analysis software.

## NMR

Isolated compounds were analyzed by ¹H NMR, ¹³C NMR, and 2D NMR spectroscopy (COSY, HSQC, HMBC, and ROESY) in methanol-d₄. The spectra were recorded on an AC 300 MHz and Avance III 600 MHz Bruker spectrometers (Wissembourg, France).

# Results and discussion

## Purification of tetramer and pentamers

After tetramer/pentamer purification from approximately 4 L of red wine, the yield of the purification protocol was: 5.6 mg of tetramer, 1.5 mg of pentamer 1 and 2 mg of pentamer 2. The lower relative concentration of pentamers in wine could be the explanation of the lower yield achieved in the purification protocol. The purity of these fractions was estimated to be up to 99% according to the chromatograms obtained at 280 nm.

## NMR and phloroglucinolysis results

1D (¹H and ¹³C) and 2D (HSQC, HMBC, ROESY) NMR analyses were carried out on the 5.6 mg of tetramer in methanol-d₄. All proton and carbon resonances were assigned according to solvent chemical shift (Table 1). NMR data are consistent with a procyanidin tetramer constituted by two symmetric procyanidin dimer units (Figure 1). By comparison with literature, the carbon signals of the procyanidin tetramer in the range δC 155-160 ppm were assigned to 5-, 7- and 8a-position carbon signals. These assignments were performed thanks to long-range correlations between carbons 8a and protons 2 of the heterocyclic rings, correlations between carbon 5A and protons 6A and 4C, correlations between carbon 5D and 4F. The HMBC correlations between proton 4C and carbons 7D, 8D and 8aD indicate that the inter-flavanoid bond between the C and D units was 4→8. Furthermore, the HMBC correlations between proton 4F and carbons 5A, 6A and 7A proved the presence of a cyclic and symmetric structure. These last correlations also confirm the 4→6 inter-flavanoid bond between the flavanol unit II and III (Figure 1). The configuration of the carbon 2, 3, and 4 of the C and F ring were determined using the ROESY spectrum. Strong NOE correlations between aromatic protons of the B ring and protons 4C suggest that this proton is on the same side as the B ring, proving that the inter-flavanoid linkage between unit I and II is β. Similarly, a strong correlation between the protons 2C and 3C proved that these protons are on the same side, indicating that unit I and III are (-)-epicatechin. Similar strong NOE correlations between aromatic protons of E ring and proton 4F as well as NOE correlation between the protons 2F and 3F proved that the flavan-3-ol unit II and IV are (-)-epicatechin and the inter-flavanoid linkage between unit II and IV is also β. Therefore, all units of this cyclic procyanidin tetramer are (-)-epicatechin and all the inter-flavanoid linkage are β leading to the structure in figure 1.

<figure><img src="image_380.png" /><figcaption>Fig. 1 - Cyclic tetramer of condensed tannins detected in red wine.</figcaption></figure>
To confirm this new cyclic structure observed in the NMR analysis, a depolymerization by phloroglucinolysis was carried out. The results obtained by phloroglucinolysis on UPLC-Q-TOF showed that after chemical depolymerization of a pure aliquot of the tetramer, no terminal units were observed and detected. The lack of release terminal unit is one more strong argument proving that all units have to be connected by inter-flavanoid bound, hence confirming the cyclic structure. So the NMR and phloroglucinolysis results show that the structure of this more polar procyanidin tetramer is cyclic. Since such carbon skeleton has never been reported before for procyanidins in wine, or in the plant kingdom, we decided to name this new group of procyanidins "crown procyanidins". Data concerning the two pentamers are not presented in this article.

<table><caption>**Table 1** - ¹H- and ¹³C-NMR data and HMBC correlations (C → H) of cyclic tetramer</caption><thead><tr><th>ring</th><th>no.</th><th>δC</th><th>δH m(J in Hz)</th><th>HMBC (Unit: C)</th></tr></thead><tbody><tr><td colspan="5">Unit I</td></tr><tr><td rowspan="3">C</td><td>2</td><td>78.5</td><td>5.17 brs</td><td>B2', B6', C4</td></tr><tr><td>3</td><td>73.0</td><td>4.29 m</td><td>C4</td></tr><tr><td>4</td><td>37.7</td><td>4.22 brd (5.2)</td><td>C2</td></tr><tr><td rowspan="7">A</td><td>4a</td><td>104.9</td><td>-</td><td>A8, C3, C4</td></tr><tr><td>5</td><td>155.5</td><td>-</td><td>F4*, C4</td></tr><tr><td>6</td><td>106.3</td><td>-</td><td>F4*, A8</td></tr><tr><td>7</td><td>155.5</td><td>-</td><td>F4*, A8</td></tr><tr><td>8</td><td>96.1</td><td>6.13 s</td><td>-</td></tr><tr><td>8a</td><td>155.1</td><td>-</td><td>C2, C4, A8</td></tr><tr><td>1'</td><td>131.9</td><td>-</td><td>B5', C2, C3</td></tr><tr><td rowspan="6">B</td><td>2'</td><td>115.4</td><td>6.99 d (1.8)</td><td>B6', C2</td></tr><tr><td>3'</td><td>145.4</td><td>-</td><td>B5'</td></tr><tr><td>4'</td><td>145.5</td><td>-</td><td>B2', B6'</td></tr><tr><td>5'</td><td>115.5</td><td>6.72 d (8.1)</td><td>-</td></tr><tr><td>6'</td><td>119.8</td><td>6.84 dd (1.8; 8.1)</td><td>B2', B5', C2</td></tr><tr><td>Unit II</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">F</td><td>2</td><td>75.2</td><td>4.45 s</td><td>E2', E6', F3, F4</td></tr><tr><td>3</td><td>67.9</td><td>4.48 d (2.1)</td><td>F2, F4</td></tr><tr><td>4</td><td>37.8</td><td>4.56 d (2.5)</td><td>F2, F3</td></tr><tr><td rowspan="7">D</td><td>4a</td><td>98.6</td><td>-</td><td>D6, F3, F4</td></tr><tr><td>5</td><td>157.1</td><td>-</td><td>D6, F4</td></tr><tr><td>6</td><td>97.0</td><td>6.10 s</td><td>-</td></tr><tr><td>7</td><td>157.3</td><td>-</td><td>D6, C4</td></tr><tr><td>8</td><td>109.0</td><td>-</td><td>D6, C3, C4</td></tr><tr><td>8a</td><td>157.2</td><td>-</td><td>F4, C4</td></tr><tr><td>1'</td><td>131.3</td><td>-</td><td>E5', F2</td></tr><tr><td rowspan="5">E</td><td>2'</td><td>114.4</td><td>6.21 d (1.8)</td><td>E6', F2</td></tr><tr><td>3'</td><td>144.8</td><td>-</td><td>E5'</td></tr><tr><td>4'</td><td>144.6</td><td>-</td><td>E2', E6'</td></tr><tr><td>5'</td><td>115.5</td><td>6.43 d (8.1)</td><td>-</td></tr><tr><td>6'</td><td>119.7</td><td>5.76 d (1.8; 8.1)</td><td>E2', F2</td></tr></tbody></table>
**Conclusion**

For the first time in red wine, a new cyclic procyanidin sub-class that we named “crown procyanidin” was discovered, purified and structurally characterized.

One crown procyanidin tetramer and two crown procyanidin pentamers were purified by a “three steps-two gels” strategy. Their identity belonging to the procyanidin family was confirmed by high resolution mass spectrometry. The inter-flavanoid linkages, sub-unit information, as well as overall configuration of the tetramer were obtained by 1D and 2D NMR. These crown procyanidins showed high polarity, partial resistance to phloroglucinolysis condition and high stability during red wine aging. Their origins, health effect and sensory properties are to be clarified.

**Acknowledgements**

The authors thank the French Ministry of High Education and Research for providing doctoral support and the Regional Council of Aquitaine for equipment funding support (UPLC-UV-Q-TOF system).

**Abbreviations**

UPLC, ultra performance liquid chromatography; DAD, diode array detector; Q-TOF, quadruple-time of flight; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation; NOESY, nuclear overhauser effect spectroscopy.

**References**

1. Chira K, Jourdes M, Teissedre P-L (2011) Cabernet sauvignon red wine astringency quality control by tannin characterization and polymerization during storage. *Eur Food Res Technol* **234**: 253-261.

2. Petti S, Scully C (2009) Polyphenols, oral health and disease: a review. *J Dent* **37**: 413-423.

3. Es-Safi N-E, Fulcrand H, Cheynier V et al (1999) Competition between (+)-catechin and (−)-epicatechin in acetaldehyde-induced polymerization of flavanols. *J Agric Food Chem* **47**: 2088-2095.
# Rosé wines: impact of storage conditions in tank on polyphenol composition and color

M.-A. DUCASSE<fnref n="1" />*, L. CAYLA<fnref n="2" />, G. MASSON<fnref n="2" />, C. ANNERAUD<fnref n="3" />, P. CHRETIEN<fnref n="4" />, F. DAVAUX<fnref n="5" />, J.-C. BOULET<fnref n="6" />, E. MEUDEC<fnref n="6" />, A. VERBAERE<fnref n="6" />, N. SOMMERER<fnref n="6" />, V. CHEYNIER<fnref n="6" />

Rosé wine – storage – color – polyphenols

## Introduction

The color of Rosé wines is an important criterion for consumer choice. This color depends on the concentration of anthocyanins and also of other pigments derived from them. Production conditions (grape variety, winemaking) and storage conditions have an impact on the polyphenol composition, in particular on anthocyanins and other pigments which can be formed by complex chemical reactions [1, 2].

In this work, the effect of conditions of wine storage in tank (temperature, level of sulfite, oxygen exposure) on the color and phenolic composition of Rosé wines was investigated.

## Materials and methods

### Rosé wine samples

Rosé wines were made in four different vine-growing areas: Val de Loire, Provence, Tarn, and Bordeaux. In each region, common practices of storage conditions in tank during six months were compared: temperature regimes, two levels of sulfite addition, two levels of oxygen exposure. Sample description is provided in Table 1.

### Polyphenol analysis

Polyphenols were quantified by ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC- QqQ-MS) used in multiple reaction monitoring (MRM) mode. This method [3] enabled quantification of over one hundred phenolic compounds in Rosé wines.

### Color analysis

Color characteristics were determined by several color indices and chromatic coordinates (L*, a*, b*) deduced from absorbance measurements [2]. Three bottles were analyzed for each sample after bottling in the same conditions and after storage during one year at 20°C.

## Results and discussion

The polyphenol composition (quantities and proportions) was different between the four series. The concentrations of the main groups of grape compounds (hydroxycinnamic acids, anthocyanins and tannins) are presented in Figure 1. Derived pigments resulting from complex chemical mechanisms [4-8] were also found in different proportions in the four series:

- caftaric-anthocyanin adducts formed by the addition of anthocyanins onto the quinone of caftaric acid, depending on enzymatic oxidation at the must stage.

<footnote n="1">¹French Vine & Wine Institute, UMT Qualinnov, Pôle Rhône-Méditerranée, F-11430 Gruissan, France</footnote>

<footnote n="2">²Centre du Rosé, French Vine & Wine Institute, Pôle National Rosé, France</footnote>

<footnote n="3">³French Vine & Wine Institute, Pôle Bordeaux Aquitaine, France</footnote>

<footnote n="4">⁴French Vine & Wine Institute, Pôle Val de Loire, France</footnote>

<footnote n="5">⁵French Vine & Wine Institute, Vinnopole, Sud-Ouest, France</footnote>

<footnote n="6">⁶INRA UMR 1083 SPO Sciences pour l'Œnologie, Plate-forme d'analyse des polyphénols, 2 place Viala, 34060 Montpellier cedex 2, France</footnote>

<footnote>*Corresponding author: marie-agnes.ducasse@vignevin.com</footnote>
- carboxypyranoanthocyanins (e.g., vitisin A) and pyranoanthocyanins (e.g., vitisin B) formed by reaction of anthocyanins with pyruvic acid and acetaldehyde, respectively, depending on yeast metabolism and oxidation.

- phenyl-, catechyl- and guaiacyl-pyranoanthocyanins, formed by reaction of anthocyanins with hydroxycinnamic acids or vinylphenols.

<table><caption>**Table 1** - Code and storage conditions in tank of studied Rosé wines</caption><thead><tr><th rowspan="2">TRIALS</th><th rowspan="2">Code</th><th rowspan="2">Grape variety</th><th colspan="3">Storage conditions in tank</th></tr><tr><th>SO<sub>2</sub> (mg/L)</th><th>Temperature</th><th>Oxygen exposure</th></tr></thead><tbody><tr><td rowspan="4">Val de Loire</td><td>6012NO</td><td rowspan="4">Cabernet</td><td>60</td><td>12°C</td><td>normal</td></tr><tr><td>30VANO</td><td>30</td><td>variable</td><td>normal</td></tr><tr><td>6012HI</td><td>60</td><td>12°C</td><td>high</td></tr><tr><td>3012NO</td><td>30</td><td>12°C</td><td>normal</td></tr><tr><td rowspan="4">Bordeaux</td><td>18VANO</td><td rowspan="4">Cabernet franc</td><td>18</td><td>variable</td><td>normal</td></tr><tr><td>3012NO</td><td>30</td><td>12°C</td><td>normal</td></tr><tr><td>3012HI</td><td>30</td><td>12°C</td><td>high</td></tr><tr><td>30VANO</td><td>30</td><td>variable</td><td>normal</td></tr><tr><td rowspan="4">Tarn</td><td>28VANO</td><td rowspan="4">Négrette</td><td>28</td><td>variable</td><td>normal</td></tr><tr><td>3812NO</td><td>38</td><td>12°C</td><td>normal</td></tr><tr><td>38VANO</td><td>38</td><td>variable</td><td>normal</td></tr><tr><td>3812HI</td><td>38</td><td>12°C</td><td>high</td></tr><tr><td rowspan="4">Provence</td><td>2520NO</td><td rowspan="4">Grenache-Shiraz</td><td>25</td><td>20°C</td><td>normal</td></tr><tr><td>3512NO</td><td>35</td><td>12°C</td><td>normal</td></tr><tr><td>3520NO</td><td>35</td><td>20°C</td><td>normal</td></tr><tr><td>3512HI</td><td>35</td><td>12°C</td><td>high</td></tr></tbody></table>

<figure><img src="image_381.png" /><figcaption>**Fig. 1** - Composition in hydroxycinnamic acids (HA), anthocyanins and tannins of each series of Rosé wines.</figcaption></figure>

A principal component analysis was performed on all the data (color data in green, native anthocyanins in red, derived pigments in blue and other polyphenols in black) for each trial: Val de Loire (Figure 2), Bordeaux (Figure 3), Tarn (Figure 4), and Provence (Figure 5).
## Val de Loire trials

They contained a very high concentration of hydroxycinnamic acids, probably due to a varietal characteristic and a limited oxidation of must (Figure 1). A low level of sulfite and a variable temperature regime led to a loss of anthocyanins and the formation of catechyl-pyranoanthocyanins (Figure 2). Absorbance at 520 nm due to sulfite bleaching resistant pigments (SBRPigments) and the coordinate b* (yellow) were correlated with catechylpyranomalvidin-3Glc (pinotin A) (R=0.67 and R=0.72, respectively).

<figure><img src="image_382.png" /><figcaption>**Fig. 2** - Principal component analysis of the polyphenol and color data of Val de Loire trials: (A) projection of the wines on principal component 1 (PC1) and principal component 2 (PC2), (B) correlation scatterplot of the polyphenol and color variables with PC1 and PC2.</figcaption></figure>

## Bordeaux trials

They contained a low concentration of hydroxycinnamic acids (Figure 1) but relatively high concentrations of GRP (Grape React Product), flavanols (monomers, dimers, and tannins analyzed by HPLC after phloroglucinolysis), anthocyanins and tannin-anthocyanin adducts. This reflects a higher extraction rate than in the other sets and oxidation of a must with a high glutathione to caftaric acid ratio. A low level of sulfite and a variable temperature regime led mainly to an increase of color intensity, of a* and b*, of absorbance at 520 nm due to SBRPigments, phenyl- and catechyl-pyranoanthocyanins, and a loss of anthocyanins (Figure 3). In this series, SBRPigments and the coordinates a* and b* were correlated together and negatively correlated with the anthocyanin contents. However, a low correlation between phenyl- and catechyl-pyranoanthocyanins and data color (SBRPigments, a* and b*) suggests an involvement of additional pigments presumably resulting from anthocyanin reactions with tannins (not analyzed).

<figure><img src="image_383.png" /><figcaption>**Fig. 3** - Principal component analysis of the polyphenol and color data of Bordeaux trials: (A) projection of the wines on principal component 1 (PC1) and principal component 2 (PC2), (B) correlation scatterplot of the polyphenol and color variables with PC1 and PC2.</figcaption></figure>
## Tarn trials

They contained a relatively high concentration of hydroxycinnamic acids (Figure 1) but a low concentration of GRP and no caftaric-anthocyanin adducts, indicating a good protection of musts against oxidation. A low level of sulfite and a variable temperature regime led mainly to a loss of anthocyanins and an increase of color (Figure 4). This was linked to an increase of colored pigments such as vitisins and a decrease of bisulfite adducts. SBRPigments and the coordinate b* were negatively correlated with the anthocyanin concentration (malvidin-3-Glc, R=-0.83) and b* positively with carboxypyranomalvidin-3Glc (vitisin A) (R=0.76) and pyranomalvidin-3Glc (vitisin B) (R=0.72).

<figure><img src="image_384.png" /><figcaption>**Fig. 4** - Principal component analysis of the polyphenol and color data of Tarn trials:
(A) projection of the wines on principal component 1 (PC1) and principal component 2 (PC2),
(B) correlation scatterplot of the polyphenol and color variables with PC1 and PC2.</figcaption></figure>

## Provence trials

These very light Rosé wines contained a low concentration in polyphenols due to a low extraction (Figure 1). However, they had a concentration of catechyl-pyranoanthocyanins similar to the other trials. A low level of sulfite and a variable temperature regime led to increased formation of catechyl-pyranoanthocyanins (Figure 5). Color due to the derived pigments and the coordinate b* were correlated together and especially b* with p-hydroxyphenylpyranomalvidin-3Glc (R=0.72) and catechylpyranomalvidin-3Glc (Pinotin A) (R=0.69).

<figure><img src="image_385.png" /><figcaption>**Fig. 5** - Principal component analysis of the polyphenol and color data of Provence trials:
(A) projection of the wines on principal component 1 (PC1) and principal component 2 (PC2),
(B) correlation scatterplot of the polyphenol and color variables with PC1 and PC2.</figcaption></figure>
Conclusion

The polyphenol composition of Rosé wines depended of the origin, grape variety and winemaking process.
This study showed that the storage conditions in tank impacted the derived pigment composition of Rosé
wines, also depending on the proportions of native compounds (hydroxycinnamic acids, anthocyanins, and
tannins) in the wines. In particular, variable temperature regimes and low levels of sulfite led to an increase
of color attributable to a lower extent of sulfite bleaching and an increased formation of sulfite bleaching
resistant pigments. Sulfite bleaching resistant pigments and chromatic coordinate b* were correlated,
underlining the contribution of these derived pigments to the color of Rosé wines. The proportion and
quantity of the different families of derived pigments (pyranoanthocyanins, carboxypyranoanthocyanins,
phenyl- and catechyl-pyranoanthocyanins, etc.) were influenced by the concentration of native compounds
combined with the winemaking process (yeasts, storage conditions).

Acknowledgements

This study was financed by FranceAgrimer (national group “Rosé Wine conservation”).

References

1. Cheynier V, Wirth J, Morel-Salmi C et al (2013) Les composés responsables de la couleur des vins Rosés. Interaction avec l'oxygène. *Rev Fr Oenol* **260**: 30-33.

2. Wirth J, Caillé S, Souquet JM et al (2012) Impact of post-bottling oxygen exposure on the sensory characteristics and phenolic composition of Grenache rosé wines. *Food Chem* **132**: 1861-1871.

3. Lambert ML, Meudec E, Verbaere A et al (2015) A high-throughput UHPLC-QqQ-MS method for polyphenol profiling in rosé wines. *Molecules* **20**: 7890-7914.

4. Cheynier V, Rigaud J, Souquet J-M et al (1989) Effect of pomace contact and hyperoxidation on the phenolic composition and quality of Grenache and Chardonnay wines. *Am J Enol Vitic* **40**: 36-42.

5. Sarni-Manchado P, Cheynier V, Moutounet M (1997) Reaction of enzymatically generated quinones with malvidin-3-glucoside. *Phytochemistry* **45**: 1365-1369.

6. Atanasova V, Fulcrand H, Cheynier V et al (2002) Effect of oxygenation on polyphenol changes occurring in the course of wine-making. *Anal Chim Acta* **458**: 15-27.

7. Cheynier V, Dueñas-Paton M, Salas E et al (2006) Structure and properties of wine pigments and tannins. *Am J Enol Vitic* **57**: 298-305.

8. Fulcrand H, Duenas-Paton M, Salas E et al (2006) Phenolic reactions during winemaking and aging. *Am J Enol Vitic* **57**: 289-297.
# Identification and quantification of stilbenes in some Tunisian red wines using UPLC-MS and HPLC-DAD

K. ARRAKI*, E. RENOUF, P. WAFFO-TEGUO, J.M. MERILLON, T. RICHARD, A. DECENDIT

Université de Bordeaux, ISVV, EA 3675 GESVAB, 33882 Villenave d'Ornon, France

Stilbenes – red wines – UPLC-MS

## Introduction

Numerous epidemiological studies since the 90s have shown that a very moderate wine consumption may be beneficial to health [1]. The presence of polyphenols and particularly stilbenes in wine may explain these beneficial effects. Among stilbenes, resveratrol has demonstrated a number of promising biological activities, particularly in regards to disease prevention and anti-aging activities [2, 3]. Many studies have examined the resveratrol content in wine [4, 5]. In contrast, few studies have investigated stilbene oligomers in wine. However, some studies indicate that the wine contains many other stilbenes [6, 7].

The aim of the present work was to develop a method to characterize stilbene content in Tunisian red wine. While many wines, especially French wines, have been studied for their contents in resveratrol, to our knowledge, no Tunisian wine has yet been explored in this regards. The presence of various stilbenes has been studied using UPLC-MS analysis on wine extracts. These compounds have been quantified by HPLC-DAD and different stilbenes have been identified, from the simplest monomers, such as resveratrol and piceid, to the more complex oligomers, such as ε- and α-viniferin, hopeaphenol and isohopeaphenol. Concerning standards, if the simpler molecules may be obtained commercially, the complex stilbenes need to be purified from different plant sources. In this study, the standards were purified from vine parts and by-products from Carex species [8, 9].

In this preliminary study, seven Tunisian red wines mainly in the Mornag appellation were investigated. Seven stilbenes were identified in these wines: three monomers (resveratrol, piceid and piceatannol), one dimer (ε-viniferin), one trimer (α-viniferin) and two tetramers (hopeaphenol and isohopeaphenol). Regarding the presence of resveratrol derivatives, we found that one of the wines, Sidi Zahia, was the richest qualitatively. These first results are very encouraging. The study should be extended to a larger number of wines and appellations.

## Materials and methods

### Wine samples

Seven Tunisian red wines from the Mornag appellation (Clipea, Distinto, Mornag, Selian, Sidi Brahim, Sidi Rais and Sidi Zahia) were examined for their stilbene content, by using UPLC-MS and HPLC-DAD methods. The wines were purchased from local stores and kept in obscurity until analyzed.

### Stilbene purification

In order to remove sugars and part of anthocyanins and proanthocyanidins, the different wines were evaporated and purified on XAD16 and DOWEX columns (Fig. 1). Each 750-mL bottle of wine was concentrated down to 650 mL *in vacuo* at 30°C and diluted with 100 mL of H₂O. The concentrated wine was poured over 500 grams of Amberlite XAD-16 resin (Sigma-Aldrich), in an open column (5.1 × 35 cm), and rinsed with 5 L of water to remove sugars, small organic acids and other non-polyphenolic compounds. The polyphenols were then eluted with 3 L of MeOH and 1 L of acetone. The solvents were removed *in vacuo* and the samples were then lyophilized. The concentrated XAD16 residue was then chromatographed over a cation-exchange resin column (DOWEX, Sigma-Aldrich), rinsed with distilled water (4 L), and eluted with 75% (v/v) aqueous MeOH (2 L) to yield a solid powder after lyophilization.
## Stilbene identification

Monitoring of the collected Tunisian red wines was achieved by UPLC-MS. The UPLC analyses were carried out using an UPLC system model 1290 Infinity (Agilent Technologies) coupled to an Esquire 3000plus mass spectrometer (Bruker Daltonics GmbH) with electrospray ionization and ion trap analyzer. Quantification was performed by analytical HPLC-DAD using a HPLC system model 1200 (Agilent technologies).

<figure><img src="image_386.png" /><figcaption>Fig. 1 - Flow diagram of primary method used for the different stages of pre-purification of wine stilbenoids.</figcaption></figure>

## Results and discussion

In this study, stilbenes were identified by UPLC-MS/MS analysis. This combination provided an accurate method for the characterization of stilbenes [10]. To illustrate the analysis of stilbenes in wine, the UPLC chromatogram of the Sidi Zahia red wine is presented in Fig. 2. Stilbenes were tentatively identified by examining the mass spectra (MS and MS/MS). The presence of stilbene oligomers was monitored via extracted ion chromatograms in negative ion mode. Typical *m/z* values for these compounds were used, including [*M*−*H*]⁻ ions of *m/z* 227 or 242 (monomers), 453 (dimer), 677 (trimer), and 905 (tetramer). The corresponding values for these compounds were also evaluated in positive mode. To confirm the identification of the stilbenes, standards were used [8, 9].

<figure><img src="image_387.png" /><figcaption>Fig. 2 - UPLC chromatogram recorded at 290 nm showing the stilbenes detected in Sidi Zahia red wine. Peak numbering as in Table 1.</figcaption></figure>
Using this procedure, seven stilbenes (Fig. 3) were identified in Tunisian red wines, including three monomers (resveratrol, piceid and piceatannol), one dimer (ε-viniferin), one trimer (α-viniferin) and two tetramers (hopeaphenol and isohopeaphenol). Retention time (t<sub>R</sub>) and molecular ions (positive and negative modes) are shown in Table 1.

<figure><img src="image_388.png" /><figcaption>Fig. 3 - Stilbene structures.</figcaption></figure>

<table><caption>Table 1 - Chromatographic and mass data of stilbenes</caption><thead><tr><th>No</th><th>t<sub>R</sub> (min)</th><th>[M-H]<sup>-</sup></th><th>[M+H]<sup>+</sup></th><th>Compounds</th></tr></thead><tbody><tr><td>1</td><td>3.7</td><td>389</td><td>391</td><td>(E)-piceid</td></tr><tr><td>2</td><td>4.2</td><td>242</td><td>244</td><td>piceatannol</td></tr><tr><td>3</td><td>4.8</td><td>227</td><td>229</td><td>(E)-resveratrol</td></tr><tr><td>4</td><td>5.0</td><td>677</td><td>679</td><td>α-viniferin</td></tr><tr><td>5</td><td>5.3</td><td>905</td><td>907</td><td>unknown</td></tr><tr><td>6</td><td>5.4</td><td>905</td><td>907</td><td>hopeaphenol</td></tr><tr><td>7</td><td>5.6</td><td>453</td><td>455</td><td>ε-viniferin</td></tr><tr><td>8</td><td>6.2</td><td>905</td><td>907</td><td>isohopeaphenol</td></tr></tbody></table>

Concerning the stilbene content in the Tunisian red wine, the Sidi Zahia red wine was qualitatively and quantitatively the richest one (Fig. 3). The effective quantification of the stilbene content is in progress. Nevertheless, primary results indicated that, among all studied wines, only the Sidi Zahia wine contains the seven stilbenes (Table 2). Isohopeaphenol is present in all wines except Distinto. The Sidi Rais wine contains six stilbenes and does not contain ε-viniferin.

<table><caption>Table 2 - Average detected stilbenes for Tunisian varietal wines (D: detected; ND: not detected)</caption><thead><tr><th>Compound</th><th>Sidi Zahia</th><th>Sidi Rais</th><th>Mornag</th><th>Selian</th><th>Distinto</th><th>Sidi Brahim</th><th>Clipea</th></tr></thead><tbody><tr><td>(E)-piceid</td><td>D</td><td>D</td><td>D</td><td>ND</td><td>ND</td><td>D</td><td>D</td></tr><tr><td>piceatannol</td><td>D</td><td>D</td><td>ND</td><td>ND</td><td>ND</td><td>D</td><td>ND</td></tr><tr><td>(E)-resveratrol</td><td>D</td><td>D</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>α-viniferin</td><td>D</td><td>D</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>hopeaphenol</td><td>D</td><td>D</td><td>ND</td><td>ND</td><td>D</td><td>D</td><td>ND</td></tr><tr><td>ε-viniferin</td><td>D</td><td>ND</td><td>ND</td><td>ND</td><td>D</td><td>ND</td><td>D</td></tr><tr><td>isohopeaphenol</td><td>D</td><td>D</td><td>D</td><td>D</td><td>ND</td><td>D</td><td>D</td></tr></tbody></table>
**Conclusion**

In this preliminary study, we were able to obtain 7 Tunisian wines mainly in the Mornag appellation. We were able to settle in these wines the content of 7 stilbenoids: (E)-resveratrol, (E)-piceid, piceatannol, ε-viniferin, α-viniferin, hopeaphenol and isohopeaphenol. From the point of view of the presence of resveratrol derivatives, we found that one of the wines, Sidi Zahia, was the richest qualitatively. These first results are very encouraging. The study should be extended to a larger number of wines and appellations.

**Acknowledgements**

The authors wish to thank the Ministère de la Recherche et de l'Enseignement Supérieur de la Tunisie for
financial support of this research.

**References**

1. Renaud S, Guéguen R, Siest G et al (1999) Wine, beer, and mortality in middle-aged men from eastern France. *Arch Intern Med* **159**: 1865-1870.

2. Vang O, Ahmad N, Baile CA et al (2011) What is new for an old molecule? Systematic review and recommendations on the use of resveratrol. *PLoS One* **6**: e19881.

3. Catalgol B, Batirel S, Taga Y et al (2012) Resveratrol: French paradox revisited. *Front Pharmacol* **3**: 141.

4. Goldberg DM, Yan J, Ng E et al (1995) A global survey of trans-resveratrol concentrations in commercial wines. *Am J Enol Vitic* **46**: 159-165.

5. Mattivi F, Nicolini G (1997) Analysis of polyphenols and resveratrol in Italian wines. *BioFactors* **6**: 445-448.

6. Pawlus AD, Cantos-Villar E, Richard T et al (2013) Chemical dereplication of wine stilbenoids using high performance liquid chromatography-nuclear magnetic resonance spectroscopy. *J Chromatogr A* **1289**: 19-26.

7. Moss R, Mao Q, Taylor D et al (2013) Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry. *Rapid Commun Mass Spectrom* **27**: 1815-1827.

8. Arraki K, Richard T, Badoc A et al (2013) Isolation, characterization and quantification of stilbenes from some *Carex* species. *Rec Nat Prod* **7**: 281-291.

9. Papastamoulis Y, Richard T, Nassra M et al (2014) Viniphenol A, a complex resveratrol hexamer from *Vitis vinifera* stalks: structural elucidation and protective effects against amyloid-β-induced toxicity in PC12 cells. *J Nat Prod* **77**: 213-217.

10. Stecher G, Huck CW, Popp M et al (2001) Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS. *Anal Bioanal Chem* **371**: 73-80.
# Study of the colour and phenolic compounds in grapes and wines elaborated from two canopy treatments of Vitis vinifera L. cv Shiraz

G. GARRIDO-BAÑUELOS, A. BUICA, W.J. DU TOIT*

Department of Viticulture and Oenology, University of Stellenbosch, Private Bag X1, Matieland (Stellenbosch) 7602, South Africa

Phenolics compounds – canopy – vigour – wine ageing

## Introduction

Phenolic compounds are important contributors to the quality of red wines. Anthocyanins, along with flavanols and flavanols, play an important role in the colour stability of red wines as during vinification and ageing monomeric forms are progressively replaced by polymeric structures [1-3]. These compounds can also influence the sensory characteristics of red wines [4].

The concentration of phenolic compounds in wine depends mainly on the potential of the grapes and the winemaking practices employed [5]. In recent years, several studies have shown how environmental factors and viticultural practices may influence the accumulation of these compounds in grapes and how these are reflected in young wines [6-8]. Furthermore, berry ripening may also affect not only the major components of the grapes, such as sugar or acids, but also the secondary metabolites like phenolic compounds and their extractability into the wine [9-11].

The aim of the present work was to evaluate the effect of different trellising systems, vigour and ripening levels on the colour and phenolic composition of Shiraz grapes and their corresponding wines during ageing.

## Materials and methods

### Vineyard treatments and winemaking

This study was conducted in 2014 at the Welgevallen farm (33°56'S, 18°52'E), with a characterised Shiraz experimental vineyard of Stellenbosch University. This research project was carried on two different trellising systems, being Vertical Shoot Positioning (VSP) and Smart Dyson (SD). The VSP system consists of a non-divided canopy where the fruit zone is restricted to a small vertical zone along a single trellis wire, while the SD system entails a divided canopy system with shoots being trained upwards and downwards from the same cordon. The SD system may achieve an increase in grape yield.

The grapes were harvested at 23 °Brix and 25 Brix from each canopy treatment in two different vigour zones (high vigour, HV and low vigour, LV). All vinifications were made in small scale (40 kg) in triplicate. Every wine was made following the standard winemaking procedure of the Department of Viticulture and Oenology of Stellenbosch University. Samples for phenolic and colour analyses were taken after malolactic fermentation as well as after 6 months and 12 months bottle ageing, while samples for sensory analysis were collected after 6 months bottle ageing.

### Grape analysis

Frozen grapes were weighed and grinded for 3 minutes with a homogeniser. The grape homogenate was extracted in acidified methanol for 45 minutes in an ultrasonic bath. The samples were centrifuged and the supernatant was retained for High Performance Liquid Chromatography (HPLC) analysis [12].
### Wine analysis

The evolution of wine colour and general phenolic composition during time was evaluated by spectrophotometric analyses [13]. Tannin levels were quantified by the Methyl Cellulose Precipitation (MCP) assay [14]. The individual quantification of the wine phenolic compounds during ageing was also performed with HPLC [12].

### Sensory analysis

The sensory evaluation was done with Descriptive Analysis (DA) method [15]. Eleven judges were selected as panellist on the basis of availability and previous experience in wine evaluation. Training in mouth feel and aroma characteristics was performed using the relevant standards.

### Statistical analyses

All analyses were done using Statistica 12. Mixed model repeated measures ANOVAs were used and Fisher's least significant difference (LSD) corrections were used for posthoc analyses. Significant differences were judged on a 5% significance level (p < 0.05).

## Results and discussion

Grape yield differed between the two trellising systems due to the larger number of grape bunches in the SD system. The yield produced in the SD system was 8.0 kg per vine, compared to the 4.5 kg per vine of the VSP system.

Table 1 shows the polymeric phenol and malvidin-3-glucoside concentrations of the grapes. No clear trends could be observed. However, in certain treatments the tannin concentrations were significantly higher (results not shown).

<table><caption>Table 1 - Grape phenolic composition (mg/g of berry)</caption><thead><tr><th rowspan="3"></th><th rowspan="3"></th><th colspan="8">VSP</th><th colspan="8">SD</th></tr><tr><th colspan="4">23 °Brix</th><th colspan="4">25 °Brix</th><th colspan="4">23 °Brix</th><th colspan="4">25 °Brix</th></tr><tr><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th></tr></thead><tbody><tr><td rowspan="2">Pol. ph</td><td>Mean</td><td>0.91</td><td>abc</td><td>0.75</td><td>c</td><td>1.03</td><td>ab</td><td>1.14</td><td>a</td><td>0.89</td><td>bc</td><td>0.90</td><td>bc</td><td>1.01</td><td>ab</td><td>1.04</td><td>ab</td></tr><tr><td>S.dev</td><td>0.04</td><td></td><td>0.18</td><td></td><td>0.05</td><td></td><td>0.06</td><td></td><td>0.04</td><td></td><td>0.06</td><td></td><td>0.05</td><td></td><td>0.02</td><td></td></tr><tr><td rowspan="2">Malvidin-3-gluc</td><td>Mean</td><td>0.17</td><td>ab</td><td>0.19</td><td>bd</td><td>0.16</td><td>ac</td><td>0.21</td><td>b</td><td>0.17</td><td>ab</td><td>0.19</td><td>bc</td><td>0.15</td><td>a</td><td>0.16</td><td>acd</td></tr><tr><td>S.dev</td><td>0.02</td><td></td><td>0.01</td><td></td><td>0.01</td><td></td><td>0.00</td><td></td><td>0.01</td><td></td><td>0.01</td><td></td><td>0.00</td><td></td><td>0.01</td><td></td></tr></tbody></table>

Table 2 & Table 3 show the effect of vigour on the colour and polymeric pigment composition of the wines, which was also found by Cortell *et al.* [6]. Only the VSP wines at the earliest ripening stage did not show a significant difference between colour densities. This effect was often continued during wine ageing as well.

Contrary to the results observed in grapes, the vigour influenced the concentration of the polymeric phenols in wine. The HPLC results in Table 3 show a greater concentration in wines produced from low vigour vines, increasing as well with the grapes' maturity level were.

The results obtained from sensory evaluation six months after the alcoholic fermentation showed significant differences between the ripeness stages. The 23 °Brix wines were generally described as sour, while in the wines from the riper stage more differences between the treatments appeared. The SD-LV and SD-HV wines were described as sweet while VSP wines were "fuller" and more astringent.

Further sensory studies after twelve months could help to further elucidate the impact of the polymeric phenols on the taste and mouth-feel of the wines.
<table><caption>**Table 2** - Colour density of the wines during ageing</caption><thead><tr><th rowspan="3">Colour density (AU)</th><th colspan="8">VSP</th><th colspan="8">SD</th></tr><tr><th colspan="4">23 °Brix</th><th colspan="4">25 °Brix</th><th colspan="4">23 °Brix</th><th colspan="4">25 °Brix</th></tr><tr><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th></tr></thead><tbody><tr><td rowspan="2">MLF</td><td>Mean</td><td>8.37</td><td>bc</td><td>7.94</td><td>bc</td><td>8.60</td><td>b</td><td>14.24</td><td>a</td><td>11.95</td><td>a</td><td>13.45</td><td>a</td><td>6.14</td><td>c</td><td>8.71</td><td>b</td></tr><tr><td>S.dev</td><td>2.52</td><td></td><td>0.44</td><td></td><td>0.71</td><td></td><td>1.59</td><td></td><td>0.53</td><td></td><td>1.14</td><td></td><td>1.57</td><td></td><td>0.62</td><td></td></tr><tr><td rowspan="2">6 months</td><td>Mean</td><td>6.39</td><td>cb</td><td>7.75</td><td>ac</td><td>6.34</td><td>cb</td><td>9.28</td><td>a</td><td>7.71</td><td>ac</td><td>9.56</td><td>a</td><td>5.28</td><td>c</td><td>8.12</td><td>ab</td></tr><tr><td>S.dev</td><td>1.89</td><td></td><td>1.25</td><td></td><td>0.54</td><td></td><td>0.95</td><td></td><td>0.60</td><td></td><td>3.08</td><td></td><td>0.32</td><td></td><td>0.35</td><td></td></tr><tr><td rowspan="2">12 months</td><td>Mean</td><td>4.50</td><td>cb</td><td>4.49</td><td>cb</td><td>6.01</td><td>ab</td><td>7.20</td><td>a</td><td>5.51</td><td>ab</td><td>6.46</td><td>a</td><td>3.74</td><td>c</td><td>5.79</td><td>ab</td></tr><tr><td>S.dev</td><td>1.19</td><td></td><td>0.14</td><td></td><td>2.21</td><td></td><td>1.15</td><td></td><td>0.07</td><td></td><td>0.57</td><td></td><td>0.31</td><td></td><td>0.13</td><td></td></tr></tbody></table>

<table><caption>**Table 3** - Phenolic composition of the wines (mg/L)</caption><thead><tr><th rowspan="3"></th><th rowspan="3"></th><th colspan="8">VSP</th><th colspan="8">SD</th></tr><tr><th colspan="4">23 °Brix</th><th colspan="4">25 °Brix</th><th colspan="4">23 °Brix</th><th colspan="4">25 °Brix</th></tr><tr><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th><th>HV</th><th>LV</th></tr></thead><tbody><tr><td rowspan="2">Pol. ph MLF</td><td>Mean</td><td>305.6</td><td>f</td><td>433.5</td><td>e</td><td>629.4</td><td>b</td><td>675.9</td><td>b</td><td>426.9</td><td>e</td><td>570.5</td><td>c</td><td>500.6</td><td>d</td><td>789.7</td><td>a</td></tr><tr><td>S.dev</td><td>14.7</td><td></td><td>12.8</td><td></td><td>17.7</td><td></td><td>22.7</td><td></td><td>16.3</td><td></td><td>29.0</td><td></td><td>12.5</td><td></td><td>12.1</td><td></td></tr><tr><td rowspan="2">Pol. ph 6m</td><td>Mean</td><td>329.6</td><td>e</td><td>453.8</td><td>d</td><td>656.1</td><td>cb</td><td>680.3</td><td>a</td><td>502.0</td><td>d</td><td>595.1</td><td>c</td><td>501.0</td><td>d</td><td>754.7</td><td>a</td></tr><tr><td>S.dev</td><td>19.4</td><td></td><td>19.7</td><td></td><td>29.5</td><td></td><td>25.2</td><td></td><td>6.0</td><td></td><td>30.8</td><td></td><td>41.3</td><td></td><td>15.1</td><td></td></tr><tr><td rowspan="2">Pol. pg MLF</td><td>Mean</td><td>31.5</td><td>e</td><td>41.6</td><td>d</td><td>54.8</td><td>c</td><td>62.7</td><td>b</td><td>45.2</td><td>d</td><td>55.9</td><td>c</td><td>44.2</td><td>d</td><td>74.3</td><td>a</td></tr><tr><td>S.dev</td><td>1.2</td><td></td><td>1.3</td><td></td><td>3.0</td><td></td><td>1.4</td><td></td><td>2.3</td><td></td><td>2.1</td><td></td><td>2.4</td><td></td><td>2.7</td><td></td></tr><tr><td rowspan="2">Pol. pg 6m</td><td>Mean</td><td>40.0</td><td>d</td><td>47.2</td><td>dc</td><td>57.7</td><td>b</td><td>57.9</td><td>b</td><td>53.7</td><td>bc</td><td>55.8</td><td>c</td><td>45.3</td><td>d</td><td>73.0</td><td>a</td></tr><tr><td>S.dev</td><td>4.1</td><td></td><td>0.5</td><td></td><td>3.4</td><td></td><td>2.5</td><td></td><td>2.3</td><td></td><td>1.9</td><td></td><td>2.0</td><td></td><td>3.5</td><td></td></tr></tbody></table>

## Conclusion

Different viticultural treatments seems to affect the colour, phenolic and sensory profiles of Shiraz wines. Some of these changes all seem to be carried over during wine ageing.

## Acknowledgements

Winetech and Thrip for financial support.

## Abbreviations

Vertical Shoot Positioning, VSP; Smart-Dyson, SD; high vigour, HV; low vigour, LV; High Performance Liquid Chromatography, HPLC; Methyl Cellulose Precipitation, MCP; polymeric phenols, Pol. ph; polymeric pigments, Pol. pg; Descriptive Analysis, DA.

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7. Río Segade S, Soto-Vazquez E, Vazquez-Rodriguez EI et al (2009) Influence of training system on chromatic characteristics and phenolic composition in red wines. *Eur Food Res Technol* **229**: 763-770.

8. Mota RV, de Amorim DA, Favero AC et al (2011) Effect of trellising system on grape and wine composition of Syrah vines grown in the cerrado region of Minas Gerais. *Food Sci Technol* **31**: 967-972.

9. Adams DO (2006) Phenolics and ripening in grape berries. *Am J Enol Vitic* **57**: 249-256.

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# Investigations on the green aromas in French red wines related to grape ripening

X. POITOU<fnref n="1" />,<fnref n="2" />*, P. DARRIET<fnref n="1" />,<fnref n="2" />

Green aromas – volatile compounds – red wines – aroma characterisation

## Introduction

Wine aroma is due to a complex combination of volatile molecules from grapes, fermentation processes and ageing belonging to different chemical families. According to sensory perception, it is possible to categorise it in several groups [1]. Particularly, the "vegetative" or "green" class associated to different notes has major importance in red wine aroma by perceptive antagonism with fruity nuances [2]. A better characterisation of this class is an important issue. Within the "vegetative" and "green" class, it is commonly accepted that "fresh green" nuances (cut grass, minty, green bell pepper) in red wines have a varietal origin related to the grape ripeness. To date, compounds belonging to the 2-methoxypyrazine family, and particularly 3-isobutyl-2-methoxypyrazine (IBMP), first identified in bell pepper (*Capsicum annum* var. *grossum*) [3], have been identified as key compounds of wines associated with unripe grapes and many studies have shown their contribution to the herbaceous aromas of wines from different varieties, in particular Cabernet Sauvignon [4].

However, several studies have shown that the levels of IBMP in wines do not always correlate well with perceived green notes during wine tasting [5] and the hypothesis for a contribution of other volatile compounds, alone or in combination with 2-methoxypyrazine, has to be considered. The objective of this study was therefore to characterise other volatile compounds involved in "fresh green" nuances of red wines in relation to grape ripeness. A strategy applied to model wines was considered, combining both analytical and sensory approaches through a multiple-step protocol combining both HPLC fractionation, GC-olfactometry and MDGC-O/TOF-MS.

## Materials and method

### Winemaking

30 kg of Cabernet Sauvignon and Merlot grapes were harvested at two stages, spaced one month apart, during the 2014 and 2015 vintages, in the same rows from a vineyard plot in Haut Medoc (Bordeaux) (Table 1). After destemming, grapes were micro vinified in 20-L stainless steel tanks.

### Aroma characterisation

750 mL of wine were extracted using dichloromethane. After concentration of the organic extract, reversed-phase (RP) HPLC semi preparative was performed with a water:ethanol gradient. 50 fractions of 1 mL were collected and evaluated for their sensory properties. The impact of selected fractions was evaluated by an omission and a supplementation test. All samples were evaluated on the green aromas in an orthonasal way by 14 expert judges in a temperature-controlled room. Fractions of interest were re-extracted and analysed by multidimensional gas chromatography coupled with olfactometry and a time-of-flight mass spectrometer (MDGC-O-MS/TOF). Separation was performed on fused-silica polar capillary column in first dimension (BP20) and a non-polar (BP1).

## Results and discussion

The sensory evaluation of red wines elaborated from grapes harvested at two ripening stages showed a direct relation between the ripening stage and the perception of green aromas (Table 1). Indeed, unripe wines of Cabernet Sauvignon expressed in 2014 a strong green aroma in comparison to the ripe wine but

<footnote n="1">¹Univ. de Bordeaux, ISVV, EA 4577, Unité de recherche œnologie, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²INRA, ISVV, USC 1366, Unité de recherche œnologie, F-33882 Villenave d'Ornon, France</footnote>
this was not the case in 2015 and for wines elaborated with Merlot variety. This could be related to the climatic conditions depending on the vintage as no green aromas were observed in the earlier harvest of 2015 vintage (earlier) both on Merlot and Cabernet Sauvignon wines.

<table><caption>**Table 1** - Harvest date, technological parameters and sensory descriptions of Merlot (M) and Cabernet-Sauvignon (CS) model wines in the 2014 and 2015 vintages</caption><thead><tr><th>Modality</th><th>Varieties</th><th>Harvest date</th><th>% TAV<sup>a</sup></th><th>pH<sup>a</sup></th><th>Aromas</th></tr></thead><tbody><tr><td>Ripeness-</td><td>M</td><td>05/09/14</td><td>9.7</td><td>3.25</td><td>Fresh red fruits: cherry, raspberry</td></tr><tr><td><strong>Ripeness-</strong></td><td>CS</td><td>12/09/14</td><td>9.5</td><td>3.51</td><td><strong>Strong green</strong>, green bell pepper, fresh</td></tr><tr><td>Ripeness+</td><td>M</td><td>03/10/14</td><td>13.0</td><td>3.53</td><td>Red fruits: cherry, raspberry, strawberry</td></tr><tr><td><strong>Ripeness+</strong></td><td>CS</td><td>12/10/14</td><td>12.0</td><td>3.74</td><td>Fruits: blackcurrant, blackberry; <strong>green</strong></td></tr><tr><td>Ripeness-</td><td>CS</td><td>01/09/15</td><td>9.5</td><td>3.42</td><td>Fresh fruits: raspberry, blackcurrant</td></tr><tr><td>Ripeness+</td><td>CS</td><td>01/10/15</td><td>11.9</td><td>3.70</td><td>Black fruits: blackberry, blackcurrant</td></tr></tbody></table>

Further investigations were therefore mainly performed with Cabernet Sauvignon wines from the 2014 vintage. HPLC fractionation of 2014 Cabernet Sauvignon wine concentrated extracts was carried out on 50 fractions which were submitted to three expert judges. Four fractions (31, 32, 35, 36) obtained in hydro-alcoholic solution were particularly highlighted, presenting strong differences between unripe and ripe wines particularly regarding the green aromas (Table 2). These fractions were grouped in F1 and F2 to performed sensory tests because of their aromatic similarities.

<table><caption>**Table 2** - Sensory evaluation of HPLC fractions from Cabernet Sauvignon wines at two ripeness stages (Ripeness- and Ripeness+) in the 2014 vintage</caption><thead><tr><th>HPLC fraction</th><th>« Ripeness- »</th><th>« Ripeness+ »</th><th>Group</th></tr></thead><tbody><tr><td>31</td><td>Cut grass, fruity</td><td>Saffron, spicy, fruity</td><td rowspan="2">F1</td></tr><tr><td>32</td><td>Cut grass, green wood</td><td>Fruity</td></tr><tr><td>35</td><td>Fresh green</td><td>Fruity</td><td rowspan="2">F2</td></tr><tr><td>36</td><td>Strong green, minty</td><td>Fresh fruit, slightly green</td></tr></tbody></table>

Reconstitution of the 50 fractions with omission of F1 group showed no effect on the overall green perception of the mixture. On the contrary, the omission of F2 group generated a significant diminution of the green aromas (Table 3). The addition of F2 in neutral red wine confirmed this impact because an important increase in green aroma score was noted (7,7 ± 1,2) relatively to the control (4,2 ± 1,5). No synergistic effect between F1 and F2 was then observed.

<table><caption>**Table 3** - Mean score on green aromas (bell pepper, cut grass, leaf and minty) evaluated by an expert panel for omission and supplementation test</caption><thead><tr><th colspan="2">Omission test</th><th colspan="2">Supplementation test</th></tr><tr><th>Sample</th><th>Mean ± CI</th><th>Sample</th><th>Mean ± CI</th></tr></thead><tbody><tr><td>TAR</td><td>6,4 ± 1,2 a</td><td>R-</td><td>8,9 ± 0,8 d</td></tr><tr><td>TAR-F2</td><td>3,8 ± 1,3 b</td><td>R+</td><td>4,2 ± 1,5 ab</td></tr><tr><td>TAR-[F1+F2]</td><td>3,8 ± 1,1 b</td><td>R+ [+F1]</td><td>3,7 ± 1,1 a</td></tr><tr><td>TAR-F1</td><td>6,8 ± 1,2 a</td><td>R+ [+F1+F2]</td><td>5,6 ± 1,3 bc</td></tr><tr><td></td><td></td><td>R+ [+F2]</td><td>7,7 ± 1,2 c</td></tr></tbody></table>

Omission test: TAR (Total Aromas Reconstitution), TAR-F1 (without fraction 31 and 32), TAR-F2 (without fraction 35 and 36) and TAR-[F1+F2] (without fraction 31, 32, 35 and 36). Addition test in wine: R- (Ripeness-), R+ (Ripeness+), R+ [+F1] (R+ supplemented with fraction 31 and 32), R+ [+F2] (R+ supplemented with fraction 35 and 35), R+ [+F1+F2] (R+ supplemented with fraction 31, 32, 35, 36). Letters indicate statistical groups by ANOVA (p value < 0.05). CI: confidence interval at 95% level.

<footnote><sup>a</sup>Fourier Transform Infrared spectroscopy (OenoFoss™).</footnote>
To identify the volatile compounds associated with this sensory perception, F1 and F2 fractions were re-extracted and analysed both on a GC polar capillary column (BP20) through a monodimensional chromatography and by bidimensional GC with BP20 as first capillary and DB1 as analytical column coupled to mass spectrometry or to olfactometry detection.

Doing so, several known compounds could be first evidenced, such as 1-hexanol, associated with herbaceous and cut grass odours, which is a major compound of F1 group. As F1 group could not contribute to the enhancement of green flavour when supplemented to neutral red wine, this result raises questions about the real contribution of this C6 alcohol to herbaceous notes in red wine. Then, characterisation of aromatic compounds in F2 group by GC-olfactometry highlighted several green odorous zones and specifically three odorous zones of interest (Table 4). An odorous zone of interest was defined as a green odour at a chromatographic time with a greater intensity in the unripe wine.

The compound responsible for the first zone (1525 and 1180 LRI) associated with a strong "green bell pepper" aroma in the unripe wine was the well-known 3-isobutyl-2-methoxypyrazine, or IBMP. A second zone at 1430 and 1035 LRI with "pea" and "earthy" odours was associated with 3-isopropyl-2-methoxypyrazine or IPMP, a potent odorous compound associated with the "ladybug taint" due to the contamination of grapes by *Harmonia axyridis* [6]. Unlike IBMP, the link between this compound and the ripeness of the grapes has not been proved [7] and this result allows to formulate the hypothesis of a relationship between IPMP and Cabernet Sauvignon grape ripeness. In view of its very low detection threshold, it is also possible to consider a sensory contribution in association with IBMP.

Several odorous zones of interest associated with a strong geranium leaf odour were also evidenced, but associated compounds could not be identified (Table 4).

<table><caption>**Table 4** - Main green odorous zones determined by MDGC-O-MS/TOF in (F2) group extract</caption><thead><tr><th>LRI 1D</th><th>LRI 2D</th><th>Odorous zone « Unripe wine »</th><th>Odorous zone «Ripe wine »</th><th>Compounds</th><th>Identification</th></tr></thead><tbody><tr><td>1183</td><td>1015</td><td>minty, fresh, herbal (+++)</td><td>N.D.</td><td>1,8-cineole</td><td>LRI, MS, IS</td></tr><tr><td>1430</td><td>1085</td><td>pea, earthy (++)</td><td>green (+)</td><td>IPMP</td><td>LRI, IS</td></tr><tr><td>1525</td><td>1180</td><td>bell pepper (+++)</td><td>green (+)</td><td>IBMP</td><td>LRI, MS, IS</td></tr><tr><td>1537</td><td>1090</td><td>geranium leaf (+++)</td><td>geranium leaf (+++)</td><td>unknown</td><td>x</td></tr><tr><td>1592</td><td>1127</td><td>cucumber (+++)</td><td>cucumber (+++)</td><td>(E,Z)-2,4-nonadienal</td><td>LRI, MS</td></tr><tr><td>1696</td><td>1090<br>1251<br>1313</td><td>geranium leaf (+++)</td><td>geranium leaf (+++)</td><td>unknown</td><td>x</td></tr><tr><td>1709</td><td>1215</td><td>cucumber (+++)</td><td>cucumber (+++)</td><td>(E,E)-2,4-nonadienal</td><td>LRI, MS</td></tr></tbody></table>

LRI, linear retention index in BP20 (1D) and BP1 (2D) compared with those found in literature; IS, injection of pure commercial standard; MS, mass spectrum obtained with electronic impact (70eV) and compared to NIST library. Bracketed, evaluation of odour intensity: +++ for strong, ++ for medium and + for low intensity.

A third odorous zone, which was not detected in ripe wine, was associated with very fresh vegetal notes, such as herbs and mint. It was therefore likely that the compounds associated with this odorous zone were related to the ripeness of grapes and responsible for part of the green aroma of the fraction F2 in unripe wine. The compound was identified by comparing the mass spectrum with the NIST 2005 library and injecting its commercial standard. It was identified as 1,8-cineole, well known as eucalyptol, the major component and surprisingly detected in Bordeaux red wines. This molecule has been first identified in Californian Merlot wine, with a detection threshold at 1,1 µg/L [8]. Then, 1,8-cineole was the subject of several studies in recent years, in particular as regards to its origin in red wines [9], showing that the first
source of 1,8-cineole found in significant concentrations in Australian wines was the proximity of eucalyptus trees in the vineyard. The contribution of this compound was shown to be possible by airborne transmission or incorporation of matter other than grapes (Eucalyptus or grape leaves) in the harvest.

Regarding the varietal aspect, Farina et al. [10] suggested the formation of eucalyptol in Tannat wines (Uruguay) from other terpenes (limonene and α-terpineol) by hydrolysis reaction and rearrangement under conditions related to those of red wine ageing. The low conversion rates (around 0,6%) over a 2-year period established that these compounds were not significant precursors of 1,8-cineole [11]. Kalua et al. [12] then suggested 1,8-cineole to be the major monoterpene formed in floral tissues but showed a total disappearance early in the berry cycle development (7 weeks after flowering). Furthermore, it was also found that 1,8-cineole concentration in grapes at the beginning of the ripening was very low, but showed an important increase throughout ripening [10].

This study therefore contributes to the clarification of the varietal origin of 1,8-cineole in Cabernet Sauvignon and Merlot, and indicates an abundance of 1,8-cineole in French wines related to grape ripeness.

## Acknowledgements

We thank the Château Lagrange, who agreed to make available plots of vines for sampling. We are grateful to all participants of sensory analysis sessions.

## References

1. Noble C, Arnold RA, Buechsenstein J et al (1987) Modification of a standardized system of wine aroma terminology. *Am J Enol Vitic* **38**: 143-146.

2. Hein K, Ebeler SE, Heymann H (2009) Perception of fruity and vegetative aromas in red wine. *J Sens Stud* **24**: 441-455.

3. Buttery RG, Seifert RM, Guadagni DG et al (1969) Characterization of some volatile constituents of bell peppers. *J Agric Food Chem* **17**: 1322-1327.

4. Robinson AL, Boss PK, Solomon PS et al (2013) Origins of grape and wine aroma. Part 1. Chemical components and viticultural impacts. *Am J Enol Vitic* **65**: 1-24.

5. Preston LD, Block DE, Heymann H et al (2008) Defining vegetal aromas in cabernet sauvignon using sensory and chemical evaluations. *Am J Enol Vitic* **59**: 137-145.

6. Pickering GJ, Lin Y, Reynolds A et al (2005) The influence of *Harmonia axyridis* on wine composition and aging. *J Food Sci* **70**: 128-135.

7. Sala C, Busto O, Guasch J et al (2005) Contents of 3-alkyl-2-methoxypyrazines in musts and wines from *Vitis vinifera* variety Cabernet Sauvignon: influence of irrigation and plantation density. *J Sci Food Agric* **85**: 1131-1136.

8. Herve E, Price S, Burns G (2003) Eucalyptol in wines showing a "eucalyptus" aroma. *In Proceedings of the VIIeme Symposium International d'Oenologie, Actualités Oenologiques*, Bordeaux; Lonvaud A, De Revel G, Darriet P (Eds); Tec & Doc; Lavoisier: Paris, France; pp 598-600.

9. Capone DL, Jeffery DW, Sefton MA (2012) Vineyard and fermentation studies to elucidate the origin of 1,8-cineole in Australian red wine. *J Agric Food Chem* **60**: 2281-2287.

10. Fariña L, Boido E, Carrau F et al (2005) Terpene compounds as possible precursors of 1,8-cineole in red grapes and wines. *J Agric Food Chem* **53**: 1633-1636.

11. Capone DL, van Leeuwen K, Taylor DK et al (2011) Evolution and occurrence of 1,8-cineole (Eucalyptol) in Australian wine. *J Agric Food Chem* **59**: 953-959.

12. Kalua CM, Boss PK (2009) Evolution of volatile compounds during the development of Cabernet sauvignon grapes (*Vitis vinifera* L.). *J Agric Food Chem* **57**: 3818-3830.
# The effect of supplementation with three commercial inactive dry yeasts on the chemical composition and astringency of model wine solution and red wine

E. GONZÁLEZ-ROYO, M. ESTERUELAS, N. KONTOUDAKIS, F. FORT, J.M. CANALS, F. ZAMORA*

Departament de Bioquímica i Biotecnologia, Universitat Rovira i Virgili,
C/Marcel.li Domingo s/n, 43007 Tarragona, Spain

Inactive yeasts – polysaccharides – proanthocyanidins – astringency

## Introduction

It is widely acknowledged that high quality red wines have a balanced level of astringency. When there is excessive astringency, wines are considered aggressive and/or rough. In contrast, when there is too little astringency they may taste flat, insipid and uninteresting. Proanthocyanidins have been widely associated to astringency perception [1]. In this regard, several variables involving proanthocyanidin composition have been related to astringency, such as their total concentration, the mean degree of polymerization (mDP) [2] and monomeric composition [3]. More specifically, the greater degree of polymerization and the greater percentage of galloylation will cause a greater sensation of astringency [4].

Yeast mannoproteins have been described for their positive influence on a number of technological and quality properties of red wines, such as inhibition of tartrate salt crystallization [5], reduction of protein haze [6] and color stabilization [7]. Recent studies have also reported that yeast mannoproteins can increase body and mouthfeel [8], and also smooth the astringency of red wines [9]. For this reason the use of inactive dry yeasts as a source of mannoproteins has been introduced into the market during recent years [10, 11]. In the light of all this we may wonder what the mechanism is whereby supplementation with inactive dry yeasts reduces wine astringency. Hence the aim of this article was to study - in a red wine and also in a model wine solution - how supplementation with three commercial inactive dry yeasts affects the chemical composition of wine in order to better understand its action mechanism.

## Materials and methods

### Yeasts and winemaking

300 mg/L of three commercial inactive yeasts (CIY), Optired®, Booster Rouge® and Noblesse® (Lallemand Inc ), were macerated in triplicate in 1.5 L of each of the maceration media for 15 days at 18 °C. Maceration were performed in two model wine solutions (A and B) and in a red wine. Solution A was composed of 13% (v/v) ethanol and 4 g/L tartaric acid adjusted to pH 3.5 with sodium hydroxide in high purity water. Solution B was identical to Solution A but supplemented with 2 g/L of a commercial seed proanthocyanidin (Protan Pépin, AEB).

### Chemical analyses

Color intensity (CI) and total phenolic index (TPI) were estimated according with [12]. Anthocyanidins were measured by HPLC-DAD [13]. Polysaccharides were analyzed by HRSEC-RID [14]. Proanthocyanidin concentration as well as their mDP and monomeric composition were determined by HPLC after acid-catalysis cleavage in the presence of excess phloroglucinol [15].

## Results and discussion

Table 1 shows the effects that the supplementation with three CIY had on the color, TPI and anthocyanidins of the different samples.
<table><caption>**Table 1** - Influence of CIY supplementation on color intensity, TPI, anthocyanins and polysaccharides</caption><thead><tr><th>Medium</th><th>Parameter</th><th>Control</th><th>Optired®</th><th>Noblesse®</th><th>Booster Rouge®</th></tr></thead><tbody><tr><td rowspan="2">Synthetic wine B with tannins</td><td>CI</td><td>4,9 ± 0,0 a</td><td>3,8 ± 0,1 b</td><td>4,1 ± 0,1 b</td><td>3,6 ± 0,2 b</td></tr><tr><td>TPI</td><td>20,7 ± 0,2 a</td><td>18,5 ± 0,4 b</td><td>18,8 ± 0,5 b</td><td>18,2 ± 0,2 b</td></tr><tr><td>Synthetic wine A without tannins</td><td>Polysaccharides (mg/L)</td><td>0,0 ± 0,0 aα</td><td>15,2 ± 0,8 bα</td><td>3,0 ± 1,2 bα</td><td>17,4 ± 0,2 bα</td></tr><tr><td>Synthetic wine B with tannins</td><td></td><td>0,0 ± 0,0 aα</td><td>8,1 ± 0,9 bβ</td><td>1,2 ± 0,0 bβ</td><td>10,8 ± 3,3 bβ</td></tr><tr><td rowspan="4">Red Wine</td><td>CI</td><td>12,6 ± 0,1 a</td><td>12,3 ± 0,1 b</td><td>12,4 ± 0,0 b</td><td>12,2 ± 0,2 b</td></tr><tr><td>TPI</td><td>66,7 ± 0,3 a</td><td>65,5 ± 0,2 b</td><td>64,3 ± 0,4 b</td><td>64,0 ± 0,3 b</td></tr><tr><td>Anthocyanidins (mg/L)</td><td>249 ± 1 a</td><td>241 ± 2 a</td><td>235 ± 1 a</td><td>239 ± 2 a</td></tr><tr><td>Polysaccharides (mg/L)</td><td>575 ± 29 a</td><td>636 ± 27 b</td><td>480 ± 14 b</td><td>610 ± 30 a</td></tr></tbody></table>

Overall, all the synthetic wines and also red wines treated with the three CIY have significant lower CI and TPI. This data indicates that CIY have absorbed or precipitate some phenolic compounds. However, no differences were found in the anthocyanin concentration of treated red wines. Table 1 also shows the effects of the supplementation with inactive yeasts on polysaccharide concentration. All the inactive yeasts released polysaccharides in both synthetic solutions, but the concentrations were always significantly higher when no tannin was present. Consequently this data seems to indicate that some polysaccharides released by inactive yeasts precipitate in the presence of seed proanthocyanidins. Polysaccharides of red wine did not show a clear trend probably because of the complicated balance between the polysaccharide release and precipitation.

Table 2 shows the effects of supplementation with inactive yeasts on the proanthocyanidins of synthetic wine and red wine.

<table><caption>**Table 2** - Influence of CIY supplementation on proanthocyanidins</caption><thead><tr><th>Medium</th><th>Proanthocyanidins</th><th>Control</th><th>Optired®</th><th>Noblesse®</th><th>Booster Rouge®</th></tr></thead><tbody><tr><td rowspan="3">Synthetic wine B</td><td>PA (mg/L)</td><td>1201 ± 82 a</td><td>915 ± 59 b</td><td>980 ± 69 b</td><td>1029 ± 44 b</td></tr><tr><td>mDP</td><td>5,9 ± 0,1 a</td><td>5,3 ± 0,2 b</td><td>5,4 ± 0,1 b</td><td>5,9 ± 0,4 a</td></tr><tr><td>mDP-EPA</td><td>-</td><td>9,8 ± 0,3 b</td><td>10,3 ± 0,3 b</td><td>6,4 ± 0,3 b</td></tr><tr><td rowspan="3">Red wine</td><td>PA (mg/L)</td><td>1275 ± 66 a</td><td>1015 ± 3 b</td><td>1135 ± 85 a</td><td>1015 ± 67 b</td></tr><tr><td>mDP</td><td>6,0 ± 0,1 a</td><td>5,3 ± 0,1 b</td><td>5,6 ± 0,1 b</td><td>5,3 ± 0,1 b</td></tr><tr><td>mDP-EPA</td><td>-</td><td>14,2 ± 0,3 b</td><td>20,7 ± 1,3 b</td><td>14,1 ± 0,4 b</td></tr></tbody></table>

In the synthetic wine, the addition of the three inactive yeasts significantly decreased the total proanthocyanidin concentration. This trend was also observed in the red wine with the exception of Noblesse. The mDP of proanthocyanidins decreased significantly when the synthetic wine and the red wine were treated with all the inactive yeasts, the exception being Booster Rouge in synthetic wine. This tendency seems to indicate that the use of inactive yeasts brings about a loss of proanthocyanidins of high molecular weight. A basic mathematical approach using a simple equation was performed to calculate the mDP of the eliminated proanthocyanidins (mDP-EPA) by the supplementation with CIY. In both media, synthetic wine and red wine, the mDP-EPA was higher than the original mDP. This data indicates that higher polymeric tannins, precisely the most astringent, were preferentially eliminated.

## Conclusion

Overall these results seem to indicate that the mechanism of action by which the inactive dry yeast can enhance the mouthfeel and smooth astringency perception is related to two different phenomena. The first is that inactive yeasts release polysaccharides which can increase mouthfeel and act to inhibit interactions between salivary protein and tannins, while the second involves the direct effect they have on the precipitation of proanthocyanidins, especially the larger polymers, which have been described as the most astringent. It can be concluded that supplementation with CIY is a useful tool for smoothing the astringency of red wines.
# Acknowledgements

The authors are grateful to Lallemand Inc. for its financial support.

## References

1. Kennedy JA, Ferrier J, Harbertson JF et al (2006) Analysis of tannins in red wine using multiple methods: correlation with perceived astringency. *Am J Enol Vitic* **57**: 481-485.

2. Preys S, Mazerolles G, Courcoux P et al (2006) Relationship between polyphenolic composition and some sensory properties in red wines using multiway analyses. *Anal Chim Acta* **563**: 126-136.

3. Quijada-Morín N, Regueiro J, Simal-Gándara J et al (2012) Relationship between the sensory-determined astringency and the flavanolic composition of red wines. *J Agric Food Chem* **60**: 12355-12361.

4. Vidal S, Francis L, Guyot S et al (2003) The mouth-feel properties of grape and apple proanthocyanidins in a wine-like medium. *J Sci Food Agric* **83**: 564-573.

5. Waters EJ, Pellerin P, Brillouet JM (1994) A *Saccharomyces* mannoprotein that protects wine from protein haze. *Carbohydr Polym* **23**: 185-191.

6. Guilloux-Benatier M, Guerreau J, Feuillat M (1995) Influence of initial colloid content on yeast macromolecule production and on the metabolism of wine microorganisms. *Am J Enol Vitic* **46**: 486-492.

7. Guadalupe Z, Palacios A, Ayestarán B (2007) Maceration enzymes and mannoproteins: a possible strategy to increase colloidal stability and color extraction in red wines. *J Agric Food Chem* **55**: 4854-4862.

8. Fornairon-Bonnefond C, Camarasa C, Moutounet M et al (2002) New trends on yeast autolysis and wine ageing on lees: a bibliographic review. *J Int Vigne Vin* **36**: 49-69.

9. Quijada-Morín N, Williams P, Rivas-Gonzalo JC et al (2014) Polyphenolic, polysaccharide and oligosaccharide composition of tempranillo red wines and their relationship with the perceived astringency. *Food Chem* **154**: 44-51.

10. Pozo-Bayón MA, Andujar-Ortiz I, Alcaide- Hidalgo JM et al (2009) Characterization of commercial inactive dry yeast preparations for enological use based on their ability to release soluble compounds and their behavior toward aroma compounds in model wines. *J Agric Food Chem* **57**: 10784-10792.

11. González-Royo E, Urtasun A, Gil M et al (2013) Effect of yeast strain and supplementation with inactive yeast during alcoholic fermentation on wine polysaccharides. *Am J Enol Vitic* **64**: 268-273.

12. Ribéreau-Gayon P, Glories Y, Maujean A et al (2006) *The Chemistry of Wine Stabilization and Treatments in Phenolic Compounds - Handbook of Enology*, Wiley, Chichester, UK.

13. Gil M, Kontoudakis N, González E et al (2012) Influence of grape maturity and maceration length on color, polyphenolic composition, and polysaccharide content of cabernet sauvignon and tempranillo wines. *J Agric Food Chem* **60**: 7988-8001.

14. Ayestarán B, Guadalupe Z, León D (2004) Quantification of major grape polysaccharides (Tempranillo V.) released by maceration enzymes during the fermentation process. *Anal Chim Acta* **513**: 29-39.

15. Kennedy JA, Jones GP (2001) Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. *J Agric Food Chem* **49**: 1740-1746.
# Volatile aroma compounds related to 'stone fruit' aroma in Viognier and Chardonnay wine

T.E. SIEBERT<fnref n="1" />*, W.P. PEARSON<fnref n="1" />, A. BARKER<fnref n="1" />, M.A. DE BARROS LOPES<fnref n="2" />, P. DARRIET<fnref n="3" />, M.J. HERDERICH<fnref n="1" />, I.L. FRANCIS<fnref n="1" />

Volatile compounds – wine – sensory descriptive analysis – partial least squares regression

## Introduction

Different wine grape varieties have diverse aromas and flavours, with distinct differences within varieties resulting from vineyard origin and winemaking or viticultural practices [1]. However, often the volatile compositions of wines made from different varieties only vary in the proportions of a number of key aroma-active compounds. Relating the compositional differences among wines to their sensory attributes can help determine if sensory difference is due to variation in concentrations of aroma compounds, an absence/presence of certain aroma compounds or an additive effect of several aroma compounds [2].

In fresh apricots and other yellow stone fruit, the n-alkyl γ-lactones (octa-, deca- and dodeca-) and δ-decalactone are considered to be impact aroma compounds [3] but very little is known about the chemical basis of 'stone fruit' aromas in wine.

Previous studies involving gas chromatography-olfactometry-mass spectrometry (GC-O-MS) have suggested that 'apricot' or 'stone fruit' aroma in wine may be due to the n-alkyl lactones that were detected. However, quantitative analytical data has shown that generally sub-aroma detection threshold concentrations of these are present in white wines [4]. These earlier studies did not focus on wines with clear 'stone fruit' aroma attributes. Therefore, the focus of this study were Viognier and Chardonnay wines from France and Australia with differing levels of 'stone fruit' character, ranging from low to high.

## Materials and methods

### Wines

Suitable subsets of wines with a range of 'stone fruit' aroma intensities were selected, by a group of experienced wine tasters, from approximately 100 commercially available wines: Australian Chardonnay; Australian Viognier; French Viognier; Australian botrytised sweet Semillon; and Sauternes.

### Sensory analysis

A set of 10 commercial wines (four Australian Chardonnay wines; three Australian Viognier wines; one French Viognier wine; and two Australian botrytised Semillon) were analysed by HS-SPME-GC-O-MS analysis using a panel of four assessors in a method similar to [5].

Descriptive sensory analysis was conducted with a set of 18 commercial wines (six wines of: Australian Chardonnay; Australian Viognier; and French Viognier) in a comparable manner to [5]. A sensory panel, consisting of 12 trained panellists, rated the intensity of sensory attributes of the wines in triplicate under controlled conditions (based on QDA® methodology). The panel attended four training sessions before undertaking the formal assessment of the wines. The panellists generated 18 aroma attributes appropriate for describing the wines. Reference standards were presented to the panel for training and aligning the panellists terminology. FIZZ software was used for the collection of sensory data.

<footnote n="1">¹The Australian Wine Research Institute, P.O. Box 197 Glen Osmond (Adelaide) 5064, Australia</footnote>

<footnote n="2">²University of South Australia, School of Pharmacy and Medical Science, City East Campus, Adelaide, 5000, Australia</footnote>

<footnote n="3">³University of Bordeaux, Unité de Recherche Œnologie EA 4577, USC 1366 INRA, ISVV, Villenave d'Ornon cedex, 33882, France</footnote>

<footnote>*Corresponding author: tracey.siebert@awri.com.au</footnote>
## Chemical analysis

Volatile compounds were quantified in the wines by targeted analyses using previously published methods (Table 1). All targeted analysis methods, except one [6], used stable isotope dilution analysis (SIDA) with deuterated analogues as internal standards and the MS in selected ion monitoring (SIM) mode or MS/MS with multiple reaction monitoring (MRM). The wines were also analysed for basic chemical composition.

<table><caption>**Table 1** - Volatile aroma compounds quantified by targeted analyses</caption><thead><tr><td>Group of compounds</td><td>Compounds</td><td>Analysis type</td></tr></thead><tbody><tr><td>Fermentation derived</td><td>11 ethyl and 5 acetate esters, 6 alcohols, 9 fatty acids</td><td>SIDA-HS-SPME-GC-MS [7]</td></tr><tr><td>Sulfur</td><td>3 thiols, 2 sulfides, 3 disulfides, 2 thioacetate esters</td><td>HS-COC-GC-SCD [6]</td></tr><tr><td>Lactones</td><td>C<sub>8</sub>-C<sub>12</sub> γ- and δ-lactones, 3 other lactones</td><td>SIDA-SPE-GC-MS [4]</td></tr><tr><td>Oxidation related</td><td>4 (E)-2-alkenals, 4 Strecker aldehydes, 4 other aldehydes, 3 furans, 3 alcohols</td><td>SIDA-deriv-SPE-GC-MS/MS [8]</td></tr><tr><td>Polyfunctional thiols</td><td>3MH, 3MHA, 4MMP, furfuryl thiol, benzenemethanethiol</td><td>SIDA-deriv-SPE-HPLC-MS/MS [9]</td></tr><tr><td>C<sub>6</sub> volatiles</td><td>3 alcohols, mesityl oxide</td><td>SIDA-HS-SPME-GC-MS [10]</td></tr><tr><td>Acetaldehyde related</td><td>acetaldehyde, diacetyl, acetoin, 2,3-butanediol</td><td>SIDA-HS-SPME-GC-MS [11]</td></tr><tr><td>Oak derived</td><td>8 oak compounds, 4-EP and 4-EG</td><td>SIDA-LLE-GC-MS [12]</td></tr><tr><td>Grape derived</td><td>4 monoterpenes, 3 norisoprenoids, TDN, naphthalene</td><td>SIDA-LLE-GC-MS [13]</td></tr><tr><td>Other</td><td>2 terpenoids, ethyl cinnamate, ethyl 9-decenoate</td><td>SIDA-HS-SPME-GC-MS*</td></tr></tbody></table>

## Data analysis

The sensory attribute ratings were related to volatile composition by partial least squares regression (PLSR). The Unscrambler software was used for the multivariate data analysis.

# Results and discussion

The aroma profiles of a set of 10 wines with high or low 'stone fruit' character were deconstructed by HS-SPME-GC-O-MS analysis. However, there were no clear regions that gave obvious 'stone fruit' aroma.

For the descriptive sensory analysis, the sensory panel determined that the 'apricot' aroma attribute was reminiscent of preserved apricots and the 'peach' aroma attribute was reminiscent of fresh white peach. The sensory data showed the selected wines had varied ratings of 'apricot' and 'peach' aroma attributes and the two attributes were not closely correlated.

Of the 108 targeted wine aroma compounds, 78 were quantified in some or all of the 18 wines. The remaining 30 aroma compounds were either below the limit of quantitation or not detected.

Relating the sensory attribute ratings to volatile composition by partial least squares regression (Figure 1), γ-nonalactone and γ-decalactone were associated with the 'apricot' sensory attribute, together with the monoterpenes linalool, geraniol and nerol, and the compounds E-2-hexenal and E-2-hexenol. The polyfunctional thiol 3-mercaptohexyl acetate (3MHA), and also ethyl cinnamate were negatively related to the 'apricot' attribute. The 'peach' aroma attribute was also associated with 2-methylpropyl acetate, 3-methyl butyl acetate, and benzaldehyde, and the lactones were not important to the model for this attribute (Figure 2).

<footnote>* Unpublished, in-house method</footnote>
Although γ-nonalactone and γ-decalactone were highlighted as being of importance to ‘apricot’ aroma, when comparing the individual reported sensory aroma detection thresholds of these compounds in model wine (Table 2), it could be considered that the concentrations are too low to be of sensory significance. However, the combination of these lactones with the other compounds implicated in this study may give rise to the ‘apricot’ aroma attribute.

<figure><img src="image_389.png" /><figcaption>Fig. 1 - PLSR of X and Y loadings for chemical composition and sensory aroma attribute ratings.</figcaption></figure>

<figure><img src="image_390.png" /><figcaption>Fig. 2 - Volatile compounds from the PLS model of greatest importance to the ‘apricot’ and ‘peach’ aroma attributes.</figcaption></figure>
<table><caption>**Table 2** - Concentration ranges of the two lactones associated with the 'apricot' aroma attribute in the 18 wines studied</caption><thead><tr><th></th><th>γ-Nonalactone (µg/L)</th><th>γ-Decalactone (µg/L)</th></tr></thead><tbody><tr><td>Minimum-maximum</td><td>2.0 - 6.1</td><td>0.4 - 1.3</td></tr><tr><td>Aroma threshold in model wine</td><td>76</td><td>10</td></tr></tbody></table>

## Conclusion

γ-Nonalactone and γ-decalactone were associated with 'apricot' aroma in the wines studied, together with several monoterpenes. Further work will include reconstitution, addition and omission studies to confirm the importance of these compounds to 'stone fruit' aroma.

## Acknowledgements

The Australian Wine Research Institute (AWRI), a member of the Wine Innovation Cluster at the Waite Precinct in Adelaide, is supported by Australian grapegrowers and winemakers through their investment body, the Australian Grape and Wine Authority (AGWA), with matching funds from the Australian government. The assistance of Panagiotis Stamatopoulos and Pascaline Redon, ISVV, and Sheridan Barter and sensory panellists, AWRI, is gratefully acknowledged.

## References

1. Noble AC, Ebeler SE (2002) Use of multivariate statistics in understanding wine flavor. *Food Rev Int* 18: 1-20.

2. Polášková P, Herszage J, Ebeler SE (2008) Wine flavor: chemistry in a glass. *Chem Soc Rev* 37: 2478-2489.

3. Greger V, Schieberle P (2007) Characterization of the key aroma compounds in apricots (*Prunus armeniaca*) by application of the molecular sensory science concept. *J Agric Food Chem* 55: 5221-5228.

4. Cooke RC, Capone DL, van Leeuwen KA et al (2009) Quantification of several 4-alkyl substituted γ-lactones in Australian wines. *J Agric Food Chem* 57: 348-352.

5. Mayr CM, Geue JP, Holt HE et al (2015) Characterization of the key aroma compounds in Shiraz wine by quantitation, aroma reconstitution, and omission studies. *J Agric Food Chem* 62: 4528-4536.

6. Siebert TE, Solomon MR, Pollnitz AP et al (2010) Selective determination of volatile sulfur compounds in wine by gas chromatography with sulfur chemiluminescence detection. *J Agric Food Chem* 58: 9454-9462.

7. Siebert TE, Smyth HE, Capone DL et al (2005) Stable isotope dilution analysis of wine fermentation products by HS-SPME-GC-MS. *Anal Bioanal Chem* 381: 937-947.

8. Mayr CM, Capone DL, Pardon KH et al (2015) Quantitative analysis by GC-MS/MS of 18 aroma compounds related to oxidative off-flavor in wines. *J Agric Food Chem* 63: 3394-3401.

9. Capone DL, Ristic R, Pardon KH et al (2015) Simple quantitative determination of potent thiols at ultratrace levels in wine by derivatization and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. *Anal Chem* 87: 1226-1231.

10. Capone DL, Black CA, Jeffery DW (2012) Effects on 3-mercaptohexan-1-ol precursor concentrations from prolonged storage of Sauvignon blanc grapes prior to crushing and pressing. *J Agric Food Chem* 60: 3515-3523.

11. Varela C, Kutyna DR, Solomon MR et al (2012) Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts. *Appl Environ Microbiol* 78: 6068-6077.

12. Pollnitz AP, Pardon KH, Sykes M et al (2004) The effects of sample preparation and gas chromatograph injection techniques on the accuracy of measuring guaiacol, 4-methylguaiacol and other volatile oak compounds in oak extracts by stable isotope dilution analyses. *J Agric Food Chem* 52: 3244-3252.

13. Pedersen DS, Capone DL, Skouroumounis GK et al (2003) Quantitative analysis of geraniol, nerol, linalool, and α-terpineol in wine. *Anal Bioanal Chem* 375: 517-522.
# Wine oxygen and organoleptic property evolutions in classified near infra red oak wood barrel during aging

J. MICHEL<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, M. JOURDES<fnref n="1" />,<fnref n="2" />, T. GIORDANENGO<fnref n="3" />, N. MOUREY<fnref n="3" />, P.L. TEISSEDRE<fnref n="1" />,<fnref n="2" />*

Oxygen – oak wood – wine organoleptic properties

## Introduction

The aging in wooden barrels is essential to achieve high wine quality. Vescalagin, castalagin, roburins A, B, C, D, E and grandinin are solubilized during wine aging in oak barrels. Their concentration in wine ranges between 5 mg/L and 30 mg/L [1]. Indeed, wood ellagitannin content varies greatly, from 8 mg/g to more than 120 mg/g of dry oak wood [2, 3]. This variability is influenced by many factors such as ecological factors, tree age [2], wood piece location in the trees as well as wood processing in cooperage [4]. Several studies have examined the ellagitannin levels and evolutions in wine because their content is an important parameter in wine aging. In fact, these highly reactive molecules can react with wine phenolic compounds and contribute to wine sensory characteristics, in particular to color stability, modulation of astringency, bitterness [3, 5] as well as protection of wine against oxidation [6].

Another important parameter for wine quality is the dissolved oxygen because some chemical reactions are impacted by oxygen such as the aroma and taste complexity or color stabilization. In fact, when O₂ is added at a proper level, it will improve red wine organoleptic properties and the obtained wine will be perceived as more balanced compared to those added with excessive amount of O₂ in which oxidative character appears, thus decreasing wine quality. During aging, small but constant oxygen quantities penetrate through wood pores: between 1.7 and 2.5 mL/L/month for French oak wood (*Quercus robur* and *Quercus petraea*) and even lower for *Quercus alba*. However, oxygen is still consumed rapidly and this could explain the low dissolved oxygen values in aged wines (between 10 and 50 µg/L) [7].

The aim of this study was to monitor oxygen consumption by a French red wine during aging in NIRS classified oak barrels and then to correlate these measurements with the ellagitannin levels and the wood morphology (grain or growth ring width).

## Materials and methods

### Barrel making

During the barrel making process, each stave was firstly classified in three polyphenol index (PI) groups: PI 18 (PI = 18 ± 3), PI 41 (PI = 41 ± 8), and PI 61 (PI = 61 ± 4) by Oakscan®, a NIRS apparatus [8]. Indeed, earlier studies have shown that ellagitannin levels were directly correlated with the PI [5, 9]. Then, a second visual classification by grain type [i.e., growth ring width (GRW)] was carried out: extra fine grain (EFG, GRW < 1.5 mm), fine grain (FG, GRW ≈ 2 mm), and medium grain (MG, GRW > 3 mm). This double classification results in five duplicate 225 L barrel groups: PI 18-FG, PI 41-FG, PI 61-FG, PI 41-EFG, PI 41-MG. Each barrel was equipped with small windows to measure the oxygen in wine using luminescence technology (PreSens®).

### Winemaking and wine aging

100 hL of must from *V. vinifera* L. cv. Cabernet Sauvignon (CS) and Cabernet Franc (CF) were collected in September 2011 from the Pessac-Léognan region in France. Alcoholic fermentation was processed at 25°C with *Saccharomyces cerevisiae*, malolactic fermentation was carried out and wines were assembled

<footnote n="1">¹Univ. Bordeaux, ISVV, EA 4577 Œnologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">²INRA, ISVV, USC 1366 Œnologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="3">³Tonnellerie RADOUX, 10 avenue Faidherbe, BP 113 – 17503 Jonzac Cedex, France</footnote>

<footnote>*Corresponding author: pierre-louis.teissedre@u-bordeaux.fr</footnote>
[CS/CF (94/6; v/v)] in inox tanks (156 hL). Then, the wine was aged in new barrels (cf previously) closed by a hermetic silicon bung, in duplicate. Then, every month during 12 months, a wine sample (750 mL) was collected in each barrel and stored at 16°C until chemical and sensory evaluations.

## Oxygen measurement

A PreSens Fibox 3 LCD-trace sensors using luminescence as measurement principle was used to analyze wine dissolved oxygen levels without opening the barrel. Two parts composed the apparatus: the first part is a sensor foil (PSt3 type) with a luminescence complex (ruthenium complex) glued on a plain glass in contact with red wine and the second part is the analyzer which translates the luminescence signal in oxygen quantities thanks to an optical fiber (Figure 1). Wine oxygen levels were evaluated twice a day during the first days after wine was put into barrels, and then the oxygen measurements were spaced in time according to the oxygen consumption kinetics.

## Wine ellagitannin level determination

The chemical analyses were performed on wine collected from each barrel. An adapted method [3] from wine was used for the quantification of ellagic acid, released by ellagitannins during the acidic hydrolysis [10]. Red wine was evaporated and re-dissolved in methanol. Then, this mixture was put in hydrolysis tubes with HCl 37%, and placed at 100°C for 2 h. In the same time, control tubes stayed at room temperature. After hydrolysis, internal standard was added prior to UPLC-UV/MS analysis. UPLC-UV/MS analysis used a Thermo scientific Accela composed by an autosampler, a quaternary pump and an UV-vis detector, controlled by Xcalibur data system. A reverse phase 50 x 2.1 mm, 1.9 µm Hypersil GOLD column was used to separate ellagic acid with a flow rate set at 0.4 mL/min. The mass spectra used to quantify total ellagitannins were obtained on a Thermo scientific MSQ Plus mass spectrometer. The electrospray ionization and mass detection was performed in negative ion mode for the detection of ellagic acid (m/z = 301) and in positive mode for the 1-naphthol (m/z = 145) with optimized parameters.

## Sensory evaluation

Tasting was focused on profiling wine aromas and flavors in mouth at 6 and 12 months. Sensory analyses were organized in a standard tasting room under white lights in separate booths. Before sensory evaluation, wines collected from the duplicate barrels were assembled (50/50, v/v). Several series of 3 wines were presented in clear, randomly coded glasses. Each judge was asked to hold a sample in his mouth, spit it out and rate each descriptor using a 1-7 point scale, where 1 indicated no perception of the descriptor and 7 indicated high intensity of the descriptor. Judges were asked to evaluate fruity and woody intensity, balance, spicy and smoked/toasted aromas, bitterness and astringency.

## Data analysis

Sensorial analyses were performed using FIZZ treatment (v2.41B, Biosystemes, Couternon France), and R software (v 2.15.0) was used for the chemical analysis and oxygen kinetics. When the ANOVAs were significant (p < 5%), a post-hoc test (Newman test) was processed to estimate the ellagitannin concentrations and their effects on wines. Tasting data were expressed as arithmetic average for tasted wines.

<figure><img src="image_391.png" /><figcaption>Fig. 1 - Experimental barrels.</figcaption></figure>
# Results and discussion

## Red wine oxygen consumption in NIRS classified barrels

Red wine oxygen concentration average (average of eight data points per barrel) showed oxygen consumption in three steps for the barrels (Figure 2). Oxygen level decreased quickly in the first eight days after wine was put into barrels, from 5138.65 ± 341.37 to 187.29 ± 25.08 µg of dissolved oxygen/L of red wine, which indicated a consumption average rate of 618.92 ± 4.63 µg dissolved oxygen/L/day. This consumption represented 96.36% of total dissolved oxygen at T0. In a second step, consumption was slower. Indeed, oxygen level reduced from 187.29 ± 25.08 to 76.90 ± 4.37 µg dissolved oxygen/L in 97 days with an average speed of 1.14 ± 0.38 µg dissolved oxygen/L/day. Then, oxygen level seemed to be lower than the limit of detection of the Presens sensor, which is 1 µg dissolved oxygen/L (Figure 2).

These profiles showed the major role played by grape polyphenols in wine oxygen consumption. Red wine antioxidant molecules which came from grape as gallic acid, quercetin, anthocyanins and tannins consumed quickly the dissolved oxygen [11]. Then, an oxygen concentration equilibrium takes place around 50 µg dissolved oxygen/L.

<figure><img src="image_392.png" /><figcaption>Fig. 2 - Red wine oxygen kinetics.</figcaption></figure>

## Wood PI influences on red wine oxygen consumption

Significant differences (p < 2%) were observed for dissolved oxygen rate when the wood PI classification differed. First, these variabilities were observed at day 6 of wine aging. This oxygen consumption difference increased until day 8 and they were respectively 611.64, 616.65 and 623.44 µg dissolved oxygen/L/day for PI 18-FG, PI 41-FG and PI 61-FG barrels (Figure 3). Wine aged in PI 61 barrels has higher oxygen consumption than wine aged in PI 41. Indeed, wine aged in PI 41 barrels has 35.96% more dissolved oxygen/L than wine aged in PI 61 barrels, with 151.10 ± 13.16 for PI 61 and 205.43 ± 27.58 µg dissolved oxygen/L for PI 41. Similar difference between PI 18 and PI 41 barrels was observed (245.57 ± 36.30 µg dissolved oxygen/L for PI 18 and 205.43 ± 27.58 µg dissolved oxygen/L for PI 41) (Figure 3).

<figure><img src="image_393.png" /><figcaption>Fig. 3 - Significant zone of red wine oxygen consumption and ellagitannin concentrations in the first months of aging for the PI modalities.</figcaption></figure>
These results showed that wood PI influenced dissolved oxygen consumption due to ellagitannin molecules extracted from oak wood during aging [12]. The extraction kinetics and evolutions of ellagitannins in wine were monitored and differences were already evidenced in the first month due to the wood classification (Figure 3). Indeed, PI 61 wood was richer in ellagitannins than PI 41 wood, which was also more concentrated than PI 18 wood. Thus, wine aged in contact to the wood with the lowest ellagitannin concentration exhibited also the lowest ellagitannin level in wine (1.28 ± 0.21 mg equiv ellagic acid/L), whereas the same wine aged in contact to the highest PI has the highest ellagitannin concentration (2.70 ± 0.13 mg equiv ellagic acid/L) (Figure 3). These differences were correlated with the difference in oxygen consumption and may result from the antioxidant properties of the ellagitannins [9, 13].

*Wood grain influences on red wine oxygen consumption*

When wine was aged with the same PI but with three different grain (EFG, FG and MG), significant differences in the oxygen consumption were also observed (p < 0.4%) between day 5 and day 8 of aging (Figure 4). A faster consumption of oxygen in wine aged in barrels classified as MG was observed compared to the other wines aged in barrels FG and EFG. After eight days, wine oxygen concentration in barrels MG was 249.39 ± 134.48 µg dissolved oxygen/L, representing about -73% compared to FG and EFG with respectively 429.80 ± 39.59 and 436.56 ± 30.53 µg dissolved oxygen/L (Figure 4). These results were due to oxygen consumption rate differences, with 624.25, 616.65 and 618.62 µg dissolved oxygen/L/day for MG, FG and EFG, respectively.

These differences could be due to wine ellagitannin extraction influenced by wood grain as demonstrated by recent studies [9] or different wood oxygen permeability related to this morphological difference.

Thus, the level of extracted ellagitannin was monitored (Figure 4). Wine aged in barrel with the largest grain (MG) was richer in ellagitannins (2.14 ± 0.74 mg equiv ellagic acid/L) than the modality with the smallest grain (1.02 ± 0.11 mg equiv ellagic acid/L for EFG). This faster ellagitannin extraction could be explained by wood morphology. Indeed, when grain increased, wine could penetrate more easily in the wood and allowed a faster extraction of ellagitannins.

<figure><img src="image_394.png" /><figcaption>**Fig. 4** - Significant zone of red wine oxygen consumption and ellagitannin concentrations in the first months of aging for the grain modalities.</figcaption></figure>

*Impact of wood NIRS classification on red wine organoleptic perceptions*

Sensory profiles were carried out at 6 and 12 months of aging. As demonstrated in previous studies [3, 5], some descriptors such as woody intensity in nose and mouth, smoked/toasted aromas, bitterness and astringency in mouth were influenced by Oakscan classification and correlated significantly with wood PI (p < 5%). Indeed, after 6 months of aging, the aromatic profile of wine aged in low content ellagitannin barrels (PI 18) was evaluated as less woody and smoked/toasted (p < 5%) than the same wine aged in barrels richer in ellagitannins (PI 41 and PI 61) (Figure 5). These wood aromas could impact the fruity intensity aromas (p < 1%) by mask effect as found in some recent studies [5, 14] or by wine redox potential modification due to a significant increase of ellagitannin level (Figure 5). Oakscan classification was only based on the wood polyphenolic content, so these effects on wine aromas were probably due to some wood aroma markers (aldehyde furanics, whiskey lactone, etc.) and could be correlated with NIRS classification as observed in recent studies [5, 9].
The mouth characteristics were also impacted by wood classification. Wine aged in barrels classified by Oakscan as PI 61 and PI 41 was evaluated as more woody, more bitter and more astringent (p< 5%) than wine aged in PI 18 barrels (Figure 5). Direct impacts of the ellagitannins were showed here and correlated with wood PI and oxygen consumption (Figure 5). Indeed, the ellagitannin level increased during wine aging and reached after 6 months 1.55 ± 0.22 and 6.29 ± 0.15 mg equiv ellagic acid/L for wine aged in PI 18-FG and PI 61-FG barrels, respectively (p < 0.04%).

<figure><img src="image_395.png" /><figcaption>**Fig. 5** - Red wine tasting results of wine aged in classified PI barrels after 6 and 12 months of aging and wine ellagitannin levels according to PI barrels at 6 and 12 months of aging.</figcaption></figure>

After 12 months of aging, PI impacts were important especially on mouth characteristics. Wines aged in PI 61 and PI 41 barrels were noted as more woody, bitter, and astringent (p< 5%). The impacts on wine aromas were less important and only the smoked/toasted characteristic for wine aged in PI 61 barrels was influenced (p < 5%) (Figure 5). At the end of aging (12 months), ellagitannin level exhibited significant differences (p < 0.09%) and reached maximum levels with 0.89 ± 0.26 for PI 18-FG and 5.81 ± 0.66 mg equiv ellagic acid/L for PI 61-FG (Figure 5).

## Conclusion

This study showed the oxygen consumption kinetics by wine aged in oak barrels. In the first eight days, oxygen was quickly consumed in majority by wine constituents (96% of total oxygen). However, small differences in the consumption rate can be observed. In fact, wine aged in contact with wood richer in ellagitannins consumed more quickly the dissolved oxygen. The size of wood grain was another parameter which impacted oxygen consumption. Indeed, wine aged in contact with larger grain type consumed oxygen faster. These differences can be due to the ellagitannin extraction kinetics as demonstrated in previous studies [3, 5, 9]. When wine was more concentrated in ellagitannins, its oxygen consumption was higher. Nevertheless, these differences did not impact wine organoleptic properties. In fact, the organoleptic properties were mainly influenced by ellagitannin concentrations notably on fruity and woody intensity, smocked/toasted aromas and woody, bitterness and astringency in mouth.
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# Antioxidant activities and presence of bioactive micronutrients in new South Korean wines: effect of winemaking processes and chemical composition

H.W. CHOI¹,², J.P. SOUCHARD²,³*

¹Dept of Wine and Food Fermentation Technology, Young-Dong University, 310, Daehakro, Yeungdong-eup, Chungbuk, 370-701, Korea

²Lab. des IMRCP, UMR CNRS 5623, Faculté des Sciences Pharmaceutiques, Université Paul Sabatier, 35 ch. des Maraîchers, 31062 Toulouse cedex 09, France

³Centre de Viticulture et d'Œnologie de Midi-Pyrénées, Avenue de l'Agrobiopôle, 31320 Auzeville-Tolosane, France

Antioxidant activity – polyphenols – nutraceuticals – oenological practices – sensory analysis – *Vitis labrusca*

## Introduction

For nearly three decades the assumption of a moderate consumption of wine has been advanced to explain the low mortality from cardiovascular disease in France despite a high intake of fat (French paradox) [1, 2]. Since then, many epidemiological studies have been conducted in different countries. They compared mortality between consumers of wines, beer or alcohol, and abstainers. Recent meta-analysis studies revealed that a moderate wine consumption preserved from cardiovascular disease [3] and the development of certain cancers, particularly rectal [4], gastric [5], lung [6, 7] and mouth [7]. These diet effects were attributed to antioxidant, anti-inflammatory and anti-cancerous activities of polyphenols, molecules particularly present in red wine [8, 9].

The aim of this study was to investigate the antioxidant capacity of South Korean wines (*Vitis labrusca*, Campbell Early) obtained with different oenological practices (hot or cold maceration, before or after fermentation, using enzymes or not) and their correlation with polyphenolic composition (tannins, anthocyanins, flavonoids, total polyphenols) and the nutraceuticals quercetin and resveratrol. Moreover, sensory analysis has been carried on to determine the sensory profile of wines and to validate the concept.

## Materials and methods

### Winemaking

Characterization of ten wines, vintage 2012, named A(1-5) or B(1-5) has been carried on for this study. They correspond to different winemaking processes (1-5) without enzymes (A) or with pectolytic enzymes and anthocyanases (0.5 g/L Lafase®, B):

1. classical alcoholic fermentation with punching

2. cold maceration before fermentation (5°C, 3 days)

3. hot maceration before fermentation (70°C, 30 minutes)

4. cold maceration after fermentation (5°C, 3 days)

5. hot maceration after fermentation (70°C, 30 minutes)

### Tannin treatments

In vintage 2013, A3 wine was treated with condensed tannins 0.5 g/L (A3C) or hydrolysable tannins 0.5 g/L (A3H) or a mixture of condensed 0.25 g/L and hydrolysable tannins 0.25 g/L (A3CH).