Résultats et discussion

Présents à l'état de trace dans les vins, le 3MH et le 3MHA sont des molécules très réactives à cause de la fonction chimique thiol. En effet, les thiols peuvent réagir avec de nombreuses espèces électrophiles du vin pour former soit des thioéthers [1], soit des complexes avec des métaux lourds [3], soit des disulfures [4]. Dans les deux cas, le piégeage des thiols est responsable d'une perte aromatique [5]. Cependant, l'évolution des thiols sous forme de disulfure est un processus réversible selon le potentiel redox du vin, alors que l'addition nucléophile, notamment sur les quinones d'ortho-diphénols est irréversible [6]. Il est donc pertinent de pouvoir connaître la part de thiol piégée sous forme de disulfures puisqu'elle peut s'apparenter à une réserve aromatique dont la teneur évolue de la fin de la fermentation alcoolique jusqu'au vieillissement du vin.

Pour cela, une méthode d'analyse consistant en l'analyse de toutes les formes disulfures possibles du 3MH et du 3MHA a été développée afin de caractériser le réservoir aromatique du vin. Ce nouvel indicateur analytique est appelé : « le potentiel thiol » et correspond à l'addition des formes libres et disulfures du 3MH et 3MHA. D'un point de vue analytique, la méthode a été validée selon les recommandations de l'Organisation Internationale de la Vigne et du Vin et affiche une grande sensibilité (LOD inférieure à 3 ng/L), exactitude (95-110%) et répétabilité (CV<15%) toutes matrices de vins confondues.

<figure><img src="image_203.png" /><figcaption>Fig. 1 - Répartition des formes libres, disulfures et autres du 3MH dans les vins rouge (A), rosé (B) et blanc (C). Les valeurs représentées ici sont la moyenne de triplicat d'échantillon.</figcaption></figure>
Trois types de vin ont ensuite subis divers traitements œnologiques afin de mimer le devenir des thiols en conditions plus ou moins oxydantes. L'ensemble des résultats analytiques est présenté dans le Tableau 1. De façon générale, les échantillons analysés sont des vins jeunes dont la majorité des thiols sont sous forme libre (plus de 90%). La part de disulfure est minime avant quel que traitement que ce soit. Par comparaison avec l'échantillon témoin, il a été calculé pour chaque essai la proportion de disulfures et de pertes aromatiques non-réversibles formées (Figure 1).

Les modalités oxygénations affichent des teneurs moindres en thiols libres alors que la proportion de formes disulfures reste voisine à celles rencontrées dans le témoin. Par conséquent, l'oxygénation des vins induit préférentiellement une perte aromatique de type non-réversible (jusqu'à 20% des teneurs initiales en thiols).

Les modalités CuSO₄ et CuSO₄/GSH conduisent à la disparition quasi-totale des thiols libres (Tableau 1). En revanche, la proportion des formes disulfures du 3MH et du 3MHA oscille entre 15 et 50% des teneurs initiales en thiols. En d'autres termes, des vins ayant subi des traitements au cuivre possèdent toujours un réservoir aromatique pouvant se révéler au cours du vieillissement du vin.

Les modalités comportant un traitement au GSH affichent des proportions en pertes irréversibles moindres que les autres essais et cette observation est d'autant plus vérifiée sur les vins rouges. En se combinant préférentiellement sur des espèces électrophiles, le GSH joue un rôle de protecteur (scavenger). En revanche, les ajouts de GSH ne modulent en rien les teneurs de disulfures. Dans nos conditions, le GSH agit davantage comme un protecteur plutôt qu'un antioxydant.

## Conclusion

La mesure du potentiel thiol permet d'avoir une vision plus globale de l'arôme d'un vin puisqu'elle permet de quantifier précisément la part de disulfures qui s'apparente à une réserve aromatique. Cet indicateur devrait à terme permettre de prédire la conservation maximale d'un vin en ce qui concerne la composante thiol. Il ouvre également la voie à une meilleure gestion du type d'embouteillage, du mode de vieillissement et tout autre paramètre influant sur le potentiel redox du vin.

## Remerciements

Nous remercions l'IFV de Pech Rouge qui nous a aidés à la mise en œuvre des essais.

## Abréviations

3MH, 3-mercaptohexanol; 3MHA, acétate de 3-mercaptohexyme; nanoLC-MS/MS, nano liquid chromatography – tandem mass spectrometry; GSH, glutathion; NPM, N-phénylmaléimide; TCEP, tris(carboxyethyl)-phosphine; LOD, limite de détection; CV, coefficient de variation.

## Références

1. Nikolantonaki M, Jourdes M, Shinoda K et al (2012) Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions. *J Agric Food Chem* **60**: 2647-2656.

2. Centre de Recherche et d'Expérimentation sur le Vin Rosé (2014) *Conservation des Vins Rosés*. Programme France AgriMer.

3. Franco-Luesma E, Ferreira V (2014) Quantitative analysis of free and bonded forms of volatile sulfur compounds in wine. Basic methodologies and evidences showing the existence of reversible cation-complexed forms. *J Chromatogr A* **1359**: 8-15.

4. Bencomo-Rodriguez JJ, Gambetta J, Rigou P et al (2011) Quantitative determination of varietal disulfides in wine and their behavior during alcoholic fermentation, pp. 656-662. *In Proceedings of Oeno 2011* (Bordeaux, France), Dunod.

5. Nikolantonaki M, Chichuc I, Teissedre P-L et al (2009) Reactivity of volatile thiols with polyphenols in a wine-model medium: impact of oxygen, iron, and sulfur dioxide. *Anal Chim Acta* **660**: 102-109.

6. Cheynier VF, Trousdale EK, Singleton VL et al (1986) Characterization of 2-S-glutathionyl caftaric acid and its hydrolysis in relation to grape wines. *J Agric Food Chem* **34**: 217-221.
# Recherche des marqueurs moléculaires de l'arôme de fruits cuits des raisins et des vins rouges de Merlot et de Cabernet Sauvignon : incidence de la date de récolte sur la massoia lactone et le furanéol

L. ALLAMY<fnref n="1" />,<fnref n="3" />*, P. DARRIET<fnref n="3" />, D. DUBOURDIEU<fnref n="3" />, A. PONS<fnref n="2" />,<fnref n="3" />

Arômes – maturation – Merlot – vins

## Introduction

L'expression aromatique des vins rouges de Bordeaux est intimement liée à l'état de maturité des raisins. Les modifications climatiques au cours de cette dernière décennie [1], constituent l'un des facteurs à l'origine du changement des conditions de maturation des raisins. Au cours de la maturation, le caractère végétal des raisins s'estompe puis disparait lorsque ceux-ci atteignent leur maturité pour ensuite laisser place à des nuances de fruits cuits (figue, pruneau) lorsque les raisins sont récoltés plus tardivement en état de sur-maturation. En effet, à la faveur d'étés chauds et secs, il est empiriquement admis que la maturation excessive des baies s'accompagne du développement de ces nuances de fruits cuits. Celles-ci sont alors retrouvées à la fois dans les moûts et les vins jeunes, et souvent dans ceux élaborés à partir de raisins de Merlot. A ce jour, les composés responsables de ces nuances fruits sont inconnus. Aussi, nous présentons les premiers résultats de l'identification puis du dosage de marqueurs moléculaires pertinents retrouvés dans les mouts et les vins.

## Matériel et méthodes

### Chromatographie en phase gazeuse couplée à l'olfactométrie et à la spectrométrie de masse (GC-O-MS)
Les analyses par GC-O-MS sont effectuées à partir d'extraits de moûts et de vins (marqués ou non par des odeurs de fruits cuits) obtenu par 3 extractions liquide-liquide successives au dichlorométhane (100mL d'échantillon pour 10-5-5mL de dichlorométhane). La recherche de zones odorantes (ZO) caractéristiques des nuances recherchées ainsi que leurs identifications ont été réalisées à l'aide d'un chromatographe Trace GC Ultra (Thermo Scientific) équipé d'un sniffing port et couplé à un spectromètre de masse DSQ II (Thermo Scientific) fonctionnant en impact électronique. La quantification du furanéol (étalon interne : ethyl maltol) et de la massoia lactone (étalon interne : 3-octanol) est réalisée à partir d'un extrait liquide-liquide et a été validée en terme de répétabilité, linéarité et sensibilité.

### Effet de la date de récolte : expérience en champ

Une parcelle de Merlot (millésime 2014, appellation Pauillac) a été récoltée à quatre dates de récoltes espacées de 6, 4 et 9 jours correspondants à D1, D2, D3, D4. Ces quatre lots ont été vinifiés de la même façon à la propriété dans des cuves de 25hL. Les lots ont été ensemencés en levures (S. cerevisiae) et levains malolactiques (O. oeni). Des prélèvements ont été effectués afin de suivre l'évolution des paramètres œnologiques courants ainsi que les composés volatils d'intérêt caractéristiques des moûts et des vins. Un travail d'analyse sensoriel est venu compléter l'évaluation des mouts et des vins issus des différentes dates de récolte.

<footnote n="1">¹Château Latour, Saint-Lambert, 33250 Pauillac, France</footnote>

<footnote n="2">²Seguin Moreau France, Z.I. Merpins, B.P. 94, 16103 Cognac, France</footnote>

<footnote n="3">³Université Bordeaux, ISVV, EA 4577, Unité de recherche Œnologie, F-33882 Villenave d'Ornon, France</footnote>

<footnote>*Auteur principal : lucile.allamy@bbox.fr</footnote>
# Résultats et discussion

## Recherche et identification par GC-O-MS de composés volatils associés aux zones odorantes (ZO) caractéristiques des nuances fruits cuits retrouvées dans les moûts et les vins

Afin d'améliorer notre connaissance sur les marqueurs moléculaires de l'arôme « fruit cuit » des moûts et des vins, nous avons recherché par GC-O-MS des ZO rappelant l'odeur des fruits cuits. L'analyse de nombreux moûts et vins marqués par ces nuances nous a permis de détecter 20 ZO décrites comme spécifiques des moûts et des vins.

Dans ce travail, nous présentons à titre d'exemple les résultats concernant deux ZO. La première, aux nuances de figue ou encore de noix de coco, est la 6-pentyl-5,6-dihydropyran-2-one ou massoia lactone. Cette ZO est détectée uniquement dans les moûts et les vins évoquant des nuances de fruits cuits. L'identification a été réalisée par comparaison des indices de rétention linéaire ainsi que par co-injection avec le composé pur. Elle a déjà été identifiée et dosée dans les vins de Tokaj [2] ainsi que dans les vins liquoreux de pourriture noble du bordelais [3]. Ce composé est retrouvé dans l'écorce du bois *Cryptocaria massoia* [4] et dans l'Aeollanthus suaveolens, plante brésilienne évoquant des odeurs de noix de coco [5]. Les travaux de Pons et al. [6] ont permis de montrer grâce à l'utilisation de la chromatographie en phase gazeuse bidimensionnelle sur colonne chirale couplée à la spectrométrie de masse que seulement la (R)-massoia lactone est présente dans les moûts et les vins rouges. Les seuils de perception ont été réalisés ; dans le moût il est de 10µg/L et dans un milieu modèle de composition proche du vin il est de 11µg/L.

La deuxième ZO aux nuances de sucre cuit et de fraise cuite correspond au 4-hydroxy-2,5-dimethyl-(2H)-furan-3-one ou furanéol. Dans les moûts, ce composé est détecté uniquement dans les moûts évoquant des nuances de fruits cuits. Dans les vins, il est détecté dans les vins témoins et ceux comportant des nuances de fruits cuits, mais il est perçu comme plus intense dans ces derniers. Ce composé a été identifié comme un des contributeurs majeurs de l'arôme des cépages non *Vitis vinifera* [7]. Il est retrouvé dans les vins rouges de Cabernet Sauvignon et de Merlot [8]. Les seuils de perception du furanéol retrouvés dans la littérature sont très variés. On peut observer qu'en solution hydro alcoolique, les seuils de perception varient de 5 [9] à 70µg/L [3]. Ces grandes disparités de seuils de perception nous ont amenés par la suite, à réaliser des seuils de perception au laboratoire.

Les seuils de perception du furanéol on été réalisés dans une salle de dégustation répondant aux normes AFNOR avec un panel composé de 21 experts. Il est de 10.5µg/L en solution modèle de moût (240g/L glucose/fructose, 5g/L acide tartrique, pH3.5) et de 49µg/L en solution modèle de vin (12%.vol éthanol bi-distillé, 5g/L acide tartrique, pH3.5).

Cette première approche nous permet d'établir un lien entre la présence de ces composés et les nuances de fruits cuits des moûts et des vins liées à la maturité des raisins. Par conséquent, il nous a paru nécessaire de mettre en place une expérience en champ sur l'incidence de la date de récolte sur ces composés afin de nous permettre d'observer l'évolution des concentrations en furanéol et massoia lactone dans les vins issus de raisins présentant des maturités différentes pour au final préciser leur impact sensoriel.

## Incidence de la date de récolte sur l'arôme des vins et sur leurs teneurs en furanéol et en massoia lactone en conditions réelles de vinification

L'analyse des paramètres œnologiques (Tableau 1) montre que la date de récolte a un effet sur les teneurs en sucres réducteurs ; en revanche, il n'y a pas d'effet sur le pH. Dans les vins, la maturité entraîne une augmentation du TAV et du pH.

<table><caption>**Tableau 1** - Teneurs en sucres réducteurs et pH des moûts, TAV et pH des vins</caption><thead><tr><th>MOUTS</th><th>D1</th><th>D2</th><th>D3</th><th>D4</th></tr></thead><tbody><tr><td>Sucres réducteurs (g/L)</td><td>223</td><td>224</td><td>221</td><td>244</td></tr><tr><td>pH</td><td>3.57</td><td>3.37</td><td>3.59</td><td>3.53</td></tr><tr><th>VINS</th><th>D1</th><th>D2</th><th>D3</th><th>D4</th></tr><tr><td>TAV (%Vol.)</td><td>13.31</td><td>13.32</td><td>13.7</td><td>14.20</td></tr><tr><td>pH</td><td>3.46</td><td>3.54</td><td>3.60</td><td>3.62</td></tr></tbody></table>
Afin d'observer l'incidence de la date de récolte sur les teneurs en composés volatils d'intérêt, des dosages ont été réalisés par GC-MS sur les vins (Figure 1). Les dosages du furanéol montrent que c'est à partir de D3 que les teneurs sont supérieures au seuil de perception. Il y a un facteur 2 de concentrations entre D1 et D4. Les teneurs en furanéol dans les vins sont liées à l'état de maturité des raisins. Les dosages de la massoia lactone dans les vins indiquent qu'à partir de la D2 les teneurs sont supérieures au seuil de perception ; entre D1 et D2 les teneurs sont multipliées par un facteur 4. La massoia lactone semble être elle aussi liée à l'état de maturité des raisins.

<figure><img src="image_204.png" /><figcaption>**Fig. 1** - Teneurs en furanéol (A) et massoia lactone (B) dans les vins pour les dates de récolte D1, D2, D3 et D4 (SP : Seuil de perception).</figcaption></figure>

Enfin, sur une échelle de 0 à 10, il a été demandé au panel de 20 experts de juger l'intensité des nuances fruits cuits, végétales et fruits frais dans ces quatre vins. Afin d'analyser si des corrélations entre la dégustation et les teneurs en composés volatils pouvaient émerger de cette analyse sensorielle, une analyse en composantes principales a été réalisée (Figure 2).

<figure><img src="image_205.png" /><figcaption>**Fig. 2** - Analyse en composantes principales des intensités des descripteurs « végétal », « fruits frais », « fruits cuits » et des teneurs en furanéol et massoia lactone dosés dans les vins D1, D2, D3 et D4.
(A) Projection des vins et (B) Représentation des composés volatils et des descripteurs
(Variabilité totale : 72.95%).</figcaption></figure>

La projection des vins permet de montrer que la date de récolte, c'est-à-dire l'état de maturité des raisins, est corrélée avec les nuances fruits cuits perçues à la dégustation dans les vins. De plus, la représentation des composés volatils et des descripteurs montre que l'intensité des nuances de fruits cuits est corrélée avec les teneurs en la massoia lactone et le furanéol. Il existe donc bien un lien entre ces deux composés volatils et la date de récolte dans les vins.
Pour conclure, les analyses sensorielles et les mesures quantitatives ont permis de valider l'importance de
la date de récolte et donc de l'état de maturité des raisins sur l'expression aromatique et les teneurs en
composés volatils des vins.

Conclusion

Dans des moûts et des vins issus de raisins sur-muris, deux zones odorantes ont été détectées et les composés responsables ont été identifiés par GC-O-MS ; ils correspondent au furanéol et à la massoia lactone. Les seuils du furanéol en solution modèle ont été déterminés. Nous avons mis en évidence que la date de récolte pour le millésime 2014 sur une parcelle de Merlot a une incidence sur les teneurs dans les vins en massoia lactone et en furanéol ; elles sont liées à l'état de maturité des raisins. Pour finir, l'analyse sensorielle a confirmé également l'importance de la date de récolte par la modification de la perception des nuances de fruits cuits. L'analyse en composantes principales confirme le lien entre le furanéol et la massoia lactone avec la date de récolte et l'intensité des nuances de fruits cuits perçues dans les vins.

Références

1. Santos JA, Malheiro AC, Pinto JG et al (2012) Macroclimate and viticultural zoning in Europe: observed trends and atmospheric forcing. *Clim Res* **51**: 89-103.

2. Miklosy E, Kalmar Z, Kerenyi Z (2004) Identification of some characteristic aroma compounds in noble rotted grape berries and Aszu wines froms Tokaj by GC-MS. *Acta Aliment* **33**: 215-226.

3. Sarrazin E (2007) Recherches sur l'arôme des vins liquoreux de pourriture noble issus des cépages Sémillon et Sauvignon Blanc. *Thèse doctorale*, Université de Bordeaux II.

4. Abe S (1937) Massoia oil. *J Chem Soc Jpn* **38**: 246-251.

5. Lupe FA, Lemes AC, Augusto F et al (2007) Fragrant lactones in the steam distillation residue of *Aeollanthus suaveolens* Mart. ex Spreng and analysis by HS-SPME. *J Essent Oil Res* **19**: 271-272.

6. Pons A, Lavigne V, Darriet P et al (2011) Identification et impact organoleptique de la massoia lactone dans les mouts et les vins rouges, pp. 851-854. 9ème symposium international d'œnologie (Oeno 2011), Bordeaux, France.

7. Rapp A, Knipser W, Engel L et al (1980) Off-flavour compounds in the berry and wine aroma of grapevine hybrids. I. The strawberry-like flavour. *Vitis* **30**: 12-23.

8. Kotseridis Y, Razungles A, Bertrand A et al (2000) Differentiation of the aromas of Merlot and Cabernet sauvignon wines using sensory and instrumental analysis. *J Agric Food Chem* **48**: 5383-5388.

9. Ferreira V, Ortin N, Escudero A et al (2002) Chemical characterization of the aroma of Grenache rosé wines: aroma extract dilution analysis, quantitative determination, and sensory reconstitution studies. *J Agric Food Chem* **50**: 4048-4054.
# Investigation and sensory characterisation of 1,4-cineole: a potential marker of Australian Cabernet Sauvignon wine aroma

G. ANTALICK<fnref n="1" />*, S. TEMPERE<fnref n="3" />,<fnref n="4" />, K. ŠUKLJE<fnref n="1" />, J.W. BLACKMAN<fnref n="1" />,<fnref n="2" />, A. DELOIRE<fnref n="1" />, G. DE REVEL<fnref n="3" />,<fnref n="4" />, L.M. SCHMIDTKE<fnref n="1" />,<fnref n="2" />

Monoterpenes – grape metabolite – aromatic herbs – red wines

## Introduction

Australian Cabernet Sauvignon wine aromas have been described using specific attributes such as “eucalyptus” and “dried herbs” [1, 2]. It has been established that 1,8-cineole plays an important role in the occurrence of “eucalyptus” character in Australian red wines [3]. The relationship between the presence of eucalyptus trees in the vicinity of vineyards and the presence of 1,8-cineole in corresponding wines has also been reported [3]. In parallel, other studies have suggested that the 1,8-cineole found in Australian Cabernet Sauvignon wines could also be directly derived from grapes [4]. 1,4-cineole, a compound with very similar structure and odour to 1,8-cineole, has been recently identified in red wines [5] but quantitative data and sensory characterisation have not been reported. Its origin in wine is still unknown. This study investigated the occurrence and potential aromatic contribution of 1,4-cineole to Australian red wines.

## Materials and methods

### Wines

A range of commercial Australian red wines (104 in total), comprising of 51 Cabernet Sauvignon (mean age 3.5 years), 4 Cabernet Sauvignon/Merlot blends (mean age 7 years), 27 Shiraz (mean age 3.5 years) and 22 Pinot Noir wines (mean age 2 years) were purchased. The wines originated from different Australian regions, including important regions for Cabernet Sauvignon wine production: Barossa, Coonawarra, McLaren Vale and the Margaret River. Twelve commercial French wines, predominantly Cabernet Sauvignon-Merlot blends (mean age 6.5 years) from the Bordeaux region, were also analysed.

### Cineole analysis

The method for quantifying 1,4-cineole and 1,8-cineole by HS-SPME-GC-MS was developed in combination of previously published method for wine volatiles [3, 6]. The method was validated with a limit of quantification for 1,4-cineole calculated at 4 ng/L using octan-2-ol as internal standard.

### Sensory analysis

Sensory analysis was carried out by orthonasal evaluation. Sixteen to thirty-three volunteers participated in the different sensory panels. The contribution of 1,4-cineole to Cabernet Sauvignon wine aroma was assessed quantitatively using a series of triangle tests, covering a concentration range from 0.13 to 0.54 µg/L in red wine, that were presented in a random order. The triangle test results were analysed statistically using tables that interpret the binomial law that corresponds to the distribution of answers in this type of test.

<footnote n="1">¹National Wine and Grape Industry Centre - Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia</footnote>

<footnote n="2">²School of Agricultural and Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia</footnote>

<footnote n="3">³Université de Bordeaux, EA 4577, ISVV, Unité de recherche Œnologie, Villenave d'Ornon, France</footnote>

<footnote n="4">⁴INRA, ISVV, USC 1366 Œnologie, Villenave d'Ornon, France</footnote>
The descriptive analysis focused on the impact of 1,4-cineole, both independently and in combination with 1,8-cineole, using an adapted version of the Deviation from Reference Method [7]. These tests were performed by comparing the aromatic perception of the hay and bay leaf reference standards to the same base wine that had been spiked with 1,4-cineole (1.6 µg/L) and/or 1,8-cineole (2.5 µg/L). For each test, the panel assessed the olfactory similarity of the samples compared to the standard presented. All the descriptors were mean-centered per panellist and scaled to unit variance. The data from multiple comparison tests were processed comparing scores with a Duncan post hoc test.

## Results and discussion

The 1,4-cineole identification in red wines consisted of the analyses of different Australian red wines and solutions of 1,4-cineole in water and wine by HS-SPME-GC-MS. Peaks that displayed the identical mass spectrum of 1,4-cineole were found in all the chromatograms at the same retention time that matched the 1,4-cineole retention index. A quantitative method for 1,4-cineole and 1,8-cineole in red wines was developed and validated. This method enabled a survey of these two compounds in 104 Australian red wines to be performed (Table 1). The quantification of 1,4-cineole in red wine has been reported for the first time.

1,4-cineole was detected in all the wines analysed, with concentrations ranging from 0.023 to 1.6 µg/L. An important varietal effect was observed, with an average concentration 8.4 and 2.7 fold higher in Cabernet Sauvignon wines than in Shiraz and Pinot Noir wines respectively. The distribution of 1,4-cineole in the Australian red wines was dissimilar to the corresponding 1,8-cineole measurements (data not shown). These findings indicate that 1,4-cineole and 1,8-cineole in wines might have different origins and suggests the occurrence of 1,4-cineole is probably not due to the proximity of the vineyard to eucalyptus trees.

The analysis of different vintages of a unique Australian and French wine label from different vintages showed that the concentration of 1,4-cineole increased with wine age (Figure 1). 1,4-cineole has been previously reported in an aged Riesling wine [8], suggesting that 1,4-cineole might be chemically synthesised during wine ageing.

<figure><img src="image_206.png" /><figcaption>Fig. 1 - Effect of wine age on 1,4-cineole in Australian and French red wines.</figcaption></figure>

The average concentration of 1,4-cineole was 2.4 fold lower in the French wines in comparison to Australian Cabernet Sauvignon wines (single variety wines and blends) despite the greater age of French wines (Table 1). Significant variations in 1,4-cineole concentrations were also observed between Cabernet Sauvignon wines from different Australian regions, despite similar vintages being analysed. The wines that originated from Margaret River (0.87 ± 0.33 µg/L) exhibited higher concentrations of 1,4-cineole than wines from Barossa (0.48 ± 0.27 µg/L; p < 0.05), McLaren Vale (0.57 ± 0.28 µg/L; p < 0.05) and to a lesser extent Coonawarra (0.64 ± 0.29 µg/L; p = 0.08). It is possible regional variations of 1,4-cineole measured in wines might reflect the climatic differences that exist between regions during the corresponding vintages. Therefore the chemical composition of grapes grown under Australian climatic conditions might favour 1,4-cineole synthesis in the corresponding wines compared to French conditions.
<table><caption>**Table 1** - Mean concentrations ± SD of 1,4-cineole and 1,8-cineole in Australian Cabernet Sauvignon, Shiraz, Pinot Noir and French Cabernet Sauvignon blend wines. One way ANOVA was used to compare data. Means followed by different letters in a row are significant at p ≤ 0.05 (Fischer's LSD).</caption><thead><tr><th rowspan="2">Compounds</th><th colspan="4">Wines</th></tr><tr><th>Cab. Sauv.<br>(n = 52)</th><th>Shiraz<br>(n = 27)</th><th>Pinot Noir<br>(n = 22)</th><th>French Cab. Sauv.<br>(n = 12)</th></tr></thead><tbody><tr><td>1,4-cineole</td><td>0.59 ± 0.33a</td><td>0.07 ± 0.04c</td><td>0.22 ± 0.2b</td><td>0.25 ± 0.15b</td></tr><tr><td>1,8-cineole</td><td>2.82 ± 3.26a</td><td>1.75± 1.42a</td><td>0.99 ± 0.33b</td><td>0.20 ± 0.1c</td></tr></tbody></table>

The calculation of climatic indices [9] for the Margaret River and Bordeaux regions (Table 2) enabled a more detailed investigation of the differences in 1,4-cineole concentrations. The temperature-related indices are very similar between Margaret River and Bordeaux. The annual rainfall levels are in the same range, but with drier summers and wetter winters in Margaret River compared to Bordeaux. Light radiation has also to be considered as Australia experiences 12 to 15% higher UV radiation than Europe [10]. The impact of UV-B radiation on grape and wine composition has been recently reported for Sauvignon Blanc variety [11]. Increases in terpinolene, a potential precursor of 1,4-cineole, have been reported after UV-B radiation in grape leaf tissues [12]. Higher UV radiation observed in Australia compared to France could potentially contribute to the differences of 1,4-cineole concentrations measured between Australian and French wines.

<table><caption>**Table 2** - Average climatic indices calculated for Margaret River (Australia) and Bordeaux (France) regions from 1995 to 2014 [9]. The values were calculated for one site per region based on SILO database [13] and Meteo France data for Margaret River and Bordeaux respectively.</caption><thead><tr><th></th><th>Margaret River</th><th>Bordeaux</th></tr></thead><tbody><tr><td>Huglin index</td><td>2154 ± 118</td><td>2144 ± 133</td></tr><tr><td>Cold night index (°C)</td><td>14.9 ± 0.7</td><td>15.0 ± 0.8</td></tr><tr><td>Rain winter (mm)</td><td>813 ± 119</td><td>493 ± 127</td></tr><tr><td>Rain summer (mm)</td><td>159 ± 56</td><td>361 ± 107</td></tr><tr><td>Number of days T≥35 °C</td><td>4 ± 3</td><td>3 ± 4</td></tr></tbody></table>

The sensory characterisation of 1,4-cineole was performed in a French Cabernet Sauvignon wine that was known to contain 10 ng/L of 3-isobutyl-2-methoxypyrazine. A similar method to that described above was used in the present study to quantitatively assess the concentration range above which 1,4-cineole was perceivable in the red wine. An addition of 0.54 µg/L of 1,4-cineole, to produce a final concentration of 0.63 µg/L, was required before it was detected by the panel. This level suggests that 1,4-cineole can probably influence the aromatic profile of Australian Cabernet Sauvignon wines.

The descriptive analysis focused on attributes such as “hay” and “bay leaf” often reported in Australian Cabernet Sauvignon [2]. The ANOVA and Duncan post-hoc test showed the addition of 1,4-cineole, both independently and in combination with 1,8-cineole, enhanced hay aromas in comparison to the control wine (p < 0.05) (Figure 2). The intensity of bay leaf notes were significantly enhanced by the association of 1,4-cineole and 1,8-cineole (p < 0.05) (Figure 2).

<figure><img src="image_207.png" /><figcaption>**Fig. 2** - Evaluation of the contribution of 1,4-cineole and 1,8-cineole to hay and bay leaf aromas of Cabernet Sauvignon wines using the deviation from reference method, and expressed in normalised score. CS: control wine; 1,4c: 1,4-cineole; 1,8c: 1,8-cineole.</figcaption></figure>
These findings highlight that 1,4-cineole may contribute to the hay and dried herb aromas that have been reported in Australian Cabernet Sauvignon wines.

## Conclusion

This work contributes to the knowledge of wine aromas describing for the first time the quantification and sensory influence of 1,4-cineole in red wine. The possibility of using 1,4-cineole as a varietal marker for Australian Cabernet Sauvignon wines was discussed. Further studies that investigate the origin and influence of 1,4-cineole in wines produced from other regions in the world would be valuable.

## Acknowledgements

The authors thank the Australia's grape growers and wine makers through their investment body the Australian Grape and Wine Authority (AGWA) and the Australian federal government for their financial support. We are also grateful to all the sensory panellists who contributed to this study.

## References

1. Robinson AL, Adams DO, Boss PK et al (2011) The relationship between sensory attributes and wine composition for Australian Cabernet Sauvignon wines. *Aust J Grape Wine Res* **17**: 327-340.

2. Halliday J (2011) *Australian Wine companion*. Hardie grant books.

3. 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.

4. Kalua CM, Boss PK (2010) Comparison of major volatile compounds from Riesling and Cabernet Sauvignon grapes (*Vitis vinifera* L.) from fruitset to harvest. *Aust J Grape Wine Res* **16**: 337-348.

5. Robinson AL, Boss PK, Heymann H et al (2011) Development of a sensitive non-targeted method for characterizing the wine volatile profile using headspace solid-phase microextraction comprehensive two-dimensional gas-chromatography time-of-flight mass spectrometry. *J Chromatogr A* **1218**: 504-517.

6. Antalick G, Šuklje K, Blackman JW et al (2015) Influence of grape composition on red wine ester profile: comparison between Cabernet Sauvignon and Shiraz cultivars from Australian warm climate. *J Agric Food Chem* **63**: 4664-4672.

7. Larson-Powers N, Pangborn RM (1978) Descriptive analysis of the sensory properties of beverages and gelatins containing sucrose or synthetic sweeteners. *J Food Sci* **43**: 47-51.

8. Simpson RF, Miller GC (1983) Aroma composition of aged Riesling wine. *Vitis* **22**: 51-63.

9. Tonietto J, Carbonneau A (2004) A multicriteria climatic classification system for grape-growing regions worldwide. *Agric Forest Meteorol* **124**: 81-97.

10. Gies P, Roy C, Javorniczky J et al (2004) Global solar UV index: Australian measurements, forecasts and comparison with the UK. *Photochem Photobiol* **79**: 32-39.

11. Šuklje K, Antalick G, Coetzee Z et al (2014) Effect of leaf removal and ultraviolet radiation on the composition and sensory perception of *Vitis vinifera* L. cv. Sauvignon Blanc wine. *Aust J Grape Wine Res* **20**: 223-233.

12. Gil M, Pontin M, Berli F et al (2012) Metabolism of terpenes in the response of grape (*Vitis vinifera* L.) leaf tissues to UV-B radiation. *Phytochemistry* **77**: 89-98.

13. Jeffrey SJ, Carter JO, Moodie KB et al (2001) Using spatial interpolation to construct a comprehensive archive of Australian climate data. *Environ Model Softw* **16**: 309-330.
# The direct contribution of several volatile sulfur compounds in the aromatic expression of the wine ageing bouquet

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 – DMS – volatile thiols

## Introduction

The ageing bouquet, which defines the overall quality of fine wines, counts among the most fascinating but least known phenomena in oenology. It denotes a set of aromas which, together, form an olfactive equilibrium, where individual perceptions do not clearly dominate, analogous to a complex perfume. As a result, a wine possessing an ageing bouquet is described as a fine wine. However, the identification of compounds bearing the chemical expression of this olfactory concept is still poorly documented. In a previous work, the ageing bouquet typicality of a set of high quality red wines from the Bordeaux area was assessed and seven aromatic descriptors (undergrowth, empyreumatic, liquorice, mint, truffle, spicy, and fresh red and black-berry fruit notes) were highlighted as its best representative attributes [1]. As a second step, the current study was designed to define the potential impact of some active odorous molecules in the aroma signature of the ageing bouquet of red Bordeaux wines. Our investigations were focused on compounds formed under reducing conditions during bottle ageing whose aromatic notes are related to the previously identified aroma descriptors and which may give a positive contribution to the polymorphism of the final ageing bouquet.

## Materials and methods

### Red wines

Twenty four commercial red wines were selected from nine different Bordeaux area appellations. All wines, coming from 19 vineyards, were barrel-aged and came from various vintages from 1994 to 2005.

### Sensory analysis

Sensory analyses were focused on a typicality assessment task and were performed in a similar way than described in a previous work [1]. A Hierarchical Cluster Analysis (HCA), using the Ward method (dissimilarity criteria), was applied to classify wines according to their typicality score.

### Chemical analysis

DMS, tabanone isomers and aromatic heterocycles (thiazoles, thiophens, alkylpyrazines and furans) were quantified in all studied red wines by SPME-GC/MS developed methods [2-4]. Six volatile polyfunctional thiols (3SH, 4MSP, E2SP, E3SP, BMT and 2FMT) were specifically extracted and analyzed by GC-MS according to the methodology described by Tominaga and Dubourdieu [5].

### Data analysis

For the multivariate (chemical and sensorial data) analysis, the statistical method employed was the Factorial Discriminant Analysis (FDA). The FDA was based on the classification from the wine tasting, which allowed to group wines according to typicality score of the ageing bouquet.

## Results and discussion

HCA led to the clusterization of the 24 tasted wines into three main groups according to perceived differences in assessment of their typicality.

<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>*Corresponding author: magali.picard@u-bordeaux.fr</footnote>
Cluster 1 consisted of ten wines with the highest average typicality scores (mean value ± SD: 6.2 ± 0.6) and considered the most representative models of the wine ageing bouquet concept. Cluster 2 consisted of five wines with the lowest average typicality scores (mean value ± SD: 3.8 ± 0.9). Finally, cluster 3 included seven wines with no clearly-defined correlation with the ageing bouquet concept. As their typicality assessment varied drastically among the panel of wine professionals, they were considered intermediate models for ageing bouquet typicality.

The circle of correlation issued from the FDA (Figure 1a) showed that the first principal component, which explained 87.3% of the total variance, was highly positively loaded with the typicality score and tended to distinguish wines with and without an ageing bouquet.

Besides, the loading plots showed a good correlation between the typicality score and DMS, 2FMT and 3SH concentrations. BMT and E2SP were positioned on the same positive side than the typicality score, but farther from the correlation circle than DMS and 2FMT, showing their minor tendency to contribute to the wine ageing bouquet.

Alkypyrazines and thiophenes were situated on the negative side of the first axis, meaning that they unlikely brought any impact on the typicality. At last, as regards to 4MSP, E3SP, thiazoles, tabanone and furans, they all were positioned on the second axis (12.7% of the total variance) which was not at all correlated with the typicality score, meaning that these compounds did not contribute actively to the aroma of the ageing bouquet of red Bordeaux wines.

Figure 1b represents the projection of the wines in the space defined by the two first principal components. Nine out of ten wines initially considered as good examples of the wine ageing bouquet (represented by circles) were loaded on the positive side of the first component whereas the 5 wines considered as poor examples (represented by squares) appeared on the opposite and negative side of the first component.

These results validated the wine typicality clusterization performed by HCA and pointed out a good agreement between the two statistical methods. Importantly, within the currently analyzed series of 24 red Bordeaux wines, they highlighted that those with an ageing bouquet seemed to present specific features concerning their chemical profile in DMS, 2FMT and 3SH.

<figure><img src="image_208.png" /><figcaption>**Fig. 1** - Factorial Discriminant Analysis (FDA) according to the typicality score and the concentrations of volatile compounds: (a) correlation circle between volatile compounds and typicality score, (b) projection of wines.</figcaption></figure>

Importantly, measured concentrations of DMS, 2FMT and 3SH in wine samples (Figure 2) showed that these compounds significantly discriminated wines according to their typicality representativeness since mean concentrations were found twice higher in wines with an ageing bouquet compared to atypical ones [6].
<figure><img src="image_209.png" /><figcaption>**Fig. 2** - Mean concentration (± confidence interval) of DMS, 2FMT and 3SH in red Bordeaux wines according to their ageing bouquet typicality representativeness. (*) corresponded to a significant difference (Mann-Whitney test U; p<0.05).</figcaption></figure>

**Conclusion**

Overall, the current results provided convincing arguments for a relevant impact of DMS, 2FMT and 3SH into the global aromatic balance of the wine ageing bouquet concept of Bordeaux red wines.

**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 in the sensory sessions.

**Abbreviations**

DMS, dimethyl sulphide; BMT, benzenemethanethiol; 2FMT, 2-furanmethanethiol; 3SH, 3-sulfanylhexanol; E2SP, ethyl 2-sulfanylpropionate; E3SP, ethyl 3-sulfanylpropionate; 4MSP, 4-methyl-4sulfanyl-2pentanone.

**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. Slaghenaufi D, Perello MC, Marchand-Marion S et al (2014) Quantitative solid phase microextraction – Gas chromatography mass spectrometry analysis of five megastigmatrienone isomers in aged wine. *Anal Chim Acta* **813**: 63-69.

4. Burin VM, Marchand S, de Revel G et al (2013) Development and validation of method for heterocyclic compounds in wine: optimization of HS-SPME conditions applying a response surface methodology. *Talanta* **117**: 87-93.

5. 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.

6. 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.
# Identification of new trimeric anthocyanins occurring in Merlot red wine

C. QUAGLIERI<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, P. BRETHENOUX<fnref n="1" />,<fnref n="2" />, M. JOURDES<fnref n="1" />,<fnref n="2" />, P. WAFFO-TEGUO<fnref n="3" />, J.M. MERILLON<fnref n="3" />, P.L. TEISSEDRE<fnref n="1" />,<fnref n="2" />*

Merlot – wine fractionation – trimeric anthocyanins – CPC system

## Introduction

During all the wine-making process, red wine undergoes physical and chemical reactions involving phenolic compounds. Tannins, which are flavan-3-ol oligomers or polymers, are responsible for astringency and bitterness sensations, whereas anthocyanins are pigments mainly involved in wine colour, especially in red wine colour. Anthocyanins are responsible for red/purple hue of young red wines.

During wine ageing, molecular anthocyanin concentration decreases. These molecules are involved in several chemical reactions:

- Direct condensation reaction between anthocyanins themselves (A-A adducts) or between anthocyanins and flavan-3-ols (A-T and T-A molecules) [1-3];

- Anthocyanin condensation, with other anthocyanins or flavan-3-ol units, by the way of an ethyl-bridge (A-ethyl-T or T-ethyl-A), due to the presence of ethanal in the wine [4];

- Cycloaddition between anthocyanins and some yeast cell double-bond compounds, which leads to the formation of very stable pigments: pyranoanthocyanins [5].

Anthocyanin polymerization via direct condensation mechanism is possible in red wines following the similar mechanism as anthocyanins and flavan-3-ol addition. Anthocyanin-anthocyanin dimers with A and B-type structures have been detected in red wine fractions [1-3]. A and B-type trimeric anthocyanins have also been identified in grape skin [6]. Only one A-type anthocyanin trimeric molecule, composed of three malvidin-3-*O*-glucoside (Mv-3G) moieties, has been identified and characterized in a Port red wine [7]. However, investigations did not yet revealed the occurrence of trimeric forms including other anthocyanin monomers than Mv-3G.

Our previous study, based on wine fractionation with XAD 16N and TSK HW 40S resins, has shown the occurrence of three trimeric anthocyanins in Merlot red wines. The first one was composed of three Mv3G moieties, previously characterized [7]. Two other trimeric anthocyanins were detected, composed of two monomers of Mv3G, the third unit being either petunidin-3-*O*-glucoside or delphinidin-3-*O*-glucoside, respectively. Furthermore, a first quantification in equivalent Mv3G was established to obtain a characteristic kinetic pattern according to variable oxygenation and acidity conditions applied to a Merlot red wine.

In order to further investigate our findings and to detect other trimeric pigments, a fractionation method is developed using centrifugal partition chromatography (CPC) as the key purification technique. CPC is a support-free liquid-liquid chromatography [8], thus providing a few advantages. On one hand, the fractionation is applied on a significant quantity of product, in a short period of time, and thus leads to high purification yield [9]. On the other hand, the selectivity of the solvent systems provides efficiency for separating molecules from each other.

<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">³Univ. Bordeaux, ISVV, GESVAB, EA 3675, F-33140 Villenave d'Ornon, France</footnote>
# Materials and methods

## Wines and extract preparations

The studied wine was a young Merlot red wine purchased from Rauzan cooperative winery (Bordeaux region, France) aged in stainless steel tanks for 12 months [pH 3.5, 12%vol.]. The Merlot wine was previously conditioned under pH 3 and oxygenated during 4 months with 20 mg/L of O₂. 20-25% of 1 L of this wine was evaporated and fractionated onto a 530 × 65 mm i.d. Amberlite® XAD 16N column. The sample was first washed with 6 L of milli-Q water acidified with 1% formic acid, to elute sugars and glycerol. Then the polyphenolic fraction was eluted with 3 L of acidic acetone (1% formic acid). This fraction of interest was evaporated, re-solubilized in acidic water and lyophilized to obtain the crude extract.

## Fractionation conditions

The CPC system used was a commercial model from Kromaton® (Angers, France) fitted with a stacked disks type rotor. The characteristics of the apparatus are: total volume of the column: 204 mL, dead volume: 32.3 mL, total number of partition disks: 20, total number of partition cells: 1320 cells with a volume of 0.13 mL, distance between each cell and the middle of the rotor: 105 mm, rotation speed: from 0 to 2000 rpm. A HPLC 321-H12 binary pump (Gilson) was used to drive the solvents into the column. The sample was injected by the way of a high-pressure valve (3725(i)038 Rhéodyne), equipped with a 20 mL injection loop.

The system used was ethyl acetate/1-butanol/water 0.1% trifluoroacetic acid (2:3:5) in the descending mode (lower phase mobile) [10]. The rotor was entirely filled with the stationary phase while rotating at 500 rpm. After injection of the 1-g crude extract dissolved in 9 mL stationary phase and 9 mL mobile phase, speed was increased to 1100 rpm, and the mobile phase was pumped into the column in descending mode at a flow-rate of 3 mL/min for 240 min. Fractions of 3 mL were collected in 12 mL test tubes every minute. The back pressure was 33 bars.

Each sample collected in the test tubes was concentrated 5 times, filtered on a 0.45 µm membrane and injected on a UHPLC-UV Q-TOF system. The UHPLC Agilent 1290 Infinity series consisted of a quaternary pump, a solvent degasser, an autosampler and a diode array detector (DAD). The analysis was carried out on a 2.1 × 50 mm i.d., 1.8 µm Eclipse Plus C18 column. The UHPLC system was coupled to an ESI-Q-TOF-MS Agilent 6530 Series Accurate quadrupole time-of-flight (Q-TOF) with an Agilent Jet Stream Technology ESI. The mass spectrometer was operating in extended dynamic range of 2 GHz with the mass range up to 1700 Th, with a drying gas flow and temperature respectively set at 9 L/min and 300°C. The capillary voltage was 4 kV. The software used for the determination of the exact molecular formula of the trimeric anthocyanins was MassHunter Qualitative Analysis. The solvents used for the gradient were A: H₂O/CH₃CN/HCOOH (919.5:79.5:1) and B: H₂O/CH₃CN/HCOOH (449.5:549.5:1). The gradient consisted of 6-25% B in 2 min, 25-50% B in 6 min, 50-60% B in 1.5 min, 60% B for 0.5 min, and 60-95% B in 0.5 min. The column was washed with 95% B for 1 min and re-equilibrated with the initial conditions for 1 min.

# Results and discussion

First of all, Test tube no. 10 was recovered, concentrated 5 times and analyzed with UHPLC/Q-TOF system. Five masses which could correspond to five trimeric anthocyanins were detected (Figure 1): 1477, 1463, 1449, 1433 and 1447. The differences between the extracted ion masses and those calculated from the formulas are respectively 0.47 ppm, 4.65 ppm, 2.69 ppm, 1.88 ppm and 2.90 ppm.

These results were a first element of confirmation of the formulas potentially corresponding to five trimeric anthocyanins: 1477.3876, composed of three Mv3G moieties; 1463.3720, composed of two Mv3G moieties and a petunidin-3-O-glucoside (Pt3G) moiety; 1449.3563, composed of two Mv3G moieties and a delphinidin-3-O-glucoside (Dp3G) moiety; 1433.3614, composed of two Mv3G moieties and a cyanidin-3-O-glucoside (Cy3G) moiety; and 1447.3770, composed of two Mv3G moieties and a paeonidin-3-O-glucoside (Pn3G) moiety.
<figure><img src="image_210.png" /><figcaption>**Fig. 1** - High-resolution mass analyses (LC/Q-TOF), test tube no. 10: Mv3G-Mv3G-Mv3G, mass 1477.3876 (1); Mv3G-Mv3G-Pt3G, mass 1463.3720 (2); Mv3G-Mv3G-Dp3G, mass 1449.3563 (3); Mv3G-Mv3G-Cy3G, mass 1433.3614 (4); Mv3G-Mv3G-Pn3G, mass 1447.3770 (5).</figcaption></figure>

The first three compounds were identified in our previous study, and the last two molecules were identified for the first time in Bordeaux red wine. However these pigments were not yet visible from the 520 nm chromatogram, for they might occur in very small quantities.

Second of all, Test tube no. 33 was recovered, concentrated 5 times and analyzed with UHPLC/Q-TOF system. Only one mass, which could correspond to a trimeric anthocyanin, was detected: 1623. The difference between the extracted ion mass and thus calculated from the formula is 0.86 ppm, which was a first element in favour of the formula corresponding to a trimeric Mv3G pigment with one *p*-coumaroylglucoside moiety (mass: 1623.4244) (Figure 2).

<figure><img src="image_211.png" /><figcaption>**Fig. 2** - High-resolution mass analysis (LC/Q-TOF), test tube no. 33: MvMvMv-2G·CG (CG = *p*-coumaroylglucoside), mass 1623.4244.</figcaption></figure>

**Conclusion**

Five masses corresponding to five oligomeric anthocyanins were identified in a Merlot red wine aged under oxidative conditions. More wine fractionation will confirm that CPC is an appropriate technique to separate monoglucoside anthocyanins from *p*-coumaroylglucoside anthocyanins. Further investigation about the CPC solvent system and UV characterization will provide pure fractions of these molecules to fully characterize their molecular structure by NMR.
Acknowledgements

We gratefully thank our colleagues from the Vine and Wine Scientific Institute (ISVV): USC 1366 Œnologie research unit for the polyphenolic chemical expertise and high-resolution mass strategy, and GESVAB EA 3675 for insight and expertise about the CPC fractionation.

References

1. Salas E, Dueñas M, Schwarz M et al (2005) Characterization of pigments from different high speed countercurrent chromatography wine fractions. *J Agric Food Chem* **53**: 4536-4546.

2. Alcalde-Eon C, Escribano-Bailón MT, Santos-Buelga C et al (2007) Identification of dimeric anthocyanins and new oligomeric pigments in red wine by means of HPLC-DAD-ESI/MS". *J Mass Spectrom* **42**: 735-748.

3. Pati S, Liberatore MT, Gambacorta G et al (2009) Rapid screening for anthocyanins and anthocyanin dimers in crude grape extracts by high performance liquid chromatography coupled with diode array detection and tandem mass spectrometry. *J Chromatogr A* **1216**: 3864-3868.

4. Fulcrand H, Doco T, Es Safi NE et al (1996) Study of the acetaldehyde induced polymerisation of flavan-3-ols by liquid chromatography-ion spray mass spectrometry. *J Chromatogr A* **752**: 85-91.

5. Fulcrand H, Cameira-dos-Santos PJ, Sarni-Manchado P et al (1996) Structure of new anthocyanin-derived wine pigments. *J Chem Soc Perkin Trans* **1**: 735-739.

6. Vidal S, Meudec E, Cheynier V et al (2004) Mass spectrometric evidence for the existence of oligomeric anthocyanins in grape skins. *J Agric Food Chem* **52**: 7144-7151.

7. Oliveira J, Alhinho da Silva M, Parola AJ et al (2013) Structural characterization of a A-type linked trimeric anthocyanin derived pigment occurring in a young Port wine. *Food Chem* **141**: 1987-1996.

8. Murayama W, Kobayashi T, Kosuge Y et al (1982) A new centrifugal counter-current chromatograph and its application. *J Chromatogr A* **239**: 643-649.

9. Vidal S, Hayasaka Y, Meudec E et al (2004) Fractionation of grape anthocyanin classes using multilayer coil countercurrent chromatography with step gradient elution. *J Agric Food Chem* **52**: 713-719.

10. Degenhardt A, Hofmann S, Knapp H et al (2000) Preparative isolation of anthocyanins by high-speed countercurrent chromatography and application on the color activity concept to red wine. *J Agric Food Chem* **48**: 5812-5818.
# Does stereochemistry influence the taste of wine?
## Purification, characterization and sensory evaluation of lyoniresinol isomers

B.N. CRETIN<sup>1,2*</sup>, Q. SALLEMBIEN<sup>1,2</sup>, L. SINDT<sup>1,2</sup>, N. DAUGEY<sup>3</sup>, T. BUFFETEAU<sup>3</sup>, P. WAFFO-TEGUO<sup>4</sup>,
D. DUBOURDIEU<sup>1,2</sup>, A. MARCHAL<sup>1,2</sup>

Lignan – bitterness – vibrational circular dichroism – natural products

## Introduction

Lyoniresinol is a lignan from *Quercus* oak wood [1-3], also found in oaked wines [4]. It has been recently described to participate to wine taste by exhibiting bitterness [5]. Another study showed that lyoniresinol was present in wines aged in new oak barrels at concentrations higher than its perception threshold (1.5 mg/L in white wine), establishing its contribution to the increase in bitterness observed during oak ageing [6].

As lyoniresinol is a chiral compound, it has two enantiomers. Furthermore, among the several plant species from which lyoniresinol has been isolated, it has been observed as a mixture of both 8*R*, 8*R', 7*S*- and 8*S*, 8*S*, 7*R*- enantiomers in diverse proportions [7]. However, in *Quercus* oak wood, its specific optical rotation measurement indicates that the two enantiomers are present as a racemic mixture [2]. Nevertheless, the individual gustatory properties of lyoniresinol enantiomers have never been assessed. Considering the strong bitterness developed by lyoniresinol in racemic mixture, we decided to isolate and characterize lyoniresinol enantiomers to study their gustative impact on wine taste. In this way, we opted for an original racemic resolution method based on the hydrolysis of two natural xylose-derivatives of lyoniresinol.

## Materials and methods

### *Lyoniresinol enantiomer isolation*

Two natural xylose-derivatives of lyoniresinol, lyoniside 2 ((+)-lyoniresinol 9'-O-β-xylopyranoside) and nudiposide 3 ((-)-lyoniresinol 9'-O-β-xylopyranoside), were isolated as previously described by Marchal *et al.* [6].

### *Mass spectrometry analysis*

For the racemic resolution of lyoniresinol enantiomers, lyoniside (57.4 mg) and nudiposide (58.6 mg) were separately solubilized in a 4 mol/L trifluoroacetic acid (TFA) solution and placed under reflux at 80°C. Experiments were performed in parallel under a nitrogen atmosphere for 14 hours. After reaction, the solutions were evaporated in *vacuo* to remove TFA traces, suspended in water and freeze-dried twice. In order to remove the by-products, the crude reaction mixtures were purified using preparative HPLC equipped with a C18 column and monitored by a UV detector at 280 nm. Chromatographic peaks were collected manually just downstream the UV detector. Samples obtained after successive injections were pooled, evaporated in *vacuo* to remove acetonitrile and freeze-dried twice. The hydrolysis reaction and the purification by preparative-HPLC were monitored by U-HPLC-HRMS (Thermo Fisher Scientific, Les Ulis, France) using a C18 column with water and acetonitrile as mobile phases. Also, chiral chromatography analyses were performed on a Chiralpak® IB-3 column (2.1 mm x 150 mm, 3μm particle size). Mass acquisitions were performed in negative Fourier transform mass spectrometry (FTMS) ionization mode.

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

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

<footnote n="3"><sup>3</sup>Institut des Sciences Moléculaires, UMR 5255-CNRS, Université Bordeaux, F-33405 Talence, France</footnote>

<footnote n="4"><sup>4</sup>Univ. de Bordeaux, ISVV, GESVAB, EA 3675, F-33882 Villenave d'Ornon, France</footnote>
### Structure characterization

Characterization of lyoniresinol enantiomers structure were obtained by 1D and 2D NMR experiments performed on a Bruker Avance 600 NMR spectrometer and all NMR spectra were acquired at 300 K in methanol-d4. Specific optical rotations of lyoniresinol stereoisomers were determined with a JASCO P-2000 polarimeter with a sodium emission wavelength (λ = 589 nm) in methanol at 293 K. Lyoniresinol enantiomers **1a** and **1b** absolute configuration were determined by means of Vibrational Circular Dichroism and DFT calculations. IR and VCD spectra were recorded with a ThermoNicolet Nexus 670 FTIR spectrometer equipped with a VCD optical bench. Lyoniresinol enantiomers spectra were measured in DMSO-d6 at a concentration of 50 mM.

### Sensory analysis

To determine lyoniresinol enantiomers sensory characteristics, a tasting session was organized in a specific room air-conditioned at 20°C and equipped with individual booths and normalized glasses. Lyoniresinol enantiomers were tasted at 2 mg/L in a 12 % vol. alc. hydro-alcoholic solution composed of pure and demineralized water and distilled ethanol, as well as in a white non-oaked wine (Bordeaux 2013). Five wine-tasting experts were asked to describe the gustatory perception of each compound using wine tasting vocabulary and to evaluate the bitterness intensity.

## Results and discussion

### Isolation of lyoniresinol enantiomers

The specific optical rotation of lyoniresinol was reported to be close to zero in *Quercus* oak wood [2], suggesting that lyoniresinol was present as a mixture of two enantiomers **1a** and **1b** (Figure 1). Therefore, all sensory studies concerning lyoniresinol until now have been carried out using a racemic mixture purified from oak wood and the properties of each enantiomer have never been individually assessed.

<figure><img src="image_212.png" /><figcaption>**Fig. 1** - Chemical structure of lyoniresinol enantiomers **1a** and **1b** (absolute configuration).</figcaption></figure>

Since two enantiomers cannot be separated in a symmetric environment, we decided in this study to resolve the lyoniresinol racemic mixture by separation and hydrolysis of two natural derivatized diastereroisomers. Indeed, previous studies have established the natural presence in oak wood of lyonide **2** and nudiposide **3**, two native xylopyranoside-derivatives of (+)- and (-)-lyoniresinol, respectively [6].

For this purpose, purified lyonide and nudiposide were heated in acidic conditions under inert atmosphere in order to proceed to the hydrolysis of the xylose moiety of lyoniresinol (Figure 2). The hydrolysis was monitored by LC-HRMS in order to ensure a complete reaction with a total disappearance of lyonide **2** and nudiposide **3** through xylopyranoside cleavage from the genin, leading to lyoniresinol enantiomers **1a** and **1b** and xylopyranoside by-products. The last step consisted of a preparative HPLC on each crude reaction mixture in order to purify **1a** and **1b** by removing all by-products.

An HPLC analysis with a chiral analytical column was performed on each purified enantiomer and on racemic lyoniresinol. (±)-lyoniresinol injection revealed two peaks at 5.88 min and 7.16 min, while the purified (+)- and (-)-lyoniresinol enantiomers each presented only one peak at 5.92 min and 7.17 min respectively, confirming the enantiomers purity and the efficiency of the racemic resolution (Figure 3).
<figure><img src="image_213.png" /><figcaption>Fig. 2 - Release of lyoniresinol enantiomers **1a** and **1b** by acidic hydrolysis of lyoniside **2** and nudiposide **3**, respectively.</figcaption></figure>

<figure><img src="image_214.png" /><figcaption>Fig. 3 - LC-HRMS chromatograms of (±)-lyoniresinol, (+)-lyoniresinol and (-)-lyoniresinol (from top to bottom) on a chiral column.</figcaption></figure>
*Structure characterization of lyoniresinol enantiomers*

The enantiomers relative configuration was confirmed by 1D and 2D NMR experiments. ¹H NMR spectra of (+)- and (-)-lyoniresinol enantiomers superimposed on that of (±)-lyoniresinol and the NMR data were identical to those reported in the literature for lyoniresinol [8-11]. ROEs correlations between protons H-2'(H-6')/H-8' and H-7'/H-8 suggest that H-7' and H-8' are trans-orientated and that H-7' and H-8 are cofacial, which establish lyoniresinol enantiomer relative configuration. Specific optical rotation of each enantiomer was measured to be +43 and -45. These values, similar with opposite signs, confirm the enantiomeric relationship of the purified compounds. Lyoniresinol enantiomers absolute configuration has never been determined by chiroptic spectroscopic method. Nevertheless, it has been previously proposed as (8R, 8'R, 7'S)-(+)-lyoniresinol but only by degradation reactions and comparison with known compounds [8, 12]. In this study, we used vibrational circular dichroism (VCD) which is a well-established method for determining the absolute configuration and conformation of chiral molecules in solution [13]. The method is based on the comparison between VCD spectra measured for both purified compounds and the spectrum calculated at the density functional theory (DFT) level for a specified absolute configuration [14]. Because enantiomers have a VCD band of opposite sign for each vibrational mode, the VCD spectrum provides a unique rich signature of the absolute configuration.

The two spectra were opposite with respect to the baseline, confirming that the two molecules were enantiomers (Figure 4). Theoretical calculation of the (8R, 8'R, 7'S)-lyoniresinol was performed and its VCD spectrum was predicted. This theoretical VCD spectrum reproduced fairly well the intensity and the sign of most bands observed in the experimental spectrum of (+)-lyoniresinol, confirming the absolute configuration as (8R, 8'R, 7'S)-(+)-lyoniresinol and (8S, 8'S, 7'R)-(-)-lyoniresinol.

<figure><img src="image_215.png" /><figcaption>**Fig. 4** - Comparison of experimental VCD spectra of (+)- and (-)-lyoniresinol recorded in DMSO-d6 solution (50 mM, 100 µm path length, bottom) with the predicted VCD spectrum of (8R, 8'R, 7'S) lyoniresinol isomer calculated using DFT at the B3PW91/6-311G** level (top).</figcaption></figure>

*Sensory characterization of lyoniresinol enantiomers*

To measure the gustatory impact of each lyoniresinol enantiomer on the bitter perception of their racemic mixture, enantiomers **1a** and **1b** were tasted at 2 mg/L in a 12 % vol. alc. hydro-alcoholic solution and compared to the control medium solution. The same concentrations were tasted in a non-oaked white wine. Five experts described for the first time that lyoniresinol **1a** exhibited a strong bitterness equivalent to the bitterness of (±)-lyoniresinol at 4 mg/L, while lyoniresinol **1b** exhibited no taste. Thus, only (+)-lyoniresinol **1a** is responsible of (±)-lyoniresinol bitterness.

Finally, an oaked Bordeaux wine was analyzed by LC-HRMS equipped with a chiral column to search for the presence of lyoniresinol enantiomers. Two peaks were observed on the XIC for an m/z ratio specific to lyoniresinol, one at 5.94 min and the second at 7.15 min corresponding to lyoniresinol enantiomers **1a** and **1b**, respectively. This analysis confirmed that both lyoniresinol enantiomers were present in this red oaked wine with a relative abundance similar to that observed in oak wood. In view of these results and the sensory characteristics of lyoniresinol enantiomers, it appears that only (+)-lyoniresinol **1a** is likely to influence the taste of wine by increasing its bitterness.
**Conclusion**

This study reports the purification and sensory characterization of lyoniresinol enantiomers which established that only (8R, 8'R, 7'S)-(+)-lyoniresinol impacted significantly the taste of wine by its bitter taste. The racemic resolution allowed for the first time the unambiguous determination of the absolute configuration of lyoniresinol enantiomers by means of VCD measurements associated with DFT calculations. These results demonstrated that the stereochemistry strongly influences the nature and the intensity of the sensory perception and subsequently the perception of consumers.

**Acknowledgements**

NMR experiments were performed at the Plateforme Métabolome-Fluxome, Centre de Génomique Fonctionnelle de Bordeaux, Bordeaux, France. Blandine Cretin is supported by a grant from the Fondation Jean Poupelain. The authors would like to acknowledge the Conseil Interprofessionnel des Vins de Bordeaux, France AgriMer, Seguin-Moreau cooperage and Rémy-Martin for funding this project.

**References**

1. Seikel MK, Hostettler FD, Niemann GJ (1971) Phenolics of *Quercus rubra* wood. *Phytochemistry* **10**: 2249-2251.

2. Nabeta K, Yonekubo J, Miyake M (1987) Phenolic compounds from the heartwood of European oak (*Quercus robur* L.) and brandy. *J Jpn Wood Res Soc* **33**: 408-415.

3. Vivas N, Pianet I, Bourgeois G et al (1998) Characterization of heartwood lignin fractions from *Quercus robur* L. and *Quercus petraea* (Matt) Liebl., the main oak species used for barrel making. *Am J Enol Vitic* **49**: 49-55.

4. Moutounet M, Rabier P, Puech JL et al (1989) Analysis by HPLC of extractable substances in oak wood. Application to a Chardonnay wine. *Sci Aliments* **9**: 35-51.

5. Marchal A (2010) Recherches sur les bases moléculaires de la saveur sucrée des vins secs : Approches analytique et sensorielle. *Thèse de doctorat*, Université Victor Segalen Bordeaux 2.

6. Marchal A, Cretin BN, Sindt L et al (2014) Contribution of oak lignans to wine taste: chemical identification, sensory characterization and quantification. *Tetrahedron* **71**: 3148-3156.

7. Rahman MDA, Katayama T, Suzuki T et al (2007) Stereochemistry and biosynthesis of (+)-lyoniresinol, a syringyl tetrahydronaphthalene lignan in *Lyonia ovalifolia* var. *elliptica*: I: isolation and stereochemistry of syringyl lignans and predicted precursors to (+)-lyoniresinol from wood. *J Wood Sci* **53**: 161-167.

8. Kato Y (1963) Structure of lyoniresinol (dimethoxyisolariciresinol). *Chem Pharm Bull* **11**: 823-827.

9. Dadá G, Corbani A, Manitto P et al (1989) Lignan glycosides from the heartwood of European oak *Quercus petraea*. *J Nat Prod* **52**: 1327-1330.

10. Zhang X-Y, Li BG, Zhou M et al (2006) Chemical constituents of *Phacellaria compressa* Benth. *J Integr Plant Biol* **48**: 236-240.

11. Imai K, Yamauchi K, Mitsunaga T (2013) Extractives of *Quercus crispula* sapwood infected by the pathogenic fungus *Raffaelea quercivora* II: isolation and identification of phenolic compounds from infected sapwood. *J Wood Sci* **59**: 517-521.

12. Kato Y (1966) The absolute configuration of lyoniresinol. *Chem Pharm Bull* **14**: 1438-1439.

13. Freedman TB, Cao X, Dukor RK et al (2003) Absolute configuration determination of chiral molecules in the solution state using vibrational circular dichroism. *Chirality* **15**: 743-758.

14. Stephens PJ, Devlin FJ (2000) Determination of the structure of chiral molecules using ab initio vibrational circular dichroism spectroscopy. *Chirality* **12**: 172-179.
# How does chirality of substituted esters affect red wine fruity aroma?

G. LYTRA<fnref n="1" />,<fnref n="2" />*, S. TEMPERE<fnref n="1" />,<fnref n="2" />, M. CAMELEYRE<fnref n="1" />,<fnref n="2" />, G. DE REVEL<fnref n="1" />,<fnref n="2" />, J.-C. BARBE<fnref n="1" />,<fnref n="2" />

Red wine – substituted esters – enantiomers – sensory impact

## Introduction

Among red wine ethyl esters, those derived from short hydroxylated and/or branched-chain aliphatic acids constitute a family with a particular behavior and sensory importance. They have been previously discussed in the literature [1] and recent studies have established that some of them are strongly involved in the fruity aromas of red wine [2].

Some of these compounds have an asymmetrical carbon atom, indicating the possible presence of two different enantiomers which may have different olfactive properties. Therefore, it is important to separate these optical isomers to obtain an accurate assessment of their organoleptic impact. In this work, three chiral esters, with alkyl and/or hydroxyl substituents, have been studied: ethyl 2-hydroxy-4-methylpentanoate (2Me4OHC5C2), ethyl 2-methylbutanoate (2MeC4C2), and ethyl 3-hydroxybutanoate (3OHC4C2).

## Materials and methods

### *Samples*

2Me4OHC5C2, 2MeC4C2, and 3OHC4C2 were assayed in 55, 37, and 87 red wines, respectively, from several vintages and origins.

### *Enantiomer quantification in wine samples*

The optical isomers of the 3 chiral esters were separated by chiral gas chromatography–mass spectrometry on β- and γ-cyclodextrin phases. Chromatographic conditions and sample preparation were as described by Lytra et al. [3, 4].

### *Aromatic reconstitution using wine fractions (FAR<sub>1</sub>)*

A 500 mL wine sample (Vin de Pays d'Oc) was prepared as described by Lytra et al. [3] to obtain 1.25 mL wine extract. Reverse-phase (RP) HPLC was performed on this raw extract using a Nova-Pak C18 column (300 × 3.9 mm i.d., 4 µm, 60 Å, Waters, Saint-Quentin, France), without a guard cartridge. Chromatographic conditions were those optimized by Pineau et al. [1]. The 25 fractions in dilute alcohol solution were then directly evaluated by three trained assessors. For aromatic reconstitutions, fractions were retained and added individually or blended together to reproduce the initial concentrations in the original wines, adding double-distilled ethanol and microfiltered water to obtain an ethanol content of 12% (v/v).

### *Aromatic reconstitution using esters (FAR<sub>2</sub>)*

For aromatic reconstitutions, the various esters were added, individually or blended together at the average concentrations found in red wines (Table 1), to double-distilled ethanol diluted with microfiltered water to obtain an ethanol content of 12% (v/v) (pH adjusted to 3.5 with tartaric acid). The mixture containing all 13 esters constituted the fruity aromatic reconstitution (FAR<sub>2</sub>).

### *Sensory analyses*

Discriminative testing methods. The “olfactory thresholds” of specific mixtures were determined using a three alternative, forced-choice presentation (3-AFC) (NF ISO 13301: 2002). The “olfactory thresholds” of

<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>*Corresponding author: geolytra@yahoo.gr</footnote>
FAR were thus measured in different matrices: dilute alcohol solution and dilute alcohol solution containing a compound of interest. The olfactory thresholds of the following mixtures were measured:

* FAR₁ in dilute alcohol alone, or supplemented with 420 µg/L of a mixture of R- and S-2Me4OHC₅C₂ (95/5, m/m).

* FAR₂ excluding 2MeC₄C₂ in dilute alcohol solution alone or containing 50 µg/L S-2MeC₄C₂.

* FAR₂ excluding 3OHC₄C₂ in dilute alcohol solution alone or 350 µg/L of a mixture of S- and R-3OHC₄C₂ (85/15, m/m). In addition, interaction effect for the mixture of S- and R-3OHC₄C₂ (85/15, m/m) was evaluated using Feller's additive model [5]. The "olfactory threshold" of FAR was thus measured in dilute alcohol solution containing 350 µg/L of a mixture of S- and R-3OHC₄C₂ (85/15, m/m) at a fixed concentration only in the "positive sample" (the latter applied the value calculated for Feller's additive model [5]).

The results of all 3-AFC tests were statistically analyzed (NF ISO 13301: 2002).

Descriptive testing methods. Sensory profiles of aromatic reconstitutions were evaluated for red-berry, black-berry, fresh-, and jammy-fruit aroma intensity. Each sample was presented twice in each session. The subject rated the intensity of the descriptors in each sample on a 100-mm scale. Statistical data were analyzed using the Mann-Whitney statistical non-parametric test (XLSTAT software). All descriptors were mean-centered per panelist and scaled to unit variance. The statistically significant level was 5% (p<0.05).

<table><caption>**Table 1** - Composition of the fruity aromatic reconstitutions presented in the olfactory tests</caption><thead><tr><td>Compound</td><td>C (µg/L)</td></tr></thead><tbody><tr><td>ethyl propanoate</td><td>150</td></tr><tr><td>ethyl butanoate</td><td>200</td></tr><tr><td>ethyl hexanoate</td><td>200</td></tr><tr><td>ethyl octanoate</td><td>200</td></tr><tr><td>ethyl 2-methylpropanoate</td><td>250</td></tr><tr><td>ethyl 3-methylbutanoate</td><td>50</td></tr><tr><td>ethyl (2S)-2-methylbutanoate (2MeC<sub>4</sub>C<sub>2</sub>)</td><td>50</td></tr><tr><td>ethyl (2R)-2-hydroxy-4-methylpentanoate and ethyl (2S)-2-hydroxy-4-methylpentanoate (95/5, m/m) (2Me4OHC<sub>5</sub>C<sub>2</sub>)</td><td>400</td></tr><tr><td>ethyl 3-hydroxybutanoate (3OHC<sub>4</sub>C<sub>2</sub>)</td><td>300</td></tr><tr><td>butyl acetate</td><td>10</td></tr><tr><td>hexyl acetate</td><td>2</td></tr><tr><td>methylpropyl acetate</td><td>50</td></tr><tr><td>methylbutyl acetate</td><td>250</td></tr></tbody></table>

# Results and discussion

## Enantiomeric distribution and concentrations

Young red wines (after the end of alcoholic fermentation) contained only the R-2Me4OHC₅C₂ form. Aging was demonstrated to impact the R/S-2Me4OHC₅C₂ ratio with an average of approximately 95/5 (m/m). The maximum prevalence of the R-2Me4OHC₅C₂ form (R-form percentage compared to their combined concentration) was found in all wines just after the end of alcoholic fermentation with an enantiomeric ratio of 100/0 (m/m). The highest S-2Me4OHC₅C₂ levels were found in the oldest samples (maximum concentration in red wines = 62 µg/L, Haut-Médoc, 27 year-old) and the S-2Me4OHC₅C₂ form reached a maximum percentage compared to their combined concentration in a Margaux wine (19 year-old) with an R/S enantiomeric ratio of 85/15 (m/m).
$2\text{MeC}_4\text{C}_2$ concentrations were generally higher in the oldest wines than in the younger ones, with a maximum of 157 µg/L (Haut Médoc, 21 year-old), a minimum of 3 µg/L (Pessac-Léognan, after the end of alcoholic fermentation), and an average of about 50 µg/L. In light of these findings, it seems likely that only a few micrograms per liter of $2\text{MeC}_4\text{C}_2$ were formed during alcoholic fermentation. Wine aging resulted in a gradual increase in $2\text{MeC}_4\text{C}_2$ concentrations over time, stabilizing after about 5 years. With regard to the enantiomeric distribution of $2\text{MeC}_4\text{C}_2$, the S-enantiomer was found almost exclusively in all wines studied. Generally, young red wines (after the end of alcoholic fermentation) contained only the S-$2\text{MeC}_4\text{C}_2$ form, whereas very small amounts of the R-$2\text{MeC}_4\text{C}_2$ form were detected in aged red wines.

Concerning $3\text{OHC}_4\text{C}_2$, the average S/R enantiomeric ratio of this compound in all 72 red wines studied was approximately 75/25 (±13), with an average total concentration of 450 (±150) µg/L. During aging, R-$3\text{OHC}_4\text{C}_2$ levels increased gradually over time, but no differences was detected in S-$3\text{OHC}_4\text{C}_2$. The maximum S- and R-$3\text{OHC}_4\text{C}_2$ concentrations were 625 µg/L (Pessac-Léognan, after the end of alcoholic fermentation, S/R = 96/4, m/m) and 325 µg/L (Corton, 12 year-old, S/R = 50/50, m/m), respectively. The maximum percentage (compared to their combined concentration) was 100% for the S-form (Côtes-de-Bordeaux, after the end of alcoholic fermentation in two out of 18 wines, with concentrations of 130 and 37 µg/L S-$3\text{OHC}_4\text{C}_2$) and 50% for the R-form (Corton, 12 year-old, 325 µg/L R-$3\text{OHC}_4\text{C}_2$).

### Organoleptic impact of substituted esters on quantitative odor perception

The “olfactory threshold” of FAR₁ was 2.5 times higher than that of dilute alcohol solution supplemented with 420 µg/L of a mixture of R- and S-$2\text{Me4OHC}_5\text{C}_2$ (95:5, m/m) (p<0.001). The “olfactory threshold” of FAR₂ (excluding $2\text{MeC}_4\text{C}_2$) was 2.7 times higher in dilute alcohol solution alone than when it was supplemented with 50 µg/L S-$2\text{MeC}_4\text{C}_2$ (p<0.001). The “olfactory threshold” of FAR₂ (excluding $3\text{OHC}_4\text{C}_2$) was 1.5 times lower when the mixture of S- and R-$3\text{OHC}_4\text{C}_2$ (85/15, m/m) was added (p<0.05), than that of dilute alcohol solution alone. These results clearly demonstrated that esters $2\text{Me4OHC}_5\text{C}_2$, $2\text{MeC}_4\text{C}_2$, and $3\text{OHC}_4\text{C}_2$ had a synergistic effect on the perception of fruity aromas in wine. The experimental detection probability FAR₂ (excluding $3\text{OHC}_4\text{C}_2$) was higher than the value calculated using Feller’s additive model (Figure 1), revealing a hyper-addition effect for this ester, present at subthreshold concentrations.

FAR₂ (excluding $3\text{OHC}_4\text{C}_2$) + 350 µg/L of the mixture of S- and R-$3\text{OHC}_4\text{C}_2$ (85/15, m/m) in the "positive" sample

<figure><img src="image_216.png" /><figcaption>**Fig. 1** - Detection probability of FAR₂ (excluding $3\text{OHC}_4\text{C}_2$) supplemented with 350 µg/L of a mixture of S- and R-$3\text{OHC}_4\text{C}_2$ (85/15, m/m) in the “positive” sample, determined experimentally and calculated according to Feller’s additive model. *, expressed in mL FAR₂ diluted in 50 mL matrix; OTFeller, olfactory threshold estimated using Feller’s additive model; and OTExp, experimental olfactory threshold. The curves are drawn according to a sigmoid function.</figcaption></figure>
*Organoleptic impact of substituted esters on qualitative odor perception*

Sensory profile evaluations of FAR<sub>1</sub> demonstrated that the average scores for red-berry fruit intensity were identical after the addition of 2Me4OHC<sub>5</sub>C<sub>2</sub>, whereas jammy-fruit intensity was significantly lower. The average scores for black-berry and fresh-fruit aromas were significantly higher when FAR<sub>1</sub> was supplemented with 2Me4OHC<sub>5</sub>C<sub>2</sub>. These results confirmed the sensory importance of 2Me4OHC<sub>5</sub>C<sub>2</sub>, suggesting that it contributed actively to the black-berry and fresh-fruit nuances. Later evaluations revealed significant differences in black-berry-fruit intensity between FAR<sub>2</sub> alone (without 2MeC<sub>4</sub>C<sub>2</sub>) and supplemented with 50 µg/L S-2MeC<sub>4</sub>C<sub>2</sub>, whereas the average scores for red-berry-, fresh-, and jammy-fruit aroma descriptors were unchanged by the addition of this ester. These results confirmed the sensory impact of 50 µg/L S-2MeC<sub>4</sub>C<sub>2</sub>, suggesting that it contributed actively to the black-berry-fruit nuance. Finally, adding the S- and R-3OHC<sub>4</sub>C<sub>2</sub> mixture (85/15, m/m) to FAR<sub>2</sub> (without 3OHC<sub>4</sub>C<sub>2</sub>) had a significant intensifying effect on red-berry and fresh-fruit aromas.

The sensory impact of these chiral compounds has been established, revealing their role as natural enhancers of black-berry, red-berry and fresh-fruit aromas. Our data corroborate and expand the findings of Pineau et al. [1] and Lytra et al. [2-4], highlighting the impact of substituted esters on the fruity expression of red wines.

These results suggest that these compounds are initially produced during alcoholic fermentation and increase during aging due to a chemical reaction involving the corresponding direct precursors, also produced during alcoholic and malolactic fermentation. This hypothesis requires further investigation to elucidate the various pathways involved.

References

1. Pineau B, Barbe JC, van Leeuwen C et al (2009) Examples of perceptive interactions involved in specific "red-and blackberry" aromas in red wines. *J Agric Food Chem* **57**: 3702-3708.

2. Lytra G, Tempere S, Le Floch A et al (2013) Study of sensory interactions among red wine fruity esters in a model solution. *J Agric Food Chem* **61**: 8504-8513.

3. Lytra G, Tempere S, de Revel G et al (2012) Distribution and organoleptic impact of ethyl 2-hydroxy-4-methylpentanoate enantiomers in wine. *J Agric Food Chem* **60**: 1503-1509.

4. Lytra G, Tempere S, de Revel G et al (2014) Distribution and organoleptic impact of ethyl 2-methylbutanoate enantiomers in wine. *J Agric Food Chem* **62**: 5005-5010.

5. Feller W (1968) *An introduction to probability theory and its applications*. Wiley Series in Probability and Mathematical Statistics.
# Importance de la forme énantiomère ou diastéréoisomère dans les phénomènes d'interactions perceptives participant à des nuances aromatiques typiques de vins liquoreux de pourriture noble

P. STAMATOPOULOS<fnref n="1" />,<fnref n="2" />*, E. FRÉROT<fnref n="3" />, E. BROHAN<fnref n="4" />, C. PROVOST<fnref n="4" />, T. SIEBERT<fnref n="5" />,
M. HERDERICH<fnref n="5" />, P. DARRIET<fnref n="1" />,<fnref n="2" />

Enantiomères – diastéréoisomères – perception – arôme

## Introduction

Des résultats récents concernant l'arôme des vins liquoreux de pourriture noble, ont montré l'importance d'un phénomène bien connu en parfumerie, l'accord aromatique, pour interpréter la perception de nuances d'orange confite dans ces vins [1]. Ainsi, des composés associés à la fois au bois de chêne (3-méthyl-4-octanolide, eugénol) et à la botrytisation (2-nonen-4-olide, γ-nonalactone) permettent de contribuer ensemble à une nuance aromatique spécifique des grands vins liquoreux de pourriture noble au travers des phénomènes d'interactions perceptives alors même que ces composés présentent tous une odeur distincte de celle qui est perçue dans le mélange. Cette démonstration, phénomène de perception synthétique, a pu être établie à partir d'expériences de reconstitution sensorielle et des tests d'omission, avec des fractions d'extraits de vins supplémentées en les composés volatils préalablement mentionnés et aussi en solution simple [2]. Par ailleurs, si nos résultats ont montré l'impact majeur de composés tels que le 2-nonen-4-olide et le 3-méthyl-4-octanolide, d'autres composés peuvent aussi contribuer à renforcer l'expression aromatique d'orange confite tel que le 3-sulfanylhexanol, rappelant l'odeur du pamplemousse [3]. Cette observation conforte les études de Sarrazin [4, 5] qui avait établi le rôle de ce thiol dans la valorisation de l'arôme des vins liquoreux de pourriture noble. Afin d'approfondir les connaissances concernant l'implication des composés préalablement mentionnés sur la perception sensorielle de l'accord aromatique, l'axe des recherches a été mis sur l'étude des formes énantiomères du 2-nonen-4-olide et de la γ-nonalactone d'une part, et des diastéréoisomères du 3-méthyl-4-octanolide d'autre part.

## Matériel et méthodes

### Expériences des reconstitutions

Des différentes expériences de reconstitution ont été effectuées dans des solutions hydroalcooliques (12,5% vol., pH : 3,5). Les concentrations utilisées correspondent, à celles trouvés dans le vin. L'intensité de l'arôme orange confite a été évaluée sur une échelle non structurée de 0 à 10 cm. Afin d'évaluer statistiquement les modifications sensorielles en mélange, des analyses de la variance à un facteur sur les données ont été réalisées. Les conditions d'application de l'ANOVA ont été vérifiées sous le logiciel R : homogénéité des variances (test de Levene) et normalité de résidus (test de Shapiro-Wilk).

### Extraction et séparation enantiosélective des composés

Un volume de 50 mL de vin est percolé sur une cartouche pré remplie « Bond Elut-ENV » (Varian, les Ulis, France ; 200 mg ; 3 mL) à un débit de 2 mL/min. Puis la cartouche est rincée avec 20 mL d'une solution méthanol/eau 40:60 (V/V) contenant 1% NaHCO₃ (V/V ou W/V). La cartouche est séchée pendant

<footnote n="1">¹Univ. de Bordeaux, ISVV, EA4577 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">³Firmenich SA, R&D Division, 1 Route Des Jeunes, CH-1211 Genève 8, Switzerland</footnote>

<footnote n="4">⁴Sanofi-Aventis, 13 quai Jules Guesde, 94400 Vitry, France</footnote>

<footnote n="5">⁵The Australian Wine Research Institute, PO Box 197, Glen Osmond (Adelaide) SA 5064, Australia</footnote>

<footnote>*Auteur principal : panagiotis.stamatopoulos@u-bordeaux.fr</footnote>
30 minutes par circulation d'air. L'extrait récupéré est concentrée à 50 µL et est ensuite analysé par GC-GC/MS avec une précolonne polaire BP20 (50 m, 0,22 mm, 0,25 µm) et d'une seconde colonne chirale (Mega Dex Beta 25 m, 0,25 mm, 0,20 µm).

# Résultats et discussion

Les derniers résultats obtenus concernant l'implication des formes énantiomères a soulevé des questions d'un grand intérêt. Nous avons débuté les expérimentations en nous focalisant sur la distribution enantiomérique des composés associées à l'arôme d'orange confite dans les vins liquoreux de pourriture noble qui sont des lactones qui proviennent soit de l'élevage du vin en barrique soit de l'action de *Botrytis cinerea* sur les raisins. Ensuite, avec des méthodes chromatographiques adaptées (CPG-chirale), l'étude de la distribution enantiomérique des différentes formes a été effectuée.

<figure><img src="image_217.png" /><figcaption>**Fig. 1 -** Distribution énantiomérique du 2-nonen-4-olide dans des vins de millésimes variés (1966 à 2009).</figcaption></figure>

<figure><img src="image_218.png" /><figcaption>**Fig. 2 -** Distribution énantiomérique de la γ-nonalactone dans des vins de millésimes variés (1966 à 2009).</figcaption></figure>

Les premiers résultats de l'étude des lactones du raisin comme le 2-nonen-4-olide et de la γ-nonalactone ont montré des différences à l'évolution des formes enantiomériques selon le vieillissement du vin. Concernant la lactone 2-nonen-4-olide, nous constatons une plus grande abondance de la forme (R) en
corrélation avec le vieillissement du vin alors que la forme (S) est plus présente dans les vins jeunes (Figure 1). A l'inverse la γ-nonalactone présente toujours une dominance de la forme (R) sur n'importe quel millésime (Figure 2). Des études ont monté que l'abondance relative de chaque énantiomère dans la nature varie considérablement, mais il y a une tendance générale pour une dominance de la configuration (R) [6, 7]. Dans ces études, les formes énantiomères (R) semblent être plus odorantes, que les formes énantiomères (S) correspondantes. Ainsi pour un même contenu en lactones, selon la forme énantiomère, l'impact sensoriel sera différent. L'étude sur la distribution diastéréoisomérique du 3-méthyl-4-octanolide (Whisky Lactone) a montré une domination de la forme cis (Figure 3) ce qui est liée exclusivement par la gestion des barriques et le choix du bois au cours de l'élevage des vins étudiés. Le rapport entre les isomères cis et trans est caractéristique de l'origine du bois de chêne [8]. Pour le chêne américain, ce rapport varie de 5 à 8, avec une moyenne de 6,0 ± 1,3 (pour l'isomère cis). Pour le chêne européen, le ratio varie entre 1 à 1,5, avec une moyenne de 1,3 ± 0,2 (pour l'isomère cis). En analysant le rapport des isomères, il est possible de déterminer la source de bois utilisée pour l'élevage des vins. Par des travaux complémentaires il a été montré que le chêne américain présente environ 10% d'isomère trans alors que le chêne européen présent des quantités presque égales entre les isomères trans et cis [9].

<figure><img src="image_219.png" /><figcaption>Fig. 3 - Distribution énantiomérique du 3-méthyl-4-octanolide dans des vins de millésimes variés (1966 à 2009).</figcaption></figure>

Après avoir étudié la distribution énantiomérique des composés associés à l'arôme d'orange confite dans les vins liquoreux de pourriture noble, nous nous intéressons à étudier le comportement du mélange des quatre composés qui ont préalablement été associés à l'arôme d'orange confite, dans une solution simple telle que le solution hydroalcoolique (12% vol., pH 3,5). Des expériences de reconstitution avec les différents formes isomériques et diastéréoisomériques ont été effectuées en n'utilisant que les composés de référence (3-méthyl-4-octanolide, eugénol, 2-nonen-4-olide, γ-nonalactone). Les concentrations ajoutées dans les mélanges sont celles qui étaient dosées dans un vin typé. Ainsi, différents types des mélanges à partir de composés ont été préparés. Dans un premier temps, les analyses sensorielles avec reconstitution, impliquant les composés associés à l'arôme d'orange confite, et intégrant les formes énantiomères du 2-nonen-4-olide, ont montré que l'arôme est mieux perçu lors de l'utilisation de la forme (R) dans le mélange (Figure 4). Ensuite, l'étude de l'impact sensorielle concernant les formes énantiomères de la γ-nonalactone, a montré qu'il n'y a pas des différences significatives entre les échantillons (Figure 5), ainsi l'effet sensoriel des formes énantiomères de la γ-nonalactone reste limité. Enfin les analyses sensorielles sur l'effet des formes diastéréoisomères du 3-méthyl-4-octanolide, ont montré un effet significatif pour le cas du cis 3-méthyl-4-octanolide avec de moyennes plus élevées par rapport à la perception de l'arôme orangé (Figure 6). En conclusion, grâce à la caractérisation des déterminants moléculaires associés à l'arôme d'orange confite, le rôle privilégié de deux lactones, l'une provenant du bois, l'autre associé à la pourriture noble ont été soulignés. Aux concentrations considérées, la γ-nonalactone semble présenter une moindre contribution ainsi que l'eugénol.
Perception orangée (cas du 2-nonen-4-olide)

<figure><img src="image_220.png" /><figcaption>**Fig. 4** - Intensité moyenne de l'arôme orangé à partir de reconstitutions aromatiques des composés purs (cas du 2-nonen-4-olide).</figcaption></figure>

Perception orangée (cas de la γ-nonalactone)

<figure><img src="image_221.png" /><figcaption>**Fig. 5** - Intensité moyenne de l'arôme orangé à partir de reconstitutions aromatiques des composés purs (cas de la γ-nonalactone).</figcaption></figure>

Perception orangée (cas du 3-méthyl-4-octanolide)

<figure><img src="image_222.png" /><figcaption>**Fig. 6** - Intensité moyenne de l'arôme orangé à partir de reconstitutions aromatiques des composés purs (cas du 3-méthyl-4-octanolide).</figcaption></figure>
Conclusion

Après l'analyse des proportions relatives de formes énantiomères ou diastéréoisomères, par chromatographie en phase gazeuse multidimensionnelle impliquant une chromatographie énantiosélective, ou chromatographie en phase gazeuse conventionnelle, respectivement, les expériences de reconstitution ont été menées avec les composés considérés, impliquant l'une ou l'autre forme énantiomère ou diastéréoisomère. Les premiers résultats, concernant la lactone 2-nonen-4-olide, montrent une dominance de la forme (R) en corrélation avec le vieillissement du vin alors que la forme (S) est plus présente dans les vins jeunes. Les analyses sensorielles impliquant les expériences de reconstitution des composés associés à l'arôme d'orange confite, contribuent à démontrer l'importance des formes isomères dans la construction de l'image sensorielle. Plus particulièrement, les expériences de reconstitution impliquant la forme (R) du 2-nonen-4-olide et la forme cis du 3-méthyl-4-octanolide, permettent de souligner leur impact majeur en comparaison avec les formes énantiomères de la γ-nonalactone.

Remerciements

Nous tenons à remercier le Conseil Régional d'Aquitaine, le CIVB, France Agrimer et le Château d'Yquem
pour leur soutien financier.

Références

1. Stamatopoulos P, Frérot E, Tempère S et al (2014) Identification of a new lactone contributing to overripe orange aroma in Bordeaux dessert wines via perceptual interaction phenomena. *J Agric Food Chem* **62**: 2469-2478.

2. Stamatopoulos P (2013) Caractérisation des composés impliqués par des phénomènes d'interactions perceptives dans les nuances fruitées de l'arôme des vins liquoreux. *Thèse de Doctorat*, Université de Bordeaux 2.

3. Stamatopoulos P, Tempère S, Darriet P (2014) Perceptive interactions on Bordeaux dessert wines fruity aroma. *Proceedings of the XIV Weurman flavor research Symposium*, Andrew J Taylor & Donald S Mottram (Eds).

4. Sarrazin E (2007) Recherches sur l'arôme des vins liquoreux de pourriture noble issus des cépages Sémillon et Sauvignon blanc. *Thèse de Doctorat*, Université Victor Segalen Bordeaux 2.

5. Sarrazin E, Shinkaruk S, Tominaga T et al (2007) Odorous impact of volatile thiols on the aroma of young botrytized sweet wines: identification and quantification of new sulfanyl alcohols. *J Agric Food Chem* **55**: 1437-1444.

6. Mosandl A, Günther C (1989) Stereoisomeric flavor compounds. Structure and properties of γ-lactone enantiomers. *J Agric Food Chem* **37**: 413-418.

7. Cooke RC, van Leeuwen KA, Capone DL et al (2009) Odor detection thresholds and enantiomeric distributions of several 4-alkyl substituted γ-lactones in Australian red wine. *J Agric Food Chem* **57**: 2462-2467.

8. Waterhouse AL, Towey JP (1994) Oak lactone isomer ratio distinguishes between wines fermented in American and French oak barrels. *J Agric Food Chem* **42**: 1971-1974.

9. Pérez-Coello MS, Sanz J, Cabezudo MD (1999) Determination of volatile compounds in hydroalcoholic extracts of French and American oak wood. *Am J Enol Vitic* **50**: 162-165.
# Impact of grape processing and yeast strain selection on glycosylated aroma compounds and flavour of sparkling wine

D. SCHOBER*, M. WACKER, S. KLINK, H.-G. SCHMARR, U. FISCHER

Department of Viticulture and Enology, DLR Rheinpfalz, Breitenweg 71,
67435 Neustadt an der Weinstraße, Germany

Sparkling wine – glycosylated aroma compounds – yeast – sensory perception

## Introduction

The flavour of sparkling wine, which emerges during the vinification process, including grape processing, first and secondary fermentation, is significantly influenced by glycosylated aroma precursors [1]. Precursor content is basically determined by the grape variety but can also be considerably influenced by processing parameters, e.g. maceration time and pressing, as these substances are primarily located in grape skins [2]. The odourless aroma precursors are liberated by acid hydrolysis or by enzymatic hydrolysis depending on microorganisms used for fermentation [3]. Microorganisms are highly diverse in their enzymatic function and therefore in their ability to hydrolyse glycosidic linkage which includes glucose and, in the majority of cases, an additional sugar which can be rhamnose, arabinose, or apiose. β-glucosidase activity in *Saccharomyces* strains is controversially discussed as liberation of aroma-glycosides has been described on the one hand, but enzymatic function and extent of enzymatic activity remain unclear on the other [4]. In contrast, non-*Saccharomyces* yeasts and especially mould species like *Aspergillus niger* are known to possess β-D-glucosidase enzyme and also α-L-rhamnopyranosylglucopyranosidase, α-L-arabinofuranosylglucopyranosidase and β-D-apiofuranosyl glucopyranosidase which are required to cleave glycosidic linkage concerning grape aroma precursors [5].

## Materials and methods

### Grape samples

Chardonnay and Riesling grapes were processed differently using whole bunch pressing and a skin contact time of 24 h to obtain varying concentrations of aroma precursors. Muscatel grapes were only subjected to 24 h of skin contact. The three different grape varieties were fermented using various combinations of yeast strains and enzymes for first and secondary fermentation. The chosen yeast strains had either low (L) or high (H) ability to liberate aroma precursors. From these base wines, sparkling wines were produced using different yeast strains again (table 1). Each batch of sparkling wine produced was either left untreated or was additionally treated with enzyme preparation AR 2000 (DSM) originating from *Aspergillus niger*.

<table><caption>**Table 1** - Experimental design. SCT: skin contact time 24 h; WBP: whole bunch pressing. Yeast strains used: Uvaferm BC (BC), Lalvin DV10 (DV10), Erbslöh IOC 18-2007 (IOC), and Siha Aktivhefe 4 (Siha 4) with either low (L) or high (H) ability to liberate aroma precursors.</caption><thead><tr><th>Grape variety</th><th>Chardonnay (SCT/WBP)</th><th>Riesling (SCT/WBP)</th><th>Muscatel (SCT/WBP)</th></tr></thead><tbody><tr><td>First fermentation</td><td>DV10<sup>L</sup></td><td>BC<sup>H</sup> DV10<sup>L</sup></td><td>IOC<sup>H</sup> DV10<sup>L</sup> BC<sup>H</sup></td></tr><tr><td>Second fermentation</td><td>BC<sup>H</sup> IOC<sup>H</sup> Siha4<sup>H</sup></td><td>BC<sup>H</sup> IOC<sup>H</sup> Siha4<sup>H</sup></td><td>IOC<sup>H</sup> DV10<sup>L</sup> BC<sup>H</sup> DV10<sup>L</sup> BC<sup>H</sup></td></tr></tbody></table>
Sparkling wines were prepared according to the *méthode champenoise*. Additionally to usual fermentation, experimental control samples (acid hydrolysis control) without yeast or enzyme treatment were prepared to examine the effects of acid hydrolysis only.

### Descriptive analysis

Descriptive analysis (DA) was conducted with base and sparkling wines. 20 experienced judges were subjected to two training sessions. Subsequently, every sample was rated on an unscaled 10 cm line scale in triplicates with regards to colour, mouthfeel, fruity, sour, bitter, sweet and to seven aroma perceptions (peach/apricot, cantaloupe, smokey/honey, floral, lemon/grapefruit, apple, green grass). Aroma standards were prepared in neutral white wine or sparkling wine.

### GC-MS analysis

Following solid phase extraction (SPE) and enzymatic hydrolysis the extracted free aroma compounds were analysed by gas chromatography-mass spectrometry (GC-MS) supporting large volume injection. Using the concurrent solvent recondensation large volume splitless injection technique [6], 22 µl of sample were injected allowing detection of trace amounts without any further concentration step after liquid-liquid extraction from the enzyme buffer. Overall, 30 individual aroma compounds were quantified utilizing a stable isotope dilution assay (SIDA) and their quantitative data was then combined in the categories monoterpenes, C6-alcohols, benzene derivatives, C13-norisoprenoids, and volatile phenols. A structurally related deuterated standard was used for every group of substances.

## Results and discussion

In Chardonnay, which is basically characterised by very low aroma precursor contents, no significant differences in the sensory profile of the four base wines could be found caused by yeast selection or grape processing (figure 1a). Chemical analysis of precursor liberation confirmed this observation as no substance group showed any significant differences (figure 1b).

<figure><img src="image_223.png" /><figcaption>**Fig. 1** - Influence of yeast strain selection on Chardonnay sensory profile, n=20 (a) and precursor contents, n=3 (b). SCT skin contact, WBP whole bunch pressing, yeast strains: Uvaferm BC (BC), Lalvin DV10 (DV10).</figcaption></figure>

In contrast to Chardonnay, Riesling features medium levels of aroma precursors. Comparing the four Riesling base wines, the effect of yeast strains was still minor but at least obvious (figure 2). However, grape processing had a stronger influence on sensory profile and analysed precursor liberation than yeast strain selection. Sensory analysis showed that SCT base wines were rated significantly higher in the perceptions of floral notes, lemon, apple, and colour. Skin contact time most likely increased the amounts of aroma precursors which had already been detected in the must and were still detectable in the base wines.
<figure><img src="image_224.png" /><figcaption>**Fig. 2** - Influence of yeast strain selection on Riesling sensory evaluation, n=20 (a) and precursor contents, n=3 (b). SCT skin contact, WBP whole bunch pressing, yeast strains: Uvaferm BC (BC), IOC 18-2007 (IOC).</figcaption></figure>

For Muscatel, which exhibits very high amounts of glycosylated monoterpenes, a significant influence of yeast strain selection became obvious (data not shown). Using yeast strain BC, base wines were rated significantly more intense in floral and cantaloup in descriptive analysis. That could be confirmed in chemical analysis although the differences where within standard deviation.

Concluding, the influence of yeast strain selection in the first fermentation seems to be highly dependent on the concentration of the aroma precursors present, which is determined by the grape variety itself. Hence, it might be hypothised that a high content of aroma precursors stimulates enzyme activity in the yeast, however, mechanisms for this remain unclear.

Examining secondary fermentation, chemical and sensory analysis revealed generally a minor impact of yeast strains on the liberation of aroma precursors and the resulting aroma profile of the final sparkling wine for all three grape varieties. This is exemplified in figure 3 using the examples of Chardonnay trials.

<figure><img src="image_225.png" /><figcaption>**Fig. 3** - Partial Least Square Regression (PLS) of glycosylated precursors and sensory data explaining Chardonnay sparkling wines. SCT skin contact, WBP whole bunch pressing, yeast strains: Uvaferm BC (BC), Lalvin DV10 (DV), Erbslöh IOC 18-2007 (IOC) and enzyme preparation AR2000 (E).</figcaption></figure>
PLS analysis revealed that grape processing and the use of enzyme preparation had a major impact as the wines are grouped irrespective of yeast strain combinations. Sparkling wines treated with “skin contact time (SCT)” were correlated with glycosylated monoterpenes. In contrast, sparkling wines produced by “whole bunch pressing (WBP)” correlated with glycosylated norisoprenoids, hexenols and benzene derivatives. Neither SCT nor WBP sparkling wines showed any correlation with perceived sensory attributes. However, using enzyme preparation, all sparkling wines were shifted in the PLS space meaning that aroma precursors were liberated and sensory perception of green grass, floral, apple, peach/apricot (SCT) and honey and cantaloupe (WBT) was increased in intensity.

To conclude the results of secondary fermentation, grape processing might influence the accessible aroma potential of the base wine. Aroma precursors can then potentially be liberated during secondary fermentation, however, only the use of enzyme was proofed to noticeably liberate precursors while yeast selection did not have a major impact. An explanation for the minor impact of the yeast might be that a levelling effect during nine months of sparkling wine fermentation has occurred. Moreover, our observation of the experimental control samples (acid hydrolysis control) showed that the same amounts of precursors were liberated without yeast treatment compared to treatments with yeast strains (data not shown). Eventually, precursor liberation due to acid hydrolysis may have levelled out any possible differences caused by using different yeast strains.

## Conclusion

The impact of yeast strains on the chemical and sensory properties in base wines was strongly dependent on the grape variety, respectively, on the given aroma precursor content. In Chardonnay, which is usually characterised by a low amount of aroma precursors, no effects were detectable, while in Riesling and Muscatel, with higher precursor contents, the impact of yeast strains was greater. For sparkling wines, irrespective of the grape variety, the impact of yeast strains was minor. This observation might be explained by acid hydrolysis of the precursors during nine months of fermentation, which may have levelled any differences caused by enzymatic activity of the yeast. The mechanisms of enzymatic hydrolysis are still unclear. Only the use of enzyme preparation AR2000, which has a broad spectrum of enzymatic functions and activities, could liberate high amounts of precursors. Furthermore, for both, base wines and sparkling wines, grape processing had a high impact on aroma compounds and flavour.

## Acknowledgements

The project (AiF 16627 N) is supported by the German Ministry of Economics and Technology (via AiF) and the Forschungskreis der Ernährungsindustrie e.V. (FEI).

## References

1. Ganss S, Kirsch F, Winterhalter P et al '2011) Aroma changes due to second fermentation and glycosylated precursors in Chardonnay and Riesling sparkling wines. *J Agric Food Chem* **59**: 2524-2533.

2. Günata YZ, Bayonove CL, Baumes RL et al (1985) The aroma of grapes. Localisation and evolution of free and bound fractions of some grape aroma components c.v. Muscat during first development and maturation. *J Sci Food Agric* **36**: 857-862.

3. Williams PJ (1993) In: Acree TE, Teranishi R (Eds.) *Flavor Science – Sensible principles and techniques*, American Chemical Society, Washington, pp. 287-308.

4. Daenen L, Saison D, Sterckx F et al (2008) Screening and evaluation of the glucoside hydrolase activity in *Saccharomyces* and *Brettanomyces* brewing yeasts. *J Appl Microbiol* **104**: 478-488.

5. Cabaroglu T, Selli S, Canbas A et al (2003) Wine flavor enhancement through the use of exogenous fungal glycosidases. *Enzyme Microb Technol* **33**: 581-587.

6. Magni P, Porzano T (2003) Concurrent solvent recondensation large sample volume splitless injection. *J Sep Sci* **26**: 1491-1498.
# Interactions of grape proanthocyanidins and wine polyphenols with yeast protein extract, mannoproteins and β-glucans: isothermal titration calorimetry and dynamic light scattering studies

J. MEKOUE NGUELA<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="4" />*, A. VERNHET<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, N. SIECZKOWSKI<fnref n="4" />, C. PONCET-LEGRAND<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Yeast fractions – grape skin tannins – wine tannins – interactions

## Introduction

There is currently a great interest in enology for the use of yeast derived products (YDPs) as an alternative to aging on lees to improve wine mouthfeel, color and stability or as new fining aids. These YDPs are inactivated cells, cell walls and mannoproteins (MPs) in the first case, and yeast protein extracts (YPE) in the second one [1-4]. When dealing with red wines, their impact on wine taste and stability as well as their potential interest as fining aids is attributed to their interactions with polyphenols. These interactions can take place in solution with YPE or MPs, or be related to polyphenol adsorption on the yeast cell wall, which is mainly composed of mannoproteins and β-glucans. However, the exact mechanisms involved in their impact on wine composition and properties are not clear yet. To further understand the mechanisms that drive interactions between these yeast components and red wine polyphenols, interactions in solution between polyphenols and a YPE, a mannoprotein fraction (MPs) and a model linear β-glucan (laminarin) were monitored by Isothermal Titration Calorimetry (ITC) and Dynamic Light Scattering (DLS). Polyphenols were grape skin tannins, a whole polyphenol pool purified from a red wine (WP) and a wine tannin fraction (WT) purified from the pool.

## Materials and methods

### *Yeast protein extract and polysaccharides*

The yeast protein extract (YPE) used in the present work was obtained from a commercial *Saccharomyces cerevisiae* enological strain (Lallemand, Montreal, Canada). It contained an insoluble fraction (13% w/w) that was removed by centrifugation (15000 g, 15 min) before further experiments. Total Nitrogen was determined by the Kjeldahl method on the whole YPE and after fractionation using a 10 kDa ultrafiltration membrane. Proteins in the high molecular weight fraction were analyzed by 1D-SDS PAGE. Neutral sugars were analyzed after hydrolysis by converting the monosaccharides into their alditol acetate derivatives [5]. A commercial yeast mannoprotein fraction from *Saccharomyces cerevisiae* was used. Ultrafiltration (10 kDa) and extensive diafiltration were performed on the commercial solution before use to remove low molecular weight components. β-glucan (laminarin) was purchased from Sigma-Aldrich.

### *Polyphenols*

Grape skin tannins (Skin27) with an average degree of polymerization (aDP) of 27 and a % of galloylation of 16.8 were purified from the skin of Muscat berries [6]. A red wine polyphenol pool (WP) was purified from a Merlot wine, according to the procedure described in Mekoue *et al.* (2015) [6]. Wine tannins (WT) were further purified from the WP by chromatography on a Fractogel® EMD Phenyl (S) (Millipore, France). Polyphenol monomers in the WP were analyzed according to the procedure described in Ducasse *et al.* (2010) [7]. Tannins were analyzed by HPLC after acid-catalysed cleavage in the presence of excess 2-mercaptoethanol [8].

<footnote n="1">¹INRA - UMR 1083 SPO, 34060 Montpellier, France</footnote>

<footnote n="2">²Montpellier SupAgro - UMR 1083 SPO, 34060 Montpellier, France</footnote>

<footnote n="3">³Université de Montpellier - UMR 1083 SPO, 34060 Montpellier, France</footnote>

<footnote n="4">⁴Lallemand SAS, 19 rue des Briquetiers, 31702 Blagnac, France</footnote>
### Model solution

ITC and DLS experiments were performed in the same wine-like solution: 12% v/v ethanol solution with 2 g.L⁻¹ tartaric acid, pH adjusted to 3.5 with KOH 1 M, ionic strength set at 50 mM by NaCl (2.16 g.L⁻¹), SO₂ (25 mg.L⁻¹).

### Isothermal titration microcalorimetry

ITC experiments were performed with a VP-ITC instrument (MicroCal, Northampton, MA). A solution of (typically 4 g.L⁻¹) YPE, laminarin or mannoproteins was placed in the 1.448 mL sample cell of the calorimeter and the polyphenol solution (typically 5 g.L⁻¹) was loaded into the injection syringe. The polyphenol solution was titrated into the sample cell as a sequence of 48 injections of 6 µL aliquots. The duration of each injection was 12 s, and the time delay (to allow equilibration) between successive injections was 8 min.

### Dynamic light scattering

DLS experiments were carried out by mixing equal volumes of a polyphenol (0.8 g.L⁻¹) solution and of a macromolecule (0.8 g.L⁻¹) solution for a final concentration of 0.4 g.L⁻¹. Measurements were carried out on a Malvern Zetasizer 3000 HS (Malvern instruments, Malvern, U.K.), equipped with a 10 mW He-Ne laser with a wavelength of 633 nm.

## Results and discussion

### Characteristics of YPE and polyphenol fractions

The YPE soluble fraction (YPE-S) (87% w/w) was used in this work for the interactions studies. The YPE-S was mostly low molecular weight compounds (<10 kDa). Analysis indicated the presence of about 18% free amino acids and peptides and of oligosaccharides (16%) mostly composed of glucose. Proteins with molecular weight higher than 10 kDa accounted for 7.7% w/w, along with 5.9% w/w glucan and mannoprotein polymers/copolymers. 1D SDS-PAGE analysis evidenced ten protein bands with molecular weight ranging from 14 to 66 kDa.

The characteristics of the three polyphenols fractions are given in **Table 1**. Skin tannins (Skin27) were mostly native structures, as evidenced by the high yield of the depolymerization reaction. Their aDP was 27. Monomers and tannins represented 11% and 21% respectively of the total polyphenols in the WP. This low yield is related to a large proportion in this WP of derived pigments and tannins formed during winemaking. The tannin fraction purified from this pool was devoid of monomers and oligomers and represented 37% of the tannins in the initial WP. The yield of the depolymerization reaction was twice the one obtained from tannins in the WP but still characteristic of chemically modified structures.

<table><caption>**Table 1** - Analysis of the different polyphenol fractions obtained by HPLC before (monomers) and after depolymerization reactions</caption><thead><tr><th></th><th></th><th>WP</th><th>WT</th><th>Skin27</th></tr></thead><tbody><tr><td rowspan="5">Tannins</td><td>aDP</td><td>5.8 ± 0.2</td><td>7.5 ± 0.2</td><td>26.7 ± 0.5</td></tr><tr><td>% Gallate</td><td>3.9 ± 0.6</td><td>4.4 ± 0.0</td><td>5.4 ± 0.1</td></tr><tr><td>% TriOH</td><td>31.2 ± 2</td><td>26.5 ± 0.2</td><td>15 ± 1</td></tr><tr><td>Total (mg/g)</td><td>170 ± 15</td><td>428 ± 41</td><td>930</td></tr><tr><td>Yield (%)</td><td>21.2 ± 0.3</td><td>42.8 ± 4.1</td><td>93</td></tr><tr><td rowspan="6">Monomers (mg/g)</td><td>Anthocyanins</td><td>33.1</td><td></td><td></td></tr><tr><td>Flavan-3-ols</td><td>8.7</td><td></td><td></td></tr><tr><td>Phenol Acids</td><td>36.9</td><td></td><td></td></tr><tr><td>Stilbenes</td><td>27.3</td><td></td><td></td></tr><tr><td>Flavonols</td><td>6.7</td><td></td><td></td></tr><tr><td>Total (mg/g)</td><td>112.7</td><td>0</td><td>0</td></tr><tr><td>A<sub>420</sub>/A<sub>280</sub></td><td></td><td>5.3</td><td>7.3</td><td></td></tr><tr><td>A<sub>460</sub>/A<sub>280</sub></td><td></td><td>6.4</td><td>7.7</td><td></td></tr><tr><td>A<sub>520</sub>/A<sub>280</sub></td><td></td><td>9.2</td><td>7.8</td><td></td></tr></tbody></table>

<footnote>* in %: from UV-visible spectrophotometric measurements in the model wine</footnote>
Isothermal titration calorimetry

ITC experiments allowed measuring enthalpy changes associated with interactions between polyphenols (WP, WT or Skin27) and macromolecules (YPE-S, mannoproteins or laminarin). From the titration curves the heat exchange was calculated from the area under each peak and was expressed as a function of mass ratio (tannins/protein) instead of molar ratio due to the sample heterogeneity. For this reason, association constants, enthalpies and stoichiometry parameters were not calculated. Depending on the polyphenol-protein and/or polysaccharides pairs, differences were observed:

- The shape of the initial part of the integration curve suggested a strong affinity between Skin27 and YPE (Figure 1, a1b1c1), attributable to proteins in the extract. The binding sites present in YPE were saturated at a mass ratio of about 0.02, and addition of more Skin27 led to a plateau. When wine tannins (WT) were added (Figure 1, a3b3c3), the heat changes where higher. Finally, the heat changes observed with WP (Figure 1, a2b2c2) was about 2-fold lower compared to those observed with wine tannins at the same concentration, and the mass ratio higher than the one obtained with Skin27.

<figure><img src="image_226.png" /><figcaption>**Fig. 1** - Interaction of grape skin tannins (1), red wine polyphenol pool (2) and red wine tannins (3) with yeast protein extract studied by ITC at 25°C showing the thermogram and binding isotherm. (a1, a2, a3) Blank experiment for injection of polyphenols at 5 g.L⁻¹ in wine-like solution. (b1, b2, b3) Titration plots of polyphenols into 4 g.L⁻¹ yeast protein extract. The molar ratio is the number of moles of polyphenols divided by the number of moles of YPE compounds. (c1, c2, c3) Integrated peaks.</figcaption></figure>

- With mannoproteins (Figure 2), heat exchanged remained very low with the wine polyphenol pool (Figure 2, a2b2c2). With the tannins (WT and skin27), the curves were different. The shape of the initial part of the integration curve and the low exchanged heat obtained suggested a lower affinity between Skin27 and mannoproteins than that obtained with Skin27 and YPE-S.

- With β-glucan (laminarin), results evidenced initial exothermic interactions upon Skin27 addition (not observed with WP), followed by endothermic peaks (Figure 3, a1b1c1). Despite the high concentration of laminarin (20 g.L⁻¹), saturation seemed to be reached at a lower mass ratio than that observed with mannoproteins and YPE.
<figure><img src="image_227.png" /><figcaption>**Fig. 2** - Interaction of grape skin tannins (1), red wine polyphenol pool (2) and red wine tannins (3) with mannoproteins studied by ITC at 25°C showing the thermogram and binding isotherm. (a1, a2, a3) Blank experiment for injection of polyphenols at 5 g.L⁻¹ in wine-like solution. (b1, b2, b3) Titration plots of polyphenols into 10 g.L⁻¹ mannoproteins for Skin27 and 4 g.L⁻¹ mannoproteins for WT and WP. (c1, c2, c3) Integrated peaks.</figcaption></figure>

<figure><img src="image_228.png" /><figcaption>**Fig. 3** - Interaction of grape skin tannins (1) and red wine polyphenol pool (2) with β-glucan (laminarin) studied by ITC at 25°C showing the thermogram and binding isotherm. (a1, a2) Blank experiment for injection of polyphenols at 5 g.L⁻¹ in wine-like solution. (b1, b2) Titration plots of polyphenols into 20 g.L⁻¹ laminarin for Skin27 and 4 g.L⁻¹ laminarin for WP. (c1, c2) Integrated peaks.</figcaption></figure>
In accordance with previous studies, these results show that interactions with polyphenols increase with the estimated percentage of proteins or with the protein content in the polysaccharides [9]. Much lower affinities were found between polyphenols and mannoproteins than between polyphenols and YPE, and strong differences were observed between mannoproteins and the model β-glucan. Affinities were also strongly influenced by tannin structure, in accordance with previous results [9-12] and by the presence of monomers/oligomers in the fraction.

### Dynamic light scattering

Results of DLS experiments are summarized **Figure 4**. The addition of Skin27 (0.4 g.L⁻¹) to YPE-S (0.4 g.L⁻¹) led to the very fast formation of polydisperse micron-size aggregates, followed by sedimentation (Figure 4A). This was evidenced by the very large initial increase of the scattering intensity of the mixture (I<sub>S-(YPE+PP)</sub> around 1600 kC in A.U.) by comparison to that of the sum of the scattering intensity of the YPE and PP initial solutions (I<sub>S-YPE</sub> + I<sub>S-PP</sub>), followed by its decrease, and by the changes in mean hydrodynamic diameters D<sub>H</sub> values. By contrast, experiments performed with WP or WT and YPE (0.4 g.L⁻¹) indicated the formation of finite and sub-micronic aggregates (D<sub>H</sub> between 200 and 300 nm), and no enlarged aggregation was observed. Results obtained with mannoproteins are reported in Figure 4B. The scattering intensity of the mixture (I<sub>S-(MP+PP)</sub>) was found to be much higher than the sum of their own scattering intensity (I<sub>S-MP</sub> + I<sub>S-PP</sub>), indicating there were interactions between the macromolecules, leading with all polyphenol fractions to the formation of stable sub-micronic aggregates. Contrary to that observed with YPE and MPs, no interactions could be evidenced by DLS between laminarin and polyphenols (Figure 3C).

<figure><img src="image_229.png" /><figcaption>**Fig. 4** - Dynamic Light Scattering aggregation studies between YPE-S (A), mannoproteins (B), β-glucan (Laminarin) (C) and polyphenols (Skin27, WP and WT).</figcaption></figure>
Conclusion

YPE, mannoproteins and β-glucans can thus be ranked by decreasing order for their interactions with tannins as follows: YPE > MPs > β-glucans, in accordance with the protein content of these free macromolecules or fractions. ITC and DLS results also evidenced the influence of tannin structure on these interactions and on their consequences at a colloidal level. Whereas interactions between YPE and skin tannins led to enlarged aggregation and precipitation, this was not the case with the wine polyphenols and tannins studied. Tannin structural characteristics also influence the structure of the aggregates formed with mannoproteins. However, these aggregates remained stables with all the studied polyphenols in our experimental conditions.

Acknowledgements

The authors thank Stephanie Carrillo, Jean-Paul Mazauric and Jean-Marc Souquet for their precious help in
polyphenol purification/analysis and Stephanie Roi for her helpful contribution to the interaction studies.

References

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# Red wine adsorption on functionalised surfaces

A. MIERCZYNSKA-VASILEV*, P. SMITH

*The Australian Wine Research Institute, Glen Osmond, Adelaide, SA 5064, Australia*

Red wine – constituent adsorption – surface properties – polymer film

## Introduction

It is widely known that surfaces play an important role in numerous biological processes and technological applications. Thus being able to modify surface properties provides an opportunity to control many phenomena occurring at interfaces. For example interactions at interfaces are of primary importance in membrane and filtration processes [1, 2]. It is essential to understand the membrane fouling mechanisms in wine membrane bioreactor processes in order to improve fouling control and cleaning strategies since membrane efficiency and membrane fouling present a significant challenge in membrane related technologies. At present, membrane fouling is still the biggest challenge and the major obstacle for the widespread implementation of membrane processes. Thus key research is required to investigate in more detail the mechanisms causing membrane fouling, the properties of membranes that contribute to fouling and the composition and size distribution of wine colloids and their complexes that affect filtration.

The primary goal of this study was to determine how surface modification affects wine constituents adsorption. One way of controlling surface properties is to adsorb a polymer film onto the surface, for example through plasma deposition. Plasma-produced reactive surfaces with amine, carboxyl, hydroxyl and aldehyde groups have been used by many researchers because of their compatibility with well-established chemical reactions for grafting of bioactive moieties such as enzymes, antibodies and proteins [3-5]. In this paper we generate surfaces modified with functional groups such as carboxyls and hydroxyls that can be utilized for binding of wine constituents. The goal is to explore the capacity of functional surfaces to promote or impede red wine constituents' adsorption.

## Materials and methods

### Wine samples and preparation

A 2014 AWRI Red MS wine was used as received. Acrylic acid (AcrA) (99%) and ethanol (ET) (absolute 99.5% v/v) were obtained from Sigma-Aldrich (Australia) and used as supplied. Plasma polymerization was carried out in a custom-built reactor described elsewhere using a 13.56 MHz plasma generator. AcrA and ET were used as precursors for plasma deposition in order to generate thin films rich in amine carboxyl (–COOH) and hydroxyl (–OH) chemical groups, respectively. Clean silicon wafers and QCM sensors were used as substrates. The chamber was evacuated to base pressure of $1 \times 10^{-3}$ mbar before deposition. Ethanol vapour was introduced at 4 sccm, and plasma was applied at 40 W for 4 min. Deposition of acrylic acid was carried out at a flow rate of 0.5 sccm, power of 10 W for 10 min.

### XPS analysis

The surface composition of the plasma-polymerized coatings before and after wine adsorption was characterized by X-ray photoelectron spectroscopy (XPS). XPS spectra were recorded on a Specs SAGE XPS spectrometer using a MgKα radiation source (hv = 1253.6 eV) operated at 10 kV and 20 mA. Elements present in the samples' surface were identified from the survey spectrum recorded over the energy rate 0-1000 eV at a pass energy 100 eV and a resolution of 0.5 eV. The areas under selected photoelectron peaks in a wide-scan spectrum were used to calculate percentage atomic concentrations (excluding hydrogen). High-energy resolution (0.1 eV) spectra were then recorded for pertinent photoelectron peaks at a pass energy of 20 eV to identify the possible binding environments for each element. A QSenseE4 instrument (Q-Sense, Sweden) was used with a variable speed peristaltic pump (ISM 935, IDEX Health & Science, Germany). During experiments, four identical plasma-coated gold QCM sensors were equilibrated in parallel with MilliQ water delivered at 0.1 mL min$^{-1}$ until stable
frequency and dissipation traces resulted. Wine samples were introduced and remained in contact with the sensors for 40 min. In the next step washing with MilliQ water was performed to obtain the sensor readings for the final bound amount.

*Atomic force microscopic analysis*

An NT-MDT NTEGRA SPM atomic force microscope (AFM) was used in non-contact mode to provide topographical images. Silicon nitride non-contact tips coated with Au on the reflective side (NT-MDT, NSG03) were used and had resonance frequencies between 65 and 100 kHz. The amplitude of oscillation was 10 nm, and the scan rate was 1 Hz. The scanner used had a maximum range of 100 µm and was calibrated using 1.5 µm standard grids with a height of 22 nm. The root-mean squared (RMS) and the peak-to-valley (PTV) distance for the imaged surfaces were determined using standard AFM procedures.

**Results and discussion**

We have generated functional surfaces where the presence of surface groups such as carboxyls and hydroxyls were utilized to evaluate the binding of red wine constituents. AcrA and ET were used as precursors for plasma deposition to fabricate surfaces rich in carboxyl acid and hydroxyl functional groups, respectively. The XPS results are consistent with studies of plasma polymerized films deposited from AcrA (Fig. 1A) and ET (Fig. 1C) which indicate that their coatings contain a significant population of carboxyl and hydroxyl functional groups, respectively.

<figure><img src="image_230.png" /><figcaption>**Fig. 1** - XPS C1s profiles showing the peak fit analysis of (A) ppAcrA and (C) ppET as deposited, and after red wine adsorption onto ppAcrA (B) and ppET (D) surface.</figcaption></figure>

The capacity of the plasma deposited acrylic acid and ethanol films to bind wine constituents was examined in real time using QCM-D measurements. QCM sensors treated with acrylic acid and ethanol were conjugated with red wine. An example of typical raw data from the QCM-D measurements upon adsorption of wine on plasma polymer surface on one of the sensors is shown in Figure 2. Wine binds to the ethanol pp coated QCM-D crystal surface causing a negative frequency shift as mass adsorbs. After adsorption, a rigorous washing with MilliQ water was used in attempt to remove any loosely bound material. As evident from Figure 2 there is a large change to the dissipation energy loss and Voigt-based viscoelastic modeling [6] is required to determine accurate adsorbed mass values. The Q-Sense Q-Tools software was used for data analysis and Voigt modeling. The experimental data (open and filled: circles, squares and triangles, cf. Figure 2A) and the fit (filled, dashed and dotted lines) using the viscoelastic model are displayed as ΔF/n versus time and ΔD versus time for n = 3, 5 and 7. The agreement between the experimental QCM-D data and the model must be regarded as very good as the fitted curves are overlaid on the measured values for the changes in frequency and dissipation. For ppAcrA/red wine, the average amount bound was 1890 ± 140 ng cm⁻², and for ppET/red wine it was 850 ± 30 ng cm⁻². The results presented above demonstrate that modification of surface chemical states is a strategy to control the adsorption of red wine constituents such as proteins, polysaccharides and polyphenols. Figure 2B shows the variations of viscosity and shear elastic modulus of the red wine sample calculated from the Voigt model adsorbed onto ppAcrA
and ppET surfaces. On the hydroxyl-rich surface both the viscosity and the shear elastic modulus values are larger which suggest that the adsorbed wine layer is much more viscoelastic as compared to carboxyl-rich surface. Possible reasons for the differences in the adherence and viscoelasticity may be differences in the mass concentration as well as different locations and/or conformation of the wine constituents on the surface.

Sensors were then analysed by XPS. The high resolution C1s spectra for wine-bound samples were obtained by XPS as shown in Figure 1B and D. While the XPS spectra of ppAcrA and ppET had no traces of nitrogen in the N 1s spectra of the acrylic acid, showing that no contamination was present, the ppAcrA and ppET coated with red wine exhibited nitrogen peak. The presence of nitrogen in the XPS spectrum is the evidence that some of the wine nitrogen compounds adsorbed on AcrA surface. Fundamentally, total nitrogen in wine includes mineral nitrogen (inorganic form) and various forms of organic nitrogen such as amino acids, oligopeptides and polypeptides, proteins and protein casse, amide nitrogen, bioamines, nucleic nitrogen and amino sugar nitrogen.

<figure><img src="image_231.png" /><figcaption>**Fig. 2** - (A) ΔF/n and ΔD vs time and (B) changes of viscosity and shear elastic modulus of wine sample on the pp surfaces estimated from the Voigt model.</figcaption></figure>

Figure 3 shows the ex-situ tapping mode AFM height images of red wine adsorbed onto ppAcrA (A) and ppET (B) QCM sensor surface. The AFM images of the adsorbed layers allow one to make an estimate of the wine adsorbed amount. The average height multiplied by the area coverage allows one to determine the volume of adsorbed material over the area of the AFM image. This volume per unit area can then be compared with the adsorbed amount of each pp wine adsorbed surface determined from QCM data. Knowing the actual adsorbed mass from QCM and AFM experiments makes it possible to estimate the percentage of hydration water in the adsorbed layers of the different wine samples [7]. The data comparing adsorbed amounts per unit area, expressed as adsorbed volume per unit area for both AFM and QCM results, are given in Table 1.

<figure><img src="image_232.png" /><figcaption>**Fig. 3** - Ex-situ AFM height images of QCM sensors: (A) ppAcrA/red wine and (B) ppET/red wine.</figcaption></figure>

It is well established that surface -COOH groups are less acidic than those in bulk solution. The zeta potential of carboxyl-rich surface is near neutral at pH 3 and became negative with increasing pH, which is consistent with the weak acidity of -COOH groups. Shyue et al. [8] also reported that at pH 3, the zeta potential was zero, and with rising pH, the zeta potential became increasingly negative (-85 mV at pH 11). At pH 3.6, which is the pH of the investigated red wine, the -COOH groups are largely protonated and thus there is little negative charge on the ppAcrA surface.

This slightly negatively charged surface attracts positively charged wine compounds. The adsorbed amount of red wine on ppAcrA surface is 1.89 ± 0.14 µg cm⁻². The presence of nitrogen (N/C ratio of 0.038) in the XPS spectrum is the evidence of the nitrogen compounds present in wines. Besides two primary
ingredients, water and ethanol, wine constituents include glycerol, organic acids, minerals, nitrogen compounds (amino acids and proteins), phenolic compounds and polysaccharides. Depending on pH, proteins can be positively or negatively charged. They may also be at the isoelectric point (i.p.) where they are neutral. In a solution with a pH < i.p., normally the case for proteins in wine, proteins are positively charged. The positively charged components found in wine are expected to adsorb onto a negatively charged surface due to the electrostatic interactions. The presence of nitrogen in the XPS spectrum is evidence of nitrogen compounds present in wines. Fundamentally, total nitrogen in wine includes mineral nitrogen (inorganic form) and various forms of organic nitrogen such as amino acids, oligopeptides and polypeptides, proteins and protein casse, amide nitrogen, bioamines, nucleic nitrogen and amino sugar nitrogen. The QCM-D and AFM results both demonstrate that the carboxyl-rich ppAcrA surfaces enhanced the red wine adsorption. On these surfaces the red wine adsorbed amount was high. The -OH groups are neutral hydrophilic. The QCM-D and AFM results both demonstrate that the hydroxyl-rich ppET had the low adsorbed amount of wine (red wine: 0.85 ± 0.03). It is known that alcohol-like functionality reduces non-specific protein adsorption as it is a non-interacting support for the hydrophobic groups.

<table><caption>Table 1 - Adsorbed Amount (Γ) and Adsorbed Volume (V) for red wine and water on pp surfaces</caption><thead><tr><th>Sample</th><th>Γ [mg m<sup>-2</sup>] (QCM)</th><th>V x 10<sup>-8</sup> [m<sup>-3</sup>] (QCM)</th><th>V x 10<sup>-8</sup> [m<sup>-3</sup>] (AFM)</th><th>V x 10<sup>-8</sup> [m<sup>-3</sup>] volume of water (QCM)</th><th>% of water</th></tr></thead><tbody><tr><td>AcrA/Red MS</td><td>18.9</td><td>1.93</td><td>1.88</td><td>0.05</td><td>3</td></tr><tr><td>ET/Red MS</td><td>8.5</td><td>0.87</td><td>0.83</td><td>0.04</td><td>5</td></tr></tbody></table>

## Conclusion

This study demonstrates that tuning the surface functionalities is an effective way to promote or discourage adsorption of wine constituents. It is likely that functionalities affect the conformation/structure of the wine constituents and in turn regulate their binding. In this way they activate different ‘signals’ and affect the morphology, location and conformation of the wine constituents. Those results are meaningful for the development of the next generation low fouling membrane and/or new sensing platforms that will reduce cost and improve productivity in the wine and other related industries.

## Acknowledgements

This work was supported by Australian grapegrowers and winemakers through their investment body the Wine Australia, with matching funds from the Australian Government. The Australian Wine Research Institute is a member of the Wine Innovation Cluster in Adelaide.

## References

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2. Cartalade D, Vernhet A (2006) Polar interactions in flavan-3-ol adsorption on solid surfaces. *J Agric Food Chem* **54**: 3086-3094.

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4. Liu X, Feng Q, Bachhuka A et al (2014) Surface modification by allylamine plasma polymerization promotes osteogenic differentiation of human adipose-derived stem cells. *ACS Appl Mater Interfaces* **6**: 9733-9741.

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# The concentration, composition and structure of oligosaccharides in wine are affected by grape cultivar

R. APOLINAR-VALIENTE<fnref n="1" />,<fnref n="2" />*, I. ROMERO-CASCALES<fnref n="1" />, P. WILLIAMS<fnref n="2" />, E. GÓMEZ-PLAZA<fnref n="1" />, J.M. LÓPEZ-ROCA<fnref n="1" />, J.M. ROS-GARCÍA<fnref n="1" />, T. DOCO<fnref n="2" />

Wine – oligosaccharides – grape cultivar

## Introduction

Oligosaccharides can be found in important food and medicinal applications [1]. Several health benefits have been linked with these molecules [2]. In wine, they have physicochemical properties such as ability to chelate cations [3]. Concerning sensorial properties of wines, it has been observed recently a positive relation between the astringency perception and the oligosaccharide fraction from wines [4]. Cabernet Sauvignon, Syrah and Monastrell are three of the most widely used varieties around the world. We have studied the cultivar influence on the concentration, composition and structure of oligosaccharides in wines.

## Materials and methods

### Grape samples

The Cabernet Sauvignon, Syrah and Monastrell grapes were grown in Murcia (southern Spain) and carefully harvested at commercial maturity during the 2007 vintage. Three 90-kg grape samples from each variety were destemmed, crushed, and distributed into 100-L stainless steel tanks, then supplemented with potassium bisulfite.

### Wine samples

All fermentations were started by adding commercial dry yeast and were carried out in stainless steel tanks equipped with temperature control. When alcoholic fermentation was totally finished, the musts were pressed. Free-run juice and press wines of each trial were combined and stored in tanks. One month later the wines were racked. After spontaneous malolactic fermentation, the wines were racked again and supplied with sulfur dioxide. The wines were cold stabilized, bottled and stored at 18°C.

### Mass spectrometry analysis

Oligosaccharide fractions were isolated after removal of phenolic compounds by polyamide and high resolution size-exclusion chromatography and the neutral and acidic sugar composition was determined by gas chromatography [5]. The glycosyl-linkage composition was determined by GC-MS of the partially methylated alditol acetates. The MS spectra of the oligosaccharides were performed on an AccuTOF mass spectrometer. Molar-mass distributions on the oligosaccharide fraction from red wine were determined by SEC-MALLS and a DRI detector. Average values, standard deviation and statistical significance were calculated and performed with the Statgraphics Plus 5.1. package.

## Results and discussion

The fraction eluted on Superdex 30-HR column and collected between 60 and 93 minutes was taken to represent the oligosaccharide fraction of the studied wines. Syrah wine showed significantly higher values for most of the detected sugars except glucuronic acid and 4-O-Me glucuronic acid (data not shown). The lowest arabinose and glucose amounts were detected in Cabernet Sauvignon wine and the lowest

<footnote n="1">¹Departamento de Tecnología de Alimentos, Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain</footnote>

<footnote n="2">²INRA, UMR Sciences pour l'Œnologie n° 1083, Equipe Biosynthèse et Composition en Polyphénols et Polysaccharides, 2 place Viala, 34060 Montpellier, France</footnote>
xylose and xylitol amount in Monastrell wine. The total oligosaccharide amount was calculated as the sum of individual monosaccharide amounts. Oligosaccharide concentrations were significantly higher in Syrah wine (480 mg/L) than in Cabernet Sauvignon (174 mg/L) and Monastrell (244 mg/L) wines. We previously presented that oligosaccharide concentration of Monastrell wines from different origin varied between 195 and 288 mg/L [6]. Ducasse et al. [5] measured 332 mg/L of oligosaccharides in Carignan wine, although our Cabernet Sauvignon and Monastrell data were more similar than those reported for Merlot (252 mg/L) [6]. On the other hand, Bordiga et al. [7] found 127 and 102 mg/L for Grignolino and Chardonnay wines, respectively. It could be explained by differences in maturity stages of grapes according to the cultivar properties [5, 8].

<table><caption>**Table 1** - Characteristic ratios of oligosaccharides from studied wines</caption><thead><tr><th>Cultivar</th><th>Ara/Gal<sup>a</sup></th><th>Rha/Gal<sup>a</sup></th><th>(Ara+Gal)/Rha<sup>a</sup></th></tr></thead><tbody><tr><td>CA</td><td>1.18 ± 0.19 a</td><td>0.47 ± 0.07 a</td><td>4.77 ± 0.90 b</td></tr><tr><td>SY</td><td>1.96 ± 0.20 c</td><td>0.73 ± 0.06 b</td><td>2.87 ± 0.06 a</td></tr><tr><td>MO</td><td>1.53 ± 0.18 b</td><td>0.67 ± 0.07 b</td><td>5.10 ± 0.09 b</td></tr></tbody></table>

Several characteristic sugar ratios show significant differences between varieties (Table 1). The Ara/Gal ratio shows significant differences between studied wines, pointing a higher release of arabinose or oligosaccharides rich in arabinose arising from the pectic framework in Syrah wines [9]. The Rha/GalA ratio was lower in Cabernet Sauvignon oligosaccharide fraction, which could indicate a majority of homogalacturonan structures [10]. Ducasse et al. [5] found similar values for this ratio for Carignan and Merlot wines. The ratio of (Ara + Gal)/Rha was notably lower in Syrah oligosaccharide fraction, suggesting that Syrah oligosaccharides contain more structures coming from the hairy regions of pectins, that is to say rhamnogalacturonan-like structures carrying neutral lateral chains. Our values for this ratio are in general similar to those that we previously obtained in Monastrell oligosaccharides [6] and also to those showed by Ducasse et al. [5] for Carignan and Merlot oligosaccharides.

Proportions of several families of oligosaccharides (Figure 1) have been calculated from glycosyl linkage composition (data not shown) according to previous works about OligoMannans, OligoRhamnogalacturonans, OligoArabinogalactans type I and type II and OligoArabinans by Ducasse et al. [11], OligoGlucans by Ballou [12] and OligoXyloglucans by Fry et al. [13].

Regarding the oligosaccharide families, wines from Cabernet Sauvignon and Monastrell presented similar values concerning the oligosaccharides from yeasts (the sum of OligoGlucans and OligoMannans), with Syrah showing the lowest value (Figure 1). On the contrary, Syrah wines showed a relevant release of OligoRhamnogalacturonans. Monastrell had the highest release of OligoArabinans and also of OligoXyloglucans. The lowest values for OligoAG type II were detected in Syrah wines. Other oligosaccharide structures as OligoAG type I did not show differences between the studied cultivar wines.

Cabernet Sauvignon, Syrah and Monastrell oligosaccharides showed differences concerning the ratio of the terminal to the branched residues. The branching degree could be linked with certain physical-chemical properties of wine. For example, Kühnel et al. [14] reported that branched arabino-oligosaccharides from sugar beet presented more difficulties to be degraded than linear arabinan-oligomers.

Differences among the composition of released oligosaccharides in wines from Cabernet Sauvignon, Syrah and Monastrell are deduced from the ESI mass spectrometry results. The predominant ions observed in mass spectra for Cabernet Sauvignon oligosaccharides were at m/z 499 (Rha-[GalA-CH₃]-Rha), 605 (4-OMe-GlcA, two xylose residues linked in 1→4, and a xylitol residue in non-reducing position) and 737 ([4-OMe-GlcA-[Xyl]₃-Xylitol]), for Syrah oligosaccharides were at m/z 605, 661, 807, 837 and 983, whereas for Monastrell oligosaccharides they were at m/z 235, 605, 661, 737 and 983. The ion identified at m/z 499 appears only in Cabernet Sauvignon oligosaccharides and represents the structures of galacturonans coming from smooth regions. On the other hand, the ions at m/z 605 and 737 correspond to glucuronoxylans, a characteristic of xylan families [5, 11, 15].

<footnote><sup>a</sup>Average of three measurements and standard deviation.</footnote>

<footnote>Different letters within the same column represent significant differences according to a least significant difference test (P < 0.05).</footnote>
<figure><img src="image_233.png" /><figcaption>**Fig. 1** - Evolution of major oligosaccharide families (relative mole percentage) isolated from Cabernet Sauvignon (CA), Syrah (SY) and Monastrell (MO) wines.</figcaption></figure>

The ions observed in the mass spectra at m/z 661, 837 and 983 corresponded to a duplication of the basic unit [(1→4)-α-D-GalAp-(1→2)-α-L-Rhap], the pentasaccharide GalA-[Rha-GalA]₂ and [Rha-GalA]₃, respectively [5]. These three oligosaccharides detected mainly in Syrah represent rhamnogalacturonans and come from hairy regions. Oligosaccharides of Syrah wine also show the ion at m/z 807, which corresponds to α-L-Rhap-(1→4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→4)-α-D-GalAp-(1→2)-α-L-Rhap. This ion indicates the presence of rhamnogalacturonans with a residue of rhamnose in terminal position. On the other hand, the ion at m/z 605 has also been detected in Syrah wines, corresponding to the presence of glucuronoxylan-like structure [5, 11]. The ion at m/z 235 was only identified in Monastrell oligosaccharides and corresponds to GalA-COCH₃. The rest of the ions observed in Monastrell wines (at m/z 605, 661, 737 and 983) have been above described.

The oligosaccharide refractive index elution profiles by SEC-MALLS displayed two principal populations whose concentration signal peaks are in the ranges 2150-2350 sec and 2350-2600 sec (Figure 2). Oligosaccharides of Monastrell wine showed the lowest molar mass profile, Cabernet Sauvignon wine showed the highest profile. Concerning the distribution analysis of oligosaccharide fractions, large differences along five delimited molar mass ranges (range 1 = 500 to 2 500 g/mol; range 2 = 2 500 to 5 000 g/mol; range 3 = 5 000 to 7 500 g/mol; range 4 = 7 500 to 10 000 g/mol; and range 5 = up to 10 000 g/mol) were observed between cultivars. In the case of Syrah and Monastrell oligosaccharide fractions, 22% and 64% (respectively) of mass can be found in range 1, Cabernet Sauvignon showing no mass in this range. However, this variety had 20% and 15% of mass in ranges 4 and 5 (respectively), whereas Syrah and Monastrell present lower percentages of masses in these two ranges (SY: 6% and MO: 3%). Moreover, evident differences can be also detected regarding range 2 (Monastrell: 20%; Cabernet Sauvignon and Syrah: 52% and 53%). Finally, range 3 showed no clear differences between studied wines.

<figure><img src="image_234.png" /><figcaption>**Fig. 2** - SEC-MALLS chromatograms and weight-average molar mass distributions of the oligosaccharide fraction of studied wines in 0.1 LiNO₃ at 25°C. Refractive Index (DRI, thin line) and light scattering (Mw, thick line).</figcaption></figure>
**Conclusion**

Taken together, our results showed that grape cultivar impacts the concentration, composition and structure of the wine oligosaccharides. Obviously further studies should be carried out in order to better understand how the oligosaccharide content and composition in wines could influence their physical-chemical and sensory properties.

**Acknowledgements**

This work was made possible by financial assistance from the Ministerio de Ciencia e Innovación of Spain, Project AGL2006-11019-C02-01/ALI. R. Apolinar-Valiente is the holder of an FPI fellowship from the Government of Spain.

**Abbreviations**

CA, Cabernet Sauvignon; SY, Syrah; MO, Monastrell; GC-MS, gas chromatography-mass spectrometry; SEC-MALLS, size exclusion chromatography with a multi-angle light scattering; DRI, differential refractive index; Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose; Glc A, glucuronic acid; 4-OMeGlc A, 4-O methyl glucuronic acid.

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7. Bordiga M, Travaglia F, Meyrand M et al (2012) Identification and characterization of complex bioactive oligosaccharides in white and red wine by a combination of mass spectrometry and gas chromatography. *J Agric Food Chem* **60**: 3700-3707.

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# Impact of grape juice press fractioning on polysaccharide and oligosaccharide composition of Pinot meunier Champagne base wines

S. JÉGOU<fnref n="1" />*, D.A. HOANG<fnref n="1" />, T. SALMON<fnref n="1" />, P. WILLIAMS<fnref n="2" />, C. VRIGNEAU<fnref n="3" />, T. DOCO<fnref n="2" />, R. MARCHAL<fnref n="1" />

Champagne – Pinot meunier – press fractioning – poly- and oligosaccharides

## Introduction

The separation of different grape juice fractions during the pressing cycle is an important step in the production of sparkling base wines. A complete pressing cycle for this style of wine is a series of pressure increases resulting in a considerable variation in juice composition during the pressing cycle [1-4]. After alcoholic fermentation, wines obtained from “Cuvées” (first quality) and “Tailles” (second quality) grape juices also exhibit strong differences for several oenological characteristics such as pH, total acidity, and tartaric acid level. These parameters change from a wine produced with a grape juice originating from the heart of the flesh to a wine produced with the juice released at the end of the pressing cycle. Nevertheless, the impact of the pressing cycle on wine oligosaccharide fraction (OS) and wine polysaccharides (PS), implicated in wine foaming properties [5-8], has never been studied at industrial or laboratory scale. The aim of this study performed on Pinot meunier wines (vintage 2013) was to better understand wine OS and PS composition and change along the 3h30 pressing cycle.

## Materials and methods

### Grape juices and white base wines

Eleven Pinot meunier grape juice fractions (Champagne region - France) were collected during the pressing cycle (8000 kg pneumatic press). The eleven fractions (AP = autopressing, S = squeeze juices and R = return juices) were vinified separately in 1.5-L bottles. Wines were sulfited (+ ullage) after alcoholic fermentation to avoid malolactic fermentation, centrifuged (17000 g, 10 min), stored one month at cellar temperature + 2 months at 4°C and finally filtered to remove insoluble colloidal particles and tartrate crystals.

### Isolation of polysaccharide and oligosaccharide fractions

Wine polysaccharides and oligosaccharides were isolated by high resolution size-exclusion chromatography on a Superdex-30 HR column as previously described [9].

### Sugar composition of oligosaccharide fractions

The neutral and acidic glycosyl residues of wine oligosaccharides were determined after acidic methanolysis with anhydrous MeOH containing 0.5 M HCl (80°C, 16 h), by gas chromatography (GC) of their per-O-trimethylsilylated methyl glycoside derivatives as previously described [10].

### Polysaccharide analysis and concentration

Neutral monosaccharides were released after hydrolysis of the wine polysaccharides by treatment with 2 M trifluoroacetic acid (75 min at 120°C) [11]. They were then converted to the corresponding alditol acetate derivatives by reduction and acetylation and quantified by GC analysis.

<footnote n="1">¹Laboratoire d'Œnologie et Chimie Appliquée, URVVC UPRES EA 4707, Université de Reims
Champagne-Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France</footnote>

<footnote n="2">²INRA, UMR Sciences pour l'Œnologie n° 1083, Equipe BCP2, 2 place Viala, 34060 Montpellier, France</footnote>

<footnote n="3">³Institut Œnologique de Champagne, ZI de Mardeuil, Route de Cumières, 51201 Epernay, France</footnote>
The polysaccharide composition of each wine was estimated from the concentration of individual glycosyl residues, as determined by GC, and did not differ from previously described wine polysaccharide compositions [12].

## Results and discussion

The changes in total PS and OS were monitored for the 11 Pinot meunier Champagne base wines (Figure 1). The results show significant changes in PS base wine composition and concentration as the pressing cycle progressed. For the total polysaccharides, we determined a concentration of 282 mg/L in the base wine corresponding to pressing cycle beginning. This concentration decreases by 40% (167 mg/L) at the last step of the pressing cycle.

<figure><img src="image_235.png" /><figcaption>Fig. 1 - Changes in total polysaccharides (PS) and oligosaccharides (OS) of Pinot meunier Champagne base wines elaborated from grape juices collected during the press fractioning. AP, autopressing (b, beginning; e, end); S, squeeze juices; R, return juices.</figcaption></figure>

The major wine PS can be grouped into three families: (i) polysaccharides rich in arabinose and galactose (PRAGs) from the cell wall of grape berry, (ii) those rich in rhamnogalacturonans, mainly rhamnogalacturonan II (RG-II), which comes from the pecto-cellulosic cell wall of grape berry, and (iii) mannoproteins (MPs), released from the yeast during fermentation and on-lees aging. The concentration of MPs, PRAGs and RG-II in Pinot meunier Champagne base wines is shown in Figure 2. A wide variation of concentration of the four major glycosyl residues of the total polysaccharides, namely glucose, arabinose, galactose and mannose, was observed. In Figure 2, we can observe a higher MPs concentration in the base wine corresponding to pressing cycle beginning (106 mg/L) than in the base wines obtained from the different steps of the pressing cycle. This concentration decreases by around 24% (81 mg/L) at the last step of the pressing cycle. Taking into account that the yeast strain was similar in the Champagne base wines studied, the decrease during the pressing cycle of wine MPs might be attributed to initial composition of the must [13] but also to winemaking conditions such vinification temperature.

The concentration of PRAGs significantly decreased, by 52%, in the base wine from the pressing cycle beginning to the last step of pressing cycle (from 146 to 70 mg/L). At the pressing cycle beginning, higher concentration of PRAGs in wine reflected a greater release of this polysaccharide family by berry endogenous pectolytic enzymes in grape juice. The differences observed can be related to differences in maturity stages into the grape berry pulp and the cell wall. In fact, the pH increases and the total acidity decreases in the grape juice from the pressing cycle beginning to the last step of the pressing cycle. That might modulate the extraction of components such PS during pressing cycle. Furthermore, enzymatic degradation or precipitation phenomenon of some PRAGs during vinification, as suggested by previous results [14], could affect their concentration in Champagne base wine.
<figure><img src="image_236.png" /><figcaption>**Fig. 2** - Concentration of mannoproteins (MPs), polysaccharides rich in arabinose and galactose (PRAGs) and rhamnogalacturonan II (RG-II) in Pinot meunier Champagne base wines elaborated from grape juices collected during the press fractioning. AP, autopressing (b, beginning; e, end); S, squeeze juices.</figcaption></figure>

On the other hand, the concentration for RG-II was 30 mg/L in the base wine corresponding to pressing cycle beginning. This concentration decreases by 47% (16 mg/L) at the S1 step, and it remains stable until the last step of the pressing cycle. Although RG-II was present in low concentration, this polysaccharide was detected in all Pinot meunier Champagne base wines. These results contrasted with the absence of the RG-II molecule observed in Spanish monovarietal white base wines [7]. These authors attributed this absence to the winemaking process but it could be explained by varietal characteristics or by different maturity stages of grape berries.

The total oligosaccharide amount was calculated as the sum of individual monosaccharide amounts measured by GC. For the total oligosaccharides (Figure 1), we determined a concentration of 127 mg/L in the base wine corresponding to the S1 step. This concentration decreases by 23.8% (97 mg/L) at the S4 step of the pressing cycle. These results reveal, for the first time, changes in OS Champagne base wine concentration as the pressing cycle progressed.

## Conclusion

This study is the first report proving that the OS and PS composition and content of Champagne base wines are affected by press fractioning technique. The understanding of release of OS and PS into the grape juice, and during alcoholic fermentation, is a first approach to investigate the effect of these complex fractions on the organoleptic and technological properties of Champagne still and sparkling wines. Further experiments such as “champagnisation” with on-lees aging are needed to complete the impact of press fractioning on polysaccharide and oligosaccharide fraction on Champagne wine quality as foaming properties.

## Acknowledgements

We express our gratitude to the Association Recherche Oenologie Champagne et Université (Reims, France) for financial support. We gratefully thank the Champagne Bruno Paillard (Reims, France) for providing must samples.
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9. 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.

10. 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.

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13. Rosi I, Giovani G (2003) Effect of some winemaking variables on the production of exopolysaccharides by *Saccharomyces cerevisiae*, pp. 324-328. In *7th International Symposium of Oenology*, Lonvaud-Funel A (Ed.); Editions TEC and DOC: Paris.

14. Guadalupe Z, Ayestaran B (2007) Polysaccharide profile and content during the vinification and aging of Tempranillo red wines. *J Agric Food Chem* 55: 10720-10728.
# Stilbenes as inhibitors of α-synuclein aggregation and cytotoxicity

H. TEMSAMANI<fnref n="1" />*, S. KRISA<fnref n="1" />, M. DECOSSAS<fnref n="2" />, O. LAMBERT<fnref n="2" />, J.-M. MERILLON<fnref n="1" />, T. RICHARD<fnref n="1" />

Stilbene – α-synuclein – neuroprotection

## Introduction

Among all polyphenol classes, stilbenes have been shown to possess a large panel of health-related beneficial effects [1, 2]. Stilbene structure derives from resveratrol with an essential skeleton of two aromatic rings joined by an ethylene bridge [3]. Although stilbenes were identified in various plants, vine and wine constitute the main dietary sources of stilbenes [4].

The hallmarks of Parkinson's disease are the Lewy's Bodies. They are mainly composed of highly aggregated fibrillar species of α-synuclein [5]. Studies have shown that mutation of the gene encoding this protein can lead to the transcription of an aggregation-prone structure of the protein or to its overexpression, which also triggers the α-synuclein aggregation [6, 7]. Thus, α-synuclein accumulation and aggregation are suspected to be directly linked to the disease. Small α-synuclein oligomers seem to be the most toxic species, instead of the fibrils [8]. Numerous studies have highlighted that toxic oligomers can form annular rings and then permeabilize model lipidic membranes.

Many studies have focused on phenolic compounds, indicating that some of them can have strong inhibitory effect against fibrilization of α-synuclein, leading to stabilization of non-toxic oligomer species [9]. We have recently reported that stilbenes inhibit β-amyloid fibril formation [10]. Our findings suggest the formation of non-toxic soluble complexes between polyphenol and β-amyloid [11]. Here, we aim to assess the anti-aggregative behavior of resveratrol and piceatannol. We also investigated their ability to prevent α-synuclein toxicity against PC12 neural cells.

## Materials and methods

### Material

Purified recombinant human α-synuclein was purchased from Alexotech AB (Umeå, Sweden) and was used without further purification. Solutions of 140 µM α-synuclein were prepared in a 20 mM Na₂HPO₄, 140 mM NaCl buffer at pH 7.4 and sonicated 2 min prior to each experiment. Stilbenes were isolated from *Vitis vinifera* vine stalks [4]. Purity was controlled by HPLC measurements. The stilbenes were kept as 20 mM stock solutions in dimethylsulfoxide (DMSO).

### Thioflavin T based fluorescence assay

Solutions of 140 µM α-synuclein and 200 µM stilbenes were mixed with equal volumes in a 96-well plate with 20 µM Thioflavin T (ThT). The plate was incubated at 37°C, 300 rpm. Fluorescence was measured for 4 day with a Fluostar Optima plate reader (BMG Labtech, Germany) set at 450 nm for excitation and 485 nm for emission. Blanks of each compound were subtracted from the measured fluorescence. Each condition was triplicated.

### Observation on transmission electron microscopy

Drops of samples of α-synuclein aggregated alone or with stilbenes during 4 days were deposited during 2 min on carbon coated copper grids submitted to a glow discharge (0.3 mBar, 2 A). After quick washing in ultrapure water, negative staining using 4% uranyl acetate during 2 min was then performed. Transmission electron microscopy (TEM) observations were done with a CM120 electron microscope (FEI, Oregon, USA) using 2k × 2k USC1000 slow-scan CCD camera (Gatan, CA, USA).

<footnote n="1">¹Université de Bordeaux, ISVV, EA 3675 GESVAB, 33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²CBMN-UMR 5248 CNRS, Université de Bordeaux, IPB, Allée Geoffroy St. Hilaire, 33600 Pessac, France</footnote>
### MTT assay

PC12 cells, established from a rat pheochromocytoma, were obtained from the American Type Culture Collection (ATCC, Manassas, USA). PC12 cells were maintained in DMEM-Glutamax supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 15% fetal horse serum and 2.5% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2. α-synuclein was incubated at 200 µM during 2 days in order to be fully aggregated. The cells were subcultured in 96-well culture plates (30 × 103 cells/well) for 24 h and then treated with 500 nM of aggregated α-synuclein, with or without further addition of stilbenes at concentrations ranging from 5 to 30 µM, for 24 h, in serum-free culture medium. Compounds were dissolved in DMSO at a final concentration of 0.1%, which is a subtoxic concentration. Cell viability was determined by using MTT reduction assay. PC12 cells were incubated in 0.5 mg/mL MTT at 37°C for 3 h. Then, the MTT solution was removed and the resulting formazan crystals were dissolved with DMSO. Absorbance values were read at 540 nm on a microplate reader (MRII, Dynex). All samples were analyzed in triplicate.

## Results and discussion

Finding molecules to prevent the aggregation of α-synuclein could be a therapeutic goal in Parkinson's disease and relative diseases [10]. ThT fluorescence assays were used in presence or absence of stilbenes. In absence of inhibitor, α-synuclein exhibits a quasi-sigmoidal binding curve (Fig. 1).

<figure><img src="image_237.png" /><figcaption>**Fig. 1** - α-synuclein fibril formation. The α-synuclein was incubated for 4 days in presence of ThT. Results are expressed as mean fluorescence in arbitrary fluorescence unit.</figcaption></figure>

A first screening of stilbene anti-aggregative behavior was performed at a concentration of 100 µM of each compound. The results were expressed as the percentage of α-synuclein assembly in the absence of compound (taken to be 100%). A first screening of stilbene anti-aggregative behavior was performed. It appeared that most stilbenes have an inhibitory activity on the α-synuclein aggregation. Among them, piceatannol and resveratrol appeared to be two of the best inhibitors (Fig. 2). The piceatannol lowered the aggregation to an extent of 29% and resveratrol to 15%.

To observe the morphology of α-synuclein aggregates, electron microscopy studies were realized (Fig. 3). After incubation of α-synuclein alone, evident α-synuclein fibril extensions were observed. In presence of resveratrol or piceatannol only small amorphous aggregates were observed. These results confirm that stilbenes are potent inhibitors of α-synuclein aggregation.
<figure><img src="image_238.png" /></figure>

<figure><img src="image_239.png" /></figure>

<table><caption>**Fig. 2** - Structures of stilbenes and inhibition of α-synuclein fibrils. Results are expressed as percentage of aggregation, compared to the fluorescence signal of ThT when the protein is aggregated alone.</caption><thead><tr><td>compounds</td><td>% Fibril formation</td></tr></thead><tbody><tr><td>resveratrol</td><td>15 ± 5</td></tr><tr><td>piceatannol</td><td>29 ± 7</td></tr></tbody></table>

<figure><img src="image_240.png" /><figcaption>**Fig. 3** - Observation of α-synuclein aggregation states using transmission electron microscopy. The α-synuclein (A) was aggregated alone (B) or with 100 µM piceatannol (C) during 4 days. Scale bar indicates 300 nm.</figcaption></figure>

Finally cell viability measurement assays were performed using PC12 cells to assess the potent protective effects of stilbenes. As previously reported, aggregated α-synuclein reduces PC12 cell viability (Fig. 4). Dose dependency experiments demonstrate that resveratrol and piceatannol have cytoprotective effect. These findings indicate that resveratrol and piceatannol inhibited not only α-synuclein fibril formation but also the α-synuclein-induced cytotoxicity. As previously mentioned these results suggest that stilbenes lead to the formation of non-toxic α-synuclein/polyphenol complexes and may hence have therapeutic potential. Surprisingly, piceatannol is more active than resveratrol. It demonstrates complex relation between filament inhibition and cytotoxicity in this system including other potent protective effects of stilbenes.

<figure><img src="image_241.png" /><figcaption>**Fig. 4** - Protective effects of resveratrol and piceatannol against α-synuclein cytotoxicity in PC12 cells. Results are presented as a percentage of MTT reduction, with the values obtained in the absence of added α-synuclein taken as 100%.</figcaption></figure>
**Conclusion**

The inhibitory properties of stilbenes against α-synuclein fibril formation and toxicity were evaluated.
Results indicate that resveratrol and piceatannol reduce the aggregation and the toxicity of α-synuclein.

**Acknowledgements**

The authors wish to thank the *Conseil Regional d'Aquitaine* for their financial support of this research.

**References**

1. Kasiotis KM, Pratsinis H, Kletsas D et al (2013) Resveratrol and related stilbenes: their anti-aging and anti-angiogenic properties. *Food Chem Toxicol* **61**: 112-120.

2. Waffo-Téguo P, Krisa S, Pawlus AD et al (2013) Grapevine stilbenoids: bioavailability and neuroprotection, pp. 2275-2309. *In Handbook of Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes*, Springer Berlin Heidelberg, Berlin.

3. 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.

4. Rivière C, Pawlus AD, Mérillon JM (2012) Natural stilbenoids: distribution in the plant kingdom and chemotaxonomic interest in Vitaceae. *Nat Prod Rep* **29**: 1317-1333.

5. Reyes JF, Rey NL, Bousset L et al (2013) Alpha-synuclein transfers from neurons to oligodendrocytes. *Glia* **62**: 387-398.

6. Byers B, Cord B, Nguyen HN et al (2011) SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate α-synuclein and are susceptible to oxidative stress. *PLoS ONE* doi: 10.1371/journal.pone.0026159.

7. Lázaro DF, Rodrigues EF, Langohr R et al (2014) Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation. *PLoS Genet* doi: 10.1371/journal.pgen.1004741.

8. Winner B, Jappelli R, Maji SK et al (2011) In vivo demonstration that α-synuclein oligomers are toxic. *Proc Natl Acad Sci USA* **108**: 4194-4199.

9. Ardah MT, Paleologou KE, Lv G et al (2014) Structure activity relationship of phenolic acid inhibitors of α-synuclein fibril formation and toxicity. *Front Aging Neurosci* doi: 10.3389/fnagi.2014.00197.

10. Rivière C, Papastamoulis Y, Fortin PY et al (2010) New stilbene dimers against amyloid fibril formation. *Bioorg Med Chem Lett* **20**: 3441-3443.

11. Richard T, Poupard P, Nassra M et al (2011) Protective effect of ε-viniferin on β-amyloid peptide aggregation investigated by electrospray ionization mass spectrometry. *Bioorg Med Chem* **19**: 3152-3155.
# Centrifugal partition chromatography (CPC) gradient elution applied to grapevine stilbenoids purification

J. BISSON, A. PALOS-PINTO, M. BRUNEL, J.-M. MÉRILLON, P. WAFFO-TÉGUO*

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

Grapevine – CPC-MS – gradient elution – stilbenoids

## Introduction

Stilbenoids are structurally characterized by the presence of two aromatic rings joined by an ethylene bridge ($C_6-C_2-C_6$). They can be divided into two categories, monomers and oligomers which result from different oxidative couplings of the formers. While stilbenoids are found in a number of plant species [1, 2], grapes are the most significant dietary source. Currently, there are over 60 known stilbenoids found in the various parts of the grapevine (*Vitis vinifera*), the majority of which have received little or no biological testing. Since stilbenoids are important biologically active compounds, in regards to both vine and human health, they have been the subject of numerous biological studies. The stilbenoid monomer, resveratrol (*E*-3,5,4'-trihydroxystilbene), a naturally occurring phenolic compound, has been highlighted as a potential chemopreventive agent for several pathologies such as cardiovascular [3] and neurodegenerative diseases [4, 5] and different types of cancer [6].

To investigate these proposed activities further, large amounts of compounds for *in vivo* studies are required. The existing HPLC techniques are not suitable for the large-scale purification of these compounds due to irreversible adsorptions onto the solid phase material. For this reason, efforts have been devoted to the purification of these non-flavonoid polyphenols. Centrifugal Partition Chromatography (CPC), a very versatile separation technique that does not require a solid stationary phase, appeared to be the ideal tool for this purpose.

A classical isocratic elution profile has been successfully applied for the purification of phenolic compound from several natural sources [7]. This classical method was not powerful enough to separate simultaneously the major stilbenoids (*E*-piceatannol, *E*-resveratrol, and, *E*-ε-viniferin). In order to obtain an effective and efficient separation of these major compounds, CPC gradient between Arizona K [8] and Arizona K modified systems [9] was developed to achieve their separation in the same run. Moreover, following the preparative isolation, the fractions collected are usually analyzed by HPLC to evaluate their purity. These additional steps are time-consuming. To overcome this problem, a direct coupling of the CPC with a mass spectrometer was developed.

## Materials and methods

CPC separation was performed on CPC 200 apparatus from Kromaton technologies

Two biphasic systems were used to change the polarity of the mobile phase during the gradient elution. The former was Arizona K-ACN system (n-heptane/ethyl acetate/acetonitrile/water 1:2:0.82:2, v/v) and the latter was Arizona K system (n-heptane/ethyl acetate/methanol/water 1:2:1:2, v/v). The CPC column was first filled with the upper K-ACN phase and equilibrated with the lower K-ACN phase at 4 mL/min and with column rotation at 1300 rpm. The sample (1 g of crude vine extract) previously dissolved in 5 mL of each phase of Arizona K-ACN system was injected in the column in descending mode with the lower K-ACN phase during 20 min. Then mobile phase composition changed from 100% lower K-ACN phase to 100% lower K phase in 200 min. After the total time of 220 min, the extrusion step was performed with the upper K-ACN phase.
The output of the CPC was coupled to a mass-spectrometer. The CPC outgoing flow stream was divided thanks to a Rheodyne MRA100-000 active splitter [10]. This device allows a small amount of the CPC output to be mixed with the HPLC-MS solvent flow. The solvent used on the analytical flow-path of the active splitter chain is responsible both for dilution, effective phase mixing and correct ionization in the sample in the mass spectrometer. In this experiment, 0.1% formic acid in ethanol was used at the flow-rate of 1 mL/min. The active splitter was set at a 500:1 splitting ratio to provide the appropriate dilution of the highly concentrated CPC output into the DAD-MS analytical flow (Figure 1).

ESI Source parameters: drying gas was set at a flow-rate of 9.0 L/min and a temperature of 320°C, nebulizer pressure was set to 35 psi. ESI-MS parameters in negative mode: HV capillary 4300 V, end plate offset -500 V, capillary exit -176.4 V, skimmer -29.5 V, trap drive 75, scan delay 25000 µs, rolling average 10, and trap average 10.

<figure><img src="image_242.png" /><figcaption>Fig. 1 - CPC/ESI-MS workstation.</figcaption></figure>

## Results and discussion

The crude extract of grapevine contained three major stilbenoids: two monomers, *E*-piceatannol, and *E*-resveratrol, and a dimer, *E*-ε-viniferin. To separate these major stilbenoids from the grapevine crude extract in one step CPC, a rarely used approach using an elution gradient was adopted, consisting in a change of mobile phase polarity along the elution while keeping the stationary phase the same. A gradient between Arizona system K with and without acetonitrile has been used as these showed adequate and radically different separation abilities. The Arizona K system with acetonitrile (Arizona K-ACN) was first used in descending mode, then the composition of the mobile phase was changed from the Arizona K-ACN system to the Arizona K system. While gradients involving different solvent systems are usually avoided due to the belief that they could reduce the efficacy of the separation, the results obtained here are very encouraging and prove that this is a viable approach. The resulting CPC fractions are well defined (Figure 2).

<figure><img src="image_243.png" /><figcaption>Fig. 2 - CPC-UV-MS chromatogram of crude extract of grapevine cane.</figcaption></figure>
Figure 3 shows the HPLC chromatograms of pallidol, *E*-piceatannol, *E*-resveratrol, and *E*-ε-viniferin obtained by CPC by using the gradient elution. All these major stilbenoids were purified from a crude extract in only one step at a preparative-scale, and due to the nature of CPC separations can be scaled-up with the advantage of a better resolution as the column size increases. As the HPLC-purities obtained here were close or over 90%, this approach shows a significant improvement over the more classical approaches using solid phase chromatography.

<figure><img src="image_244.png" /><figcaption>**Fig. 3** - HPLC-UV chromatograms of the major stilbenes obtained from the crude extract of grapevine canes using one step CPC gradient.</figcaption></figure>

Conclusion

In the recent years, the development of gradient elution in CPC has allowed the successful separation of natural products such as stilbenoids [10] and anthocyanins [11, 12]. In this study, we have applied CPC gradient elution using a modified solvent system to successfully purify four stilbenes from a crude extract of grapevine cane in the same run with an HPLC purity close or even greater than 90%. Moreover, the output of CPC was directly coupled to the mass spectrometer. This allowed us to shorten experimentation times by optimizing the gradient profile. Due to the exceptional abilities of CPC to be scaled-up, this method can be applied with bigger column volumes thus providing both a higher throughput and a better resolution likely leading to a greater yield. Another advantage of this chromatographic approach is the reproducibility of the elution profiles even between different column sizes, thus eliminating the need of an online hyphenation during scaled-up productions.

References

1. Shen T, Wang X-N, Lou H-X (2009) Natural stilbenes: an overview. *Nat Prod Rep* **26**: 916-935.

2. Rivière C, Pawlus AD, Mérillon J-M (2012) Natural stilbenoids: distribution in the plant kingdom and chemotaxonomic interest in Vitaceae. *Nat Prod Rep* **29**: 1317-1333.

3. Vella RK, Pullen C, Coulson FR et al (2015) Resveratrol prevents cardiovascular complications in the SHR/STZ rat by reductions in oxidative stress and inflammation. *BioMed Res Int* **2015**: 918123.

4. Williams P, Sorribas A, Howes MJ (2011) Natural products as a source of Alzheimer's drug leads. *Nat Prod Rep* **28**: 48-77.

5. Singh N, Agrawal M, Doré S (2013) Neuroprotective properties and mechanisms of resveratrol in in vitro and in vivo experimental cerebral stroke models. *ACS Chem Neurosci* **4**: 1151-1162.

6. Huang X, Zhu HL (2011) Resveratrol and its analogues: promising antitumor agents. *Anticancer Agents Med Chem* **11**: 479-490.

7. Marston A, Hostettmann K (2006) Developments in the application of counter-current chromatography to plant analysis. *J Chromatogr A* **1112**: 181-194.

8. Foucault AP, Chevolot L (1998) Counter-current chromatography: instrumentation, solvent selection and some recent applications to natural product purification. *J Chromatogr A* **808**: 3-22.
9. Bisson J (2012) Développements méthodologiques en chromatographie de partage : application aux stilbénoïdes. *Thèse de Doctorat*, Université de Bordeaux 2, http://www.theses.fr/2012BOR22006.

10. Bisson J, Poupard P, Pawlus AD et al (2011) Development of hybrid elution systems for efficient purification of stilbenoids using centrifugal partition chromatography coupled to mass spectrometry. *J Chromatogr A* **1218**: 6079-6084.

11. Renault JH, Thepenier P, Zeches-Hanrot M et al (1995) Separation of the two major anthocyanins from Champagne vintage by-products by gradient elution centrifugal partition chromatography. *J Liq Chromatogr* **18**: 1663-1670.

12. Renault J-H, Thépenier P, Zéches-Hanrot M et al (1997) Preparative separation of anthocyanins by gradient elution centrifugal partition chromatography. *J Chromatogr A* **763**: 345-352.
# Caractérisation électrochimique des polyphénols
dans les vins rouges

F. GARCIA<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, E. NEWAIR<fnref n="4" />, C. SAUCIER<fnref n="1" />,<fnref n="2" />,<fnref n="3" />*, H. FULCRAND<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Voltammétrie à vague carrée – carbone – polyphénols – vin rouge

## Introduction

Les polyphénols sont des composés ayant des propriétés réductrices et peuvent par conséquent être oxydés sur une électrode inerte. Leur caractérisation par des méthodes électrochimiques est donc possible. Parmi les différentes techniques électrochimiques existantes, la voltammétrie cyclique s'est imposée comme la technique la plus utilisée permettant de caractériser les polyphénols et les antioxydants dans les boissons, et plus particulièrement dans les vins [1-4]. On peut citer également la voltammétrie impulsionnelle différentielle [3, 5]. Le but de cette étude est de montrer que la voltammétrie à vague carrée peut également être utilisée lors de la caractérisation électrochimique des polyphénols dans les vins rouges à la fois avec une électrode classique mais aussi par des électrodes sérigraphiées jetables, cette technique permettant d'obtenir un seuil de détection faible [4].

## Matériel et méthodes

### Voltammétrie

Les mesures de voltammétrie à vague carrée ont été effectuées à l'aide d'un potentiostat (Autolab PGSTAT 302N, Metrohm, Suisse) et du logiciel GPES 4.9 (Metrohm, Suisse). Les paramètres pour les voltammétries à vague carrée ont été : fréquence 25 Hz, pas de potentiel 4 mV, amplitude 20 mV, et plage de potentiels de -0,2 V à 1,2 V.

Les mesures avec l'électrode en carbone vitreux ont été effectuées dans une cellule électrochimique classique à l'aide d'un montage à 3 électrodes (Metrohm, Suisse) : une électrode de travail en carbone vitreux de diamètre 3 mm, une électrode de référence (Ag/AgCl) et une contre électrode en platine. Avant chaque mesure, la surface de l'électrode de travail a été polie, rincée à l'eau (Millipore) puis plongée dans un bain à ultra-sons. Les mesures ont été effectuées immédiatement après avoir introduit l'électrode dans la solution à étudier. Pour les mesures avec les électrodes sérigraphiées, différentes électrodes ( dropsens, Espagne) ont été utilisées : électrode de référence en argent, contre électrode en carbone et électrode de travail de diamètre 4 mm en carbone, nanotubes de carbone monofeuillets et nanotubes de carbone multifeuillets. Les mesures ont été effectuées immédiatement après le dépôt de 200 µL de solution à étudier sur les électrodes.

### Spectrophotométrie

Les mesures spectrophotométriques de l'IPT (indice des polyphénols totaux) selon [6], de l'Indice de Folin-Ciocalteu selon [5] et de la capacité antioxydante par la méthode ABTS selon [7] ont été réalisées à l'aide d'un spectrophotomètre Safas UVmc<fnref n="2" /> (Monaco). La quantité de polyphénols totaux a été dosée par mesure de l'absorbance à 280 nm après séparation des composés par HPLC (Waters 2690/Waters 996 DBD et logiciel Millenium 32, Waters, USA) selon [6].

### Molécules standards

Les molécules standards (acide caféïque, acide gallique, malvidine-3-O-glucoside et catéchine) ont été dissoutes et les vins dilués dans une solution hydro-alcoolique modèle (12% vol. d'éthanol, concentration en acide tartrique de 0,033 M et pH=3,6).

<footnote n="1">¹INRA, UMR 1083 SPO, 34060 Montpellier, France</footnote>

<footnote n="2">²Montpellier SupAgro, UMR 1083 SPO, 34060 Montpellier, France</footnote>

<footnote n="3">³Université de Montpellier, UMR 1083 SPO, 34060 Montpellier, France</footnote>

<footnote n="4">⁴Université de Sohag, 82524 Sohag, Egypte</footnote>
## Vins

5 vins rouges commerciaux issus de 2 millésimes, de différents cépages et régions viticoles du Sud de la France ont été caractérisés : AOP Coteaux du Languedoc (Syrah/Grenache, 2012), AOP Puisseguin Saint Emilion (Merlot, 2010), AOP Cahors (Malbec, 2010), AOP Côtes du Rhône (Grenache/Mourvèdre/Syrah (60%/20%/20%), 2012) et IGP Pays d'OC (Pinot noir, 2012).

# Résultats et discussion

## Voltammétrie à vague carrée des molécules standards

Concernant l'électrode en carbone vitreux (concentration 0,1 mM), pour l'acide caféique, un seul pic anodique sur les voltammogrammes à vague carrée est observé à 0,42 V dans la plage de potentiels étudiés (0 V à 1,2 V) alors que pour l'acide gallique et la catéchine (Fig. 1), 2 pics sont présents, le premier pic étant à 0,38 V pour ces 2 composés. Le pic pour l'acide caféique ainsi que le premier pic pour l'acide gallique et la catéchine correspondent à l'oxydation des groupements hydroxyles en position ortho du diphénol, du pyrogallol et du noyau B de ces composés respectivement. Pour l'acide gallique et la catéchine, un second pic anodique est observé à 0,75 V. Pour cette dernière, ce second pic correspond à l'oxydation des groupements hydroxyles en position méta sur le noyau A alors que pour l'acide gallique, le pic est du à l'oxydation du troisième groupement hydroxyle sur le pyrogallol, adjacent aux groupements hydroxyles déjà oxydés. Enfin, en ce qui concerne la malvidine-3-O-glucoside, un seul pic d'oxydation à 0,66 V est observé aux concentrations étudiées (2 µM et 4 µM). Il correspond à l'oxydation du groupement hydroxyle du noyau B. Pour les électrodes sérigraphiées (plage de potentiels étudiés de -0,2 V à 0,8 V, concentration 0,5 mM), les voltammogrammes (molécules standards et vins) les mieux définis ont été obtenus avec les nanotubes de carbone monofeuillets et des potentiels de pics de 0,16 V pour l'acide caféique, 0,15V et 0,48 V pour l'acide gallique, 0,16 V et 0,5 V pour la catéchine, et enfin 0,4 V pour la malvidine-3-O-glucoside (concentration 0,1 mM) ont été obtenus (les valeurs de potentiels de pics étant quasi identiques pour les autres matériaux). Il est à noter que les pics sont décalés vers de plus forts potentiels lorsque la concentration en molécules augmente.

<figure><img src="image_245.png" /><figcaption>**Fig. 1** - Voltammogramme à vague carrée d'une solution de catéchine 0,1 mM dans une solution hydro-alcoolique modèle (électrode en carbone vitreux).</figcaption></figure>

## Voltammétrie à vague carrée des vins

Les voltammogrammes à vague carrée pour les vins rouges dilués 60 fois présentent 3 pics caractéristiques pour les techniques électrochimiques [1-3, 5] (Fig. 2A et 2B). Pour l'électrode en carbone vitreux, le premier pic (qui représente les molécules les plus facilement oxydables) à environ 0,4 V correspond au potentiel attendu pour l'oxydation des o-diphénols (noyau B) de molécules présentes dans le vin (flavanols, polymères). Pour ce pic, l'oxydation d'acides phénols et de flavonols intervient également [2]. Le pic central à environ 0,6 V quant à lui représente l'oxydation du noyau B des anthocyanes de type malvidine. Enfin, le troisième pic à environ 0,9 V correspond à l'oxydation des m-diphénols (noyau A) des molécules présentes dans le vin. Pour ce dernier pic, l'oxydation d'acides phénols et de flavonols se produit également [2]. Il en est de même avec l'utilisation des électrodes sérigraphiées (nanotubes de carbone monofeuillets) avec 3 pics à environ 0,15 V, 0,35 V et 0,6 V.
<figure><img src="image_246.png" /><figcaption>**Fig. 2** - Voltammogrammes à vague carrée des différents vins étudiés dilués 60 fois dans une solution hydro-alcoolique modèle (A. électrode en carbone vitreux et B. électrodes sérigraphiées (nanotubes de carbone en monofeuillets)).</figcaption></figure>

*Capacité antioxydante des vins*

L'aire sous la courbe des voltammogrammes peut représenter la capacité anti-oxydante des vins. Les valeurs de cette capacité anti-oxydante ont été comparées pour les différents vins aux valeurs de l'IPT, de l'Indice de Folin-Ciocalteu (exprimé en équivalent d'acide gallique), de la capacité oxydante mesurée par la méthode ABTS (exprimée en équivalent de Trolox) ainsi qu'aux valeurs de la somme des aires des pics obtenus à 280 nm (exprimée en équivalent d'acide gallique) après séparation par chromatographie liquide. Les valeurs obtenues par voltammétrie à vague carrée suivent majoritairement le même ordre que celles des 4 grandeurs citées ci-dessus.

**Conclusion**

La voltammétrie à vague carrée est une technique électrochimique analytique qui peut être utilisée pour la caractérisation électrochimique des polyphénols dans les vins rouges à la fois avec une électrode classique en carbone vitreux mais aussi avec des électrodes sérigraphiées jetables en nanotubes de carbone monofeuillets. Les voltammogrammes présentent 3 pics d'oxydation dans la plage de potentiels étudiés. Une capacité antioxydante des vins peut être estimée à partir des voltammogrammes.

**Remerciements**

Les auteurs remercient l'Institut Français d'Egypte de son soutien financier lors de la venue du docteur Emad Newair au sein de l'UMR Sciences pour l'Œnologie.

**Références**

1. Kilmartin PA, Zou H, Waterhouse AL (2002) Correlation of wine phenolic composition versus cyclic voltammetry response. *Am J Enol Vitic* **53**: 294-302.

2. Makhotkina O, Kilmartin PA (2010) The use of cyclic voltammetry for wine analysis: determination of polyphenols and free sulfur dioxide. *Anal Chim Acta* **668**: 155-165.

3. Rebelo MJ, Rego R, Ferreira M et al (2013) Comparative study of the antioxidant capacity and polyphenol content of Douro wines by chemical and electrochemical methods. *Food Chem* **141**: 566-573.

4. Bordonaba JG, Terry LA (2012) Electrochemical behaviour of polyphenol rich fruit juices using disposable screen-printed carbon electrodes: towards a rapid sensor for antioxidant capacity and individual antioxidants. *Talanta* **90**: 38-45.

5. Seruga M, Novak I, Jakobek L (2011) Determination of polyphenols content and antioxidant activity of some red wines by differential pulse voltammetry, HPLC and spectrophotometric methods. *Food Chem* **124**: 1208-1216.

6. 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.

7. Guedes AC, Amaro HM, Giao MS et al (2013) Optimization of ABTS radical cation assay specifically for determination of antioxidant capacity of intracellular extracts of microalgae and cyanobacteria. *Food Chem* **138**: 638-643.
# Oak wood ellagitannin changes during toasting

K. CHIRA¹,²*, M. JOURDES¹,², P.L. TEISSEDRE¹,²

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

²INRA, ISVV, USC 1366 INRA, IPB, 210 Chemin de Leysotte, F-33140 Villenave d'Ornon, France

Oak wood – ellagitannin – toasting – UPLC-MS (Q-TOF)

## Introduction

Ellagitannins (ETs) have been reported to be the main phenolic compounds found in oak wood. These compounds, belonging to the hydrolysable tannin class of polyphenols, are esters of hexahydroxydiphenic acid (HHDP) and a polyol, usually glucose or quinic acid [1]. ETs can occur as complex polymers reaching high molecular weights, which makes accurate identification difficult. The toasting process during barrel's manufacturing burns the inner oak wood barrel surface with an open fire, inducing severe changes in ellagitannin structures and compositions. Up to now, only one research group using purified oak ellagitannins showed that during the toasting process castalagin is oxidized whereas its diastereomer vescalagin is reduced [2]. Therefore, thermal ellagitannin products or the reaction mechanisms underlying the ellagitannin degradation have not been well investigated. The purpose of the present study was to examine oak wood ellagitannin changes during toasting.

## Materials and methods

### Extraction and pre-purification of ellagitannins

50 g of untoasted oak chips (Quercus robur/Quercus petraea) were extracted using acetone/water (70/30, v/v, 3 times × 1 L) overnight at ambient temperature under agitation. The obtained mixture was filtrated, evaporated under reduced pressure, dissolved in water, and freeze-dried to give a crude oak ellagitannin extract. 100 mg of this crude ellagitannin extract was dissolved in 10 mL of water and then fractionated on column using Toyopearl TSK HW-40 (F) gel from Tosoh Corp. After loading the sample, the column was washed with 100 mL of distilled water then successive elutions were performed using 300 mL of water/acetone/formic acid (70/29.6/0.4, v/v/v). Ellagitannins were eluted in the acetone/water fraction. This fraction was dried under reduced pressure and then dissolved in 10 mL of water in order to be further fractionated on a C-18 column. This column was washed with 50 mL of water and eluted with 150 mL of methanol/water/formic acid (15/84.6/0.4, v/v/v). The purity of all the obtained fractions was estimated after total ellagitannin concentration and ellagitannin composition determination by HPLC-UV [3].

### Thermal treatment of ellagitannins

The above obtained fraction containing the eight main ellagitannins was dry-heated in a lab oven for 60 min, 120 min and 180 min at 220°C. After cooling, it was further fractionated on C-18 column before being injected into the UPLC-MS (Q-TOF).

### UPLC-MS (Q-TOF)

The UPLC-MS system used was an Agilent 1290 Infinity equipped with an ESI-Q-TOF mass spectrometer (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 0.3 mL/min. The gradient of solvent B was as follows: 1% to 3% of B in 5 min; 3% to 8% of B in 2 min; 8% to 12.50% of B in 7 min; 12.50% to 98% of B in 1 min; 98% to 1% of B in 4 min; stay at 1% of B during 4 min. ESI conditions were as follows: gas temperature and flow were 300°C and 9 L/min respectively; sheath gas temperature and flow were 350°C and 11 L/min respectively; capillary voltage was 3500 V. The collision energy used for MS² was 20%. The obtained data were treated by MassHunter Qualitative Analysis software.

<footnote>*Corresponding author: kleopatra.chira@u-bordeaux.fr</footnote>